Assay methods using chemiluminescent dioxetane compounds

Abstract

The presently claimed and described technology provides methods for chemiluminescence-based assays employing 1,2 dioxetane compounds. In some instances, the analyte is cardiac troponin I (TNI), Thyroid-stimulating Hormone (TSH), or procalcitonin (PCT). Methods disclosed herein comprise: exposing a biological sample to a capture antibody configured to bind to at least one portion of an analyte disclosed herein generating a first reaction mixture; exposing the first reaction mixture to an enzyme-conjugated affinity molecule, forming a second reaction mixture; exposing the second reaction mixture to a substrate formulation comprising a 1,2 dioxetane compound disclosed herein; wherein the reaction between the enzyme-conjugated affinity molecule and the substrate formulation generates a chemiluminescent detection signal; recording the detection signal generated by the reaction; and comparing the recorded signal to a calibration curve to quantify the level of the analyte in the biological sample.

Description

ASSAY METHODS USING CHEMILUMINESCENT DIOXETANE COMPOUNDSRELATED APPLICATIONS

[0001] The present patent application claims the priority benefit of U.S. Provisional Patent Application Ser. No. 63 / 625,439, filed January 26, 2024, the content of which is hereby incorporated by reference in its entirety into this disclosure.INCORPORATION BY REFERENCE OF AN ELECTRONIC SEQUENCE LISTING

[0002] This application contains a Sequence listing that has been submitted in a computer readable format and is hereby incorporated by reference in its entirety. The computer readable file, created on January 20, 2025, is named 67670W001_seq.xml and is 3,860 bytes in size.BACKGROUND

[0003] Immunoassays are an important analytical tool for the identification and detection of specific substances in a sample and are routinely used to detect and quantify clinically important blood proteins. Many medical decisions are based on the diagnostic results from these assays, making sensitivity and specificity extremely important. Chemiluminescence-based immunoassays can offer a rapid response to the presence of analytes and excellent sensitivity because unlike fluorescence and absorption-based assays, no light excitation is required. A need exists for assay methods that allow for more sensitive detection of analytes and a higher signal to background ratio than prior substrates.BRIEF SUMMARY

[0004] One aspect of the invention is a method of detecting an analyte in a biological sample, the method comprising; exposing the biological sample to a capture antibody configured to bind to at least one portion of an analyte, generating a first reaction mixture; exposing the first reaction mixture to an enzyme-conjugated antibody or an enzyme-conjugated antigen, generating a second reaction mixture; exposing the second reaction mixture to a substrate formulation comprising a 1,2 dioxetane compound and at least one phosphonium surfactant; wherein the reaction between the enzyme-conjugated antibody or the enzyme-conjugated antigen and the substrate formulationgenerates a chemiluminescent detection signal; recording the detection signal generated hy the reaction; and comparing the recorded signal to a calibration curve to quantify the level of the analyte in the biological sample.

[0005] One aspect of the invention is method of detecting an analyte in a biological sample, the method comprising exposing the biological sample to a capture antibody configured to bind to at least one portion of an analyte, generating a first reaction mixture; exposing the first reaction mixture to an enzyme-conjugated antibody or an enzyme-conjugated antigen, generating a second reaction mixture; exposing the second reaction mixture to a substrate formulation comprising a compound of Formula I or a salt thereof:

[0006] Formula I

[0007] wherein each of R1 and R2 are independently C3-C10 alkyl, or R1 and R2 taken together with the carbon to which they are attaches provide a C5-C10 cycloalkyl ring;

[0008] R3 is C1-C10 alkyl, C6-C10 aryl, or hetero aryl;

[0009] R4 is C2-CI0 alkenyl;

[0010] R5 is H or C1-C10 alkyl; and

[0011] X is a phosphate;

[0012] wherein the reaction between the enzyme-conjugated antibody or the enzyme-conjugated antigen and the substrate formulation generates a chemiluminescent detection signal; recording the detection signal generated by the reaction; and comparing the recorded signal to a calibration curve to quantify the level of the analyte in the biological sample.

[0013] In an aspect, the substrate formulation further comprises at least one phosphonium surfactant.

[0014] In an aspect, the enzyme-conjugated antibody is configured to bind at least one portion of an analyte. In an aspect, the enzyme-conjugated antigen is configured to bind at least one portion of the capture antibody.

[0015] One aspect of the invention is a method of detecting cardiac troponin I (TNI) in a biological sample, the method comprising: exposing the biological sample to a capture antibody configured to bind to at least one portion of TNI, generating a first reaction mixture; exposing the first reactionmixture to an enzyme-conjugated affinity molecule, forming a second reaction mixture; exposing the second reaction mixture to a substrate formulation comprising a 1,2 dioxctanc compound and at least one phosphonium surfactant; wherein the reaction between the enzyme-conjugated affinity molecule and the substrate formulation generates a chemiluminescent detection signal; recording the detection signal generated by the reaction; and comparing the recorded signal to a calibration curve to quantify the level of the TNI in the biological sample.

[0016] In an aspect, the levels of TNI quantified have a concentration that is at least IX greater than a limit of quantification (LoQ) for the method, alternatively at least 2X greater than the LoQ, or alternatively at least 3X greater than the LoQ.

[0017] In an aspect, the ratio of the signal produced by cleavage of the enzyme to the background noise is at least about 1, alternatively at least about 10, alternatively at least about 20, alternatively at least about 30, alternatively at least about 40, alternatively at least about 50, alternatively at least about 100, alternatively at least about 200, alternatively at least about 300, alternatively at least about 400, alternatively at least about 500, alternatively at least about 600, alternatively at least about 700, alternatively at least about 800, alternatively at least about 900, alternatively at least about 1,000, alternatively at least about 2,000, alternatively at least about 3,000, alternatively at least about 4,000, alternatively at least about 5,000, alternatively at least about 6,000, alternatively at least about 7,000, alternatively at least about 8,000, alternatively at least about 9,000, alternatively at least about 10,000, alternatively at least about 15,000, alternatively at least about 20,000, or alternatively at least about 25,000.

[0018] In an aspect, affinity molecule is an antibody, a monoclonal antibody, a polyclonal antibody, a synthetic antibody mimic, an aptamer, an affimer, DARPins, oligonucleotide, peptide, or antigen. In an aspect, the affinity molecule is an antibody, wherein the antibody is configured to bind at least one portion of TNI.

[0019] One aspect of the invention is, a method of detecting thyroid stimulating hormone (TSH) in a biological sample, the method comprising: exposing the biological sample to a capture antibody configured to bind to at least one portion of TSH, generating a first reaction mixture; exposing the first reaction mixture to an enzyme-conjugated affinity molecule, forming a second reaction mixture; exposing the second reaction mixture to a substrate formulation comprising a 1,2 dioxetane compound and at least one phosphonium surfactant; wherein the reaction between the enzyme-conjugated affinity molecule and the substrate formulation generates a chemiluminescentdetection signal; recording the detection signal generated by the reaction; and comparing the recorded signal to a calibration curve to quantify the level of the TSH in the biological sample.

[0020] In an aspect, the levels of TSH quantified have a concentration that is at least IX greater than a limit of quantification (LoQ) for the method, alternatively at least 2X greater than the LoQ, or alternatively at least 3X greater than the LoQ.

[0021] In an aspect, the ratio of the signal produced by cleavage of the enzyme to the background noise is at least about 5, alternatively at least about 10, alternatively at least about 20, alternatively at least about 30, alternatively at least about 40, alternatively at least about 50, alternatively at least about 60, alternatively at least about 70, alternatively at least about 80, alternatively at least about 90, alternatively at least about 100, alternatively at least about 200, alternatively at least about 300, alternatively at least about 400, alternatively at least about 500, alternatively at least about 600, alternatively at least about 700, alternatively at least about 800, alternatively at least about 900, alternatively at least about 1,000, alternatively at least about 2,000, alternatively at least about 3,000, alternatively at least about 4,000, alternatively at least about 5,000, alternatively at least about 6,000, alternatively at least about 7,000, or alternatively at least about 8,000.

[0022] In an aspect, the affinity molecule is an antibody, wherein the antibody is configured to bind at least one portion of TSH.

[0023] One aspect of the disclosure is a method of detecting procalcitonin (PCT) in a biological sample, the method comprising: exposing the biological sample to a capture antibody configured to bind to at least one portion of PCT, generating a first reaction mixture; exposing the first reaction mixture to an enzyme-conjugated affinity molecule, forming a second reaction mixture; exposing the second reaction mixture to a substrate formulation comprising a 1,2 dioxetane compound and at least one phosphonium surfactant; wherein the reaction between the enzyme-conjugated affinity molecule and the substrate formulation generates a chemiluminescent detection signal; recording the detection signal generated by the reaction; and comparing the recorded signal to a calibration curve to quantify the level of the PCT in the biological sample.

[0024] In an aspect, the assay has a limit of quantification (LoQ) of about 100 fg / mL or less, 50 fg / mL or less, or 34 fg / mL or less.

[0025] In an aspect, the method has a coefficient of variation (CV) of 20% or less, alternatively 15% or less, alternatively 10% or less, alternatively 5% or less, or alternatively 4% or less.

[0026] In an aspect, the levels of PCT detected have a concentration that is at least 1 X greater than a limit of quantification (LoQ) for the method, alternatively at least 2X greater than the LoQ, or alternatively at least 3X greater than the LoQ.

[0027] In an aspect, the ratio of the signal produced by cleavage of the enzyme to the background noise is at least about 2,000, alternatively at least about 3,000, alternatively at least about 4,000, or alternatively at least about 5,000.

[0028] In an aspect, the affinity molecule is an antibody, wherein the antibody is configured to bind at least one portion of PCT.

[0029] In an aspect, the affinity molecule is an antigen, wherein the antigen is configured to bind at least one portion of the capture antibody.

[0030] In an aspect, the capture antibody and / or enzyme-conjugated affinity molecule is conjugated to at least one magnetic bead.

[0031] In an aspect, the enzyme comprises an alkaline phosphatase (AP).

[0032] In an aspect, the signal produced is a least about 10X greater, alternatively at least about 20X greater, or alternatively at least about 30X greater than a signal produced by the method, wherein the substrate formulation comprises a compound of the following formula:

[0033]

[0034] In an aspect, the 1,2 dioxetane compound is a compound of Formula I or a salt thereof:Formula I

[0035] wherein each of R1and R2are independently C3-C10 alkyl, or R1and R2taken together with the carbon to which they are attaches provide a C5-C10 cycloalkyl ring;

[0036] R3is C1-C10 alkyl, Ce-Cio aryl, or heteroaryl;

[0037] R4is C2-C10 alkenyl;

[0038] R5is H or C1-C10 alkyl; and

[0039] X is a phosphate.

[0040] In an aspect, the 1,2 dioxetane compound is a compound of Formula II or a salt thereof:

[0041] Formula II

[0042] wherein each of R10and R11is independently H, halogen, C1-C10 alkyl, C2-C10 alkenyl, orCe-Cio aryl;

[0043] R3is C1-C10 alkyl, Ce-Cio aryl or heteroaryl;

[0044] R4is C2-C10 alkenyl;

[0045] R5is H or C1-C10 alkyl; and

[0046] X is a phosphate.

[0047] In an aspect, the 1,2 dioxetane compound is a compound of Formula III or a salt thereof:Formula III

[0048] wherein each of R10and R11is independently H, halogen, C1-C10 alkyl, C2-C10 alkenyl, orCe-Cio aryl;

[0049] R3is C1-C10 alkyl, Ce-Cio aryl or heteroaryl;

[0050] R5is H or C1-C10 alkyl; and

[0051] X is a phosphate.

[0052] In an aspect, the 1,2 dioxetane compound is a compound of Formula IV or a salt thereof:

[0053] Formula IV

[0054] wherein R3is Ci-Cio alkyl, Ce-Cio aryl or heteroaryl;

[0055] R5is H or Ci-Cio alkyl; and X is a phosphate.

[0056] In an aspect the wherein the 1,2 dioxetane compound is

[0057]

[0058] or a salt thereof. ’

[0059] In an aspect, the 1,2 dioxetane compound is 4-methoxy-4-(3-phosphatephenyl)spiro[l,2- dioetane-3,2’-adamantane] or a salt thereof.

[0060] In an aspect, the phosphonium surfactant is selected from the group consisting of small molecule phosphonium surfactants and polymeric phosphonium surfactants.

[0061] In an aspect, the small molecule phosphonium surfactant is a compound having the formula:

[0062]

[0063] wherein R12-R14 are each independently C1-C10 alkyl;

[0064] R15 is arylalkyl; and

[0065] X" is a counterion.

[0066] In an aspect the polymeric phosphonoium surfactant comprises repeating unit (A), repeating unit (B), or both:

[0068] wherein Bus is tributyl and Octa is trioctyl.

[0069] In an aspect, the substrate formulation further comprises a magnesium (II) salt.

[0070] In an aspect, the method is an assay and is performed using an immunoassay analyzer, wherein the immunoassay analyzer comprises: a reagent pack configured to hold a plurality of reagent vessels, wherein each reagent vessel is configured to store a volume of reagent required for at least one instance of the assay, wherein at least one reagent comprises the capture antibody and at least one reagent comprises the enzyme-conjugated affinity molecule; a pipettor arrangement comprising at least one reagent pipettor and at least one sample pipettor; and a detector arrangement.

[0071] In an aspect, the reagent vessels comprise an elastomeric self-sealing membrane.

[0072] In an aspect, the reagent pack further comprises containment walls arranged between the reagent vessels.

[0073] In an aspect, the immunoassay analyzer further comprises a reagent storage unit, wherein the reagent pack is housed in the reagent storage unit.

[0074] In an aspect, the pipettor arrangement comprises at least a first reagent pipettor, a second reagent pipettor, a third reagent pipettor and at least one sample pipettor.

[0075] In an aspect, the pipettor arrangement further comprises at least a fourth reagent pipettor.

[0076] In an aspect, the first reagent pipettor, second reagent pipettor, third reagent pipettor, and / or fourth reagent pipettor is selectively and / or simultaneously operated.

[0077] In an aspect, the first reagent pipettor, second reagent pipettor, third reagent pipettor, and / or fourth reagent pipettor, are configured to engage a dispense tip prior to aspiration.

[0078] In an aspect, the method is configured to analyze at least about 200 biological samples / hr.

[0079] In an aspect, the method is configured to analyze at least about 300 biological samples / hr.

[0080] In an aspect, the method is configured to analyze at least about 400 biological samples / hr.

[0081] In an aspect, the first reaction mixture is generated by aspirating a portion of the biological sample from a sample vessel and dispensing the aspirated biological sample into a reaction vessel of the immunoassay analyzer and aspirating a portion of a first reagent comprising the capture antibody from at least one reagent vessel and dispensing the aspirated reagent into the reaction vessel; the second reaction mixture is generated by aspirating a portion of a second reagent comprising the enzyme-conjugated affinity molecule from at least one reagent vessel and dispensing the aspirated reagent into the reaction vessel; and a detection mixture is generated byaspirating the substrate formulation and dispensing the aspirated substrate formation into the reaction vessel.

[0082] In an aspect, incubation time of the first reaction mixture is at least about 30 minutes, alternatively at least about 40 minutes, alternatively at least about 50 minutes, or alternatively at least about 60 minutes.

[0083] In an aspect, incubation time of the second reaction mixture is at least about 2 minutes, alternatively is at least about 5 minutes, alternatively at least about 8 minutes, or alternatively at least about 10 minutes.

[0084] In an aspect, the detection mixture is incubated for at least 20 seconds, alternatively at least 30 seconds, alternatively at least 40 seconds, alternatively at least 50 seconds, alternatively at least 60 seconds, alternatively at least 70 seconds, alternatively at least 80 seconds, alternatively at least 90 seconds, alternatively at least 100 seconds, alternatively at least 110 seconds, alternatively at least 120 seconds, alternatively at least 130 seconds, alternatively at least 140 seconds, alternatively at least 150 seconds, alternatively at least 160 seconds, alternatively at least 170 seconds, alternatively at least 180 seconds, alternatively at least 190 seconds, alternatively at least 200 seconds, alternatively at least 210 seconds, alternatively at least 220 seconds, alternatively at least 230 seconds, alternatively at least 240 seconds, alternatively at least 250 seconds, alternatively at least 260 seconds, alternatively at least 270 seconds, or alternatively at least 280 seconds.

[0085] In an aspect, cycle time is about 45 seconds or less, alternatively about 40 seconds or less, alternatively about 35 seconds or less, alternatively about 30 seconds or less, alternatively about 25 seconds or less, alternatively about 20 seconds or less, or alternatively about 15 seconds.

[0086] In an aspect, time to first result (TTFR) is about 60 minutes or less, alternatively about 55 minutes or less, alternatively about 50 minutes or less, 45 minutes or less, alternatively about 40 minutes or less, alternatively about 35 minutes or less, alternatively about 30 minutes or less, alternatively about 25 minutes or less, alternatively about 20 minutes or less, alternatively about 15 minutes or less, or alternatively about 10 minutes or less.

[0087] In an aspect, the detector arrangement comprises a light detector configured to sense photons emitted from assay reactions over a period of time, an analog circuit configured to provide an analog signal based on the photons emitted from the assay reactions over the period of time,and a counter circuit configured to provide a photon count based on the photons emitted from the assay reactions over the period of time.

[0088] In an aspect, the immunoassay analyzer further comprises an ultrasonic mixing module. In an aspect, the first reaction mixture, second reaction mixture, and / or detection mixture is agitated via the ultrasonic mixing module.

[0089] In an aspect, the first reaction mixture, second reaction mixture, and / or detection mixture comprise unreacted components, and the immunoassay analyzer further comprises a washing arrangement, wherein the washing arrangement is configured to perform at least one wash action to wash away at least a portion of unreacted components, alternatively configured to perform at least two wash actions, alternatively configured to perform at least three wash actions, alternatively configured to perform at least four wash actions, alternatively configured to perform at least five wash actions, alternatively configured to perform at least six wash actions, alternatively configured to perform at least seven wash actions, alternatively configured to perform at least eight wash actions, alternatively configured to perform at least nine wash actions, or alternatively configured to perform at least ten wash actions.

[0090] In an aspect, the first reaction mixture, second reaction mixture, and / or detection mixture is subjected to a magnetic field prior to performing the at least one wash action.

[0091] In an aspect, the immunoassay analyzer further comprises: a machine vision apparatus comprising an image capture device and an image interpretation device configured to monitor instrument and / or assay functionalities of the immunoassay analyzer. In an aspect, the instrument functionalities are selected from the group consisting of optical sensors, pressure sensors and thermistors. In an aspect, the assay functionalities are selected from the group consisting of sample volume monitoring, total reagent volume monitoring, residual volume monitoring, and particle retention monitoring.

[0092] In an aspect, the biological sample is serum, whole blood, plasma, and / or cerebral spinal fluid.

[0093] These and other advantages, aspects, and novel features of the present disclosure, as well as details of illustrated embodiments thereof, will be more fully understood from the following description and drawings.BRIEF DESCRIPTION OF THE FIGURES

[0094] Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures, wherein:

[0095] FIG. 1 is a signal comparison between a commercially available substrate and an exemplary substrate according to an aspect of this disclosure.

[0096] FIG. 2 is a calibration curve plot constructed for a troponin I (TNI) assay using calibrators and a commercially available substrate and an exemplary substrate according to an aspect of this disclosure.

[0097] FIG. 3 is a calibration curve plot constructed for a thyroid stimulating hormone (TSH) assay using calibrators and a commercially available substrate and an exemplary substrate according to an aspect of this disclosure.

[0098] FIG. 4 is a calibration curve plot constructed for a procalcitonin (PCT) assay using calibrators and a commercially available substrate and an exemplary substrate according to an aspect of this disclosure.DETAILED DESCRIPTIONI. Introduction

[0099] Disclosed herein arc immunoassay methods, reagents, kits, and compounds for detecting analytes in a biological sample using chemiluminescent dioxetanes.

[0100] In some instances, the analyte is cardiac troponin I (TNI). TNI is thought to be an indicator of myocardial damage. Elevated levels of TNI have been associated with pathologies including, but not limited to, congestive heart failure, acute and chronic trauma, electrical cardioversion, hypertension, hypotension, arrhythmias, pulmonary embolism, severe asthma, sepsis, critical illness, myocarditis, stroke, non-cardiac surgery, extreme exercise, drug toxicity (adriamycin, 5- fluorouracil, herceptin, snake venoms), end stage renal disease, and rhabdomyolysis with cardiac injury. Those skilled in the art understand the troponins (I, C, and T) to be members of a complex of proteins that modulate the calcium - mediated interaction between actin and myosin within muscle cells and understand there to be at least three isoforms of troponin I: one associated with fast-twitch skeletal muscle, one with slow-twitch skeletal muscle, and one with cardiac muscle.The cardiac-specific TNT isoform is thought to have a molecular weight of approximately 24,000 Da and to contain post-translational tail of 31 amino acids on the N-tcrminus of the molecule. In some cases, the amino acid sequence of cardiac TNI is SEQ ID NO: 1. TNI may refer to a full- length cardiac Troponin I, a variant of cardiac Troponin I, a fragment of cardiac Troponin I, and post-translationally modified forms of cardiac Troponin I.

[0101] Table 1

[0102] In some instances, the analyte is Thyroid-stimulating Hormone (TSH). Those skilled in the art understand TSH to have clinical utility for assessing thyroid status, including in conjunction with other thyroid hormones. TSH is understood to be a glycoprotein hormone consisting of two noncovalently-bound subunits: an a subunit, which is nearly identical to the a subunits of human luteinizing hormone (hLH), human follicle-stimulating hormone (hFSH), and human chorionic gonadotropin (hCG), and a subunit, which is responsible for immunological and biological specificity. In some instances, TSH, including in conjunction with thyroid hormones or antibodies, is used to: 1) detect or exclude hypothyroidism or hyperthyroidism; 2) monitor T4 replacement treatment in hypothyroidism or antithyroid treatment in hyperthyroidism; 3) monitor TSH suppression in thyroid cancer patients on thyroxine therapy; and 4) assess the response to TRH stimulation testing. In some instances, the amino acid sequence of TSH is that of SEQ ID NO: 2 listed in Table 2. TSH may refer to a full-length thyroid stimulating hormone, a variant of thyroid stimulating hormone, a fragment of thyroid stimulating hormone, and post-translationally modified forms of thyroid stimulating hormone.

[0103] Table 2

[0104] In some instances, the analyte is procalcitonin (PCT). PCT levels are thought to correlate with the severity of bacterial infections and are thought to be useful in the assessment of patients with possible sepsis or septic shock. It is thought that In healthy individuals, PCT - a prohormone of calcitonin - is produced in the thyroid C-cells and is subsequently converted into calcitonin in the thyroid with almost no PCT entering the circulation, but that In individuals with systemic inflammation or bacterial infections, PCT levels rise in the circulation in response to bacterial endotoxins and inflammatory cytokines. PCT is also thought to be specific to bacterial infections, and to be able to aid in the differential diagnosis between nonbacterial illnesses and bacterial illnesses and sepsis.

[0105] PCT is thought to consist of 116 amino acids with a molecular weight of approximately 13 kDa. In some instances, the amino acid sequence of PCT is that of SEQ ID NO: 3 listed in Table 3. PCT may refer to a full-length procalcitonin protein, a variant of procalcitonin, a fragment of procalcitonin, and post-translationally modified forms of procalcitonin.

[0106] Table 3

[0107] In some instances, methods disclosed herein comprise: exposing a biological sample to a capture antibody configured to bind to at least one portion of an analyte disclosed herein generating a first reaction mixture; exposing the first reaction mixture to an enzyme-conjugated affinity molecule, forming a second reaction mixture; exposing the second reaction mixture to a substrate formulation comprising a 1,2 dioxetane compound disclosed herein; wherein the reaction between the enzyme-conjugated affinity molecule and the substrate formulation generates a chemiluminescent detection signal; recording the detection signal generated by the reaction; andcomparing the recorded signal to a calibration curve to quantify the level of the analyte in the biological sample.

[0108] In some instances, methods disclosed herein are performed on an immunoassay analyzer disclosed herein, such as an automated analyzer. Automated analyzers are commonly used in clinical chemistry, immunoassay, hematology, and other biological sampling and analyzing applications. Automated analytical equipment, such as automated analytical chemistry instruments, automated analytical immunoassay instruments, automated analytical hematology instruments, etc., can efficiently perform clinical analysis on a large number of samples, with multiple tests being run concurrently or within short time intervals. Automated analytical instruments are particularly well-suited for high-volume and mid-volume testing environments.

[0109] In some instances, methods disclosed herein can measure lower levels of analyte (e.g., TNI, PCT, TSH) in blood, obtaining lower limits of detection (LoD) and limits of quantification (LoQ) than conventional methods, including in ranges that were previously undetectable, allowing smaller differences in analyte to become more apparent for physicians interpreting results. The LoD is the lowest concentration that the assay can detect the analyte within a certain degree of confidence. It may also be defined as the lowest concentration that can be distinguished from the background reliably. The LoQ is the lowest concentration of analyte that can be quantified reliably. Reliably, or a reliable way, refers to the presence of a suitable precision, trueness, and / or reproducibility.

[0110] In some instances, the signal produced by assay methods disclosed herein is up to 35 times greater than immunoassay methods where conventional or known substrate formulations are used. In some instances, methods disclosed herein result in an enhanced ratio of signal produced by the analyte to background noise when compared to conventional methods. When a detector, such as a luminometer, is used the signal, or luminescence output, is measured relative to the light output of the detector. In these instances, the signal read-out values is given as Relative Light Units (“RLU”).IL Definitions

[0111] 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 to which the methods described herein belong. Any reference to standard methods (e.g., ASTM, TAPPI, AATCC, etc.)refers to the most recent available version of the method at the time of filing of this disclosure unless otherwise indicated.

[0112] For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

[0113] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

[0114] The words "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments or aspects does not imply that other embodiments or aspects are not useful and is not intended to exclude other embodiments or aspects from the scope of the invention.

[0115] The term "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims. Such terms will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0116] By "consisting of" is meant including, and limited to, whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of" is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

[0117] The singular form "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. These articles refer to one or to more than one (i.e., to at least one). As used herein, the term "or" is generally employed in its usual sense including "and / or" unless the content clearly dictates otherwise. The term "and / or" means any one or more of the items in the list joined by "and / or". As an example, "x and / or y" means any element of the three -element set {(x), (y), (x,y)}. In other words, "x and / or y" means "one or both of x and y". As another example, "x, y, and / or z" means any clement of the scvcn-clcmcnt set {(x), (y), (z), (x, y), (x, z), (y, z), (x, y, z)}. In other words, "x, y and / or z" means "one or more of x, y and z".

[0118] Where ranges are given, endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. Herein, "up to" a number (for example, up to 50) includes the number (for example, 50). The term "in the range" or "within a range" (and similar statements) includes the endpoints of the stated range.

[0119] Reference throughout this specification to "one aspect," "an aspect," "certain aspects," or "some aspects," "one embodiment," "an embodiment," "certain embodiment," or "some embodiment," etc., means that a particular feature, configuration, composition, or characteristic described in connection with the aspect is included in at least one aspect of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more aspects.

[0120] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." As used herein in connection with a measured quantity, the term "about" refers to that variation in the measured quantity as would be expected by the skilled artisan making the measurement and exercising a level of care commensurate with the objective of the measurement and the precision of the measuring equipment used. The term "about" as used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. In general, such interval of accuracy is + / -10%. Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, andnot as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.[01211 Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0122] The term "exemplary" means serving as a non-limiting example, instance, or illustration. As utilized herein, the terms "e.g.," and "for example" set off lists of one or more non-limiting aspects, examples, instances, or illustrations.

[0123] As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. Biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. For example, "substantially" may refer to being within at least about 20%, alternatively at least about 10%, alternatively at least about 5% of a characteristic or property of interest.

[0124] The invention is defined in the claims. However, below is a non-exhaustive listing of nonlimiting exemplary aspects. Any one or more of the features of these aspects may be combined with any one or more features of another example, embodiment, or aspect described herein.

[0125] One aspect of the invention is a method for quantitatively assessing a degree of ovarian aging in a subject using an immunoassay analyzer. The term "immunoassay" may be a laboratory method that uses one or more antibodies or antigens to determine the amount of an analyte in a sample. It can be based on the interaction of antibodies with antigens, and because of the degree of selectivity for the analyte (either antigen or antibody), an immunoassay can be used to quantitatively determine very low concentrations of analyte in a test sample. An "immunoassay analyzer" can include an instrument on which immunoassays have been automated. Various immunoassay analyzer are commercially available including the Dxl® system (Beckman Coulter, CA), the ADVIA® CENTAUR® systems (Siemens Healthcare, Germany), the COBAS® system (Roche Diagnostic, Germany), the ARCHITECT® system (Abbott, IL), the VITROS® system(Ortho-clinical Diagnostic, NJ), and the VIDAS® system (Biomerieux, France). In some aspects, the immunoassay analyzer is a high-throughput immunoassay analyzer.

[0126] As used herein, the terms "subject", "individual", and "patient" are interchangeable, and relate to vertebrates, preferably mammals. For example, mammals in the context of the disclosure are humans, non-human primates, domesticated animals such as dogs, cats, sheep, cattle, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc., as well as animals in captivity such as animals in zoos. The term "animal" as used herein includes humans. The term "subject" may also include a patient, i.e., an animal, having a disease. In exemplary aspects, a subject, individual, or patient refers to a human (e.g., a man, a woman, or a child).IL Systems and Reagents for Detecting Analytes Using Chemiluminescent Dioxetanes

[0127] In an embodiment, the method includes detecting the presence of an analyte in a biological sample obtained from a subject using an immunoassay analyzer, including exposing the biological sample to an enzyme-conjugated antibody or an enzyme-conjugated antigen and a substrate formulation comprising a 1,2 dioxetane compound and at least one phosphonium surfactant. In an aspect of the methods described herein, the reaction between the enzyme-conjugated antibody or the enzyme-conjugated antigen and the substrate formulation generates a chemiluminescent detection signal which can be recorded and compared to quantify the level of analyte in the biological sample. In an embodiment, the method includes aspirating a portion of the biological sample from a sample vessel and dispensing the aspirated sample into a reaction vessel of an immunoassay analyzer. a. Reagents

[0126] In some instances, “two-site” or “sandwich” immunoassays employ a first antibody or antibody fragment, which is described as the “capture” antibody, is bound to a solid support, such as magnetic beads or particles disclosed herein, using procedures known in the art. Further, a second antibody or antibody fragment, which is described as the “detection” antibody, is coupled or conjugated with a label, such as the enzymes disclosed herein, using procedures known in the art. The label produces a detectable signal when provided with substrate(s), so that the amount of signal measured corresponds to the amount of detection antibody that is bound to the analyte.

[0127] In an aspect, the capture antibody and / or the affinity molecule is conjugated to a magnetic bead or a magnetic particle. In some instances, magnetic beads (also known as magnetic particles,paramagnetic particles, or superparamagnetic particles) consist of a polystyrene core surrounded by a thin layer of small iron oxide particles (-20-30 nm), such as magnetite. On the surface, the magnetic beads are encapsulated by, for example, a polymer, protein A, protein G, protein L, a secondary antibody, or an epoxy. Surface modification of the coating minimizes any non-specific protein binding. Antibodies targeting the analyte of interest or capture antibody can be covalently coupled to the surface of the magnetic bead. In some embodiments, there is about 2 pg or antibody per mg of magnetic bead. In some embodiments, there is about 3 pg or antibody per mg of magnetic bead. In some embodiments, there is about 4 pg or antibody per mg of magnetic bead. In some embodiments, there is about 5 pg or antibody per mg of magnetic bead. In some embodiments, there is about 6 pg or antibody per mg of magnetic bead. In some embodiments, there is about 7 pg or antibody per mg of magnetic bead. In some embodiments, there is about 8 pg or antibody per mg of magnetic bead. In some embodiments, there is about 9 pg or antibody per mg of magnetic bead. In some embodiments, there is about 10 pg or antibody per mg of magnetic bead. In some embodiments, there is about 12 pg or antibody per mg of magnetic bead. In some embodiments, there is about 13 pg or antibody per mg of magnetic bead. In some embodiments, there is about 14 pg or antibody per mg of magnetic bead. In some embodiments, there is about 15 pg or antibody per mg of magnetic bead.

[0128] In an aspect, the substrate formulation is configured to produce chemiluminescence. These substrates can produce light and thereby provide detection corresponding to a quantity of analytes captured. The term “chemiluminescent compound” refers to a compound that produces chemiluminescence in the presence of a phosphatase enzyme and oxygen under appropriate conditions as provided herein. In a non-limiting example, the substrate formulation includes a 1,2 dioxetane compound and at least one phosphonium surfactant. Chemiluminescent compounds useful in the present formulations are capable of generating chemiluminescence when contacted with an alkaline phosphatase. Such compounds can be synthesized as described in U.S. Patent Application No. 2022 / 0390459 which is incorporated by reference herein.

[0129] At operation, the chemiluminescent substrate is added to the vessel with the second reaction mixture and light generated by the reaction is measured with a luminometer. The light production is inversely proportional to the concentration of free analyte in the sample. The amount of analyte in the sample is then determined from a stored, multi-point calibration curve. In an embodiment, the detector can generate an output signal that can be processed to generate a relativelight unit (“RLU”) value (i.e., an output response) indicating a result of the assay. For example, a larger RLU value indicates more light, which indicates a larger amount of the analyte in the biological sample than a smaller RLU value indicates.

[0130] In an exemplary method of producing light from the reaction of the chemiluminescent substrate with a phosphatase enzyme (e.g., detection antibody), the reaction is performed at a temperature between 5° C and 50° C, preferably between 20° C and 40° C in an aqueous buffer solution at a pH between 7 and 12, 8 and 11, or preferably between 8.5 and 10. The enzyme is preferably an alkaline phosphatase or an alkaline phosphatase conjugate.

[0131] In an embodiment, the 1,2 dioxetane compound is a compound of Formula I or a salt thereof:

[0132] Formula I

[0133] wherein each of R1 and R2 are independently C3-C10 alkyl, or R1 and R2 taken together with the carbon to which they arc attaches provide a C5-C10 cycloalkyl ring;

[0134] R3 is C1-C10 alkyl, C6-C10 aryl, or hetero aryl;

[0135] R4 is C2-C10 alkenyl;

[0136] R5 is H or C1-C10 alkyl; and

[0137] X is a phosphate.

[0138] In an embodiment, the 1,2 dioxetane compound is a compound of Formula II or a salt thereof:Formula II

[0140] wherein each of RIO and R11 is independently H, halogen, Cl -CIO alkyl, C2-C10 alkenyl, or C6-C10 aryl;

[0141] R3 is C1-C10 alkyl, C6-C10 aryl or heteroaryl;

[0142] R4 is C2-C10 alkenyl;

[0143] R5 is H or C1-C10 alkyl; and

[0144] X is a phosphate.

[0145] In an embodiment, the 1,2 dioxetane compound is a compound of Formula III or a salt thereof:Formula III

[0147] wherein each of RIO and Rl l is independently H, halogen, C1-C10 alkyl, C2-C10 alkenyl, or C6-C10 aryl;

[0148] R3 is C1-C10 alkyl, C6-C10 aryl or heteroaryl; and

[0149] R5 is H or C1-C10 alkyl; and

[0150] X is a phosphate.

[0151] In an embodiment, the 1,2 dioxetane compound is a compound of Formula IV or a salt thereof:

[0152] Formula IV

[0153] wherein R3 is C1-C10 alkyl, C6-C10 aryl or heteroaryl; and

[0154] R5 is H or C1-C10 alkyl.

[0155] In an embodiment, the 1 ,2 the dioxetane compound is

[0156]

[0157] or a salt thereof.

[0158] In an embodiment, the 1,2 dioxetane compound is 4-methoxy-4-(3- phosphatephenyl)spiro[l,2-dioetane-3,2’-adamantane] or a salt thereof.

[0159] The substrate also includes at least one phosphonium surfactant. In some embodiments, the phosphonium surfactant operates as a luminescence enhancer. In certain embodiments, the phosphonium surfactant comprises small-molecule phosphonium surfactants or polymeric phosphonium surfactants.

[0160] In an embodiment, the small molecule phosphonium surfactant is a compound having the formula:

[0161]

[0162] wherein R12-R14 are each independently C1-C10 alkyl;

[0163] R15 is arylalkyl; and

[0164] X" is a counterion.

[0165] In an embodiment, the polymeric phosphonoium surfactant comprises repeating unit (A), repeating unit (B), or both:

[0167] wherein B113 is tributyl and Oct3 is trioctyl.

[0168] In certain embodiments, the substrate formulation further comprises a magnesium (II) salt.

[0169] Another aspect of the disclosure includes a kit for performing any of the disclosed methods. The kit may include a reagent pack. In some aspects, the reagent pack includes several reagent formulations. In one aspect, the reagent pack includes a first reagent formulation which comprises the capture antibody. In an embodiment, the capture antibody is conjugated to magnetic beads. In one aspect, the reagent pack includes reagent formulation which comprises the enzyme-conjugated affinity molecule. In an embodiment, the enzyme-conjugated affinity molecule is conjugated to magnetic beads. The reagent pack may also include other formulations which comprise buffers and / or salts needed for the methods. The kit may also include instructions for performing any one of the disclosed methods. In some aspects, the kit may include the substrate formulation, calibrators, and / or wash buffers. b. Analyzer Systems

[0170] In an aspect, an immunoassay analyzer disclosed herein includes the following basic structural and functional modules: a sample presentation unit, an analytic unit, an incubator station, a washing station, a read station, and reagent storage. In addition, the immunoassay analyzer may include a pipettor arrangement with at least one sample pipettor and at least one reagent pipettor, and at least one transport device. In an aspect, the transport device includes mechanisms, such as pick-and-place grippers, which are used to transport sample and reaction vessels among the various modules of the immunoassay analyzer.

[0171] The immunoassay analyzer may include a container carriage device which is configured to hold and carry the containers at various locations in the instrument so that the analytic unit, incubator station, wash station, and read station can use the containers in various manners.Examples of container carriage devices include vessel racks (e.g., a sample rack, a reagent rack, and a diluent rack), the sample presentation unit, vessel carriage units (e.g., a sample carriage unit, a reaction vessel carriage unit, and a reagent carriage unit), vessel transfer units (e.g., a sample transfer unit, a reagent transfer unit, an incubator transfer unit, and an reaction vessel transfer unit), and vessel holding plates or wheels (e.g., a sample wheel, an incubator, and a wash wheel), which are described herein.

[0172] The read station may include a detector arrangement. In an aspect, the detector arrangement may include a detector that is configured to detect light or luminescence, for example chemiluminescence. The detector may be a luminescence detector, a chemiluminescence detector, a luminometer, or a photomultiplier-based detection instrument. In an embodiment, the detector includes a light detector configured to sense photons emitted from assay reactions over a period of time, an analog circuit configured to provide an analog signal based on the photons emitted from the assay reactions over the period of time, and a counter circuit configured to provide a photon count based on the photons emitted from the assay reactions over the period of time. U.S. Patent No. 11,604,146, which is incorporated by reference in its entirety herein, discloses non-limiting examples of a detector that may be used in an aspect of the invention.

[0173] In an aspect, primary sample containers can be placed into an onload section (e.g., individually or on racks) of the sample presentation unit. In certain embodiments, the sample presentation unit has at least one sample rack, alternatively at least two sample racks, alternatively at least three sample racks, alternatively at least four sample racks, alternatively at least five sample racks, alternatively at least six sample racks, alternatively at least seven sample racks, alternatively at least eight sample racks, alternatively at least nine sample racks, alternatively at least ten sample racks, alternatively at least 11 sample racks, alternatively at least 12 sample racks, alternatively at least 13 sample racks, alternatively at least 14 sample racks, alternatively at least 15 sample racks, alternatively at least 16 sample racks, alternatively at least 17 sample racks, alternatively at least 18 sample racks, alternatively at least 19 sample racks, or alternatively at least 20 sample racks. In certain embodiments, each sample rack can hold at least one sample vessel, alternatively at least two sample vessels, alternatively at least three sample vessels, alternatively at least four sample vessels, alternatively at least five sample vessels, alternatively at least six sample vessels, or alternatively at least seven sample vessels. In a specific embodiment, the sample presentation unitholds about 140 sample vessels. In a specific embodiment, the sample presentation unit has 20 sample racks with each rack holding seven sample vessels.

[0174] In certain embodiments, the sample vessels include a barcode label that uniquely identifies the sample vessel in the immunoassay analyzer. The barcode label also may include alphanumeric characters that correspond to the barcode identification information. The immunoassay analyzer may include at least optical reader, such as a barcode scanner. In one embodiment, the optical readers are area scan cameras that provide a two-dimensional image of the barcode and / or sample vessel. In another embodiment, optical readers are area scan cameras that provide a three- dimensional image of the barcode and / or sample vessel.

[0175] After being fed into the onload section, the sample containers may be moved into a presentation section of the sample presentation unit. In certain embodiments, the sample presentation unit is maintained at between about 4.5°C to 14°C. The transfer station may receive primary sample containers from the sample presentation unit from a transport device. Primary sample containers delivered to the transfer station from the transport device may be initially processed in different ways. For example, the primary sample container may temporarily remain at the transfer station while the sample provided within is aspirated by the sample pipettor at the transfer station. Following such aspiration, the primary sample container may be expelled from the automated analyzer at the offload station of the sample presentation unit or may be passed on to the transport device for further processing. The instrument may also contain a sample retention unit configured to receive sample retention vessels and store, analyze, or otherwise process samples retained within the sample retention vessels. A “primary sample container” loaded into the automated analyzer with a sample may also be considered a “sample retention vessel” when placed in a sample retention unit, such as a storage unit. As used herein, “containers” or “vessels” are analogous, and can be of various types, such as specimen tubes (also referred to herein as sample tubes) and pipettor tips, such as micro or disposable tips. In certain embodiments, the vessels are tubes with diameters between about 12mm to about 16mm and / or heights between about 75mm to about 100mm. In certain embodiments, the vessels are cups with volumes of about 0.5mL, alternatively about l.OmL, alternatively about 1.5mL, alternatively about 2.0mL, alternatively about 2.5mL, or alternatively about 3.0mL.

[0176] In an aspect, the analytic unit is configured to receive and analyze samples. In an aspect, the analytic unit configured to perform an immunoassay. In certain embodiments, the analytic unit includes the pipettor arrangement. The pipettor arrangement may be configured to aliquot, aspirate, and dispense fluidic substances into various vessels, including, but not limited to sample vessels, diluent vessels, reagent vessels, and reaction vessels. Fluidic substances are substances that have fluidic characteristics. In some embodiments, the fluidic substance is a single fluidic substance. In other embodiments, the fluidic substance is a mixture of a plurality of substances.

[0177] The pipettor arrangement may contain at least one, two, three, or four reagent pipettors used to mix reagents with sample aliquots for an assay. The pipettor arrangement may also contain at least one, two, three, or four sample pipettors used to transfer sample aliquots for an assay. In some instances, the pipettor arrangement contains one sample pipettor. In some instances, the pipettor arrangement contains one sample pipettor and four reagent pipettors. In an aspect, the reagent pipettors may be arranged as dual reagent pipetting stations and are independent to each other, each having its own fluid pumps and valves, wash towers, reaction vessel carriages, and pipettor. A sample aliquot may be transferred from a sample retention vessel into a reaction vessel using the sample pipettor in order to mix the sample aliquot with one or more reagents. In an aspect, the at least one reagent pipettor and at least one sample pipettor are configured to aspirate and / or dispense less than about 10 pL. In an embodiment, the at least one reagent pipettor and at least one sample pipettor are configured to aspirate and / or dispense less than about 9.9 pL, alternatively less than about 9.5 pL, alternatively less than about 8.0 pL, alternatively less than about 7.0 pL, alternatively less than about 6.0 pL, alternatively less than about 5.0 pL, alternatively less than about 4.0 pL, alternatively less than about 3.0 pL, alternatively less than about 2.0 pL, alternatively between than about 9.9 pL and 2.0 pL.

[0178] In an aspect, the first reagent pipettor, the second reagent pipettor, the third reagent pipettor, and / or the fourth reagent pipettor are selectively and / or simultaneously operated. In certain embodiments, the sample pipettor, the first reagent pipettor, the second reagent pipettor, the third reagent pipettor, and / or the fourth reagent pipettor are configured to engage a dispense tip prior to aspiration.

[0179] The claimed configuration allows for the simultaneous performance of at least two assays for a plurality of biological samples, alternatively at least three assays for a plurality of biologicalsamples, or at least four assays for a plurality of biological samples. Depending on the analysis desired the biological samples may be from the same subject or from multiple subjects. In some aspects, the method further comprises a plurality of sample vessels. In some embodiments, the method comprises at least one sample vessel, at least two sample vessels, at least three sample vessels, at least four sample vessels, at least five sample vessels, at least ten sample vessels, at least twenty sample vessels, at least fifty sample vessels, at least one hundred sample vessels, at least two hundred sample vessels, at least three hundred sample vessels, at least four hundred sample vessels, or at least five hundred sample vessels. In some aspects, the sample vessels are housed within the immunoassay analyzer.

[0180] In a non-limiting example, if multiple assays for multiple biomarkers are being performed, the first pipettor may be configured to aspirate a reagent from a reagent vessel comprising a first capture antibody and dispense into a first reaction vessel, while a second pipettor may be configured to simultaneously aspirate a reagent from a reagent vessel comprising a second capture antibody and dispense into a second reaction vessel. Depending on the analysis desired, the third and fourth pipettor may also be configured to simultaneously aspirate a reagent from a reagent vessel comprising a third and fourth affinity molecule and dispense into a third and fourth reaction vessel.

[0181] The disclosed simultaneous and / or selective operation of the pipettors allows for a high- throughput analysis. In some embodiments, the method is configured to analyze at least about 200 plasma samples / hr., alternatively at least about 300 plasma samples / hr., alternatively at least about 400 plasma samples / hr., alternatively at least about 440 plasma samples / hr., or alternatively at least 500 plasma samples / hr.

[0182] In an aspect, the immunoassay analyzer includes a reagent pack configured to hold a plurality of reagent vessels. A “reagent pack” may include any suitable container that can store a reagent. An example of a reagent pack can include a generally rectangular elongated body formed to include multiple reagent vessels including one or more large reagent vessels, and one or more relatively smaller reagent vessels, as well as features to facilitate handling and automation. US Patent No. 9,519,000, which is incorporated by reference in its entirety herein, discloses nonlimiting examples of a reagent pack that may be used in an aspect of the invention.

[0183] In an embodiment, the reagent pack may be configured to accommodate sufficient volumes of reagents for multiple instances of an assay. In some embodiments, each reagent pack includes reagents for about 20 to about 100 instances of an assay and in some cases about 50 instances.

[0184] In some embodiments a reagent pack may supplied with empty or partially filled reagent vessels, to which reagents are subsequently transferred from bulk containers, such as bottles. Individual reagent vessels may differ in dimension to accommodate the requirements of an assay type. Factors that can determine the size of a reagent vessel include the number of uses desired for the reagent pack type, concentration-dependent stability issues with reagent components, and the need to minimize the volume of the final reaction mixture. As noted above, in some embodiments, each reagent pack can include a large reagent vessel and a plurality of small reagent vessels. Each reagent vessel may be large enough to accommodate a microtip or disposable tip (i.e., dispo- tip) of a reagent pipettor used to remove a volume of reagent for use in an assay. In certain embodiments, the reagent pack may be maintained at a temperature of between about 4°C to 10°C.

[0185] A “reagent vessel” may refer to a vessel, unit, fluid container, or the like that is configured to store reagents. In an embodiment, the reagent vessels include an elastomeric self-sealing membrane. An elastomeric self-sealing membrane may be a polymer, such as polypropylene, which is able to regain its original shape when pierced. For some embodiments, the elastomeric membrane can be a thermoplastic elastomer with hardness of 30-40 durometer (Shore) A. In other embodiments, the hardness can be 20-50 (Shore) A, or about 30 (Shore) A. Elastomers deform sufficiently to form a tight seal with the vessel base. Thermoplastic elastomers are advantageous because of their compatibility with plastics injection molding processes.

[0186] The elastomeric membrane can be large enough to provide adequate compression without bottoming on the sealing portion of the vessel. The hardness and dimensions can cooperate to allow the elastomeric membrane to the sealing portion with reasonable sealing force. In some embodiments, the elastomeric membrane diameter is small enough so that, when compressed by engagement of the pipettor tip, it conforms to the sealing portion without contacting the wall of the pipettor tip. This advantageously concentrates sealing force to the sealing portion of vessel and distributes sealing force evenly to prevent leaks. In some embodiments, the sealing force is about 44 newtons (about 9.9 lbs.) and produces a pressure on the sealing surface of about 300 (about 43.5 pounds per square inch) to about 1000 kPa (145.0 pounds per square inch).

[0187] In methods where multiple samples are analyzed, the elastomeric self-sealing membrane allows for aspiration of the reagents without concern of evaporation. In some embodiments, the reagent pack further includes containment walls arranged between the reagent vessels. In an aspect, the reagent pack may include at least one reagent vessel, alternatively at least two reagent vessels, alternatively at least three reagent vessels, alternatively at least four reagent vessels, alternatively at least five reagent vessels, or alternatively at least ten reagent vessels. In an embodiment, the immunoassay analyzer further includes a reagent storage unit, wherein the reagent pack is housed in the reagent storage unit.

[0188] At operation, a fluidic substance is dispensed to a reaction vessel. Examples of the fluidic substance include a sample, diluent, reagent, substrate, or any combination thereof, as described herein. In some embodiments, the reaction vessel already contains other fluidic substances, such as a sample, and after a fluidic substance is dispensed to a reaction vessel, the fluidic substance is mixed with the other fluidic substances in the reaction vessel. The mixing can be performed with a stirrer in direct contact with the fluidic substances, an ultrasonic probe in direct or indirect contact with the fluidic substances, or any other suitable mixing apparatus. In some aspects, the immunoassay analyzer includes an ultrasonic mixing module. For instance, a reagent pipettor may be outfitted with a tip that allows it to perform ultrasonic mixing of a reagent in a reagent pack before aspirating it for transport to a reaction vessel, thereby ensuring that the aspirated reagent would not be impacted by any settling that may have taken place in the reagent pack. Sample pipettors may similarly be specialized.

[0189] The transfer unit transfers the reaction vessels to and from the incubator station which includes an incubator. In some embodiments, the transfer unit transfers one or more of the pipetted reaction vessels from the reagent carriage unit to the incubator. Further, the transfer unit can transfer one or more reaction vessels from the incubator to the reagent carriage unit. The transfer unit can also remove from the reaction vessels that have been read or completed the incubator.

[0190] The incubator is thermally controlled to maintain a predetermined temperature. In some embodiments, the incubator is maintained about 30 °C to 40 °C. In other embodiments, the incubator is maintained about 37 °C to ensure immunological reaction and enzyme reaction, for example. By way of example, the incubator performs assay incubation.

[0191] In some embodiments, the transfer unit transfers incubated reaction vessels from the incubator to the wash unit, transfers assay reaction vessels from the wash unit to the incubator, transfers reaction vessels containing substrate from the wash unit to the incubator for substrate incubation or enzyme reaction, transfers washed reaction vessels from the incubator to detector arrangement after substrate incubation, and transfers the reaction vessels that have been read or completed from the detector arrangement to the incubator. The used reaction vessels can be delivered to a waste location.

[0192] At operation, a sample and a reagent are dispensed into a reaction vessel and mixed. The mixture is then Iran si erred to the incubator. During the incubation, the sample and the reagent interact. The resulting “first reaction mixture” is a result of the incubation between the sample and the reagent. The reagent may include a specific -binding reagent, such as a capture antibody specific to the analyte being analyzed by the immunoassay analyzer. In a non-limiting example, the incubation time of the first reaction mixture is at least about 30 minutes, alternatively at least about 40 minutes, alternatively at least about 50 minutes, alternatively at least about 55 minutes, or alternatively at least about 60 minutes.

[0193] At operation, a reagent, which is different from the reagent used in the first reaction mixture, is added to the first reaction mixture and mixed. The mixture is then transferred to the incubator. During incubation, the first reaction mixture and the reagent interact. The resulting “second reaction mixture” is a result of an incubation between the first reaction mixture and the reagent. The reagent may include an affinity molecule, which may be an antibody, a monoclonal antibody, a polyclonal antibody, a synthetic antibody mimic, an aptamer, an affimer, DARPins, oligonucleotide, peptide, or antigen. In certain embodiments, the affinity molecule is an enzyme- conjugated antibody, an enzyme-conjugated antigen, an alkaline phosphatase (AP)-conjugated secondary antibody, or a labeled antibody. The affinity molecule may be configured to bind at least one portion of an analyte or at least one portion of the capture antibody. In a non-limiting example, the incubation time of the second reaction mixture is at least about 2 minutes, alternatively at least about 5 minutes, alternatively at least about 8 minutes, or alternatively at least about 10 minutes.

[0194] The wash station receives and supports reaction vessels thereon such that various aspects of diagnostic process are performed with the immunoassay analyzer. In an embodiment, the washstation is configured to wash away at least some of the unreacted components. Unreacted components may include unrcactcd reagents (c.g., free antigens, antibodies, unbound reactants, particles, and / or fluid, etc.) and unreacted sample. The wash station may be configured to perform a set number of wash actions depending on the assay. The wash station may also be configured to perform a set number of washes within a predetermined sequence. In certain embodiments, the wash station is configured to perform at least one wash action to wash away at least a portion of unreacted components, alternatively configured to perform at least two wash actions, alternatively configured to perform at least three wash actions, alternatively configured to perform at least four wash actions, alternatively configured to perform at least five wash actions, alternatively configured to perform at least six wash actions, alternatively configured to perform at least seven wash actions, alternatively configured to perform at least eight wash actions, alternatively configured to perform at least nine wash actions, or alternatively configured to perform at least ten wash actions. In some embodiments, the wash station is a thermally controlled device to separate bound or free analytes from particles after incubation. In some embodiments, the wash unit is maintained about 30 °C to 40 °C. In other embodiments, the wash unit is maintained about 37 °C to ensure enzyme reaction, for example, U.S. Patent Publication No. 2022 / 0357352, which is incorporated by reference in its entirety herein, discloses configurable wash processes according to an aspect of the invention.

[0195] The wash station may comprise a washing arrangement which may be configured to provide a base number of wash series (or wash actions) for each reaction vessel and optionally provide an additional number(s) of wash actions. The additional number(s) of wash actions may include one, a plurality, or all of a potential number of wash actions. An additional number of wash actions beyond the base number of wash actions may be specified for certain assays in an assay protocol file. The washing arrangement may include cleaning dispense nozzle (or probe) which dispenses a rinsing fluid and a cleaning aspiration nozzle (or probe) which aspirates the unreacted components. At operation, a base number of wash actions performed may be one, two, three, four, or five and the additional number of wash action(s) may be one, two, three, four or five. At operation, a base number of wash actions performed may be three and the additional number of wash action(s) may be one or two. In this embodiment, the base number of wash actions may be performed if three probes dispense buffer solution once per vessel and the three probes aspirate the at least some of the unreacted components some of the buffer solution, and / or the at least someof the unreacted reagents once per vessel. According to the principles of the present disclosure, certain probc(s) may be selectively used to dispense clean buffer solution into the vessel and aspirate the at least some of the unreacted components of the sample, some of the buffer solution, and / or the at least some of the unreacted reagents from the vessel to perform the additional wash action(s).

[0196] In an aspect, the immunoassay analyzer further comprises a washing arrangement. In an embodiment, the washing arrangement is configured to wash away at least some of the unreacted components from the sample, first reagent, second reagent, or substrate formulation in the first reaction mixture, second reaction mixture, and / or detection mixture. Unreacted components may include unreacted reagents (e.g., free antigens, antibodies, unbound reactants, particles, and / or fluid, etc.) and unreacted sample. The washing arrangement may be configured to perform a set number of wash actions depending on the assay. The washing arrangement may also be configured to perform a set number of washes within a predetermined sequence. In certain embodiments, the washing arrangement is configured to perform at least one wash action to wash away at least a portion of unreacted components, alternatively configured to perform at least two wash actions, alternatively configured to perform at least three wash actions, alternatively configured to perform at least four wash actions, alternatively configured to perform at least five wash actions, alternatively configured to perform at least six wash actions, alternatively configured to perform at least seven wash actions, alternatively configured to perform at least eight wash actions, alternatively configured to perform at least nine wash actions, or alternatively configured to perform at least ten wash actions.

[0197] In an embodiment, the first reaction mixture or second reaction mixture is subjected to a magnetic field. The magnetic beads do not exhibit bead-to-bead attraction, only migrating when a magnetic field is applies. Captured analytes or targets are separated from the mixture and magnetization may be used to retain desired components within a reaction vessel.

[0198] At operation, the vessel containing the first reaction mixture is moved near one or more magnets. The one or more magnets attract the magnetic bead(s) to one or more sides of the reaction vessel. The reaction vessel is then subject to a wash process in which a cleaning dispense nozzle dispenses a rinsing fluid and a cleaning aspiration nozzle aspirates the unreacted components. The aspiration nozzle may be washed with a probe washer before and / or after the aspirating. Thereaction vessel may undergo a series of wash process which may include at least two series of dispensing the rinsing fluid and aspirating the uncollected fluid components, alternatively at least three series, alternatively at least four series, alternatively at least five series. As a result, an unreacted substance or substances in the vessel is removed (e.g., rinsed away) by the bound-free cleaning aspiration nozzle.

[0199] At operation, the vessel containing the second reaction mixture is moved near one or more magnets. The one or more magnets attract the magnetic bead(s) or magnetic particle(s) to one or more sides of the reaction vessel. The reaction vessel is then subject to a wash process in which a cleaning dispense nozzle dispenses a rinsing fluid and a cleaning aspiration nozzle aspirates the unreacted components. The aspiration nozzle may be washed with a probe washer before and / or after the aspirating. The reaction vessel may undergo a series of wash process which may include at least two series of dispensing the rinsing fluid and aspirating the uncollected fluid components, alternatively at least three series, alternatively at least four series, alternatively at least five series. As a result, an unreacted substance or substances in the vessel is removed (e.g., rinsed away) by the bound-free cleaning aspiration nozzle.

[0200] In an aspect, the immunoassay analyzer includes a detector arrangement. In an aspect, the detector arrangement may include a detector that is configured to detect light or luminescence, for example chemiluminescence. The detector may be a luminescence detector, a chemiluminescence detector, a luminometer, or a photomultiplier-based detection instrument. In an embodiment, the detector includes a light detector configured to sense photons emitted from assay reactions over a period of time, an analog circuit configured to provide an analog signal based on the photons emitted from the assay reactions over the period of time, and a counter circuit configured to provide a photon count based on the photons emitted from the assay reactions over the period of time. U.S. Patent No. 11,604,146, which is incorporated by reference in its entirety herein, discloses nonlimiting examples of a detector that may be used in an aspect of the invention.

[0201] At operation, a substrate is dispensed into the second reaction mixture and mixed. After a certain reaction time necessary for the substrate and the second reaction mixture to interact. The reaction vessel is then subject to a wash process in which a cleaning dispense nozzle dispenses a rinsing fluid and a cleaning aspiration nozzle aspirates the unreacted components. The aspiration nozzle may be washed with a probe washer before and / or after the aspirating. The reaction vesselmay undergo a series of wash process which may include at least two series of dispensing the rinsing fluid and aspirating the uncollected fluid components, alternatively at least three scries, alternatively at least four series, alternatively at least five series. As a result, an unreacted substance or substances in the vessel is removed (e.g., rinsed away) by the bound-free cleaning aspiration nozzle. The resulting detection mixture is transferred to the detector arrangement.

[0202] Assays including features and / or characteristics described herein may benefit from one or more additional number(s) of wash actions.

[0203] In certain embodiments, the transport device includes three pick-and-place grippers, where a first pick-and-place gripper may be used to transport sample containers among the onload section, the transfer station, and reagent pipetting stations. A second pick-and-place gripper may be used to transport reaction vessels between the reagent pipetting stations and the incubator station or read station. A third pick-and-place gripper may be used to transport reaction vessels between the incubator station and the wash station or read station. A detailed description of the configurations and functions of one embodiment of the vessel pick-and-place grippers is provided in U.S. Patent No. 7128874 and is incorporated herein in its entirety by reference. However, it should be understood that other pick-and-place mechanism that are capable of transporting sample and reaction vessels among the various modules of the immunoassay analyzer is also contemplated for the purpose of the present invention.

[0204] In an aspect, the immunoassay analyzer includes a machine vision apparatus comprising an image capture device and an image interpretation device configured to monitor instrument and / or assay functionalities of the immunoassay analyzer. In some embodiments, instrument functionalities may include optical sensors, pressure sensors and thermistors. In some embodiments, the assay functionalities may include sample volume monitoring, total reagent volume monitoring, residual volume monitoring, and particle retention monitoring. The machine vision apparatus operates to evaluate the preparation of samples for subsequent analysis. In some embodiments, the machine vision apparatus utilizes one or more image capture units to determine whether samples have been appropriately prepared for analysis. As described herein, the machine vision apparatus provides direct and simple measurements of volume or integrity of a sample to determine whether the sample is appropriately prepared so that the analytic unit produces a reliableresult. An exemplary machine vision apparatus is described in U.S. Patent No. 11 ,263,433, which is incorporated by reference herein.

[0205] In some aspects, the machine vision apparatus operates to detect a volume of a fluidic substance in a container and determine whether the volume held in the container is appropriate as targeted. As described herein, this volume detection is configured to detect a volume at a dispense tip using the dispense tip image capture unit, and a volume at a vessel using a vessel image capture unit.

[0206] In some aspects, the machine vision apparatus operates to detect any interferents, which can interfere with an analytic procedure and may generate incorrect results in the dispense tip. As described herein, this dispense tip evaluation is configured to determine a quality of a fluidic substance at a dispense tip using a dispense tip image capture unit, and an alignment of the dispense tip with respect to the dispense tip image capture unit.

[0207] In some aspects, the machine vision apparatus operates to determine a particle concentration in a fluidic substance contained in a vessel, such as a reaction vessel, a sample vessel, a dilution vessel, a cuvette, or any suitable type of vessel, which is used throughout the process in the immunoassay analyzer. In some embodiments, this reaction vessel particle concentration check uses the vessel image capture unit.

[0208] The dispense tip image capture unit operates to capture images of dispense tips in one or more locations. In some embodiments, the dispense tip image capture unit is fixed at a particular location in the instrument. In other embodiments, the dispense tip image capture unit is movably disposed in the instrument, which can move either independently from other components of the instrument or together with one or more components of the instrument. Some embodiments of the instrument include a plurality of dispense tip image capture units. As described herein, the dispense tip image capture unit can include a camera unit.

[0209] The vessel image capture unit operates to capture images of vessels in one or more locations. In some embodiments, the vessel image capture unit is fixed at a particular location in the immunoassay analyzer. In other embodiments, the vessel image capture unit is movably disposed in the immunoassay analyzer, which can move either independently from other components of the immunoassay analyzer or together with one or more components of the immunoassay analyzer. Some embodiments of the immunoassay analyzer include a plurality ofvessel image capture units. As described herein, the vessel tip image capture unit includes a camera unit.

[0210] The carriage image capture unit operates to capture images of container carriage devices with or without containers in one or more locations. In some embodiments, the carriage image capture unit is fixed at a particular location in the instrument. In other embodiments, the carriage image capture unit is movably disposed in the instrument, which can move either independently from other components of the instrument or together with one or more components of the instrument. Some embodiments of the instrument include a plurality of carriage image capture units.

[0211] All of the units of the immunoassay analyzer are connected to a controller, which can perform block control of all of the analyzer functions by using, for example, a microcomputer. The controller may contain subunits such as a data processing unit, a communication interface, and others. A controller in accordance with an exemplary embodiment of the present technology may comprise a data processor, a non-transitory computer-readable medium, and a data storage coupled to the data processor. The non-transitory computer-readable medium may comprise code, executable by the data processor, to perform the functions described herein. The data processor may store, for example, data for processing samples, sample data, or data for analyzing sample data.

[0212] The data processor may include any suitable data computation device or combination of such devices. An exemplary data processor may comprise one or more microprocessors working together to accomplish a desired function. The data processor may include a CPU that comprises at least one high-speed data processor adequate to execute program components for executing user and / or system-generated requests. The CPU may be a microprocessor such as AMD’s Athlon, Duron and / or Opteron; IBM and / or Motorola’s PowerPC; IBM’s and Sony’s Cell processor; Intel’s Celeron, Itanium, Pentium, Xeon, and / or XScale; Apple Ml, and / or the like processor(s).

[0213] The computer-readable medium and the data storage may be any suitable device or devices that can store electronic data. Examples of memories may comprise, for example, one or more memory chips, disk drives, etc. Such memories may operate using any suitable electrical, optical, and / or magnetic mode of operation.

[0214] The computer-readable medium may comprise code, executable by the data processor to perform any suitable method. For example, the computer-readable medium may comprise code, executable by the processor, to cause the controller to operate on a pre-determined schedule. In some embodiments of the presently claimed technology, the pre-determined schedule is a constituent test.

[0215] In an aspect the cycle time of an immunoassay analyzer described herein is about 45 seconds or less. The “cycle time” is the time required for all modules and / or functions of an immunoassay analyzer to complete its tasks necessary for generating a result. In certain embodiments, the cycle time is about 40 seconds or less, alternatively about 35 seconds or less, alternatively about 30 seconds or less, alternatively about 25 seconds or less, alternatively about 20 seconds or less, or alternatively about 15 seconds. At operation, the sample pipettor can complete its tasks in 8 seconds and the reagent pipettor can complete its tasks in 32 seconds. In some aspects, a task for the pipettor is defined as the time to aspirate and dispense a sample or reagent, inclusive of the time required to move from and return to the starting position. In this embodiment, to maintain a higher throughput, four reagent pipettors are present in the immunoassay analyzer (32 seconds / 4 = 8 seconds). If the incubation of one of the reaction mixtures is 5 minutes, at least 38 incubation positions are needed to support this incubation time (300 seconds / 8 seconds = 37.5 seconds). In certain embodiments, the immunoassay analyzer has at least 30 incubation positions, alternatively at least 40 incubation positions, alternatively at least 50 incubation positions, alternatively at least 60 incubation positions, alternatively at least 70 incubation positions, alternatively at least 80 incubation positions, alternatively at least 90 incubation positions, alternatively at least 100 incubation positions, alternatively at least 125 incubation positions, alternatively at least 150 incubation positions, alternatively at least 175 incubation positions, or alternatively at least 200 incubation positions.

[0216] In an aspect, the time to first result (TTFR) of an immunoassay analyzer described herein is about 60 minutes or less. The “TTFR” is a measure of time from when the sample is aspirated to when the presence and / or concentration of an analyte is determined. In certain embodiments the TTFR is about 60 minutes or less, alternatively about 55 minutes or less, alternatively about 50 minutes or less, alternatively about 45 minutes or less, alternatively about 40 minutes or less, alternatively about 35 minutes or less, alternatively about 30 minutes or less, alternatively about25 minutes or less, alternatively about 20 minutes or less, alternatively about 1 minutes or less, or alternatively about 10 minutes or less.

[0217] In an embodiment, the reagent pack may be configured to accommodate sufficient volumes of reagents for multiple instances of an assay. In some embodiments, each reagent pack includes reagents for about 1, about 5, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 instances of an assay. In some embodiments, each reagent pack includes reagent for about 50 instances of an assay. In some embodiments, each reagent pack includes reagent for about 250 instances of an assay. In some embodiments, each reagent pack includes reagent for about 500 instances of an assay. In some embodiments, each reagent pack includes reagent for about 650 instances of an assay. In some embodiments, the reagent pack may include a plurality of reagent vessels, wherein the reagent pack is configured to store a volume of reagent required for at least about one instance of an assay. In some embodiments, the reagent pack is configured to store a volume of reagent required for up to 650 instances of an assay. In a non-limiting example, the reagent pack includes at least three reagent vessels, wherein each reagent vessel is independently configured to store a volume of reagent required for at least about one instance of an assay. In an embodiment, each reagent vessel is independently configured to store a volume of reagent required for up to 650 instances of an assay. In a non-limiting example, the reagent pack includes at least four reagent vessels, wherein each reagent vessel is independently configured to store a volume of reagent required for at least about one instance of an assay. In an embodiment, each reagent vessel is independently configured to store a volume of reagent required for up to 650 instances. In a non-limiting example, the reagent pack includes at least five reagent vessels, wherein each reagent vessel is independently configured to store a volume of reagent required for at least about one instance of an assay. In an embodiment, each reagent vessel is independently configured to store a volume of reagent required for up to 650 instances.III. Assay Methods Using Chemiluminescent Dioxetanes

[0218] In an aspect of the methods disclosed herein, an analyte (e.g., TSH, TNI, PCT) is detected in a biological sample. In an aspect, the biological sample is serum, whole blood, plasma, and / or cerebral spinal fluid. In an aspect, the method includes exposing the biological sample to a capture antibody configured to bind to at least one portion of an analyte, generating a first reaction mixture.

[0219] The “first reaction mixture” is a result of an incubation between the biological sample and the capture antibody. In certain embodiments, the first reaction mixture is generated by aspirating a portion of the biological sample from a sample vessel and dispensing the aspirated biological sample into a reaction vessel of the immunoassay analyzer and aspirating a portion of a first reagent comprising the capture antibody from at least one reagent vessel and dispensing the aspirated reagent into the reaction vessel. During the incubation, the sample and the capture antibody interact. In an embodiment, the capture antibody binds to a portion of the analyte. In a non-limiting example, the incubation time of the first reaction mixture is at least about 30 minutes, alternatively at least about 40 minutes, or alternatively at least about 50 minutes.

[0220] In an embodiment, the method includes exposing the first reaction mixture to an enzyme- conjugated antibody or an enzyme-conjugated antigen, generating a second reaction mixture. In an aspect, the enzyme comprises an alkaline phosphatase (AP). The “second reaction mixture” is a result of an incubation between the first reaction mixture and the enzyme-conjugated antibody or an enzyme-conjugated antigen. In certain embodiments, the second reaction mixture is generated by aspirating a portion of a second reagent comprising the enzyme-conjugated antibody or an enzyme-conjugated antigen from at least one reagent vessel and dispensing the aspirated reagent into the reaction vessel. During the incubation, the first reaction mixture and the enzyme- conjugated antibody or an enzyme-conjugated antigen interact. In an embodiment, the enzyme- conjugated antibody is configured to bind at least one portion of an analyte. In another embodiment, the enzyme-conjugated antigen is configured to bind at least one portion of the capture antibody. In an aspect, the incubation time of the second reaction mixture is at least about 2 minutes, alternatively at least about 5 minutes, alternatively at least about 8 minutes, or alternatively at least about 10 minutes.

[0221] In an embodiment, the method exposing the second reaction mixture to a substrate formulation comprising a 1,2 dioxetane compound and at least one phosphonium surfactant. In an embodiment, a detection mixture is generated by aspirating the substrate formulation and dispensing the aspirated substrate formation into the reaction vessel. In an aspect, the detection mixture is incubated for at least 20 seconds, alternatively at least 30 seconds, alternatively at least 40 seconds, alternatively at least 50 seconds, alternatively at least 60 seconds, alternatively at least 70 seconds, alternatively at least 80 seconds, alternatively at least 90 seconds, alternatively at least 100 seconds, alternatively at least 110 seconds, alternatively at least 120 seconds, alternatively atleast 130 seconds, alternatively at least 140 seconds, alternatively at least 150 seconds, alternatively at least 160 seconds, alternatively at least 170 seconds, alternatively at least 180 seconds, alternatively at least 190 seconds, alternatively at least 200 seconds, alternatively at least 210 seconds, alternatively at least 220 seconds, alternatively at least 230 seconds, alternatively at least 240 seconds, alternatively at least 250 seconds, alternatively at least 260 seconds, alternatively at least 270 seconds, or alternatively at least 280 seconds.

[0222] In an aspect, the substrate formulation is configured to produce chemiluminescence. These substrates can produce light and thereby provide detection corresponding to a quantity of analytes captured. In certain embodiments, the reaction between the enzyme-conjugated antibody or the enzyme-conjugated antigen and the substrate formulation generates a chemiluminescent detection signal.

[0223] At operation, the substrate is added to the vessel with the second reaction mixture and light generated by the reaction is measured with a luminometer. The light generated (e.g., the detection signal) is recorded and the amount of analyte in the sample is then determined from a stored, multipoint calibration curve. In an embodiment, the detector can generate an output signal that can be processed to generate a relative light unit (“RLU”) value (i.e., an output response) indicating a result of the assay. For example, a larger RLU value indicates more light, which indicates a larger amount of the analyte in the biological sample than a smaller RLU value indicates.

[0224] In an exemplary method of producing light from the reaction of the substrate with a phosphatase enzyme, the reaction is performed at a temperature between 5° C and 50° C, preferably between 20° C and 40° C in an aqueous buffer solution at a pH between 7 and 12, 8 and 11, or preferably between 8.5 and 10.

[0224] In an aspect, capture antibody, enzyme-conjugated antibody and / or enzyme-conjugated antigen is conjugated to a magnetic bead or a magnetic particle. In an embodiment, the first reaction mixture, second reaction mixture, and / or detection mixture is subjected to a magnetic field prior to detection. Magnetization may be used to retain desired components within a reaction vessel. In certain embodiments, the reaction vessel is moved near one or more magnets after the introduction of the first reagent or second reagent. The one or more magnets attract the magnetic bead(s) or magnetic particle(s) to one or more sides of the reaction vessel. The washing arrangement is used to wash the reaction vessel for a predetermined number to times. Whilewashing the magnet(s) retain the magnetic bead(s) or magnetic particle(s) while the unreacted components arc washed away. In certain embodiments, increasing the numbers of wash actions may result in a better signal to noise ratio and increase the sensitivity of the assay.

[0225] In an aspect, the method is performed using an immunoassay analyzer herein and the cycle time is about 45 seconds or less. In certain embodiments, the cycle time is about 40 seconds or less, alternatively about 35 seconds or less, alternatively about 30 seconds or less, alternatively about 25 seconds or less, alternatively about 20 seconds or less, or alternatively about 15 seconds.

[0226] In an aspect, the method is performed using an immunoassay analyzer disclosed herein and the time to first result (TTFR) is about 60 minutes or less. In certain embodiments the TTFR is about 55 minutes or less, alternatively about 50 minutes or less, alternatively about 45 minutes or less, alternatively about 40 minutes or less, alternatively about 35 minutes or less, alternatively about 30 minutes or less, alternatively about 25 minutes or less, alternatively about 20 minutes or less, alternatively about 15 minutes or less, or alternatively about 10 minutes or less.

[0227] In an aspect, the disclosed methods include the detection of TNI using an immunoassay. Depending on the analysis desired, different enzyme-conjugated affinity molecules may be used in the TNI assay. For example, the affinity molecule may be an antibody, wherein the antibody is configured to bind at least one portion of TNI. In another non-limiting example, the affinity molecule is an antigen, wherein the antigen is configured to bind at least one portion of the capture antibody. In some aspects, TNI commercial assays were employed per manufacturers’ protocol. A non-limiting assay protocol according to an aspect of the invention is listed in Example 2.

[0228] In some aspects of the method, the ratio of the signal produced by cleavage of the enzyme to the background noise for a TNI assay is at least about 1, alternatively at least about 10, alternatively at least about 20, alternatively at least about 30, alternatively at least about 40, alternatively at least about 50, alternatively at least about 100, alternatively at least about 200, alternatively at least about 300, alternatively at least about 400, alternatively at least about 500, alternatively at least about 600, alternatively at least about 700, alternatively at least about 800, alternatively at least about 900, alternatively at least about 1,000, alternatively at least about 2,000, alternatively at least about 3,000, alternatively at least about 4,000, alternatively at least about 5,000, alternatively at least about 6,000, alternatively at least about 7,000, alternatively at least about 8,000, alternatively at least about 9,000, alternatively at least about 10,000, alternatively at least about 15,000, alternatively at least about 20,000, or alternatively at least about 25,000. Thesignal-to-noise ratio (S / SO) gives a measure of the degree of confidence that a difference in signal noise and background is real.

[0229] In some aspects, the levels of TNI quantified have a concentration that is at least IX greater than a LoQ for the method, alternatively at least 2X greater than the LoQ, or alternatively at least 3X greater than the LoQ.

[0230] In some aspects, the TNI immunoassay is used to diagnose myocardial infarction, and / or monitor symptoms of myocardial ischemia; new ischemic changes in the electrocardiogram (ECG); development of pathological Q waves in the ECG; imaging evidence of new loss of viable myocardium or new regional wall motion abnormality in a pattern consistent with an ischemic etiology; and / or identification of a coronary thrombus by angiography or autopsy. In some aspects, the TNI immunoassay is used to monitor a rise and / or fall in cardiac Tn values. In some aspects, the method may further include administering an effective amount of a pharmaceutical composition to the subject and / or identifying a course of treatment (or treatment protocol) based on the cardiac Tn values.

[0231] The terms "treat", "treating", or "treatment" refer to administering to a subject a compound or pharmaceutical composition to partially or completely alleviate, inhibit, ameliorate, or relieve the condition from which the subject is suffering. This means any manner in which one or more of the symptoms of a condition are ameliorated or otherwise beneficially altered. As used herein, amelioration of the symptoms of a particular condition refers to any lessening, whether permanent or temporary, lasting or transient, that can be attributed to or associated with treatment by the compounds, compositions, and methods of the present disclosure. For example, treating a subject can mean eliminating or reducing the clinical signs of a condition in the subject; arrest, inhibit, or slow the progression of the condition in the subject; and / or decrease the number, frequency, or severity of clinical symptoms of the condition in the subject. A “treatment protocol” is a protocol or regime developed regarding specific therapies (including pharmaceuticals or therapeutic interventions) for treatment. A “therapeutic intervention” refers to a clinical intervention intended to manage a disease, condition, disorder or injury and avoid further clinical interventions.

[0232] An "effective amount" includes a "therapeutically effective amount" and a "prophylactically effective amount." The term "therapeutically effective amount" refers to an amount effective in treating and / or ameliorating a condition in a subject. The term "prophylactically effective amount" refers to an amount effective in preventing and / orsubstantially lessening the chances of a condition in a subject. The effective amount of the pharmaceutical composition may be administered orally or via intravenous injection. The exact amount required to achieve a therapeutically effective outcome will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition, the particular composition, its mode of administration, its mode of activity, and the like.

[0233] In an aspect, the disclosed methods include the detection of TSH using an immunoassay. Depending on the analysis desired, different enzyme-conjugated affinity molecules may be used in the TSH assay. For example, the affinity molecule may be an antibody, wherein the antibody is configured to bind at least one portion of TSH. In another non-limiting example, the affinity molecule is an antigen, wherein the antigen is configured to bind at least one portion of the capture antibody. In some aspects, TSH commercial assays were employed per manufacturers’ protocol. A non-limiting assay protocol according to an aspect of the invention is listed in Example 3.

[0234] In some aspects of the method, the ratio of the signal produced by cleavage of the enzyme to the background noise for a TSH assay is at least about 5, alternatively at least about 10, alternatively at least about 20, alternatively at least about 30, alternatively at least about 40, alternatively at least about 50, alternatively at least about 60, alternatively at least about 70, alternatively at least about 80, alternatively at least about 90, alternatively at least about 100, alternatively at least about 200, alternatively at least about 300, alternatively at least about 400, alternatively at least about 500, alternatively at least about 600, alternatively at least about 700, alternatively at least about 800, alternatively at least about 900, alternatively at least about 1,000, alternatively at least about 2,000, alternatively at least about 3,000, alternatively at least about 4,000, alternatively at least about 5,000, alternatively at least about 6,000, alternatively at least about 7,000, or alternatively at least about 8,000.

[0235] In some aspects, the levels of TSH quantified have a concentration that is at least IX greater than a LoQ for the method, alternatively at least 2X greater than the LoQ, or alternatively at least 3X greater than the LoQ.

[0236] In some aspects, the TSH immunoassay is used for detecting TSH, abnormal levels of which are associated with various thyroid disorders. In certain embodiments, the TSH is measured in conjunction with thyroid hormones or antibodies to: 1) detect or exclude hypothyroidism or hyperthyroidism; 2) monitor T4 replacement treatment in hypothyroidism or antithyroid treatment in hyperthyroidism; 3) monitor TSH suppression in thyroid cancer patients on thyroxine therapy;and / or 4) assess the response to TRH stimulation testing. In certain embodiments, the TSH assay is able to distinguish different levels of TSH suppression associated with Graves’ disease and subclinical hyperthyroidism and to assist in the diagnosis of gestational and postpartum thyroid diseases. In some aspects, the method may further include administering an effective amount of a pharmaceutical composition to the subject and / or identifying a course of treatment (or treatment protocol) based on the TSH values, alone or in combination with other clinical factors.

[0237] In an aspect, the disclosed methods include the detection of PCT using an immunoassay. Depending on the analysis desired, different enzyme-conjugated affinity molecules may be used in the PCT assay. For example, the affinity molecule may be an antibody, wherein the antibody is configured to bind at least one portion of PCT. In another non-limiting example, the affinity molecule is an antigen, wherein the antigen is configured to bind at least one portion of the capture antibody. In some aspects, PCT commercial assays were employed per manufacturers’ protocol. A non-limiting assay protocol according to an aspect of the invention is listed in Example 4.

[0238] In some aspects, the ratio of the signal produced by cleavage of the enzyme to the background noise for a PCT assay is at least about 2,000, alternatively at least about 3,000, alternatively at least about 4,000, or alternatively at least about 5,000. In some aspects, the PCT assay has a LoQ of about 100 fg / mL or less, 50 fg / mL or less, or 34 fg / mL or less.

[0239] In some aspects, the PCT assay is used to measure PCT in conjunction with other laboratory findings and clinical assessments aids in the risk assessment of critically ill patients on their first day of Intensive Care Unit (ICU) admission for progression to severe sepsis and septic shock.

[0240] In some aspects, the method may further include administering an effective amount of a pharmaceutical composition to the subject and / or identifying a course of treatment (or treatment protocol) based on the PCT values in combination with other clinical factors.

[0241] Additional examples are provided below.

[0242] EXAMPLES

[0243] Example 1: Substrate Signal Comparison

[0244] The difference in the chemiluminescence produced by a substrate formulation comprising a 1,2 dioxetane compound of Formula I herein and a phosphonium surfactant according to an aspect of the disclosure (Substrate 1) and a alkaline phosphatase chemiluminescent substratecomprising the structure (Substrate 2) were compared. The signal comparison was performed using luminometer.

[0245] A Substrate 1 test solution was prepared by adding 0.125 mg / mL of Substrate 1 to a buffer solution comprising 0.25 M 2-Amino-2-methyl- 1 -propanol (AMP) buffer and 0.18 mg / mL magnesium chloride.

[0246] An aliquot of the enzyme was placed in six reaction vessels. An aliquot of the Substrate 1 test solution was added to five reaction vessels and an aliquot of Substrate 2 was added to the sixth reaction vessel. Immediately after the injection, the chemiluminescent signal produced was continually read for 280 seconds.

[0247] As shown in FIG. 1, Substrate 1 produced between 4X and 60X the signal of Substrate 2. After substrate injection, Substrate 1 continued to generate more signal the longer the incubation time.

[0248] Example 2: TNI Enhanced Signal Generation

[0249] A series of TNI calibrators were used to compare the signal produced by Substrate 1 and Substrate 2. The calibrator levels were 30 pg / mL (SI), 139 pg / mL (S2), 550 pg / mL (S3), 2,222 pg / mL (S4), 8,967 pg / mL (S5), and 26,224 pg / mL (S6). A control sample (0 pg / mL, SO) was also analyzed.

[0250] The TNI sensitivity comparison was performed using an exemplary immunoassay analyzer having (i) four reagent pipettors and one sample pipettor; (ii) reagent packs configured to store volumes of reagents for at least 50 instances of assays; (iii) an ultrasonic mixer; (iv) the capability to perform over 5 wash cycles per reaction vessel; and (v) a luminometer.

[0251] Reagents including a conjugate comprising an antibody capable of binding to an epitope of TNI linked to alkaline phosphatase (“ALP”) were added to the reaction vessel. A sample aliquot was pipetted into the reaction vessel using a sample pipettor and this first reaction mixture was incubated. Paramagnetic particles conjugated with TNI antibodies capable of binding to an epitopeof TNI were pipetted into a reaction vessel using one of four reagent pipettors. Then the reaction vessel was mixed using the ultrasonic mixer and incubated, generating a second reaction mixture. A magnetic field was applied to the reaction vessel and the first reaction mixture was washed using a wash buffer to remove any unreacted components. Substrate 1 was added to the reaction vessel, allowed to incubate, and the signal generated from the resulting reaction was read using a luminometer. This process was repeated two more times for Substrate 1 and one for Substrate 2.

[0252] Test results were determined automatically by the system software. Detection of analyte in the sample was determined from the measured light production. The signal in Relative Light Units (“RLU”) collected for each assayed sample for each substrate is presented in Table 4. A ratio of signal to noise (S / SO) was calculated at each concentration value. The Substrate 2 assay and Substrate 1 (Run 1) assay utilized the same volume of reagents and sample. The Substrate 1 (Run 2) assay utilized half of the volume of reagents and sample used in the Substrate 1 (Run 1) assay. The Substrate 1 (Run 3) assay utilized a quarter of the volume of reagents and sample used in Substrate 1 (Run 1) assay. The results of each assay are also plotted in FIG. 2.

[0253] Table 4

[0254] From the results shown in Table 4 and FIG. 2, Substrate 1 was found to produce greater signal at all calibrator concentrations as compared to Substrate 2. For example, as shown for S4, the signal produced by Substrate 1 was up to 35 times higher than the signal produced by Substrate 2.

[0255] Example 3: TSH Enhanced Signal Generation

[0256] A series of TSH calibrators were used to compare the signal produced by Substrate 1 and Substrate 2. The calibrator levels were 0.5 pIU / mL (micro-international units per milliliter) (SI), 0.3 pIU / mL (S2), 3 pIU / mL (S3), 15 pIU / mL (S4), and 50.5 pIU / mL (S5). A control sample (0 pIU / mL, SO) was also analyzed.

[0257] The TSH sensitivity comparison was performed using an exemplary immunoassay analyzer having (i) four reagent pipettors and one sample pipettor; (ii) reagent packs configured to store volumes of reagents for at least 50 instances of assays; (iii) an ultrasonic mixer; (iv) the capability to perform over 5 wash cycles per reaction vessel; and (v) a luminometer.

[0258] Paramagnetic particle conjugated with TSH antibodies capable of binding to an epitope of TSH were pipetted into a reaction vessel using one of four reagent pipettors. A sample aliquot was pipetted into the reaction vessel using a sample pipettor. Then the reaction vessel was mixed using the ultrasonic mixer and incubated, generating a first reaction mixture. A magnetic field was applied to the reaction vessel and the first reaction mixture was washed using a wash buffer to remove any unreacted components. A secondary antibody capable of binding a different epitope of TSH conjugated to alkaline phosphatase (“ALP”) was added to the reaction vessel containing the first reaction mixture. The reaction vessel was mixed ultrasonically and incubated, generating a second reaction mixture. A magnetic field was applied to the reaction vessel and the second reaction mixture was washed five times using a wash buffer to remove any unreacted components. Substrate 1 was added to the reaction vessel, allowed to incubate, and the signal generated from the resulting reaction was read using a luminometer. This process was repeated for Substrate 2.

[0259] Test results were determined automatically by the system software. Detection of analyte in the sample was determined from the measured light production by means of the stored calibration data. The RLU data collected for each assayed sample for each substrate is presented in Table 5. A ratio of signal to noise (S / SO) was calculated at each concentration value. The results are also plotted in FIG. 3. The Substrate 1 assay utilized a third of the volume of reagents and sample used in the Substrate 2 assay.

[0260] Table 5

[0261] From the results shown in Table 5 and FIG. 3, Substrate 1 was found to generate higher signal across all concentrations and produced a higher signal to noise ratio (S / SO).

[0262] Example 4: PCT Enhanced Signal Generation

[0263] A PCT calibrator (SI, 8.60 ng / mL) was used to create a series of dilutions to compare the signal produced by Substrate 1 and Substrate 2. The diluted calibrator levels were 8.60 fg / mL (Sl / 100,000), 0.086 pg / mL (Sl / 10,000), 0.86 pg / mL (S 1 / 1,000), 8.60 pg / mL (S 1 / 100), and 0.086 ng / mL (S 1 / 10). A control sample (0 fg / mL, SO) was also analyzed.

[0264] The PCT sensitivity comparison was performed using an exemplary immunoassay analyzer having (i) four reagent pipettors and one sample pipettor; (ii) reagent packs configured to store volumes of reagents for at least 50 instances of assays; (iii) an ultrasonic mixer; (iv) the capability to perform over 5 wash cycles per reaction vessel; and (v) a luminometer.

[0265] Reagents including a conjugate comprising an antibody capable of binding to an epitope of PCT linked to alkaline phosphatase (“ALP”) were added to the reaction vessel. A sample aliquot was pipetted into the reaction vessel using a sample pipettor and this first reaction mixture was incubated. Paramagnetic particles conjugated with PCT antibodies capable of binding to an epitope of PCT were pipetted into a reaction vessel using one of four reagent pipettors. Then the reaction vessel was mixed using the ultrasonic mixer and incubated, generating a second reaction mixture. A magnetic field was applied to the reaction vessel and the second reaction mixture was washed five times using a wash buffer to remove any unreacted components. Substrate 1 was added to the reaction vessel, allowed to incubate, and the signal generated from the resulting reaction was read using a luminometer. This process was repeated for Substrate 2.

[0266] Test results were determined automatically by the system software. Detection of analyte in the sample was determined from the measured light production by means of the stored calibration data. The RLU data collected for each assayed sample for each substrate is presented in Table 6.The assay included a total of 15 replicates for each calibrator dose. A CV% was calculated at each concentration value. The results arc also plotted in FIG. 4.

[0267] Table 6

[0268] From the results shown in Table 6 and FIG. 4, Substrate 1 was found to generate higher signal across all concentrations and produced a higher signal to noise ratio (S / SO).

[0269] The results presented in Table 7 were obtained using the same procedure above, but with reaction times (TTFR) of 55 minutes.

[0270] Table 7

[0271] The signal ratio between the low concentration calibrator and the zero calibrator (SI / SO) was used as a surrogate of sensitivity evaluation.

[0272] An LOQ study, which included a total of 5 replicates using the procedures described above over three days on 1 instrument, was performed. The resulting data was used to calculate the LOQ for Substrate 1 and Substrate 2. Five standard deviations of the SO replicates X concentration of S1 / (RLU Sl-RLU SO) were used to calculate the LOQ for each immunoassay analyzer, the resultsof which are summarized in the Table 7. As can be seen from Table 7, the LOQ of Substrate 1 is at least around 34 fg / mL with CV20%.

[0273] As shown in the Examples above, the unpredictable magnitude of sensitivity of Substrate 1 is superior to commercially available substrates allowing for the sensitivity and precision needed to distinguish and quantify different low levels of several different analytes.

[0274] All features disclosed in the specification, including the claims, abstracts, and drawings, and all the steps in any method or process disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. Each feature disclosed in the specification, including the claims, abstract, and drawings, can be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar- features.

[0275] It will be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

CLAIMSWhat is claimed is:

1. A method of detecting an analyte in a biological sample, the method comprising: exposing the biological sample to a capture antibody configured to bind to at least one portion of an analyte, generating a first reaction mixture; exposing the first reaction mixture to an enzyme-conjugated antibody or an enzyme- conjugated antigen, generating a second reaction mixture; exposing the second reaction mixture to a substrate formulation comprising a 1,2 dioxctanc compound and at least one phosphonium surfactant; wherein the reaction between the enzyme-conjugated antibody or the enzyme-conjugated antigen and the substrate formulation generates a chemiluminescent detection signal; recording the detection signal generated by the reaction; and comparing the recorded signal to a calibration curve to quantify the level of the analyte in the biological sample.

2. The method of claim 1, wherein the 1,2 dioxetane compound is a compound of Formula I or a salt thereof:Formula I wherein each of R1and R2are independently C3-C10 alkyl, or R1and R2taken together with the carbon to which they are attaches provide a C5-C10 cycloalkyl ring;R3is C1-C10 alkyl, Ce-Cio aryl, or heteroaryl;R4is C2-C10 alkenyl;R5is H or C1-C10 alkyl; andX is a phosphate.

3. A method of detecting an analyte in a biological sample, the method comprising: exposing the biological sample to a capture antibody configured to bind to at least one portion of an analyte, generating a first reaction mixture; exposing the first reaction mixture to an enzyme-conjugated antibody or an enzyme- conjugated antigen, generating a second reaction mixture; exposing the second reaction mixture to a substrate formulation comprising a compound ofFormula I or a salt thereof:Formula I wherein each of R1and R2are independently C3-C10 alkyl, or R1and R2taken together with the carbon to which they are attaches provide a C5-C10 cycloalkyl ring;R3is C1-C10 alkyl, Ce-Cio aryl, or heteroaryl;R4is C2-C10 alkenyl;R5is H or C1-C10 alkyl; andX is a phosphate; wherein the reaction between the enzyme-conjugated antibody or the enzyme-conjugated antigen and the substrate formulation generates a chemiluminescent detection signal; recording the detection signal generated by the reaction; and comparing the recorded signal to a calibration curve to quantify the level of the analyte in the biological sample.

4. The method of claim 3, wherein the substrate formulation further comprises at least one phosphonium surfactant.

5. The method of any one of claims 1 to 4, wherein the enzyme-conjugated antibody is configured to bind at least one portion of the analyte.

6. The method of any one of claims 1 to 4, wherein the enzyme-conjugated antigen is configured to bind at least one portion of the capture antibody.

7. The method of any one of claims 1 to 6, wherein the 1,2 dioxetane compound is a compound of Formula II or a salt thereof:Formula II wherein each of R10and R11is independently H, halogen, Ci-Cio alkyl, C2-C10 alkenyl, or Ce-Cio aryl;R3is C1-C10 alkyl, Ce-Cio aryl or heteroaryl;R4is C2-C10 alkenyl;R5is H or C1-C10 alkyl; andX is a phosphate.

8. The method of any one of claims 1 to 7, wherein the 1,2 dioxetane compound is a compound of Formula III or a salt thereof:Formula III wherein each of R10and R11is independently H, halogen, C1-C10 alkyl, C2-C10 alkenyl, or Ce-Cio aryl;R3is C1-C10 alkyl, Ce-Cio aryl or heteroaryl;R5is H or C1-C10 alkyl; andX is a phosphate.

9. The method of any one of claims 1 to 8, wherein the 1,2 dioxetane compound is a compound of Formula IV or a salt thereof:wherein R3is C1-C10 alkyl, Ce-Cio aryl or heteroaryl;R5is H or C1-C10 alkyl; and X is a phosphate.

10. The method of any one of claims 1 to 9, wherein the 1,2 dioxetane compound isor a salt thereof.11 . The method of any one of claims 1 to 10, wherein the 1 ,2 dioxetane compound is 4- mcthoxy-4-(3-phosphatcphcnyl)spiro[l,2-dioctanc-3,2’-adamantanc] or a salt thereof.

12. The method of any one of claims 1 to 11, wherein the phosphonium surfactant is selected from the group consisting of small-molecule phosphonium surfactants and polymeric phosphonium surfactants.

13. The method of claim 12, wherein the small molecule phosphonium surfactant is a compound having the formula:Wherein R12-R14are each independently Ci-Cio alkyl;R15is arylalkyl; andX" is a counterion.

14. The method of claim 12, wherein the polymeric phosphonoium surfactant comprises repeating unit (A), repeating unit (B), or both:wherein Bua is tributyl and Oct3 is trioctyl.

15. The method of any one of claims 1 to 14, wherein the substrate formulation further comprises a magnesium (II) salt.

16. The method of any one of claims 1 to 15, wherein the capture antibody, enzyme- conjugated antibody and / or enzyme-conjugated antigen is conjugated to at least one magnetic bead.

17. The method of any one of claims 1 to 16, wherein the enzyme comprises an alkaline phosphatase (AP).

18. The method of any one of claims 1 to 17, wherein the method is an assay and is performed using an immunoassay analyzer, wherein the immunoassay analyzer comprises: a reagent pack configured to hold a plurality of reagent vessels, wherein each reagent vessel is configured to store a volume of reagent required for at least one instance of the assay, wherein at least one reagent comprises the capture antibody and at least one reagent comprises the enzyme-conjugated antibody or an enzyme-conjugated antigen; a pipettor arrangement comprising at least one reagent pipettor and at least one sample pipettor; and a detector arrangement.

19. The method of claim 18, wherein the reagent vessels comprise an elastomeric self-sealing membrane.

20. The method of claim 18 or claim 19, wherein the reagent pack further comprises containment walls arranged between the reagent vessels.

21. The method of any one of claims 18 to 20, wherein the immunoassay analyzer further comprises a reagent storage unit, wherein the reagent pack is housed in the reagent storage unit.

22. The method of any one of claims 18 to 21, wherein the pipettor arrangement comprises at least a first reagent pipettor, a second reagent pipettor, a third reagent pipettor and at least one sample pipettor.

23. The method of claim 22, wherein the pipettor arrangement further comprises at least a fourth reagent pipettor.

24. The method of claim 22 or claim 23, wherein the first reagent pipettor, second reagent pipettor, third reagent pipettor, and / or fourth reagent pipettor is selectively and / or simultaneously operated.

25. The method of any one of claims 22 to 24, wherein the first reagent pipettor, second reagent pipettor, third reagent pipettor, and / or fourth reagent pipettor are configured to engage a dispense tip prior to aspiration.

26. The method of any one of claims 18 to 25, wherein the method is configured to analyze at least about 200 biological samples / hr.

27. The method of any one of claims 18 to 25, wherein the method is configured to analyze at least about 300 biological samples / hr.

28. The method of any one of claims 18 to 25, wherein the method is configured to analyze at least about 400 biological samples / hr.

29. The method of any one of claims 18 to 28, wherein: the first reaction mixture is generated by aspirating a portion of the biological sample from a sample vessel and dispensing the aspirated biological sample into a reaction vessel of the immunoassay analyzer and aspirating a portion of a first reagent comprising the capture antibody from at least one reagent vessel and dispensing the aspirated reagent into the reaction vessel; the second reaction mixture is generated by aspirating a portion of a second reagent comprising the enzyme-conjugated antibody or an enzyme-conjugated antigen from at least one reagent vessel and dispensing the aspirated reagent into the reaction vessel; and a detection mixture is generated by aspirating the substrate formulation and dispensing the aspirated substrate formation into the reaction vessel.

30. The method of claim 29, wherein incubation time of the first reaction mixture is at least about 30 minutes, alternatively at least about 40 minutes, alternatively at least about 50 minutes, or alternatively at least about 60 minutes.

31. The method of claim 29 or claim 30, wherein incubation time of the second reaction mixture is at least about 2 minutes, alternatively is at least about 5 minutes, alternatively at least about 8 minutes, or alternatively at least about 10 minutes.

32. The method of any one of claims 29 to 31, wherein the detection mixture is incubated for at least 20 seconds, alternatively at least 30 seconds, alternatively at least 40 seconds, alternatively at least 50 seconds, alternatively at least 60 seconds, alternatively at least 70 seconds, alternatively at least 80 seconds, alternatively at least 90 seconds, alternatively at least 100 seconds, alternatively at least 110 seconds, alternatively at least 120 seconds, alternatively at least 130 seconds, alternatively at least 140 seconds, alternatively at least 150 seconds, alternatively at least 160 seconds, alternatively at least 170 seconds, alternatively at least 180 seconds, alternatively at least 190 seconds, alternatively at least 200 seconds, alternatively at least 210 seconds, alternatively at least 220 seconds, alternatively at least 230 seconds,alternatively at least 240 seconds, alternatively at least 250 seconds, alternatively at least 260 seconds, alternatively at least 270 seconds, or alternatively at least 280 seconds.

33. The method of any one of claim 18 to 32, wherein cycle time is about 45 seconds or less, alternatively about 40 seconds or less, alternatively about 35 seconds or less, alternatively about 30 seconds or less, alternatively about 25 seconds or less, alternatively about 20 seconds or less, or alternatively about 15 seconds.

34. The method of any one of claim 18 to 33, wherein time to first result (TTFR) is about 60 minutes or less, alternatively about 55 minutes or less, alternatively about 50 minutes or less, 45 minutes or less, alternatively about 40 minutes or less, alternatively about 35 minutes or less, alternatively about 30 minutes or less, alternatively about 25 minutes or less, alternatively about 20 minutes or less, alternatively about 15 minutes or less, or alternatively about 10 minutes or less.

35. The method of any one of claims 18 to 34, wherein the detector arrangement comprises a light detector configured to sense photons emitted from assay reactions over a period of time, an analog circuit configured to provide an analog signal based on the photons emitted from the assay reactions over the period of time, and a counter circuit configured to provide a photon count based on the photons emitted from the assay reactions over the period of time.

36. The method of any one of claim 18 to 35, wherein the immunoassay analyzer further comprises an ultrasonic mixing module.

37. The method of claim 36, wherein the first reaction mixture, second reaction mixture, and / or detection mixture is agitated via the ultrasonic mixing module.

38. The method of any one of claims 29 to 37, wherein the first reaction mixture, second reaction mixture, and / or detection mixture comprises unreacted components, and the immunoassay analyzer further comprises a washing arrangement, wherein the washing arrangement is configured to perform at least one wash action to wash away at least a portion of unreacted components, alternatively configured to perform at least two wash actions, alternatively configured to perform at least three wash actions, alternatively configured to perform at least four wash actions, alternatively configured to perform at least five wash actions, alternatively configured to perform at least six wash actions, alternatively configured to perform at least seven wash actions, alternatively configured to perform at least eight wash actions, alternatively configured to perform at least nine wash actions, or alternatively configured to perform at least ten wash actions.

39. The method of claim 38, wherein the first reaction mixture, second reaction mixture, and / or detection mixture is subjected to a magnetic field prior to performing the at least one wash action.

40. The method of any one of claims 18 to 39, wherein the immunoassay analyzer further comprises: a machine vision apparatus comprising an image capture device and an image interpretation device configured to monitor instrument and / or assay functionalities of the immunoassay analyzer.

41. The method of claim 40, wherein the instrument functionalities are selected from the group consisting of optical sensors, pressure sensors and thermistors.

42. The method of claim 41 , wherein the assay functionalities are selected from the group consisting of sample volume monitoring, total reagent volume monitoring, residual volume monitoring, and particle retention monitoring.

43. The method of any one of claim 1 to 42, wherein the biological sample is serum, whole blood, plasma, and / or cerebral spinal fluid.

44. A method of detecting cardiac troponin I (TNI) in a biological sample, the method comprising: exposing the biological sample to a capture antibody configured to bind to at least one portion of TNI, generating a first reaction mixture; exposing the first reaction mixture to an enzyme-conjugated affinity molecule, forming a second reaction mixture; exposing the second reaction mixture to a substrate formulation comprising a 1,2 dioxetane compound and at least one phosphonium surfactant; wherein the reaction between the enzyme-conjugated affinity molecule and the substrate formulation generates a chemiluminescent detection signal; recording the detection signal generated by the reaction; and comparing the recorded signal to a calibration curve to quantify the level of the TNI in the biological sample.

45. The method of claim 44, wherein the chemiluminescent detection signal produced is at least about 10X greater, alternatively at least about 20X greater, or alternatively at least about 30X greater than a signal produced by the method wherein the substrate formulation comprises a compound of the following formula:

46. The method of claim 44 or claim 45, wherein the levels of TNI quantified have a concentration that is at least IX greater than a limit of quantification (LoQ) for the method, alternatively at least 2X greater than the LoQ, or alternatively at least 3X greater than the LoQ.

47. The method of any one of claims 44 to 46, wherein the ratio of the signal produced by cleavage of the enzyme to the background noise is at least about 1, alternatively at least about 10, alternatively at least about 20, alternatively at least about 30, alternatively at least about 40, alternatively at least about 50, alternatively at least about 100, alternatively at least about 200, alternatively at least about 300, alternatively at least about 400, alternatively at least about 500, alternatively at least about 600, alternatively at least about 700, alternatively at least about 800, alternatively at least about 900, alternatively at least about 1,000, alternatively at least about 2,000, alternatively at least about 3,000, alternatively at least about 4,000, alternatively at least about 5,000, alternatively at least about 6,000, alternatively at least about 7,000, alternatively at least about 8,000, alternatively at least about 9,000, alternatively at least about 10,000, alternatively at least about 15,000, alternatively at least about 20,000, or alternatively at least about 25,000.

48. The method of any one of claims 44 to 47, wherein the affinity molecule is an antibody, a monoclonal antibody, a polyclonal antibody, a synthetic antibody mimic, an aptamer, an affimer, DARPins, oligonucleotide, peptide, or antigen.

49. The method of claim 48, wherein the affinity molecule is an antibody, wherein the antibody is configured to bind at least one portion of TNI.

50. The method of claim 48, wherein the affinity molecule is an antigen, wherein the antigen is configured to bind at least one portion of the capture antibody.

51. The method of any one of claims 44 to 50, wherein the capture antibody and / or enzyme- conjugated affinity molecule is conjugated to at least one magnetic bead.

52. The method of any one of claims 44 to 51, wherein the enzyme comprises an alkaline phosphatase (AP).

53. The method of any one of claims 44 to 52, wherein the 1,2 dioxetane compound is a compound of Formula I or a salt thereof:Formula I wherein each of R1and R2are independently C3-C10 alkyl, or R1and R2taken together with the carbon to which they are attaches provide a C5-C10 cycloalkyl ring;R3is Ci-Cio alkyl, Ce-Cio aryl, or heteroaryl;R4is C2-C10 alkenyl;R5is H or Ci-Cio alkyl; andX is a phosphate.

54. The method of any one of claims 44 to 53, wherein the 1,2 dioxetane compound is a compound of Formula II or a salt thereof:Formula II wherein each of R10and R11is independently H, halogen, C1-C10 alkyl, C2-C10 alkenyl, or Ce-Cio aryl;R3is C1-C10 alkyl, Ce-Cio aryl or heteroaryl;R4is C2-C10 alkenyl;R5is H or C1-C10 alkyl; andX is a phosphate.

55. The method of any one of claims 44 to 54, wherein the 1,2 dioxetane compound is a compound of Formula III or a salt thereof:Formula III wherein each of R10and R11is independently H, halogen, C1-C10 alkyl, C2-C10 alkenyl, or Ce-Cio aryl;R3is C1-C10 alkyl, Ce-Cio aryl or heteroaryl;R5is H or C1-C10 alkyl; andX is a phosphate.

56. The method of any one of claims 44 to 55, wherein the 1,2 dioxetane compound is a compound of Formula IV or a salt thereof:Formula IV wherein R3is C1-C10 alkyl, C6-C10 aryl or heteroaryl;R5is H or Ci -C 10 alkyl, X is a photphate.

57. The method of any one of claims 44 to 56, wherein the 1 ,2 dioxetane compound isor a salt thereof.

58. The method of any one of claims 44 to 57, wherein the 1,2 dioxetane compound is 4- methoxy-4-(3-phosphatephenyl)spiro[l,2-dioetane-3,2’-adamantane] or a salt thereof.

59. The method of any one of claims 44 to 58, wherein the phosphonium surfactant is selected from the group consisting of small molecule phosphonium surfactants and polymeric phosphonium surfactants.

60. The method of claim 59, wherein the small molecule phosphonium surfactant is a compound having the formula:Wherein R12-R14 are each independently Cl -CIO alkyl;R15 is arylalkyl; andX’ is a counterion.

61. The method of claim 59, wherein the polymeric phosphonoium surfactant comprises repeating unit (A), repeating unit (B), or both:wherein Bu3 is tributyl and Oct3 is trioctyl.

62. The method of any one of claims 44 to 61, wherein the substrate formulation further comprises a magnesium (II) salt.

63. The method of any one of claims 44 to 62, wherein the method is an assay and is performed using an immunoassay analyzer, wherein the immunoassay analyzer comprises: a reagent pack configured to hold a plurality of reagent vessels, wherein each reagent vessel is configured to store a volume of reagent required for at least one instance of the assay, wherein at least one reagent comprises the capture antibody and at least one reagent comprises the enzyme-conjugated affinity molecule; a pipettor arrangement comprising at least one reagent pipettor and at least one sample pipettor; and a detector arrangement.

64. The method of claim 63, wherein the reagent vessels comprise an elastomeric self-sealing membrane.

65. The method of claim 63 or claim 64, wherein the reagent pack further comprises containment walls arranged between the reagent vessels.

66. The method of any one of claims 63 to 65, wherein the immunoassay analyzer further comprises a reagent storage unit, wherein the reagent pack is housed in the reagent storage unit.

67. The method of any one of claims 63 to 66, wherein the pipettor arrangement comprises at least a first reagent pipettor, a second reagent pipettor, a third reagent pipettor and at least one sample pipettor.

68. The method of claim 67, wherein the pipettor arrangement further comprises at least a fourth reagent pipettor.

69. The method of claim 67 or claim 68, wherein the first reagent pipettor, second reagent pipettor, third reagent pipettor, and / or fourth reagent pipettor is selectively and / or simultaneously operated.

70. The method of any one of claims 67 to 69, wherein the first reagent pipettor, second reagent pipettor, third reagent pipettor, and / or fourth reagent pipettor are configured to engage a dispense tip prior to aspiration.

71. The method of any one of claims 67 to 70, wherein the method is configured to analyze at least about 200 biological samples / hr.

72. The method of any one of claims 67 to 70, wherein the method is configured to analyze at least about 300 biological samples / hr.

73. The method of any one of claims 67 to 70, wherein the method is configured to analyze at least about 400 biological samples / hr.

74. The method of any one of claims 63 to 73, wherein: the first reaction mixture is generated by aspirating a portion of the biological sample from a sample vessel and dispensing the aspirated biological sample into a reaction vessel of the immunoassay analyzer and aspirating a portion of a first reagent comprising the capture antibody from at least one reagent vessel and dispensing the aspirated reagent into the reaction vessel; the second reaction mixture is generated by aspirating a portion of a second reagent comprising the enzyme-conjugated affinity molecule from at least one reagent vessel and dispensing the aspirated reagent into the reaction vessel; and a detection mixture is generated by aspirating the substrate formulation and dispensing the aspirated substrate formation into the reaction vessel.

75. The method of claim 74, wherein incubation time of the first reaction mixture is at least about 30 minutes, alternatively at least about 40 minutes, alternatively at least about 50 minutes, or alternatively at least about 60 minutes.

76. The method of claim 74 or claim 75, wherein incubation time of the second reaction mixture is at least about 2 minutes, alternatively is at least about 5 minutes, alternatively at least about 8 minutes, or alternatively at least about 10 minutes.

77. The method of any one of claims 74 to 76, wherein the detection mixture is incubated for at least 20 seconds, alternatively at least 30 seconds, alternatively at least 40 seconds, alternatively at least 50 seconds, alternatively at least 60 seconds, alternatively at least 70 seconds, alternatively at least 80 seconds, alternatively at least 90 seconds, alternatively at least 100 seconds, alternatively at least 110 seconds, alternatively at least 120 seconds, alternatively at least 130 seconds, alternatively at least 140 seconds, alternatively at least 150 seconds, alternatively at least 160 seconds, alternatively at least 170 seconds, alternatively at least 180 seconds, alternatively at least 190 seconds, alternatively at least 200 seconds, alternatively at least 210 seconds, alternatively at least 220 seconds, alternatively at least 230 seconds, alternatively at least 240 seconds, alternatively at least 250 seconds, alternatively at least 260 seconds, alternatively at least 270 seconds, or alternatively at least 280 seconds.

78. The method of any one of claim 63 to 77, wherein cycle time is about 45 seconds or less, alternatively about 40 seconds or less, alternatively about 35 seconds or less, alternatively about 30 seconds or less, alternatively about 25 seconds or less, alternatively about 20 seconds or less, or alternatively about 15 seconds.

79. The method of any one of claim 63 to 78, wherein time to first result (TTFR) is about 60 minutes or less, alternatively about 55 minutes or less, alternatively about 50 minutes or less, 45 minutes or less, alternatively about 40 minutes or less, alternatively about 35 minutes or less, alternatively about 30 minutes or less, alternatively about 25 minutes or less, alternatively about 20 minutes or less, alternatively about 15 minutes or less, or alternatively about 10 minutes or less.

80. The method of any one of claims 63 to 79, wherein the detector arrangement comprises a light detector configured to sense photons emitted from assay reactions over a period of time, an analog circuit configured to provide an analog signal based on the photons emitted from the assay reactions over the period of time, anda counter circuit configured to provide a photon count based on the photons emitted from the assay reactions over the period of time.

81. The method of any one of claim 63 to 80, wherein the immunoassay analyzer further comprises an ultrasonic mixing module.

82. The method of claim 81, wherein the first reaction mixture, second reaction mixture, and / or detection mixture is agitated via the ultrasonic mixing module.

83. The method of any one of claims 63 to 82, wherein the first reaction mixture, second reaction mixture, and / or detection mixture comprise unreacted components, and the immunoassay analyzer further comprises a washing arrangement, wherein the washing arrangement is configured to perform at least one wash action to wash away at least a portion of unreacted components, alternatively configured to perform at least two wash actions, alternatively configured to perform at least three wash actions, alternatively configured to perform at least four wash actions, alternatively configured to perform at least five wash actions, alternatively configured to perform at least six wash actions, alternatively configured to perform at least seven wash actions, alternatively configured to perform at least eight wash actions, alternatively configured to perform at least nine wash actions, or alternatively configured to perform at least ten wash actions.

84. The method of claim 83, wherein the first reaction mixture, second reaction mixture, and / or detection mixture is subjected to a magnetic field prior to performing the at least one wash action.

85. The method of any one of claims 63 to 84, wherein the immunoassay analyzer further comprises:a machine vision apparatus comprising an image capture device and an image interpretation device configured to monitor instrument and / or assay functionalities of the immunoassay analyzer.

86. The method of claim 85, wherein the instrument functionalities are selected from the group consisting of optical sensors, pressure sensors and thermistors.

87. The method of claim 85, wherein the assay functionalities are selected from the group consisting of sample volume monitoring, total reagent volume monitoring, residual volume monitoring, and particle retention monitoring.

88. The method of any one of claim 44 to 87, wherein the biological sample is serum, whole blood, plasma, and / or cerebral spinal fluid.

89. A method of detecting thyroid stimulating hormone (TSH) in a biological sample, the method comprising: exposing the biological sample to a capture antibody configured to bind to at least one portion of TSH, generating a first reaction mixture; exposing the first reaction mixture to an enzyme-conjugated affinity molecule, forming a second reaction mixture; exposing the second reaction mixture to a substrate formulation comprising a 1,2 dioxetane compound and at least one phosphonium surfactant; wherein the reaction between the enzyme-conjugated affinity molecule and the substrate formulation generates a chemiluminescent detection signal; recording the detection signal generated by the reaction;and comparing the recorded signal to a calibration curve to quantify the level of the TSH in the biological sample.

90. The method of claim 89, wherein the signal produced is a least about 10X greater, alternatively at least about 20X greater, or alternatively at least about 30X greater than a signal produced by the method, wherein the substrate formulation comprises a compound of the following formula:

91. The method of claim 89 or claim 90, wherein the levels of TSH quantified have a concentration that is at least IX greater than a limit of quantification (LoQ) for the method, alternatively at least 2X greater than the LoQ, or alternatively at least 3X greater than the LoQ.

92. The method of any one of claims 89 to 91, wherein the ratio of the signal produced by cleavage of the enzyme to the background noise is at least about 5, alternatively at least about 10, alternatively at least about 20, alternatively at least about 30, alternatively at least about 40, alternatively at least about 50, alternatively at least about 60, alternatively at least about 70, alternatively at least about 80, alternatively at least about 90, alternatively at least about 100, alternatively at least about 200, alternatively at least about 300, alternatively at least about 400, alternatively at least about 500, alternatively at least about 600, alternatively at least about 700, alternatively at least about 800, alternatively at least about 900, alternatively at least about 1,000, alternatively at least about 2,000, alternatively at least about 3,000, alternatively at least about 4,000, alternatively at least about 5,000, alternatively at least about 6,000, alternatively at least about 7,000, or alternatively at least about 8,000.

93. The method of any one of claims 89 to 92, wherein the affinity molecule is an antibody, a monoclonal antibody, a polyclonal antibody, a synthetic antibody mimic, an aptamer, an affimcr,DARPins, oligonucleotide, peptide, or antigen.

94. The method of claim 93, wherein the affinity molecule is an antibody, wherein the antibody is configured to bind at least one portion of TSH.

95. The method of claim 93, wherein the affinity molecule is an antigen, wherein the antigen is configured to bind at least one portion of the capture antibody.

96. The method of any one of claims 89 to 95, wherein the capture antibody and / or enzyme- conjugated affinity molecule is conjugated to at least one magnetic bead.

97. The method of any one of claims 89 to 96, wherein the enzyme comprises an alkaline phosphatase (AP).

98. The method of any one of claims 89 to 97, wherein the 1,2 dioxetane compound is a compound of Formula I or a salt thereof:Formula I wherein each of R1and R2are independently C3-C10 alkyl, or R1and R2taken together with the carbon to which they are attaches provide a C5-C10 cycloalkyl ring;R3is C1-C10 alkyl, Ce-Cio aryl, or heteroaryl;R4is C2-C10 alkenyl;R5is H or C1-C10 alkyl; andX is a phosphate.

99. The method of any one of claims 89 to 98, wherein the 1,2 dioxetane compound is a compound of Formula II or a salt thereof:Formula II wherein each of R10and R11is independently H, halogen, C1-C10 alkyl, C2-C10 alkenyl, or C6-C10 aryl;R3is C1-C10 alkyl, C6-C10 aryl or heteroaryl;R4is C2-C10 alkenyl;R5is H or C1-C10 alkyl; andX is a phosphate.

100. The method of any one of claims 89 to 99, wherein the 1,2 dioxctanc compound is a compound of Formula III or a salt thereof:Formula III wherein each of R10and R11is independently H, halogen, C1-C10 alkyl, C2-C10 alkenyl, or C6-C10 aryl;R3is C1-C10 alkyl, Ce-Cio aryl or heteroaryl;R5is H or Ci-Cio alkyl; and X is a phosphate.

101. The method of any one of claims 89 to 100, wherein the 1,2 dioxetane compound is a compound of Formula IV or a salt thereof:Formula IV wherein R3is Ci-Cio alkyl, Ce-Cio aryl or heteroaryl;R5is H or Ci-Cio alkyl; and X is a phosphate.

102. The method of any one of claims 89 to 101, wherein the 1,2 dioxetane compound isor a salt thereof.

103. The method of any one of claims 89 to 102, wherein the 1,2 dioxetane compound is 4- mcthoxy-4-(3-phosphatcphcnyl)spiro[l,2-dioctanc-3,2’-adamantanc] or a salt thereof.

104. The method of any one of claims 89 to 103, wherein the phosphonium surfactant is selected from the group consisting of small molecule phosphonium surfactants and polymeric phosphonium surfactants.

105. The method of claim 104, wherein the small molecule phosphonium surfactant is a compound having the formula:wherein R12-R14are each independently C’-C10alkyl;R15is arylalkyl; andX" is a counterion.

106. The method of claim 104, wherein the polymeric phosphonoium surfactant comprises repeating unit (A), repeating unit (B), or both:wherein Bus is tributyl and Oct is trioctyl.

107. The method of any one of claims 89 to 106, wherein the substrate formulation further comprises a magnesium (II) salt.

108. The method of any one of claims 89 to 107, wherein the method is an assay and is performed using an immunoassay analyzer, wherein the immunoassay analyzer comprises: a reagent pack configured to hold a plurality of reagent vessels, wherein each reagent vessel is configured to store a volume of reagent required for at least one instance of the assay,wherein at least one reagent comprises the capture antibody and at least one reagent comprises the enzyme-conjugated affinity molecule; a pipettor arrangement comprising at least one reagent pipettor and at least one sample pipettor; and a detector arrangement.

109. The method of claim 108, wherein the reagent vessels comprise an elastomeric selfsealing membrane.

110. The method of claim 108 or claim 109, wherein the reagent pack further comprises containment walls arranged between the reagent vessels.

111. The method of any one of claims 108 to 110, wherein the immunoassay analyzer further comprises a reagent storage unit, wherein the reagent pack is housed in the reagent storage unit.

112. The method of any one of claims 108 to 111, wherein the pipettor arrangement comprises at least a first reagent pipettor, a second reagent pipettor, a third reagent pipettor and at least one sample pipettor.

113. The method of claim 112, wherein the pipettor arrangement further comprises at least a fourth reagent pipettor.

114. The method of claim 112 or claim 113, wherein the first reagent pipettor, second reagent pipettor, third reagent pipettor, and / or fourth reagent pipettor is selectively and / or simultaneously operated.

115. The method of any one of claims 1 12 to 1 14, wherein the first reagent pipettor, second reagent pipettor, third reagent pipettor, fourth reagent pipettor, and / or arc configured to engage a dispense tip prior to aspiration.

116. The method of any one of claims 108 to 115, wherein the method is configured to analyze at least about 200 biological samples / hr.

117. The method of any one of claims 108 to 115, wherein the method is configured to analyze at least about 300 biological samples / hr.

118. The method of any one of claims 108 to 115, wherein the method is configured to analyze at least about 400 biological samples / hr.

119. The method of any one of claims 108 to 118, wherein: the first reaction mixture is generated by aspirating a portion of the biological sample from a sample vessel and dispensing the aspirated biological sample into a reaction vessel of the immunoassay analyzer and aspirating a portion of a first reagent comprising the capture antibody from at least one reagent vessel and dispensing the aspirated reagent into the reaction vessel; the second reaction mixture is generated by aspirating a portion of a second reagent comprising the enzyme-conjugated affinity molecule from at least one reagent vessel and dispensing the aspirated reagent into the reaction vessel; and a detection mixture is generated by aspirating the substrate formulation and dispensing the aspirated substrate formation into the reaction vessel.

120. The method of claim 119, wherein incubation time of the first reaction mixture is at least about 30 minutes, alternatively at least about 40 minutes, alternatively at least about 50 minutes, or alternatively at least about 60 minutes.

121. The method of claim 119 or claim 120, wherein incubation time of the second reaction mixture is at least about 2 minutes, alternatively is at least about 5 minutes, alternatively at least about 8 minutes, or alternatively at least about 10 minutes.

122. The method of any one of claims 119 to 121, wherein the detection mixture is incubated for at least 20 seconds, alternatively at least 30 seconds, alternatively at least 40 seconds, alternatively at least 50 seconds, alternatively at least 60 seconds, alternatively at least 70 seconds, alternatively at least 80 seconds, alternatively at least 90 seconds, alternatively at least 100 seconds, alternatively at least 110 seconds, alternatively at least 120 seconds, alternatively at least 130 seconds, alternatively at least 140 seconds, alternatively at least 150 seconds, alternatively at least 160 seconds, alternatively at least 170 seconds, alternatively at least 180 seconds, alternatively at least 190 seconds, alternatively at least 200 seconds, alternatively at least 210 seconds, alternatively at least 220 seconds, alternatively at least 230 seconds, alternatively at least 240 seconds, alternatively at least 250 seconds, alternatively at least 260 seconds, alternatively at least 270 seconds, or alternatively at least 280 seconds.

123. The method of any one of claim 108 to 122, wherein cycle time is about 45 seconds or less, alternatively about 40 seconds or less, alternatively about 35 seconds or less, alternatively about 30 seconds or less, alternatively about 25 seconds or less, alternatively about 20 seconds or less, or alternatively about 15 seconds.

124. The method of any one of claim 108 to 123, wherein time to first result (TTFR) is about 60 minutes or less, alternatively about 55 minutes or less, alternatively about 50 minutes or less, 45 minutes or less, alternatively about 40 minutes or less, alternatively about 35 minutes or less,alternatively about 30 minutes or less, alternatively about 25 minutes or less, alternatively about 20 minutes or less, alternatively about 15 minutes or less, or alternatively about 10 minutes or less.

125. The method of any one of claims 108 to 124, wherein the detector arrangement comprises a light detector configured to sense photons emitted from assay reactions over a period of time, an analog circuit configured to provide an analog signal based on the photons emitted from the assay reactions over the period of time, and a counter circuit configured to provide a photon count based on the photons emitted from the assay reactions over the period of time.

126. The method of any one of claim 108 to 125, wherein the immunoassay analyzer further comprises an ultrasonic mixing module.

127. The method of claim 126, wherein the first reaction mixture, second reaction mixture, and / or detection mixture is agitated via the ultrasonic mixing module.

128. The method of any one of claims 108 to 127, wherein the first reaction mixture, second reaction mixture, and / or detection mixture comprise unreacted components, and the immunoassay analyzer further comprises a washing arrangement, wherein the washing arrangement is configured to perform at least one wash action to wash away at least a portion of unreacted components, alternatively configured to perform at least two wash actions, alternatively configured to perform at least three wash actions, alternatively configured to perform at least four wash actions, alternatively configured to perform at least five wash actions, alternatively configured to perform at least six wash actions, alternatively configured to perform at least seven wash actions, alternatively configured toperform at least eight wash actions, alternatively configured to perform at least nine wash actions, or alternatively configured to perform at least ten wash actions.

129. The method of claim 128, wherein the first reaction mixture, second reaction mixture, and / or detection mixture is subjected to a magnetic field prior to performing the at least one wash action.

130. The method of any one of claims 108 to 129, wherein the immunoassay analyzer further comprises: a machine vision apparatus comprising an image capture device and an image interpretation device configured to monitor instrument and / or assay functionalities of the immunoassay analyzer.

131. The method of claim 130, wherein the instrument functionalities are selected from the group consisting of optical sensors, pressure sensors and thermistors.

132. The method of claim 131, wherein the assay functionalities are selected from the group consisting of sample volume monitoring, total reagent volume monitoring, residual volume monitoring, and particle retention monitoring.

133. The method of any one of claim 89 to 132, wherein the biological sample is serum, whole blood, plasma, and / or cerebral spinal fluid.

134. A method of detecting procalcitonin (PCT) in a biological sample, the method comprising: exposing the biological sample to a capture antibody configured to bind to at least one portion of PCT, generating a first reaction mixture;exposing the first reaction mixture to an enzyme-conjugated affinity molecule, forming a second reaction mixture; exposing the second reaction mixture to a substrate formulation comprising a 1,2 dioxetane compound and at least one phosphonium surfactant; wherein the reaction between the enzyme-conjugated affinity molecule and the substrate formulation generates a chemiluminescent detection signal; recording the detection signal generated by the reaction; and comparing the recorded signal to a calibration curve to quantify the level of the PCT in the biological sample.

135. The method of claim 134, wherein the signal produced is a least about 10X greater, alternatively at least about 20X greater, or alternatively at least about 30X greater than a signal produced by the method, wherein the substrate formulation comprises a compound of the following formula:

136. The method of claim 134 or claim 135, wherein the assay has a limit of quantification (LoQ) of about 100 fg / mL or less, 50 fg / mL or less, or 34 fg / mL or less.

137. The method of any one of claims 134 to 136, wherein the method has a coefficient of variation (CV) of 20% or less, alternatively 15% or less, alternatively 10% or less, alternatively 5% or less, or alternatively 4% or less.

138. The method of any one of clams 135 to 138, wherein the levels of PCT detected have a concentration that is at least IX greater than a limit of quantification (LoQ) for the method, alternatively at least 2X greater than the LoQ, or alternatively at least 3X greater than the LoQ.

139. The method of any one of claims 134 to 136, wherein the ratio of the signal produced by cleavage of the enzyme to the background noise is at least about 2,000, alternatively at least about 3,000, alternatively at least about 4,000, or alternatively at least about 5,000.

140. The method of any one of claims 134 to 139, wherein the affinity molecule is an antibody, a monoclonal antibody, a polyclonal antibody, a synthetic antibody mimic, an aptamer, an affimer, DARPins, oligonucleotide, peptide, or antigen.

141. The method of claim 140, wherein the affinity molecule is an antibody, wherein the antibody is configured to bind at least one portion of PCT.

142. The method of claim 140, wherein the affinity molecule is an antigen, wherein the antigen is configured to bind at least one portion of the capture antibody.

143. The method of any one of claims 134 to 142, wherein the capture antibody and / or enzyme-conjugated affinity molecule is conjugated to at least one magnetic bead.

144. The method of any one of claims 134 to 143, wherein the enzyme comprises an alkaline phosphatase (AP).

145. The method of any one of claims 134 to 144, wherein the 1,2 dioxetane compound is a compound of Formula I or a salt thereof:Formula I wherein each of R1and R2are independently C3-C10 alkyl, or R1and R2taken together with the carbon to which they are attaches provide a C5-C10 cycloalkyl ring;R3is C1-C10 alkyl, Ce-Cio aryl, or heteroaryl;R4is C2-C10 alkenyl;R5is H or C1-C10 alkyl; andX is a phosphate.

146. The method of any one of claims 134 to 145, wherein the 1,2 dioxetane compound is a compound of Formula II or a salt thereof:Formula II wherein each of R10and R11is independently H, halogen, C1-C10 alkyl, C2-C10 alkenyl, or Ce-Cio aryl;R3is C1-C10 alkyl, Ce-Cio aryl or heteroaryl;R4is C2-C10 alkenyl;R5is H or C1-C10 alkyl; andX is a phosphate.

147. The method of any one of claims 134 to 146, wherein the 1 ,2 dioxetane compound is a compound of Formula III or a salt thereof:Formula III wherein each of R10and R11is independently H, halogen, C1-C10 alkyl, C2-C10 alkenyl, or Ce-Cio aryl;R3is C1-C10 alkyl, C6-C10 aryl or heteroaryl;R5is H or C1-C10 alkyl; andX is a phosphate.

148. The method of any one of claims 134 to 147, wherein the 1,2 dioxetane compound is a compound of Formula IV or a salt thereof:Formula IV wherein R3is C1-C10 alkyl, C6-C10 aryl or heteroaryl;R5is H or C1-C10 alkyl; and X is a phosphate.

149. The method of any one of claims 134 to 148, wherein the 1 ,2 dioxetane compound isor a salt thereof.

150. The method of any one of claims 134 to 149, wherein the 1,2 dioxctanc compound is 4- methoxy-4-(3-phosphatephenyl)spiro[l,2-dioetane-3,2’-adamantane] or a salt thereof.

151. The method of any one of claims 134 to 150, wherein the phosphonium surfactant is selected from the group consisting of small molecule phosphonium surfactants and polymeric phosphonium surfactants.

152. The method of claim 151, wherein the small molecule phosphonium surfactant is a compound having the formula:Wherein R12-R14 are each independently C1-C10 alkyl;R15 is arylalkyl; andX" is a counterion.

153. The method of claim 151, wherein the polymeric phosphonoium surfactant comprises repeating unit (A), repeating unit (B), or both:wherein Bus is tributyl and Oct is trioctyl.

154. The method of any one of claims 134 to 153, wherein the substrate formulation further comprises a magnesium (II) salt.

155. The method of any one of claims 134 to 154, wherein the method is an assay and is performed using an immunoassay analyzer, wherein the immunoassay analyzer comprises: a reagent pack configured to hold a plurality of reagent vessels, wherein each reagent vessel is configured to store a volume of reagent required for at least one instance of the assay, wherein at least one reagent comprises the capture antibody and at least one reagent comprises the enzyme-conjugated affinity molecule; a pipettor arrangement comprising at least one reagent pipettor and at least one sample pipettor; and a detector arrangement.

156. The method of claim 155, wherein the reagent vessels comprise an elastomeric selfsealing membrane.

157. The method of claim 155 or claim 156, wherein the reagent pack further comprises containment walls arranged between the reagent vessels.

158. The method of any one of claims 155 to 157, wherein the immunoassay analyzer further comprises a reagent storage unit, wherein the reagent pack is housed in the reagent storage unit.

159. The method of any one of claims 155 to 158, wherein the pipettor arrangement comprises at least a first reagent pipettor, a second reagent pipettor, a third reagent pipettor and at least one sample pipettor.

160. The method of claim 159, wherein the pipettor arrangement further comprises at least a fourth reagent pipettor.

161. The method of claim 1 9 or claim 160, wherein the first reagent pipettor, second reagent pipettor, third reagent pipettor, and / or fourth reagent pipettor is selectively and / or simultaneously operated.

162. The method of any one of claims 159 to 161, wherein the first reagent pipettor, second reagent pipettor, third reagent pipettor, and / or fourth reagent pipettor are configured to engage a dispense tip prior to aspiration.

163. The method of any one of claims 155 to 162, wherein the method is configured to analyze at least about 200 biological samples / hr.

164. The method of any one of claims 155 to 162, wherein the method is configured to analyze at least about 300 biological samples / hr.

165. The method of any one of claims 155 to 162, wherein the method is configured to analyze at least about 400 biological samples / hr.

166. The method of any one of claims 155 to 165, wherein: the first reaction mixture is generated by aspirating a portion of the biological sample from a sample vessel and dispensing the aspirated biological sample into a reaction vessel of the immunoassay analyzer and aspirating a portion of a first reagent comprising the capture antibody from at least one reagent vessel and dispensing the aspirated reagent into the reaction vessel;the second reaction mixture is generated by aspirating a portion of a second reagent comprising the cnzymc-conjugatcd affinity molecule from at least one reagent vessel and dispensing the aspirated reagent into the reaction vessel; and a detection mixture is generated by aspirating the substrate formulation and dispensing the aspirated substrate formation into the reaction vessel.

167. The method of claim 166, wherein incubation time of the first reaction mixture is at least about 30 minutes, alternatively at least about 40 minutes, alternatively at least about 50 minutes, or alternatively at least about 60 minutes.

168. The method of claim 166 or claim 167, wherein incubation time of the second reaction mixture is at least about 2 minutes, alternatively is at least about 5 minutes, alternatively at least about 8 minutes, or alternatively at least about 10 minutes.

169. The method of any one of claims 166 to 168, wherein the detection mixture is incubated for at least 20 seconds, alternatively at least 30 seconds, alternatively at least 40 seconds, alternatively at least 50 seconds, alternatively at least 60 seconds, alternatively at least 70 seconds, alternatively at least 80 seconds, alternatively at least 90 seconds, alternatively at least 100 seconds, alternatively at least 110 seconds, alternatively at least 120 seconds, alternatively at least 130 seconds, alternatively at least 140 seconds, alternatively at least 150 seconds, alternatively at least 160 seconds, alternatively at least 170 seconds, alternatively at least 180 seconds, alternatively at least 190 seconds, alternatively at least 200 seconds, alternatively at least 210 seconds, alternatively at least 220 seconds, alternatively at least 230 seconds, alternatively at least 240 seconds, alternatively at least 250 seconds, alternatively at least 260 seconds, alternatively at least 270 seconds, or alternatively at least 280 seconds.

170. The method of any one of claim 155 to 169, wherein cycle time is about 45 seconds or less, alternatively about 40 seconds or less, alternatively about 35 seconds or less, alternativelyabout 30 seconds or less, alternatively about 25 seconds or less, alternatively about 20 seconds or less, or alternatively about 15 seconds.

171. The method of any one of claim 155 to 170, wherein time to first result (TTFR) is about 60 minutes or less, alternatively about 55 minutes or less, alternatively about 50 minutes or less, 45 minutes or less, alternatively about 40 minutes or less, alternatively about 35 minutes or less, alternatively about 30 minutes or less, alternatively about 25 minutes or less, alternatively about 20 minutes or less, alternatively about 15 minutes or less, or alternatively about 10 minutes or less.

172. The method of any one of claims 155 to 171, wherein the detector arrangement comprises a light detector configured to sense photons emitted from assay reactions over a period of time, an analog circuit configured to provide an analog signal based on the photons emitted from the assay reactions over the period of time, and a counter circuit configured to provide a photon count based on the photons emitted from the assay reactions over the period of time.

173. The method of any one of claim 155 to 172, wherein the immunoassay analyzer further comprises an ultrasonic mixing module.

174. The method of claim 173, wherein the first reaction mixture, second reaction mixture, and / or detection mixture is agitated via the ultrasonic mixing module.

175. The method of any one of claims 155 to 174, wherein the first reaction mixture, second reaction mixture, and / or detection mixture comprise unreacted components, and the immunoassay analyzer further comprises a washing arrangement,wherein the washing arrangement is configured to perform at least one wash action to wash away at least a portion of unrcactcd components, alternatively configured to perform at least two wash actions, alternatively configured to perform at least three wash actions, alternatively configured to perform at least four wash actions, alternatively configured to perform at least five wash actions, alternatively configured to perform at least six wash actions, alternatively configured to perform at least seven wash actions, alternatively configured to perform at least eight wash actions, alternatively configured to perform at least nine wash actions, or alternatively configured to perform at least ten wash actions.

176. The method of claim 175, wherein the first reaction mixture, second reaction mixture, and / or detection mixture is subjected to a magnetic field prior to performing the at least one wash action.

177. The method of any one of claims 155 to 176, wherein the immunoassay analyzer further comprises: a machine vision apparatus comprising an image capture device and an image interpretation device configured to monitor instrument and / or assay functionalities of the immunoassay analyzer.

178. The method of claim 177, wherein the instrument functionalities are selected from the group consisting of optical sensors, pressure sensors and thermistors.

179. The method of claim 177, wherein the assay functionalities are selected from the group consisting of sample volume monitoring, total reagent volume monitoring, residual volume monitoring, and particle retention monitoring.

180. The method of any one of claim 134 to 179, wherein the biological sample is serum, whole blood, plasma, and / or cerebral spinal fluid.

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