Method of detecting analyte in sample

Abstract

A method of detecting an extracellular analyte in a sample is provided. In some examples, there is provided a method of detecting an extracellular analyte in a sample, the method comprising: providing a biosensor system selected from the group consisting of (i) an extracellular bioluminescent sensor molecule comprising a donor luminophore, an analyte-sensitive domain, and an acceptor fluorophore; and (ii) a precursor system thereof, the precursor system comprising a pair of molecules adapted to react in solution to form the extracellular bioluminescent sensor molecule; incubating an assay composition comprising the sample and the biosensor system for an incubation period sufficient to cause the extracellular analyte to interact with the analyte-sensitive domain if the extracellular analyte is present in the sample; and detecting light emitted from the assay composition after the incubation period, where a ratio of a luminosity associated with the acceptor luminophore to a luminosity associated with the donor luminophore is indicative of an interaction of the extracellular analyte with the analyte-sensitive domain, wherein at least one caged substrate compound is caged off in the assay composition during the incubation period to produce a caged off substrate compound that excites the donor luminophore to perform the detection.

Description

Technical Field

[0001] The present invention relates to a method for detecting an extracellular analyte in a sample. The present invention further relates to a system for detecting an extracellular analyte in a sample, a BRET reagent system, a solid biosensor composition, a substrate dispensing system, and a substrate composition. Although not limited thereto, the present invention is particularly interested in point-of-care (bio)chemical assays for use in production or other non-laboratory environments, such as in the analysis of food and beverage compositions. Background Art

[0002] Point-of-care biochemical tests are needed in many fields such as human healthcare, environmental monitoring, managing agricultural production, and supporting the manufacture of food, drugs, and personal care products. As an example, in the food industry, point-of-care tests can, in principle, provide the quickest information on the (bio)chemical composition of food ingredients and finished products for quality control and cost-effectiveness across the entire value chain. The desired goal is that point-of-care test methods can quickly provide accurate and precise results, even when performed by minimally trained personnel in a production environment.

[0003] Chemiluminescence-based techniques, such as bioluminescence resonance energy transfer (BRET), are attractive for biochemical assays, potentially allowing for highly sensitive real-time optical detection of multiple analytes in many different types of samples. Since the detected light is generated internally within the biosensor, chemiluminescence assays can be used for complex fluid samples containing autofluorescent molecules that would interfere with assays relying on an external light source. BRET assays often use BRET bioluminescence sensor molecules that include a donor luminophore and an acceptor fluorophore linked by a linker including an analyte-sensitive domain. The donor luminophore and the acceptor fluorophore are held close enough (usually spaced <10 nm) and aligned to allow Förster resonance energy transfer from the donor luminophore to the acceptor fluorophore. When the donor luminophore (e.g., luciferase such as Renilla luciferase; RLuc) acts on a suitable substrate (e.g., luciferin such as coelenterazine molecules), light is thus emitted from both the donor luminophore and the acceptor fluorophore, where the relative intensity (luminescence) is highly dependent on the spacer distance and relative orientation of these two moieties. Thus, the interaction of an analyte with the analyte-sensitive domain, such as by binding or cleavage, can be detected by a change in the ratio (BRET ratio) of the luminescence associated with the acceptor fluorophore to the luminescence associated with the donor luminophore.

[0004] BRET biosensor systems containing various donor luminophores, acceptor fluorophores, analyte-sensitive domains, and substrates have been reported. For example, the BRET 2 platform uses RLuc as the donor luminophore, green fluorescent protein (GFP2 ) has been used in biosensing techniques for detecting proteases (e.g., plasmin in milk; Australian Patent 2015243093) or sugars (e.g., maltose or lactose; Australian Patent 2018315053), with a donor fluorophore and coelenterazine (coelenterazine 400a) as the substrate.

[0005] One difficulty with using BRET technology is the instability of the reagents, including substrates that are prone to oxidation, which can affect the accuracy of test results. Another problem is the difficulty in matching the kinetics of the bioluminescence reaction (i.e., the enzymatic oxidation of the substrate by the donor fluorophore) with the kinetics of the interaction of the analyte with the bioluminescence sensor molecule (e.g., by binding to or cleaving an analyte-sensitive domain). As the substrate is consumed, the decay in luminescence may affect the accuracy of the analysis, since the BRET ratio is usually not completely independent of the total luminescence, especially for assays that require a long incubation time for the analyte to interact with the bioluminescence sensor molecule.

[0006] These problems can be addressed in a laboratory setting through cryogenic storage of the reagents, complex and rigorous assay protocols, or the use of microfluidic techniques, but such measures are impractical for widespread use in many point-of-care testing applications where less sophisticated infrastructure and operators are available. More importantly, by optimizing the bioluminescence sensor molecule and the corresponding substrate to enhance the stability and persistence of the luminescence, the robustness of the BRET assay can be improved to some extent, but this may compromise the sensitivity of the sensor and thus its usefulness for detecting certain analytes.

[0007] There is a desire to develop chemiluminescence-based assay techniques that utilize reagents stable at room temperature and minimize the complexity of each assay without unacceptably compromising the reliability and sensitivity of the analysis.

[0008] Accordingly, there has been a continuing need for methods and systems for detecting analytes in a sample that at least partially address one or more of the above disadvantages or provide a useful alternative.

[0009] The citation of patent documents or other matters given herein as prior art should not be taken as an admission that such documents or matters are known or that the information contained therein is part of the common general knowledge as of the priority date of any one of the claims. SUMMARY OF THE INVENTION

[0010] According to a first aspect, the present invention provides a method for detecting an extracellular analyte in a sample, the method comprising: providing a biosensor system selected from (i) an extracellular bioluminescence sensor molecule comprising a donor luminophore, an analyte-sensitive domain, and a receptor fluorophore; and (ii) a precursor system thereof, the precursor system comprising a pair of molecules adapted to react in solution to form the extracellular bioluminescence sensor molecule; incubating a assay composition comprising the sample and the biosensor system for an incubation period sufficient for the extracellular analyte to interact with the analyte-sensitive domain, if the extracellular analyte is present in the sample; and detecting light emitted from the assay composition after the incubation period, wherein the ratio of the luminescence associated with the receptor fluorophore to the luminescence associated with the donor luminophore indicates the interaction of the extracellular analyte with the analyte-sensitive domain, wherein at least one caged substrate compound uncages in the assay composition during the incubation period to produce an uncaged substrate compound that excites the donor luminophore for the detection.

[0011] It has been found that the addition of one or more caged (i.e., protected) substrate compounds, which are configured to uncage (i.e., deprotect) in situ to the substrate of the (BRET) bioluminescence sensor molecule, to the assay composition provides significant and surprising benefits in the methods disclosed herein, making them particularly suitable for point-of-care diagnostic testing in production and other non-laboratory environments. The uncaging of the substrate compound in the assay allows the luminescence generated by the bioluminescence sensor molecule to be maximized and sustained over an extended detection window that comfortably spans the detection time at the end of the incubation period. This ensures that the accuracy of the analysis is not affected by luminescence decay and is insensitive to operator-specific variability when performing the test. Depending on the nature of the analyte-biosensor interaction, the luminescence curve can be optimized for analyte detection after short (e.g., 1-5 minutes) or long (e.g., 10-30+ minutes) incubation times by selecting appropriate caged substrates and uncaging agents. In some embodiments, two or more caged substrate compounds with different uncaging kinetics can be used in combination to further extend and customize the detection window.

[0012] Furthermore, it has been surprisingly found by experiment that solutions of caged substrate compounds can be stored at room temperature for extended periods, for some compounds over one year, without compromising the performance of subsequent BRET assays. This advantageously allows the substrate to be stored in solution for dispensing when needed, thereby minimizing the number of steps involved in the test method while avoiding errors caused by reagent degradation.

[0013] As a result of these advantages, the method of the present invention can be implemented in a simple and robust workflow in a production or other non-laboratory environment.

[0014] In some embodiments, the at least one caged substrate compound is present in the assay composition from the start of the incubation period.

[0015] In some embodiments, the at least one caged substrate compound is uncaged by an uncaging agent present in the assay composition. The uncaging agent can be present in the assay composition from the start of the incubation period.

[0016] In some embodiments, the uncaging agent is independent of the sample. The uncaging agent can be an esterase that is independent of the sample.

[0017] In some embodiments, the uncaging agent is endogenous to the sample. The uncaging agent can be an esterase that is endogenous to the sample.

[0018] In some embodiments, the incubation period is between 1 minute and 30 minutes. In some embodiments, the incubation period is between 5 minutes and 15 minutes.

[0019] In some embodiments, the at least one caged substrate compound is uncaged to provide a luminescence associated with the acceptor fluorophore that is greater than 50% of the maximum value or greater than 80% of the maximum value upon detection of the light in the absence of the extracellular analyte. In the absence of the extracellular analyte, the luminescence associated with the acceptor fluorophore can be greater than 80% of the maximum value in a detection window of at least 3 minutes or at least 5 minutes, wherein the light is detected during the detection window.

[0020] In some embodiments, at least two different caged substrate compounds present in the assay composition from the start of the incubation period are uncaged during the incubation period, thereby producing the uncaged substrate compounds at different reaction rates.

[0021] In some embodiments, each caged substrate compound is a caged coelenterazine compound comprising a carbonyl protecting group at the 3'-position.

[0022] In some embodiments, each caged substrate compound has a structure according to Formula 1 or Formula 2:

[0023]

[0024] Wherein: each R 1 is independently selected from H, OH, OR alk and NH2, wherein R alkis a C1-C6 alkyl group, each R 2 is independently selected from H, Ar, and (CH2) n Ar, where each Ar is independently a carbocyclic aromatic group optionally substituted with OH, C1-C6 alkyl, or halogen, and n is 1 or 2, each L 1 is independently selected from CH2 or S, each R 3 is independently selected from C6H5, CH2C6H5, C1-C6 alkyl, and C1-C6 cycloalkyl, R 4 is independently selected from H and CH3, L 2 is independently selected from (CH2) n and CH═CH, where n is 1-3, and each X is independently a carbonyl protecting group.

[0025] In some embodiments, each X has a structure according to Formula 3, Formula 4, or Formula 5:

[0026]

[0027] where R 5 、R 6 and R 7 are each independently an optionally substituted C1-C 12 alkyl, C1-C 12 cycloalkyl, or C1-C 12 aryl.

[0028] In some embodiments, the uncaged substrate compound is a coelenterazine compound. In some embodiments, the coelenterazine compound is bisdeoxycoelenterazine.

[0029] In some embodiments, in a substrate solution stable at room temperature, the at least one caged substrate compound is introduced into the assay composition, optionally by dropwise dispensing the substrate solution into the assay composition. The substrate solution can be a non-aqueous solution containing a water-miscible aprotic solvent such as DMSO. The method can further comprise storing the substrate solution for at least one week, or at least one month, or at least six months before introducing the substrate solution into the assay composition.

[0030] In some embodiments, the method comprises preparing the assay composition by a method comprising dispersing a solid composition comprising the biosensor system in a liquid. The solid composition can be a lyophilized solid composition. The liquid can comprise the sample, or consist of the sample. The solid composition can further comprise a caging agent for uncaging the at least one caged substrate compound in the assay composition. The assay composition can be prepared and incubated in an assay vessel in which the solid composition was previously lyophilized.

[0031] In some embodiments, the assay composition is prepared and incubated in an assay vessel, and the light emitted from the assay composition is detected passing through the wall of the assay vessel.

[0032] In some embodiments, the extracellular bioluminescence sensor molecule is BRET 2 biosensor.

[0033] In some embodiments, the extracellular analyte is selected from the group consisting of enzymes and carbohydrates. In some embodiments, the extracellular analyte is a protease.

[0034] In some embodiments, the sample is a food or beverage composition, such as a dairy composition.

[0035] In some embodiments, the method comprises:

[0036] ● providing the sample as a liquid for analysis;

[0037] ● providing an assay vessel containing a solid composition comprising an extracellular bioluminescence sensor molecule and optionally a caged reagent;

[0038] ● providing a substrate storage container containing a substrate solution storable at room temperature, the substrate solution storable at room temperature comprising the at least one caged substrate compound;

[0039] ● adding an aliquot of the sample to the assay vessel;

[0040] ● thereafter, transferring an aliquot of the substrate solution storable at room temperature from the substrate storage container to the assay vessel and mixing the assay composition contained therein;

[0041] ● incubating the assay composition in the assay vessel for the incubation period; and

[0042] ● detecting, with a photoelectronic device, the light emitted from the assay composition passing through the wall of the assay vessel.

[0043] According to a second aspect, the present invention provides a system for detecting an extracellular analyte in a sample, the system comprising: a biosensor system selected from (i) an extracellular bioluminescent sensor molecule comprising a donor luminophore, an analyte-sensitive domain, and an acceptor fluorophore; and (ii) a precursor system thereof, the precursor system comprising a pair of molecules adapted to react in solution to form the extracellular bioluminescent sensor molecule, the biosensor system being contained in an assay vessel configured to receive the sample for combination with the biosensor system in an assay composition; a room-temperature stable substrate solution comprising at least one caged substrate compound for dispensing into the assay composition in the assay vessel, the substrate solution being contained in a substrate storage vessel; and a optoelectronic device configured to detect light emitted from the assay composition passing through the wall of the assay vessel, wherein the ratio of the luminescence associated with the acceptor fluorophore to the luminescence associated with the donor luminophore indicates the interaction of the extracellular analyte with the analyte-sensitive domain, wherein the at least one caged substrate compound is adapted to uncage in the assay composition, thereby producing an uncaged substrate compound that excites the donor luminophore.

[0044] According to some embodiments, the optoelectronic device is configured to receive the assay vessel and incubate the assay composition contained therein for an incubation period sufficient for the extracellular analyte to interact with the analyte-sensitive domain, if the extracellular analyte is present in the sample. The optoelectronic device may be configured to incubate the assay composition at a predetermined incubation temperature.

[0045] According to some embodiments, the biosensor system is present in a solid composition. The solid composition may be a lyophilized solid composition. The solid composition may further comprise an uncaging agent for uncaging the at least one caged substrate compound in the assay composition.

[0046] According to some embodiments, the substrate solution comprises at least two different caged substrate compounds adapted to uncage in the assay composition to produce the uncaged substrate compounds at different reaction rates.

[0047] According to some embodiments, each caged substrate compound is a caged coelenterazine compound comprising a carbonyl protecting group at the 3'-position.

[0048] According to some embodiments, each caged substrate compound has a structure according to Formula 1 or Formula 2:

[0049]

[0050] Wherein: each R 1 is independently selected from H, OH, OR alk and NH2, wherein R alk is a C1-C6 alkyl group, each R 2 is independently selected from H, Ar, and (CH2) n Ar, wherein each Ar is independently a carbocyclic aromatic group optionally substituted with OH, C1-C6 alkyl, or halogen, and n is 1 or 2, each L 1 is independently selected from CH2 or S, each R 3 is independently selected from C6H5, CH2C6H5, C1-C6 alkyl, and C1-C6 cycloalkyl, R 4 is independently selected from H and CH3, L 2 is independently selected from (CH2) n and CH=CH, wherein n is 1-3, and each X is independently a carbonyl protecting group.

[0051] According to some embodiments, each X has a structure according to Formula 3, Formula 4, or Formula 5:

[0052]

[0053] Wherein R 5 、R 6 and R 7 are each independently an optionally substituted C1-C 12 alkyl, C1-C 12 cycloalkyl, or C1-C 12 aryl.

[0054] According to some embodiments, the uncaged substrate compound is a coelenterazine compound. The coelenterazine compound can be bisdeoxycoelenterazine.

[0055] According to some embodiments, the substrate solution is a non-aqueous solution containing a water-miscible aprotic solvent, such as DMSO.

[0056] According to some embodiments, the substrate storage vessel is configured to dispense an aliquot of the substrate solution into the assay vessel. The substrate storage vessel can be configured to dispense the substrate solution dropwise into the assay vessel.

[0057] According to some embodiments, the extracellular bioluminescence sensor molecule is a BRET 2 biosensor.

[0058] According to some embodiments, the optoelectronic device includes a first photodetector and a second photodetector. The first photodetector includes a microphotonic multiplier tube equipped with a bandpass filter to selectively allow transmission of light wavelengths associated with the acceptor fluorophore. The second photodetector includes a microphotonic multiplier tube equipped with a bandpass filter to selectively allow transmission of light wavelengths associated with the donor luminophore.

[0059] According to some embodiments, the optoelectronic device is configured to incubate the assay composition at a predetermined incubation temperature.

[0060] According to a third aspect, the present invention provides a BRET reagent system for combining with a sample to produce a bioluminescence assay composition which, after incubation of the bioluminescence assay composition, has a BRET ratio indicative of an extracellular analyte in the sample. The BRET reagent system comprises: (a) a solid composition comprising a biosensor system selected from (i) an extracellular bioluminescence sensor molecule comprising a donor luminophore, an analyte-sensitive domain adapted to interact with the extracellular analyte, and an acceptor fluorophore; and (ii) a precursor system thereof comprising a pair of molecules adapted to react in solution to form the extracellular bioluminescence sensor molecule, wherein the solid composition is capable of being dispersed in a liquid comprising the sample to produce the assay composition; and (b) a room-temperature stable substrate solution comprising at least one caged substrate compound for dispensing into the assay composition, wherein the at least one caged substrate compound is adapted to uncage in the assay composition to produce an uncaged substrate compound that excites the donor luminophore.

[0061] According to some embodiments, the solid composition is contained within an assay vessel configured to receive the liquid comprising the sample and to permit light from the assay composition to transmit through the assay vessel wall for detection.

[0062] According to some embodiments, the solid composition is a lyophilized composition prepared by lyophilizing an aqueous precursor composition in the assay vessel.

[0063] According to some embodiments, the solid composition further comprises an uncaging agent for uncaging the at least one caged substrate compound in the assay composition.

[0064] According to some embodiments, the room temperature stable substrate solution is contained in a substrate storage vessel configured to dispense aliquots of the substrate solution into the assay composition. The substrate storage vessel can be configured to dispense the substrate solution dropwise into the assay composition.

[0065] According to some embodiments, the room temperature stable substrate solution comprises at least two different caged substrate compounds, the at least two different caged substrate compounds being adapted to uncage in the assay composition to produce the uncaged substrate compounds at different reaction rates.

[0066] According to some embodiments, the BRET reagent system is in the form of a kit, the kit comprising instructions for using the BRET reagent system in the method according to any embodiment of the first aspect.

[0067] According to a fourth aspect, the present invention provides a solid biosensor composition for performing BRET detection of an extracellular analyte in a sample, the solid biosensor composition comprising: (a) a biosensor system selected from (i) an extracellular bioluminescent sensor molecule comprising a donor luminophore, an analyte-sensitive domain adapted to interact with the extracellular analyte, and a receptor fluorophore; and (ii) a precursor system thereof, the precursor system comprising a pair of molecules adapted to react in solution to form the extracellular bioluminescent sensor molecule; and (b) an uncaging agent, wherein the solid biosensor composition is capable of being dispersed in a liquid containing the sample to produce an assay composition, and wherein the uncaging agent is adapted to uncage at least one caged substrate compound in the assay composition, thereby producing an uncaged substrate compound that excites the donor luminophore.

[0068] According to a fifth aspect, the present invention provides a substrate dispensing system for performing BRET detection of an extracellular analyte in a sample, the substrate dispensing system comprising a room temperature stable substrate solution comprising at least one caged substrate compound, wherein the substrate solution is contained in a substrate storage vessel configured to dispense a predetermined aliquot of the substrate solution into an assay composition comprising the sample and a BRET bioluminescent sensor molecule.

[0069] In some embodiments, the room temperature stable substrate solution comprises at least two different caged substrate compounds, the at least two different caged substrate compounds being adapted to uncage at different reaction rates.

[0070] According to a sixth aspect, the present invention provides a substrate composition for BRET detection of extracellular analytes in a sample, the substrate composition comprising at least two different caged substrate compounds, the at least two different caged substrate compounds being adapted to uncage in an assay composition comprising the sample and a BRET bioluminescence sensor molecule, thereby producing uncaged substrate compounds at different reaction rates.

[0071] Where the terms "comprise", "comprises" and "comprising" are used in this specification (including the claims), they should be construed as specifying the stated features, integers, steps or components, but not precluding the presence of one or more other features, integers, steps or components or groups thereof.

[0072] Further aspects of the present invention appear in the detailed description of the present invention below.

[0073] Unless otherwise explicitly stated, any example herein should be considered applicable to any other example with the necessary modifications. In non-limiting examples, other embodiments of the third, fourth, fifth, sixth and other aspects may include features disclosed in the context of the first or second aspect.

[0074] The scope of the present invention is not limited by the specific embodiments described herein, which are intended for illustrative purposes only. Functionally equivalent products, compositions and methods are clearly within the scope of the present invention as described herein.

[0075] Throughout this specification, unless otherwise explicitly stated or the context otherwise requires, references to a single step, composition of matter, group of steps or group of compositions of matter shall be considered to cover one and more (i.e., one or more) of these steps, compositions of matter, groups of steps or groups of 20 compositions of matter. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Embodiments of the present invention will be illustrated herein by way of example only with reference to the accompanying drawings, in which:

[0077] Figure 1 An assay vessel containing a luminescence assay composition held in a light detection portion of an optoelectronic device is schematically depicted according to some embodiments of the present invention.

[0078] Figure 2 An optoelectronic device is schematically depicted according to some embodiments of the present invention, which comprises Figure 1 a light detection portion.

[0079] Figure 3Shows the analyte-free BRET excited with uncaged coelenterazine substrate (CLZ400a in ethanol). 2 Graph showing the luminescence of the acceptor fluorophore measured as a function of storage time of the substrate solution at different temperatures (Example 1).

[0080] Figure 4 Shows the analyte-free BRET excited with uncaged coelenterazine substrate (CLZ400a in ethanol). 2 Measured BRET 2 Ratio as a function of storage time of the substrate solution at different temperatures (Example 1).

[0081] Figure 5 Shows the analyte-free BRET excited with uncaged coelenterazine substrate (CLZ400a-HCl in DMSO). 2 Graph showing the luminescence of the acceptor fluorophore measured as a function of storage time of the substrate solution at different temperatures (Example 1).

[0082] Figure 6 Shows the analyte-free BRET excited with uncaged coelenterazine substrate (CLZ400a-HCl in DMSO). 2 Measured BRET 2 Ratio as a function of storage time of the substrate solution at different temperatures (Example 1).

[0083] Figure 7 Shows the analyte-free BRET excited by in situ uncaging of caged coelenterazine compound 7 2 Graph showing the luminescence of the acceptor fluorophore measured as a function of storage time of the substrate solution at different temperatures (Example 1).

[0084] Figure 8 Shows the analyte-free BRET excited by in situ uncaging of caged coelenterazine compound 7 2 Measured BRET 2 Ratio as a function of storage time of the substrate solution at different temperatures (Example 1).

[0085] Figure 9 Shows the analyte-free BRET excited by in situ uncaging of caged coelenterazine compound 8 2 Graph showing the luminescence of the acceptor fluorophore measured as a function of storage time of the substrate solution at different temperatures (Example 1).

[0086] Figure 10 Shows the analyte-free BRET excited by in situ uncaging of caged coelenterazine compound 8 2 Measured BRET 2Graph showing the ratio as a function of the storage time of the substrate solution at different temperatures (Example 1).

[0087] Figure 11 Is a graph showing the analyte-free BRET excited by the in situ uncaging of the caged coelenterazine compound 10 2 Graph showing the luminescence of the acceptor fluorophore measured in the BRET assay as a function of the storage time of the substrate solution at different temperatures (Example 1).

[0088] Figure 12 Is a graph showing the analyte-free BRET excited by the in situ uncaging of the caged coelenterazine compound 10 2 Measured BRET 2 Graph showing the ratio as a function of the storage time of the substrate solution at different temperatures (Example 1).

[0089] Figure 13 Is a graph showing the analyte-free BRET excited by the in situ uncaging of a mixture of caged coelenterazine compounds 8 and 10 2 Graph showing the luminescence of the acceptor fluorophore measured in the BRET assay as a function of the storage time of the substrate solution at different temperatures (Example 1).

[0090] Figure 14 Is a graph showing the analyte-free BRET excited by the in situ uncaging of a mixture of caged coelenterazine compounds 8 and 10 2 Measured BRET 2 Graph showing the ratio as a function of the storage time of the substrate solution at different temperatures (Example 1).

[0091] Figure 15 Is a graph showing the analyte-free BRET in a buffer matrix containing caged coelenterazine compounds 7, 8, or 10 without an uncaging agent or with added porcine liver esterase 2 Graph showing the luminescence of the acceptor fluorophore measured in the assay (Example 2).

[0092] Figure 16 Is a graph showing the analyte-free BRET in a milk matrix containing caged coelenterazine compounds 7, 8, or 10 without an exogenous uncaging agent or with added porcine liver esterase 2 Graph showing the luminescence of the acceptor fluorophore measured in the assay (Example 2).

[0093] Figure 17 Is a graph showing the analyte-free BRET in a milk matrix containing different mixtures of caged coelenterazine compounds and different amounts of porcine liver esterase compounds 2 Graph showing the luminescence of the acceptor fluorophore measured in the assay (Example 2; Table 9).

[0094] Figure 18Is a graph showing the analyte-free BRET of different mixtures containing a caged coelenterazine compound and different amounts of porcine liver esterase compound in a milk matrix 2 Measured BRET 2 Ratio (Example 2; Table 9).

[0095] Figure 19 Is a graph showing the Figure 17 Receptor fluorophore luminescence as shown in, each normalized to 100% and depicted only during the window where the luminescence is 80 - 100% of the maximum value.

[0096] Figure 20 Is a graph showing the measured normalized Figure 17 Maximum receptor fluorophore luminescence of the measurements presented in, and depicting the window where the luminescence is 80 - 100% of the maximum value.

[0097] Figure 21 Is a graph showing that during the window where the luminescence is 80 - 100% of the maximum value Figure 17 Measured BRET of the measurements presented in 2 Ratio standard deviation.

[0098] Figure 22 Is a graph showing the analyte-free BRET 2 Measured receptor fluorophore luminescence as a function of storage time of the lyophilized BRET 2 Biosensor composition at different temperatures (Example 3).

[0099] Figure 23 Is a graph showing the analyte-free BRET 2 Measured BRET 2 Ratio as a function of storage time of the lyophilized BRET 2 Biosensor composition at different temperatures (Example 3).

[0100] Figure 24 Is a graph showing the analyte-free BRET 2 Measured receptor fluorophore luminescence in raw milk, UHT milk, and fresh (pasteurized) milk matrices containing caged coelenterazine compound 10 and added porcine liver esterase (Example 4).

[0101] Figure 25 Is a graph showing the analyte-free BRET in raw milk, heat-treated raw milk, and sonication-treated raw milk matrices containing caged coelenterazine compound 10 and added porcine liver esterase2 Graph of the measured acceptor fluorophore luminescence (Example 4).

[0102] Figure 26 Shows analyte-free BRET in UHT milk matrix containing caged coelenterazine compound 10 and different amounts of porcine liver esterase 2 Graph of the measured acceptor fluorophore luminescence (Example 4).

[0103] Figure 27 Shows analyte-free BRET in raw milk matrix containing caged coelenterazine compound 10 and added porcine liver esterase 2 During the BRET measurement 2 Graph of the change in BRET ratio with acceptor fluorophore luminescence (Example 4).

[0104] Figure 28 Is for raw milk samples spiked with known amounts of AprX for 10-minute and 15-minute BRET 2 Calibration graph of the BRET ratio obtained from the measurement (caged coelenterazine compound 10, not uncaged in situ by porcine liver esterase) (Example 5). 2 Calibration graph of the BRET ratio obtained from the measurement (caged coelenterazine compound 10, not uncaged in situ by porcine liver esterase) (Example 5).

[0105] Figure 29 Is for UHT raw milk samples spiked with known amounts of AprX for 10-minute and 15-minute BRET 2 Calibration graph of the BRET ratio obtained from the measurement (caged coelenterazine compound 10, not uncaged in situ by porcine liver esterase) (Example 5). 2 Calibration graph of the BRET ratio obtained from the measurement (caged coelenterazine compound 10, not uncaged in situ by porcine liver esterase) (Example 5).

[0106] Figure 30 Shows an example of a sample preparation station for preparing milk analysis samples. A provides an overview of the preparation station. B is an image of the storage space for the sample dropper bottle holder located within the airtight cavity. C is a graph showing the dispensed volume of DMSO if the dropper bottle is operated through its holder system or if operated by hand (20 repetitions). D is a graph showing the volume of whole milk dispensed using a burette dispenser or a 1 mL fixed volume pipette (20 repetitions). E is a graph showing the coefficient of variation of the measurements shown in C and D. F and G are graphs of the average maximum signal (F) and BRET ratio (G) of the blue and green channels on the optoelectronic device, comparing FD-B4 (1 U / assay esterase) reagent prepared with UHT whole milk on the preparation station or by pipette (3 repetitions). H and I show the results of the stability test (Example 6) of compound 10 substrate (4.3 mM in anhydrous DMSO) stored in the dropper bottle holder cavity of the preparation station at room temperature for more than 16 weeks (3 repetitions). Graph of the volume of whole milk dispensed using a burette dispenser or a 1 mL fixed volume pipette (20 repetitions). Graph of the coefficient of variation of the measurements shown in C and D. F and G are graphs of the average maximum signal (F) and BRET ratio (G) of the blue and green channels on the optoelectronic device, comparing FD-B4 (1 U / assay esterase) reagent prepared with UHT whole milk on the preparation station or by pipette (3 repetitions). H and I show the results of the stability test (Example 6) of compound 10 substrate (4.3 mM in anhydrous DMSO) stored in the dropper bottle holder cavity of the preparation station at room temperature for more than 16 weeks (3 repetitions).

[0107] Figure 31 Development of lactose sensing assays using lyophilized BRET 2 -lactose sensors on optoelectronic devices. A is a graph showing CLARIOstar plate reader analysis of wild-type lactose sensors and three sensor variants. B is a calibration graph of lactose sensors (wild-type and variants) in PBS buffer supplemented with serial dilutions of lactose. C is a graph showing green / blue channel bioluminescence counts of lyophilized lactose biosensors analyzed on optoelectronic devices reconstituted with 10% lactose-free milk (diluted in dH2O) and supplemented with Compound 10 substrate. D is a graph of the BRET ratio of lyophilized lactose biosensors analyzed on optoelectronic devices reconstituted with 10% lactose-free milk (diluted in dH2O) and supplemented with Compound 10 substrate. E is a graph showing BRET ratio analysis of various lyophilized lactose sensor formulations on optoelectronic devices. F is a graph showing the difference in BRET ratio between lactose-free milk samples with or without 5% (w / v) lactose analyzed using various lyophilized lactose sensor formulations on optoelectronic devices. BRET ratio endpoints were taken every 1 minute. G shows the optoelectronic device analysis (BRET ratio) of lactase-treated lactose-free milk with or without 5% (w / v) lactose in a 4-minute assay (triplicate) using FD-L3-13 reagent. H is a calibration graph of lactose sensing for lactase-treated lactose-free milk samples supplemented with serial dilutions of lactose (duplicate). (Example 7).

[0108] Figure 32 Graph of the BRET ratio of a lyophilized plasmin sensor reconstituted with raw milk and supplemented with Compound 10 substrate analyzed on an optoelectronic device (Example 8).

[0109] Figure 33 Shows the analysis of raw milk samples using an optoelectronic device and FD-plasminogen-1 assay reagent. A is a graph of the BRET ratio of raw milk or raw milk heat-treated at 90° for 20 minutes over 900 seconds. B is a graph of the BRET ratio of a 15-minute assay of raw milk and heat-treated raw milk. C is a graph of the BRET ratio of a 15-minute assay of raw milk and heat-treated raw milk samples spiked with 3 - 100 mU / mL human plasminogen (Example 9).

[0110] Figure 34Shows the analysis of UHT milk samples spiked with subtilisin using FD-B4 (5 units of esterase per determination) and a photoelectronic device. A is a graph of the BRET ratio results for 10-minute and 15-minute determinations using UHT milk spiked with 0 μU / mL, 100 μU / mL, or 1000 μU / mL subtilisin. B is a graph of the BRET ratio of UHT milk samples spiked with 500 μU / mL subtilisin or 1000 μU / mL AprX over 800 seconds. The samples were either untreated before analysis or supplemented with 0.8 mM PMSF and incubated on ice for 30 minutes (Example 10).

[0111] Figure 35 Shows the calibration of the micro-photomultiplier tube (μPMT) of a photoelectronic device using a gaseous tritium light source (GTLS). A shows the emission spectra (UV blue, white, and green) of GTLS micro-markers. The horizontal lines indicate the photoelectronic device filters for 'blue' and 'green' photons. B is a graph of the 'blue' and 'green' channel photon counts at the upper end of the tube holder placed in the photoelectronic device . C is a graph of the blue counts for four runs (2 minutes at 37 °C) at the bottom of a 2 mL tube placed on the photoelectronic device . After each run, the tube was removed and reinserted into the tube holder. D shows the signal counts for the 'green' and 'blue' channels over 20 minutes at different temperatures. G shows the CT 2 measurements of three photoelectronic devices (B0001 - B0003) using the same calibration tool (Example 11).

[0112] Figure 36 Shows an example schematic diagram of the Cybertongue calibration tool (CT 2 ). Although not shown, the screw cap form may also include lugs that mate with notches located at the end of the photoelectronic device, thereby improving the reproducibility of calibration measurements by ensuring that the tool is placed in the same position for each run (Example 11).

[0113] Figure 37 Shows an example schematic diagram of the Cybertongue calibration tool (CT 2 ) without positioning notches and lugs (Example 11).

[0114] Symbolic Explanation of the Sequence Listing

[0115]

[0116]

[0117] Detailed implementation mode

[0118] General techniques and definitions

[0119] Unless otherwise specifically stated, all technical and scientific terms used herein shall be considered to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in chemiluminescence-based sensor technology, particularly bioluminescence-based sensor technology, molecular biology, protein chemistry, bioconjugation technology, biochemistry, etc.). Unless otherwise indicated, the recombinant protein, cell culture, bioconjugation, and immunological techniques used in the present invention are standard procedures well known to those skilled in the art.Such techniques are described and explained in all the literature in the sources, such as J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984); J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989); T. A. Brown (ed.), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991); D. M. Glover and B. D. Hames (eds.), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996); and F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date); Ed Harlow and David Lane (eds.) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory Press, (1988); and J. E. Coligan et al. (eds.) Current Protocols in Immunology, John Wiley and Sons (including all updates to date), Greg T. Hermanson, Bioconjugate Techniques. Elsevier Inc. (2013).

[0120] The term “and / or”, e.g., “X and / or Y” shall be understood to mean “X and Y” or “X or Y” and shall be taken as providing explicit support for both meanings or either meaning.

[0121] Unless the context otherwise indicates, terms recited in the singular (such as sensor and substrate) clearly also mean the plural. For example, logically, many individual molecules rather than a single molecule will flow through the device or be contained within a pore.

[0122] As used herein, unless stated to the contrary, the term “about” means + / - 10% of the specified value, more preferably + / - 5%, even more preferably + / - 1%.

[0123] Methods for detecting extracellular analytes

[0124] The present invention relates to a method for detecting an extracellular analyte in a sample. In some instances, the method comprises incubating an assay composition comprising the sample and a biosensor system selected from (i) an extracellular bioluminescent sensor molecule comprising a donor luminophore, an analyte-sensitive domain, and a receptor fluorophore; and (ii) a precursor system thereof comprising a pair of molecules adapted to react in solution to form the extracellular bioluminescent sensor molecule. The assay composition is incubated for an incubation period sufficient for the extracellular analyte to interact with the analyte-sensitive domain, if the extracellular analyte is present in the sample. At least one caged substrate compound is uncaged in the assay composition during the incubation period to produce an uncaged substrate compound that excites the donor luminophore for a subsequent detection step. Then, after the incubation period, light emitted from the assay composition is detected, including emissions associated with the receptor fluorophore and the donor luminophore. The ratio of the luminescence (i.e., intensity) associated with the receptor fluorophore to the luminescence (i.e., intensity) associated with the donor luminophore indicates the interaction of the extracellular analyte with the analyte-sensitive domain.

[0125] Samples and extracellular analytes

[0126] A sample can be any sample that contains or may contain an extracellular analyte of interest or concern. The methods disclosed herein are particularly useful for measuring enzyme activity or the concentration of multiple analytes in complex biological matrices. Thus, in some embodiments, the sample is a food or beverage composition, including food or beverage intermediates, products, and derivatives. For example, a food or beverage derivative can be a water extract or other processed water sample derived from a food or beverage composition for analytical purposes. In some embodiments, the sample is a dairy composition. For example, it can be a sample of raw milk or processed milk.

[0127] The milk can be cow milk, goat milk, sheep milk, buffalo milk, camel milk or human milk. The milk can be colored and flavored, including, for example, vanilla, chocolate, coffee or strawberry milk, it can be lactose-modified, such as "low-lactose" or "lactose-free", and it can be sweetened or unsweetened. The sample can be whey or reconstituted from skim or whole milk powder or whey powder by adding water. It can also be a milk-based product containing other ingredients designed to provide a meal or snack alternative. The sample can be a fermented or plant-based product sold as a milk substitute, including those containing casein and / or whey protein prepared by fermentation or made from soy, almond, oat, coconut, etc.

[0128] The sample can be another beverage, including fruit juice, liqueur, beer, wine or spirits or coffee, tea, etc. The sample can be a soup, sauce or condiment. The sample can also be a thickened product, or an extract or dilution thereof, such as yogurt or custard. The sample can also be an extract of almost any dry or semi-solid food, made from or including grains, meat, baked products, chocolate, meat, eggs, fish, cheese, nuts, fruits, herbs, spices, snacks, etc. In such cases, the sample can be prepared by extracting the food with an aqueous buffer, with or without mechanical grinding, and can include the addition of detergents, emulsifiers and enzymes to aid in the extraction of the analyte.

[0129] The sample can be an extract or preparation of a prescription or over-the-counter drug. The sample can be an extract or preparation of a cosmetic or personal care product.

[0130] The sample can be a biological fluid from a human or other animal, including blood, serum, plasma, tears, saliva, urine, feces, cerebrospinal fluid, lymph fluid, concentrated exhaled fluid, etc. The sample can be an extract or preparation of a solid tissue sample from a human or other animal that has been ground or homogenized to provide a liquid sample. The sample can be an extract of a plant, including the juice or ground leaves or other plant tissues. The sample can be a culture medium for mammalian or other animal cells or tissues, or for plant cells or tissues, or a fermentation medium for bacteria, fungi or other microorganisms.

[0131] The sample can be an environmental sample, including wash water, wastewater, surface water, groundwater, tap water, rainwater, leachate, or an aqueous extract of a soil sample. The sample can be an environmental sample collected by swabbing a natural or artificial surface (including indoor surfaces) and extracting the aqueous rinse from the swab. For example, a sample can be prepared by swabbing a surface with a moistened swab (e.g., a cotton swab) and making an aqueous rinse extract from the swab. A sample can also be prepared by equilibrating air with an aqueous buffer and testing the buffer (e.g., using a wet-wall cyclone device such as the SASS2400 or a spray device). The sample can be any ingredient or raw material used in a manufacturing process (including food and beverage manufacturing), or an aqueous extract thereof.

[0132] The sample can be liquid or solid. In some embodiments, the sample is liquid, specifically an aqueous liquid.

[0133] The methods disclosed herein are applicable to detecting a variety of extracellular analytes. In some embodiments, the extracellular analytes include, but are not limited to, extracellular analytes for which a BRET-based biosensing system has been previously reported. In some embodiments, the extracellular analytes include, but are not limited to, extracellular analytes for which a chemiluminescent biosensing system has been previously reported.

[0134] As used herein, an extracellular analyte is a molecule of interest and includes extracellular molecules (e.g., compounds including sugars, lipids, and proteins, as well as intermediate metabolites, including those derived from and secreted / transported / effluxed from cells, tissues, or organs), and intracellular molecules that become extracellular due to disruption of the cell (e.g., by cell death). The term extracellular analyte does not include molecules detected within the cell and also encompasses BRET reporter molecules expressed within the cell.

[0135] Examples of suitable extracellular analytes that can be detected by the methods disclosed herein include:

[0136] ● Disaccharides and monosaccharides, including glucose, sucrose, lactose, maltose, etc. and their derivatives (e.g., lactulose).

[0137] ● Oligopeptides, polypeptides, and proteins, including food allergens, nutritional proteins such as whey protein, casein, gluten, and peptides therefrom

[0138] ● Linear and conformational epitopes (e.g., β-casein A1 / A2 dimorphism)

[0139] ● Other antigens

[0140] ● Other proteins, including those validated as health biomarkers, such as ferritin, hemoglobin A1c

[0141] ● All major classes of antibiotics

[0142] ● Mycotoxins, such as aflatoxins, fumonisins, patulin, etc.

[0143] ● Heavy metals, including lead, zinc, cadmium, arsenic, etc.

[0144] ● Other toxins, including cyclic peptides (e.g., cereulide)

[0145] ● Adenosine triphosphate

[0146] ● Other enzymes, such as alkaline phosphatase, esterase

[0147] ● Amino acids and dipeptides

[0148] ● Hormones, including steroid hormones, such as cortisol, testosterone, estrogen, progesterone and their derivatives

[0149] ● Peptide hormones, including follicle-stimulating hormone, thyroid-stimulating hormone

[0150] ● Anions for food preservation, such as nitrite, nitrate, benzoate, sulfite, propionate, sorbate

[0151] ● High-intensity sweeteners

[0152] ● Spice active compounds, such as curcumin, capsaicin and capsanthin

[0153] ● Therapeutic drugs

[0154] ● Vitamins and vitamin precursors, such as vitamin B12 and 1a,25(OH)2-vitamin D3 and their biological precursors

[0155] In some embodiments, the extracellular analyte is selected from the group consisting of: enzymes, zymogens, and carbohydrates. In some embodiments, the extracellular analyte is selected from the group consisting of: enzymes and carbohydrates. In some embodiments, the enzyme is a protease or a zymogen. In some embodiments, the enzyme is a protease.

[0156] In some embodiments, the extracellular analyte is a protease, such as alkaline metalloprotease (AprX), another metalloprotease, or other proteases, including plasmin, thrombin, caspases (e.g., caspase III), Bacillus serine proteases such as subtilisin, etc.

[0157] Proteases are typically produced as inactive precursors (also known as proenzymes or zymogens). A zymogen or proenzyme is a protein that does not display catalytic activity but can be processed (e.g., by proteolytic processing) into an active enzyme. In some embodiments, the extracellular analyte is a proenzyme or zymogen, such as plasminogen, prothrombin, caspase zymogen, etc. The proenzyme or zymogen can be converted into an active enzyme, and the active enzyme can be analyzed using the methods described herein. As understood by those skilled in the art, proteases include activated zymogens. For example, plasminogen can be activated to form plasmin, and then the plasmin can be detected / analyzed using the methods described herein.

[0158] In some exemplary embodiments, the extracellular analyte is an alkaline metalloprotease (AprX) secreted by Pseudomonas species such as Pseudomonas fluorescens. This protease has significant economic implications because it can cause spoilage of otherwise quality-acceptable ultra-high temperature (UHT) milk and other dairy products. After UHT processing, even very low residual levels of this protease are potentially harmful.

[0159] In some exemplary embodiments, the extracellular analyte is plasmin. Plasmin is present in human milk and cow's milk. Plasmin in cow's milk has been shown to degrade β-casein, α s1 -casein, and α s2 -casein into peptides. Like AprX, this protease has significant economic implications because it can cause spoilage of otherwise quality-acceptable ultra-high temperature (UHT) milk and other dairy products. After UHT processing, even very low residual levels of this protease are potentially harmful.

[0160] In some embodiments, the extracellular analyte is plasminogen. Plasminogen is an inactive precursor of plasmin and is processed to form plasmin. Both urokinase-type plasminogen activator and tissue-type plasminogen activator cleave plasminogen to form plasmin. It has been reported that a mixture of active and inactive plasmin is present in milk. In some embodiments, plasminogen can be converted to plasmin by including an activator in the biosensor composition. In some embodiments, the biosensor composition comprises urokinase-type plasminogen activator or tissue-type plasminogen activator. In some embodiments, the biosensor composition comprises urokinase-type plasminogen activator. The amount of activator included in the biosensor composition should be sufficient to convert all zymogens to active proteins. In some embodiments, the biosensor composition comprises at least 10 U / mL, at least 100 U / mL, at least 200 U / mL, at least 300 U / mL, at least 400 U / mL, at least 500 U / mL, at least 600 U / mL, at least 700 U / mL, at least 800 U / mL, at least 900 U / mL, at least 1000 U / mL or more of the activator.

[0161] Biosensor system

[0162] The biosensor system is selected from extracellular bioluminescence sensor molecules and precursor systems thereof, the precursor system comprising a pair of molecules adapted to react in solution to form in situ the extracellular bioluminescence sensor molecule. In some embodiments, the extracellular bioluminescence sensor molecule (also referred to herein as a BRET biosensor) comprises a donor luminophore, an analyte-sensitive domain, and a receptor fluorophore. The analyte-sensitive domain can be present on a linker that holds the donor luminophore and the receptor fluorophore close enough (usually spaced <10 nm) and in a dipole orientation to allow Förster resonance energy transfer from the donor luminophore to the receptor fluorophore. When the donor luminophore (e.g., luciferase) acts on a suitable substrate (e.g., luciferin), light is thus emitted from both the donor luminophore and the receptor fluorophore, where the relative intensity (luminescence) is highly dependent on the separation distance and relative orientation of these two moieties. Thus, the interaction of an analyte with the analyte-sensitive domain, such as by binding or cleavage, can be detected by a change in the ratio (BRET ratio) of the luminescence associated with the receptor fluorophore to the luminescence associated with the donor luminophore. The efficiency of (Förster) resonance energy transfer and the BRET ratio decrease with the sixth power of the donor-receptor separation. Thus, a properly designed BRET-based sensor system is highly sensitive to the presence or concentration of an analyte that interacts with the selected analyte-sensitive domain.

[0163] An extracellular bioluminescence sensor molecule can comprise any suitable compatible donor luminophore and acceptor fluorophore pair, i.e., when the donor luminophore is excited, the acceptor fluorophore is adapted for Förster resonance energy transfer and emits a distinguishable light signal.

[0164] The donor luminophore can be a bioluminescent protein. Examples include but are not necessarily limited to luciferase, β-galactosidase, lactamase, horseradish peroxidase, alkaline phosphatase, β-glucuronidase, or β-glucosidase. Examples of luciferase include but are not necessarily limited to Renilla luciferase, firefly luciferase, coelenterazine luciferase, Photinus pyralis luciferase, Pyrophorus or other beetle luciferases, Rhagoletis pomonella luciferase, bacterial luciferase, Gaussia luciferase, Cypridina luciferase, or a bioactive variant or fragment of any of these, or a chimera of two or more of them.

[0165] In some embodiments, the donor luminophore is Renilla luciferase (RLuc). Native RLuc and its variants catalyze a luminescence reaction in which the emission spectrum is within the blue portion of the spectrum. The Renilla luciferase can be a variant of the native Renilla luciferase with enhanced thermal stability, such as RLuc2 or RLuc8. In some embodiments, the donor luminophore is RLuc2. In some embodiments, the donor luminophore is RLuc8.

[0166] The donor luminophore can be excited by a suitable corresponding substrate (i.e., the molecule on which the donor luminophore acts to produce bioluminescence). In the methods of the present disclosure, as will be described below, the substrate is provided in situ in the assay by uncaging the corresponding caged substrate compound.

[0167] The acceptor fluorophore can be a protein. Examples include but are not necessarily limited to green fluorescent protein (GFP), blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), TagRFP, TurboFP635, red fluorescent protein (RFP), GFPS65T, Emerald, Venus, mOrange, Topaz, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEYFP), HcRed, t-HcRed, DsRed, DsRed2, t-Dimer2, t-Dimer2(12), mRFPl, pocilloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein, or phycobiliprotein, or a bioactive variant or fragment of any of these. In some embodiments, the acceptor fluorophore is GFP or a variant thereof. In some embodiments, the acceptor fluorophore is GFP 2。

[0168] In other embodiments, the acceptor fluorophore is non - protein. Examples include, but are not limited to, Alexa Fluor dyes, Bodipy dyes, Cy dyes, fluorescein, dansyl, umbelliferone, fluorescent microspheres, luminescent microspheres, fluorescent nanocrystals, dock blue, cascade blue, cascade yellow, pacific blue, oregon green, tetramethylrhodamine, rhodamine, texas red, rare earth element chelates, or any combination or derivative thereof.

[0169] In some embodiments, the extracellular bioluminescence sensor molecule can be selected from BRET 1 , BRET 2 , BRET 3 , BRET 4 , BRET 5 or BRET 6 biosensors. In the BRET 1 system, the donor luminophore is RLuc, which acts on native coelenterazine (CLZ) or coelenterazine h (CLZ - h), and the acceptor fluorophore is yellow fluorescent protein (YFP). In the BRET 2 system, the donor luminophore is RLuc, which acts on coelenterazine 400a (CLZ 400a), and the acceptor fluorophore is green fluorescent protein or its variant (GFP 2 ). In the BRET 3 system, the donor luminophore is RLuc8, which acts on CLZ - h, and the acceptor fluorophore is mOrange. In the BRET 4 system, the donor luminophore is RLuc8, which acts on CLZ - h or coelenterazine v (CLZ - v), and the acceptor fluorophore is TagRFP. In the BRET 5 system, the donor luminophore is RLuc8.6, which acts on CLZ - h, and the acceptor fluorophore is TagRFP. In the BRET 6 system, the donor luminophore is RLuc8.6, which acts on CLZ - h or CLZ - v, and the acceptor fluorophore is TurboFP635.

[0170] In an exemplary embodiment, the extracellular bioluminescence sensor molecule is a BRET 2 biosensor. Such biosensors advantageously provide long Stokes shift bioluminescence resonance energy transfer, with peak donor emission and acceptor emission at 395 nm and 515 nm, respectively. A sensitive BRET 2A biosensor for detecting a range of analytes, including proteases (e.g., plasmin in milk; Australian Patent 2015243093) or sugars (e.g., maltose or lactose; Australian Patent 2018315053) or hydrolases (e.g., esterase or phosphatase; Australian Patent Application 2018321580).

[0171] In some embodiments, an extracellular bioluminescence sensor molecule refers to a BRET biosensor that operates in an assay composition, in a cell-free environment, or outside of any cells present. This does not mean that the bioluminescence sensor molecule is produced in an extracellular environment. In fact, the extracellular bioluminescence sensor molecule can be an engineered protein. For example, the extracellular bioluminescence sensor molecule can be a recombinant protein. Suitable engineered biosensor proteins can be expressed in Escherichia coli and purified from Escherichia coli. For example, the biosensor can be a recombinant protein expressed in Escherichia coli BL21(DE3) using an IPTG-inducible pRSET expression system. The biosensor can be translationally fused to a polyhistidine tag for purification by immobilized metal (e.g., cobalt or nickel) affinity chromatography.

[0172] In some embodiments, the extracellular bioluminescence sensor molecule can be selected from cleavable, allosteric, or competitive BRET biosensors. BRET biosensors are classified into these types based on the nature of the interaction between an extracellular analyte and an analyte-sensitive domain. In a cleavable BRET biosensor, the analyte-sensitive domain on the linker between the donor fluorophore and the acceptor fluorophore contains a specific peptide target sequence that is cleaved by a protease analyte, thereby disconnecting the donor fluorophore and the acceptor fluorophore and thus causing a change in the BRET ratio. Examples of cleavable BRET biosensors are disclosed in AU2015243093, Australian Patent Application 2017358067A1, Dacres et al., Sensors and Actuators B: Chemical 2019 301 127141, and Dacres et al., Analytical Biochemistry 2009 385(2) 194-202. In some embodiments, the extracellular bioluminescence sensor molecule is thus a protease sensor, for example, for AprX, plasmin, thrombin, caspase III, etc.

[0173] In an allosteric BRET biosensor, an analyte-sensitive domain on a linker between a donor fluorophore and an acceptor fluorophore contains a binding site for a ligand analyte (e.g., a specific saccharide such as maltose or lactose). When bound by the ligand analyte, the binding site changes shape, reorienting the donor fluorophore relative to the acceptor fluorophore and thus causing a change in the BRET ratio. Examples of allosteric BRET biosensors are disclosed in Australian Patent 2018315053, Dacres et al., Biosensors & Bioelectronics 2013 41 459 - 464, and Caron et al., Anal Chem 2018 90(21)12986 - 12993. In some embodiments, the extracellular bioluminescence sensor molecule is thus a carbohydrate sensor, e.g., for lactose, maltose, sucrose, etc.

[0174] In a competitive BRET biosensor, a BRET-labeled ligand (labeled with one of a donor fluorophore and an acceptor fluorophore) binds to a BRET-labeled binding protein (labeled with the other of the donor fluorophore and the acceptor fluorophore). The BRET-labeled ligand is competitively displaced by an unlabeled ligand analyte, disconnecting the donor fluorophore and the acceptor fluorophore and thus causing a change in the BRET ratio. Thus, in such embodiments, the BRET-labeled binding protein, although initially occupied, is the analyte-sensitive domain. Examples of competitive BRET biosensors are disclosed in Australian Patent Application 2020265898.

[0175] In an assay using a competitive BRET biosensor, a diagnostic BRET ratio is typically detected when the system reaches equilibrium between a BRET-labeled binding protein bound to an analyte and a BRET-labeled binding protein bound to a BRET-labeled ligand. It should be appreciated that the same equilibrium can be achieved by introducing the BRET-labeled binding protein and the BRET-labeled ligand as separate species into the assay composition, i.e., as a precursor system comprising a pair of molecules (the BRET-labeled binding protein and the BRET-labeled ligand) adapted to react (by binding) in solution to form a competitive BRET biosensor. Indeed, in some embodiments, this may be preferred because the equilibrium can be reached more quickly when starting from initially unbound species, especially if the dissociation kinetics are slow.

[0176] Preparing and incubating the assay composition

[0177] The methods disclosed herein include the step of incubating an assay composition comprising a sample and a biosensor system. The assay composition may optionally comprise other components such as (i) one or more uncaging agents, such as esterases, for uncaging a caged substrate compound in the assay composition; (ii) a buffer; (iii) one or more salts; (iv) one or more metal ions (if required, for example, for metalloprotease assays); (v) one or more carrier proteins, such as fish skin gelatin; and (vi) one or more cryoprotectants, such as trehalose, sucrose, and pullulan. These other components may form part of a solid (biosensor) composition, be added to the sample prior to combination with the biosensor composition, or be added directly to the assay composition.

[0178] The assay composition to be incubated can be prepared by any suitable method. In some embodiments, the assay composition is prepared by dispersing a solid composition in a liquid, the solid composition comprising at least a bioluminescent sensor molecule or a precursor system thereof, and typically also comprising various optional components (such as uncaging agents). The solid composition can be dispersed directly in the liquid sample (undiluted or diluted), but alternatively it can be dispersed in another aqueous liquid prior to addition of the sample. A solid composition containing a sufficient amount of biosensor for a single assay can be provided in an assay vessel for the incubation and detection steps, thus simplifying the method and avoiding or minimizing errors caused by inaccurate dispensing of reagents into the assay vessel.

[0179] The solid composition can be a lyophilized solid composition, which can be suitably prepared by dispersing or dissolving an extracellular bioluminescent sensor molecule or a precursor system thereof and other optional components in an aqueous liquid and lyophilizing the composition by conventional methods. Advantageously, lyophilization allows the extracellular bioluminescent sensor molecule to be stored for extended periods prior to reconstitution in the assay composition. The solid composition can be lyophilized in an assay vessel for the incubation and detection steps.

[0180] In some embodiments, a lyophilized solid composition containing a biosensor system is prepared by filling an aqueous solution (e.g., 200 μL) comprising the biosensor system and other components into a suitable assay vessel (e.g., QSP TMin a natural screw cap microtube 520-GRD (ThermoFisher Scientific), and the aqueous solution is lyophilized. After lyophilization, the assay vessel is sealed and preferably stored in the dark, at room temperature or colder, until analysis is required. The inventors have demonstrated that such lyophilized compositions are stable over the long term even at elevated storage temperatures and thus are expected to have a shelf life of up to one year or even longer. In addition, the lyophilized compositions can be readily reconstituted in aqueous liquid samples such as milk, thus facilitating reproducible analysis.

[0181] It should be understood that the amount of biosensor (e.g., BRET biosensor) present in the solid composition will depend on the particulars of the embodiment. It should also be understood that the presence and relative amounts of optional components present in the solid composition will depend on the particulars of the embodiment. For example, the solid composition may contain a caged reagent, but in some embodiments it does not contain a caged reagent because this component may be endogenous to the sample in which the solid composition is reconstituted or may be added separately to the assay composition.

[0182] The amount of BRET biosensor is typically sufficient to result in a low variation in the luminescence intensity to produce a BRET signal under assay conditions (e.g., coefficient of variation of the BRET ratio ≤ 1% within a measurement window of 10 - 20 seconds). Other factors affecting the optimal BRET biosensor concentration include, (i) in the case of a ligand: the affinity of the biosensor for the ligand (e.g., Kd), the concentration range of the ligand expected or required to be detected in the sample, and the concentration of any competing binding proteins or ligands normally present in the sample, and (ii) in the case of measuring enzyme activity: the concentration and specific activity of the enzyme normally found or to be measured, the Michaelis constant (Km) and Kcat of the target enzyme of the biosensor target, the concentration of any alternative substrates of the enzyme that will compete with the target site of the biosensor, and the concentration of other inhibitors normally found in the sample. The appropriate amount of BRET biosensor for any particular assay can be determined by routine experimentation (e.g., by titrating down the amount of biosensor to give a high proportion of analyte binding to the biosensor, or dissociation in a competitive assay, or cleavage of the linker under assay conditions when using a representative sample).

[0183] In some embodiments, the concentration of the BRET biosensor in the assay composition is from about 10 ng to 2000 ng / mL, from about 10 ng / mL to 1600 ng / mL, from about 10 ng / mL to 1200 ng / mL, from about 10 ng / mL to 1000 ng / mL, 10 ng / mL to 900 ng / mL, 50 ng / mL to 800 ng / mL, 50 ng / mL to 700 ng / mL, 50 ng to 600 ng / L, 50 ng to 500 ng / L, 100 ng / mL to 900 ng / mL, 200 ng / mL to 900 ng / mL, 300 ng / mL to 900 ng / mL, 400 ng / mL to 900 ng / mL, 500 ng / mL to 800 ng / mL, or 600 ng / mL to 800 ng / mL. In some embodiments, the concentration of the BRET biosensor in the assay composition is from about 600 ng / mL to 800 ng / mL. In some embodiments, the concentration of the BRET biosensor in the assay composition is about 690 ng / mL.

[0184] The assay composition can include one or more uncaging agents, such as esterases, for uncaging a caged substrate compound in the assay composition. Any suitable uncaging agent can be used based on the caged substrate used in the method. In some embodiments, the uncaging agent is an esterase, such as porcine esterase. In some embodiments, the concentration of the uncaging agent in the assay composition is from about 0.2 U / mL to 50 U / mL, 0.2 U / mL to 40 U / mL, 0.2 U / mL to 20 U / mL, 0.2 U / mL to 18 U / mL, 0.2 U / mL to 16 U / mL, 0.2 U / mL to 14 U / mL, 0.2 U / mL to 12 U / mL, 0.2 U / mL to 10 U / mL, 1 U / mL to 20 U / mL, 1 U / mL to 15 U / mL, or 1 U / mL to 10 U / mL. In some embodiments, the concentration of the uncaging agent in the assay composition is about 1 U / mL, 5 U / mL, or 10 U / mL.

[0185] The assay composition can include one or more buffers. Any suitable buffer can be used. Suitable buffers can include, but are not limited to, sodium phosphate, sodium citrate, sodium succinate, histidine (or histidine-HCl), sodium carbonate, CAPS, CAPSO, tris, and / or sodium malate. As will be understood by those skilled in the art, the preferred pH (and thus the buffer) will depend on the assay being performed. In some embodiments, the concentration of the buffer in the assay composition is sufficient to provide the desired buffering capacity. In some embodiments, the concentration of the buffer in the assay composition is between about 2 mM and 100 mM, such as 50 mM.

[0186] The assay composition comprises one or more cryoprotectants. Suitable cryoprotectants are known to those skilled in the art. Suitable cryoprotectants can include, but are not limited to, sucrose, trehalose, lactose, glycerol, glucose, raffinose, pullulan, and / or mannitol. In some embodiments, the cryoprotectant is pullulan and / or trehalose. In some embodiments, the cryoprotectant is pullulan. In some embodiments, the cryoprotectant is trehalose. The concentration of the cryoprotectant can be determined by those skilled in the art. In some embodiments, the concentration of each cryoprotectant in the assay composition ranges from about 0.2 mg / mL to 20 mg / mL, such as about 2.5 mg / mL or 5 mg / mL. In some embodiments, the total concentration of the cryoprotectant in the assay composition ranges from about 0.4 mg / mL to 40 mg / mL, such as about 5 mg / mL.

[0187] In some embodiments, the assay composition comprises a carrier protein, such as fish gelatin. In some embodiments, the concentration of the carrier protein in the assay composition ranges from about 0.02% to 0.2% (v / v), such as about 0.02%, 0.04%, 0.06%, 0.08%, 0.09%, 0.1%, 0.12%, 0.14%, 0.16%, 0.18%, or 0.2% (v / v). In some embodiments, the concentration of the carrier protein is about 0.09% (v / v). In some embodiments, %(v / v) refers to the volume of the fish gelatin stock solution containing 0.45 g / mL (45% w / v) fish skin gelatin added to the assay composition.

[0188] In some embodiments, the assay composition comprises one or more salts, such as sodium chloride. In some embodiments, the concentration of the one or more salts in the assay composition ranges from about 2 mM to 100 mM, such as 50 mM.

[0189] In some embodiments, the assay composition comprises one or more metal ions (if required, for example, for metalloprotease assays or for the activity of enzymes). The metal ions can be provided in the form of salts. For example, in the case of the assay for AprX, the biosensor system can optionally comprise magnesium chloride and / or calcium chloride. In some embodiments, the concentration of the metal ions in the assay composition ranges from about 0.2 mM to 20 mM, such as 5 mM.

[0190] In some embodiments, the assay composition comprises one or more additional components. The additional components include, but are not limited to, activators, inhibitors, enhancers, detergents, reducing agents, oxidizing agents, etc. For example, in the case of a zymogen assay, the assay composition may optionally comprise an agent that activates the zymogen. For example, in the case of an assay for plasmin that has been disclosed to associate with casein micelles, the assay composition may optionally comprise an agent that dissociates plasmin from the casein micelles. For example, the assay composition may comprise 1 mM aminocaproic acid and 80 mM trisodium acetate, which helps to dissociate plasmin from the casein micelles. For example, in the case of a plasmin assay, the assay composition may optionally comprise L-lysine that supports plasmin activity. In some embodiments, the concentration of L-lysine in the assay composition is between 2 mM and 100 mM, such as 25 mM. In some embodiments, the assay composition comprises a protease inhibitor, such as a protease inhibitor that inhibits a specific class of proteases (e.g., serine protease inhibitor, cysteine protease inhibitor, metalloprotease inhibitor). Suitable protease inhibitors include PMSF (phenylmethylsulfonyl fluoride), EDTA, AEBSF (4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride), aprotinin, bestatin, E-64, leupeptin, pepstatin, etc. A protease inhibitor may be included to selectively inhibit the activity of proteases that are not assayed.

[0191] Incubate the assay composition for an incubation period sufficient for the extracellular analyte to interact with the analyte-sensitive domain, if the extracellular analyte is present in the sample. As used herein, the incubation period begins when the extracellular bioluminescent sensor molecule and the sample first come into contact under conditions that permit continuous interaction of the extracellular analyte with the analyte-sensitive domain, and ends when light from the assay composition is detected to determine the interaction. For example, the incubation period may begin when a (lyophilized) solid composition comprising the bioluminescent sensor molecule is dispersed in a liquid containing the sample (or consisting of the sample), or when the sample is mixed into a liquid that already contains the bioluminescent sensor molecule. Then, the assay composition may be incubated for the required incubation period before light emitted from the resulting assay composition is detected. Of course, light may be detected continuously or periodically within an extended detection window, where the relevant incubation period increases gradually at each detection time.

[0192] The assay composition can be incubated at any suitable temperature (e.g., 0 °C to 100 °C, more typically 10 °C to 50 °C, such as room temperature (about 22 °C) to 40 °C). In some embodiments, the assay composition is incubated at about 37 °C. It should be understood that higher temperatures can accelerate the interaction between the extracellular analyte and the analyte-sensitive domain, thus requiring a shorter incubation period. However, higher temperatures may also render one or more components of the assay composition unstable. In some embodiments, the incubation temperature is controlled at the target temperature or within a narrow (e.g., ±2 °C) temperature range. In some embodiments, incubation with associated temperature control can be carried out in a optoelectronic device for detecting light emitted from the assay composition.

[0193] For any given embodiment, the preferred incubation period can depend on the nature of the interaction between the extracellular analyte and the analyte-sensitive domain of the BRET biosensor. Certain cleavable BRET biosensors may require a longer incubation time to detect the interaction, e.g., in the range of 10 minutes to 30 minutes or even longer. Such biosensors can be used to directly measure enzyme activity, where the analyte (protease) acts on the biosensor. The ultimate modification of the biosensor produces a progressive change in the BRET signal over an extended period of time, corresponding to the kinetics of the enzymatic conversion. Similar considerations will apply to other biosensors containing analyte-sensitive domains vulnerable to modification by low levels of enzymatic analyte.

[0194] On the other hand, certain allosteric and competitive BRET biosensors may only require a short incubation time, e.g., in the range of 1 minute to 10 minutes, such as 2 minutes to 5 minutes. Such biosensors can be used in ligand-binding assays, in which the analyte is a small or large molecule ligand and can be detected once equilibrium binding to the analyte-sensitive domain (i.e., binding site) is achieved. This is typically rapid, depending on the "association rate" of the equilibrium binding reaction. In principle, the minimum incubation time in such embodiments can be very short, i.e., detection can be carried out practically immediately after the sample and the extracellular bioluminescent sensor molecules are mixed and sufficient caged substrate compound has been uncaged in the assay composition to excite the donor luminophore.

[0195] However, ligand-binding assays are not limited to short incubation time assays. For example, they can also be used to measure the accumulation or consumption of a ligand over time in the assay composition by another process (e.g., an enzymatic process), thereby indirectly estimating enzyme activity. In such cases, a longer incubation time may be required since the relevant process being detected is also an enzymatic conversion.

[0196] A particular advantage of the methods disclosed herein is that by adjusting various parameters, including the choice of caged substrate molecule and the amount or activity of the uncaging agent, a range of different incubation periods can be accommodated, thereby ensuring that the biosensor is appropriately excited at the end of the incubation time. In some embodiments, the incubation period is between 1 minute and 60 minutes, or between 1 minute and 30 minutes, or between 5 minutes and 15 minutes or between 10 minutes and 30 minutes. In some embodiments, the incubation period is 10 minutes or less, such as 2 minutes, 4 minutes, 6 minutes, 8 minutes or 10 minutes.

[0197] The methods disclosed herein can be performed as a batch assay in a batch assay vessel and do not require microfluidics or other flow technologies to mediate the incubation of the assay composition or the uncaging of the caged substrate therein. Thus, in some embodiments, the assay composition is incubated without flowing through a flow channel, such as in a microfluidic device.

[0198] Uncaging a caged substrate compound in an assay composition

[0199] In embodiments of the methods disclosed herein, during the incubation period, at least one caged substrate compound is uncaged in the assay composition to produce an uncaged substrate compound that excites a donor fluorophore when light emitted from the assay composition is to be detected.

[0200] As used herein, a caged substrate compound is a protected form of a substrate of a donor fluorophore, wherein the reactive functional group of the substrate is reversibly protected by a protecting group. Thus, the caged substrate compound may be non-reactive with or insensitive to reaction with the donor fluorophore until it is deprotected to reform the substrate (also referred to herein as the uncaged substrate compound) in the assay composition. Additionally, the caged substrate compound is less sensitive to other unwanted reactions, such as auto-oxidation reactions. Advantageously, the caged substrate compound can be stored in solution for extended periods, making it more amenable to various point-of-care diagnostic test applications. Further, the uncaging of at least one caged substrate compound can be coordinated with the interaction of the analyte with the BRET biosensor to ensure that the BRET biosensor is appropriately excited at the desired detection time.

[0201] In some embodiments, only one caged substrate compound uncages in the assay composition. In other embodiments, two or more caged substrate compounds uncage in the assay composition. In the latter case, the two or more caged substrate compounds are typically protected forms of the same uncaged substrate compound, i.e., they produce the same substrate when uncaged. Different caged forms of the substrate can be selected to uncage at different reaction rates, thereby extending the period during which the uncaged substrate is available to sufficiently activate the BRET biosensor. This can advantageously ensure that the BRET assay is less sensitive to precise incubation times or temperature profiles, or allow repeated BRET assays within an extended detection window while reducing the risk that the observed BRET ratio is confounded by variable luminescence.

[0202] In some embodiments, the caged substrate compound is present in the assay composition from the start of the incubation period. This means that the caged substrate compound is present in the assay composition when the extracellular bioluminescence sensor molecule first contacts the sample or immediately thereafter, e.g., within one to two minutes, as is typical when preparing the assay composition by sequential combination of the various components. Thus, for example, an assay composition can be prepared by dispersing a (lyophilized) solid composition containing the BRET biosensor in a liquid sample and then immediately adding the caged substrate compound (e.g., within two minutes, preferably within one minute or less). The combined liquid assay composition is then incubated for the desired incubation period.

[0203] It may be preferred to combine all components in the assay composition (including the caged substrate compound and any uncaging agent) from the start of the incubation period, as this simplifies the assay protocol. However, it is not excluded that the caged substrate compound is added to the assay composition one or more times during the incubation period (i.e., midway). Such methods may be useful when a very long incubation period (e.g., greater than 30 minutes) is required to detect the interaction of the analyte with the biosensor. The use of the caged substrate remains advantageous in such cases because the activation of the BRET biosensor can be sustained over a longer period (the concentration of the uncaged substrate compound in solution is controlled by both uncaging and consumption kinetics), and because of the enhanced storability of the caged substrate molecules.

[0204] A caged substrate compound can be introduced into an assay composition in a solution (referred to herein as the substrate solution). Preferably, the solution is a room-temperature stable substrate solution, such that a single solution can be stored for an extended period of time and reused in a BRET assay. As used herein, a room-temperature stable substrate solution is substantially stable for at least one week, preferably at least one month, when stored at about 22-25 °C while excluding air and light exposure. A solution is considered to be substantially stable if, in a blank (analyte-free) BRET assay, the emission amplitude of the solution is not less than 10% of its original value and the BRET ratio remains within ±5% of its original value. In contrast, the inventors have found that the uncaged substrate compound degrades rapidly in solution, even when light and air are rigorously excluded.

[0205] The substrate solution can be a non-aqueous solution. The non-aqueous solution can comprise a polar aprotic solvent that is water-miscible and preferably of low volatility, such as dimethyl sulfoxide (DMSO). In some embodiments, the solvent is dry DMSO, such as ultra-dry DMSO.

[0206] The substrate solution can be introduced into the assay composition by any method suitable for accurate dispensing. In some embodiments, for example, the substrate solution is dispensed dropwise into the assay composition using a precision pipette. As an example, a diluted DMSO solution can be accurately dispensed as droplets of a known volume (e.g., 28 μL droplets), such that the substrate solution can be formulated so that a desired amount of the caged substrate compound is dispensed in one or more such droplets. Advantageously, this allows a predetermined amount of the caged substrate to be dispensed into the assay composition in a simple and sufficiently reproducible manner for point-of-care diagnostic testing. The substrate solution can be contained and stored in a substrate storage vessel for dispensing into the assay composition. In some embodiments, the substrate storage vessel is configured to dispense an accurately predetermined amount of the substrate solution into the assay composition, such as dropwise.

[0207] In some embodiments, the substrate solution is stored for at least one week, or at least one month, or at least six months before being introduced into the assay composition. This advantageously allows the methods disclosed herein to be used in point-of-care diagnostic testing in environments where it would be unacceptable or undesirable to prepare fresh reagent solutions for each test.

[0208] When introduced into the substrate solution, the caged substrate is added to the assay as a component separate from the BRET biosensor. Alternatively, the caged substrate can be formulated with the BRET biosensor, such as a component of a (lyophilized) solid composition dispersed in the sample. In such embodiments, the solid composition typically does not contain a uncaging agent, which can then be added to the assay as a separate component (e.g., in solution) or as part of the sample itself.

[0209] The uncaged substrate compound can be any suitable substrate for a BRET biosensor, i.e., a substrate on which the donor luminophore acts to produce (bio)luminescence. Typically, the uncaged substrate compound is a high-energy luciferin molecule that is oxidatively enzymatically converted by the donor luminophore with molecular oxygen. Examples of luciferins that occur naturally in bioluminescent organisms include coelenterazine in seapansy (Renilla reniformis), D-luciferin (in combination with adenosine triphosphate) in fireflies, and other luciferins such as ostracod luciferin, furimazine, and coelenteramine. The uncaged substrate compounds used in the presently disclosed methods can be such naturally occurring luciferin molecules or their synthetic analogs.

[0210] In some embodiments, the uncaged substrate compound is selected from coelenterazine, ostracod luciferin, furimazine, and synthetic analogs thereof having the same imidazopyrazinone-based core structure. Such imidazopyrazinone-based substrates are all capable of exciting RLuc. In some embodiments, the uncaged substrate compound is a coelenterazine compound. As used herein, a coelenterazine compound refers to natural coelenterazine or its natural or synthetic analogs having the same imidazopyrazinone core structure. In some embodiments, the uncaged substrate compound thus has a structure according to Formula 1a or Formula 2a:

[0211]

[0212] where each R 1 is independently selected from H, OH, OR alk and NH2, where R alk is a C1-C6 alkyl group, each R 2 is independently selected from H, Ar, and (CH2) n Ar, where each Ar is independently a carbocyclic aromatic group optionally substituted with OH, C1-C6 alkyl, or halogen, and n is 1 or 2, each L 1 is independently selected from CH2 or S, each R 3 is independently selected from C6H5, CH2C6H5, C1-C6 alkyl, and C1-C6 cycloalkyl, R 4 is independently selected from H and CH3, and L 2 is independently selected from (CH2) n and CH═CH, where n is 1-3.

[0213] In some embodiments, the uncaged substrate compound has a structure according to Formula 1a, where R 1 is selected from H and OH, R 2 is selected from C6H5 (phenyl) and p-hydroxyphenyl, R 3 is C6H5, L1 is CH2, and R 4 is H. In some embodiments, the uncaged substrate compound is native coelenterazine, which is a compound according to Formula 1a, where R 1 is hydroxy, R 2 is p-hydroxyphenyl, R 3 is phenyl, R 4 is H, and L 1 is CH2. In other embodiments, the uncaged substrate compound is coelenterazine h, which is a compound according to Formula 1a, where R 1 is hydroxy, R 2 and R 3 are phenyl, R 4 is H, and L 1 is CH2.

[0214] In some embodiments, the uncaged substrate compound is bisdeoxycoelenterazine (also known as coelenterazine 400a, Clz400a, Deep Blue C; or 2,8-dibenzyl-6-phenylimidazo[1,2-a]pyrazin-4-ium-3-one). Bisdeoxycoelenterazine is a compound according to Formula 1a, where R 1 is H, R 2 and R 3 are each phenyl, R 4 is H, and L 1 is CH2. Bisdeoxycoelenterazine has a significant blue-shifted emission relative to native coelenterazine, which enables long Stokes shift resonance energy transfer such as bioluminescence resonance energy transfer 2 (BRET 2 ). It has higher sensitivity than more red-shifted versions of BRET. Unfortunately, bisdeoxycoelenterazine is highly prone to oxidation both as a solid and in solution, and thus, formulations of this compound are inherently unstable under ambient conditions. Therefore, bisdeoxycoelenterazine is not suitable as an off-the-shelf long shelf-life reagent.

[0215] It is believed that the first step in the enzymatic oxidation of coelenterazine compounds involves O2 binding at the C2 position of the imidazopyrazinone core structure adjacent to the carbonyl at the C3 position (3'-carbonyl). The C2 and C3 carbons are labeled in Formulas 1a and 2a above. Thus, by reacting the 3'-carbonyl with a suitable carbonyl protecting group, coelenterazine compounds (including coelenterazine, furimazine, and their synthetic analogs) can be caged (i.e., protected from oxidation). In some embodiments, each caged substrate compound for use in the methods of the present disclosure is a caged coelenterazine compound that includes a carbonyl protecting group at the 3' position. For example, the 3'-carbonyl can be protected as a 3'-ether functional group, a 3'-ester functional group, or a 3'-carbonate functional group.

[0216] Thus, each caged substrate compound can have a structure according to Formula 1 or Formula 2:

[0217]

[0218] where each R 1 is independently selected from H, OH, OR alk and NH2, where R alk is a C1-C6 alkyl group, each R 2 is independently selected from H, Ar, and (CH2) n Ar, where each Ar is independently a carbocyclic aromatic group optionally substituted with OH, C1-C6 alkyl, or halogen, and n is 1 or 2, each L 1 is independently selected from CH2 or S, each R 3 is independently selected from C6H5, CH2C6H5, C1-C6 alkyl, and C1-C6 cycloalkyl, R 4 is independently selected from H and CH3, L 2 is independently selected from (CH2) n and CH=CH, where n is 1-3, and each X is independently a carbonyl protecting group.

[0219] In some embodiments, each caged substrate compound has a structure according to Formula 1, where R 1 is selected from H and OH, R 2 is selected from C6H5 (phenyl) and p-hydroxyphenyl, R 3 is C6H5, L 1 is CH2, and R 4 is H.

[0220] In the compounds of Formula 1 and Formula 2, the 3'-carbonyl (present in the compounds of Formula 1a and Formula 2a, respectively) is protected as a functional group of the form C3-O-X (where C3 is the C3 carbon of the imidazopyrazinone core structure). Similarly, other coelenterazine compounds can be caged by converting the 3'-carbonyl of the imidazopyrazinone core to C3-O-X, where X is a carbonyl protecting group. In some embodiments, the C3-O-X functional group is selected from ethers (e.g., C3-O-CH2-), esters (i.e., C3-O-C[=O]-C), and carbonates (i.e., C3-O-C[=O]-O-).

[0221] In some embodiments, X in the compounds of Formula 1 and Formula 2 has a structure according to Formula 3 (thus forming an ester functional group), Formula 4 (thus forming a carbonate functional group), or Formula 5 (thus forming an ether functional group):

[0222]

[0223] where R 5 、R6 and R 7 each independently is an optionally substituted C1-C 12 alkyl, C1-C 12 cycloalkyl or C1-C 12 aryl, preferably an optionally substituted C1-C 12 alkyl or C1-C 12 cycloalkyl, and represents the point of attachment to the 3'-oxygen atom. When the C1-C 12 alkyl, cycloalkyl or aryl is substituted, it may be substituted by one or more, for example one to three, groups selected from the following: halogen, =O, =S, -CN, -NO2, -CF3, -OCF3, alkyl, alkenyl, alkynyl, haloalkyl, haloalkenyl, haloalkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkoxyaryl, alkoxyheteroaryl, alkenoxy, alkynoxy, cycloalkoxy, cycloalkenoxy, heterocycloalkoxy, heterocycloalkenoxy, aryloxy, heteroaryloxy, arylalkyl, heteroarylalkyl, arylalkoxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, aminoalkyl, COOH, SH and acyl. Optionally, such substituents may have no more than 12 carbon atoms.

[0224] In some embodiments, R 5 , R 6 and R 7 are sterically hindered C1-C 12 alkyl or cycloalkyl, such as secondary or tertiary alkyl substituents or bicyclic cycloalkyl substituents, more preferably tertiary substituents such as tert-butyl or adamantyl.

[0225] Non-limiting examples of suitable cage substrate compounds are disclosed in Jiang et al., Journal of Photochemistry and Photobiology Science (Photochem. Photobiol. Sci.), 2016, 15, 466 and Yuan et al., Organic and Biomolecular Chemistry (Org. Biomol. Chem.), 2017, 15, 10238-10244.

[0226] The present inventors have found that caged coelenterazine compounds having an ester, carbonate or ether protecting group according to Formula 3 (Compound 7 below), Formula 4 (Compound 8 below) or Formula 5 (Compound 10 below) provide enhanced stability in non-aqueous solutions, thus facilitating their use in point-of-care diagnostic tests where storage stability of the reagent is important. DMSO solutions of each of these compounds were found to be stable substrate solutions at room temperature, and each compound could be stored at room temperature for at least 1 month (Example 2). Compound 10 was found to have the greatest stability, and a DMSO solution of this compound could be stored at room temperature for well over a year (Example 1).

[0227]

[0228] The present inventors have also found that caged coelenterazine compounds having an ester, carbonate or ether protecting group (i.e., Compounds 7, 8 and 10, respectively) can be uncaged in situ in an assay composition, where the uncaging rate depends on the nature of the protecting group and the type of uncaging agent present (Example 3). In each case, uncaging produces the corresponding uncaged substrate compound coelenterazine-h, thus activating the extracellular BRET 2 biosensor. Compound 7, which has an acyl protecting group with low steric requirements, is rapidly uncaged by exogenous esterase (e.g., porcine liver esterase) or esterases naturally present in milk samples. Compounds 8 and 10, which have highly sterically demanding tert-butylcarbonyl (O-Boc) and pivaloyloxymethyl protecting groups, respectively, are uncaged more slowly in the presence of exogenous esterase, thus exciting the extracellular BRET 2 biosensor for an extended period of time (up to about 30 minutes). By selecting a caged substrate or combination of caged substrates having appropriate uncaging kinetics, the extracellular BRET biosensor can be appropriately excited, when mediated by the uncaging agent provided in the assay composition, to detect analytes that require multiple different incubation periods, or to continuously or periodically detect analyte-biosensor interactions within a broad detection window. In some embodiments, the caged substrate is Compound 10. In some embodiments, the caged substrate is Compound 8. In some embodiments, the caged substrate is Compound 7.

[0229] When detecting a diagnostic BRET ratio, the caged substrate compound should be uncaged to provide a suitably strong signal from the BRET biosensor at the time or within the time range. For a given assay protocol, or when developing a new assay protocol to meet this requirement, one way to verify this is to continuously or semi - continuously determine the luminescence associated with the acceptor fluorophore (or donor luminophore) during a blank BRET assay, i.e., under conditions representing a true assay but in the absence of any analyte that would affect the BRET ratio. When measuring the diagnostic BRET ratio in an actual assay, the observed luminescence should be suitably high at the time or over the entire time range, e.g., greater than 50% of its maximum value, or greater than 80% of its maximum value.

[0230] Thus, in some embodiments of the methods disclosed herein, the at least one caged substrate compound is uncaged to provide luminescence associated with the acceptor fluorophore (alternatively with the donor luminophore), and in the absence of extracellular analyte, when light is detected, the luminescence is greater than 50% of the maximum value, or greater than 80% of the maximum value. In a detection window of at least 3 minutes or at least 5 minutes, the analyte - free luminescence is preferably greater than 50% of the maximum value, or greater than 80% of the maximum value, during which the light emitted from the assay composition is detected one or more times. Such methods can advantageously minimize the risk that the observed BRET ratio is confounded by low or variable total luminescence.

[0231] While the methods disclosed herein encompass any method of in - situ uncaging of the caged substrate compound, the caged substrate compound is most typically uncaged by a uncaging agent or combination of uncaging agents present in the assay composition. Here, it should be understood that the uncaging agent is not the BRET biosensor itself.

[0232] In some embodiments, the uncaging agent is present in the assay composition from the start of the incubation period. Again, this means that the uncaging agent is present in the assay composition when the extracellular bioluminescence sensor molecule first contacts the sample or immediately thereafter, e.g., within one to two minutes. In some embodiments, the uncaging agent is formulated with the BRET biosensor. For example, it can be present with the BRET biosensor in a lyophilized solid composition adapted to be dispersed in a liquid sample. Alternatively, it can be present in the sample or provided as a separate formulation to be introduced independently into the assay composition.

[0233] While it is generally advantageous to combine all components from the start of the incubation time, it is not excluded to add the uncaging agent to the assay composition one or more times during the incubation period (i.e., midway). Such methods can be used to initiate uncaging of the caged substrate when a very long incubation period (e.g., greater than 30 minutes) is required to detect the interaction of the analyte with the biosensor.

[0234] In some embodiments, the uncaging agent is independent of the sample. In other embodiments, the uncaging is endogenous to the sample, i.e., it is naturally present in the sample. Some biological matrices, such as milk or other dairy products, contain naturally occurring esterases that can uncage certain caged substrate compounds.

[0235] The mechanical action of the uncaging agent will depend on the chemical form of the protecting group in the caged substrate compound. In some embodiments, the uncaging agent is an esterase or other chemical agent capable of deprotecting a carbonyl protecting group, particularly the 3'-carbonyl protecting group of a caged coelenterazine compound as disclosed herein. The inventors have found that porcine liver esterase, an industrially available low-cost enzyme, is suitable as an uncaging agent in such embodiments. The inventors have also found that the amount of uncaging agent included in the assay composition can vary to match the type of biosensor (e.g., allosteric versus cleavable versus competitive) and the desired incubation period. In some embodiments, the amount of uncaging agent in the assay composition is from 1 U / mL to 100 U / mL, 1 U / mL to 90 U / mL, 1 U / mL to 80 U / mL, 1 U / mL to 70 U / mL, 1 U / mL to 60 U / mL, 1 U / mL to 50 U / mL, 1 U / mL to 40 U / mL, 1 U / mL to 30 U / mL, 1 U / mL to 20 U / mL, or 1 U / mL to 10 U / mL. In some embodiments, the amount of uncaging agent in the assay composition is from 1 U / mL to 10 U / mL, or 1 U / mL, 5 U / mL, or 10 U / mL.

[0236] The present inventors have discovered that by selecting a caged substrate or combination of caged substrates with appropriate uncaging kinetics, optimal timed light production for assays of different durations can be achieved. This has the advantage of exciting extracellular biosensors to detect analytes that require multiple different incubation periods, or to continuously or periodically detect analyte-biosensor interactions within a broad detection window. In some embodiments, the caged substrate comprises two or more caged substrates of Formula 1, two or more caged substrates of Formula 2, or at least one caged substrate of Formula 1 and at least one caged substrate of Formula 2. In some embodiments, the caged substrate is selected from the group consisting of Compound 7, Compound 8, and Compound 10 or combinations thereof. In some embodiments, the caged substrate comprises (i) Compound 7 and Compound 8, (ii) Compound 7 and Compound 10, (iii) Compound 8 and Compound 10 or (iv) Compound 7, Compound 8, and Compound 10. In some embodiments, the caged substrate comprises Compound 8 and Compound 10. In embodiments where the caged substrate comprises two compounds (e.g., Compound 8 and Compound 10), the ratio of the two compounds can be between 100:1 and 1:100, or between 50:1 and 1:50, or between 10:1 and 1:10, or between 5:1 and 1:5, or between 3:1 and 1:3, or between 2:1 and 1:3 or between 1:1 and 1:2. In some embodiments, the molar ratio of Compound 8 to Compound 10 is from about 1:1 to 1:5, or 1:1 to 1:4, or 1:1 to 1:3 or 1:1 to 1:2. In some embodiments, the molar ratio of Compound 8 to Compound 10 is about 1:2. In embodiments where the caged substrate comprises Compound 7, 8, and 10, the molar ratio of Compound 7:Compound 8:Compound 10 can be from 1:1:1 to 1:100:100, such as 1:1:1, 1:2:2, 1:6:6, 1:12:12 or 1:60:60.

[0237] By providing substrates in the form of one or more caged substrate compounds formulated in a non-aqueous solution for in situ uncaging in an assay composition, the present inventors have demonstrated that the following balance of properties required for a successful BRET-based point-of-care diagnostic method can be achieved:

[0238] ● A substrate formulation suitable for cell-free biochemical assays (i.e., rather than intracellular assays);

[0239] ● The substrate formulation is stable upon storage at room temperature for several months, in some cases up to one year;

[0240] ● The substrate formulation is miscible with water, does not interfere with the BRET biosensor, and is not highly flammable or toxic;

[0241] ● The substrate formulation is reproducible and provides consistent results at different times, by different operators, and at different locations;

[0242] ● A substrate formulation that uses industrially available uncaging agents, such as enzymes, and is compatible with a range of sample matrices (e.g., milk);

[0243] ● Substrates provided in situ by an effective uncaging process conducted under incubation conditions suitable for the interaction of the analyte with the BRET biosensor; and

[0244] ● Based on the uncaging kinetics of different uncaged substrate compounds, the substrate formulation can be customized to match assays with different time courses.

[0245] Detected light

[0246] The methods disclosed herein include the step of detecting light emitted from the assay composition after an incubation period. As required for BRET assays, the detected light includes distinguishable emissions associated with the acceptor fluorophore and the donor luminophore. Thus, the ratio of the luminescence associated with the acceptor fluorophore to the luminescence associated with the donor luminophore (e.g., the BRET ratio) indicates the interaction of the extracellular analyte with the analyte-sensitive domain.

[0247] During detection, the assay composition can be present in an assay vessel, wherein light emitted from the assay composition is detected passing through the wall of the assay vessel. Thus, a suitable assay vessel can be transparent at least for the emission wavelengths of the donor and acceptor luminophores and at least in the region of the wall configured to allow light transmission for detection. In some embodiments, the assay composition persists in the same assay vessel during the incubation period and during detection. In some embodiments, the assay composition is prepared in the same assay vessel.

[0248] Light can be detected by any suitable means (in principle including by the human eye, which is sensitive enough to detect the luminescence and color changes of some BRET assays). More typically, the light is detected by a photoelectronic device capable of distinguishing the emissions associated with the acceptor fluorophore and the donor luminophore. The photoelectronic device can be a sophisticated laboratory instrument, such as a microplate spectrometer (plate reader). However, the methods disclosed herein can also be implemented with a simple dual-channel photoelectronic device configured to quantify the luminescence of the emissions associated with the acceptor fluorophore and the donor luminophore, which will be described in more detail below.

[0249] The method can include the steps of calculating a BRET ratio of light emitted from an assay composition and determining the activity or concentration of an extracellular analyte in a sample based on this BRET ratio. This can be done by comparing the observed BRET ratio to a predetermined calibration curve. As will be understood by those skilled in the art, the calibration curve can be obtained by measuring the BRET ratios of assays performed with a series of externally defined calibration standards and optionally fitting the calibration curve to the calibration data using a non-linear curve fitting equation.

[0250] System for detecting an extracellular analyte in a sample

[0251] The present invention further relates to a system for detecting an extracellular analyte in a sample. The system comprises a biosensor system selected from an extracellular bioluminescence sensor molecule or a precursor system, as disclosed herein, the biosensor system being contained in an assay vessel configured to receive the sample for combination with the biosensor system in an assay composition. The system further comprises a room temperature stable substrate solution, as disclosed herein, the room temperature stable substrate solution comprising at least one caged substrate compound for dispensing into the assay composition in the assay vessel. The substrate solution is contained in a substrate storage vessel. The system further comprises a photoelectronic device configured to detect light emitted from the assay composition passing through the wall of the assay vessel, including emissions associated with a receptor fluorophore and a donor luminophore. The ratio of the luminescence associated with the receptor fluorophore to the luminescence associated with the donor luminophore indicates the interaction of the extracellular analyte with the analyte-sensitive domain. The at least one caged substrate compound is adapted to uncage in the assay composition upon use, thereby producing an uncaged substrate compound that excites the donor luminophore.

[0252] The extracellular bioluminescence sensor molecule (or BRET biosensor) and the room temperature stable substrate solution comprising at least one caged substrate compound can generally be according to any of the embodiments described herein in the context of the methods of the present invention. Specifically, the extracellular bioluminescence sensor molecule (or its precursor system) can be present in a solid composition, optionally together with one or more additional components such as an uncaging agent. The solid composition can be a lyophilized solid composition, which can be prepared by lyophilizing an aqueous precursor composition in the same assay vessel. The room temperature stable substrate solution can be a non-aqueous solution, preferably comprising a water-miscible low volatility polar aprotic solvent such as DMSO. The substrate solution can be formulated to a suitable concentration for dispensing (e.g., dropwise) into the assay composition in the assay vessel.

[0253] Assay vessel

[0254] The assay vessel of the system contains a biosensor system, typically the solid compositions described herein. The assay vessel is configured to: (i) receive a sample for combination with the extracellular bioluminescence sensor molecule in the assay composition, and (ii) permit detection of light emitted from the assay composition that passes through the wall of the assay vessel. As used herein, the wall of the assay vessel through which light is transmitted is not necessarily a sidewall, but can include any part of the housing, such as the bottom or cap / cover.

[0255] The assay vessel typically contains sufficient extracellular bioluminescence sensor molecule (or precursor) for only a single assay, the amount of which will depend on the particulars of the embodiment. In one exemplary embodiment disclosed herein, for the analysis of AprX in 1 ml of milk sample, the assay vessel contains a lyophilized solid composition containing approximately 690 ng of BRET 2 biosensor and other components including porcine liver esterase. In another exemplary embodiment disclosed herein, for the analysis of plasmin or plasminogen in 1 ml of milk sample, the assay vessel contains a lyophilized solid composition containing approximately 690 ng of BRET 2 biosensor and other components including porcine liver esterase. In another exemplary embodiment disclosed herein, for the analysis of lactose in 1 ml of milk sample, the assay vessel contains a lyophilized solid composition containing approximately 500 ng of BRET 2 biosensor and other components including porcine liver esterase.

[0256] The assay vessel can be in the form of a sealable tube, for example fitted with a screw cap, and having a suitable capacity to receive the sample, for example at least 1 ml of sample. The tube can be transparent, at least to the light emitted by the BRET biosensor, thus permitting detection of light passing through the sidewall. In some embodiments, the tube is precision molded from an optically transparent thermoplastic such as polypropylene and has no markings or color on the tube wall. An example of a suitable tube is the sealable ThermoScientific TM QSP TM Natural Screw Cap Microtube 520 - GRD tube, which has a diameter of 1 cm and a height (without cap) of 4.4 cm. Although assay vessels made of different materials and having different shapes or sizes can be used, in the preferred embodiments, they can be sealed, permit efficient transmission of visible light through the wall, are optically identical under rotation, and closely match the dimensions of the tube holder in the optoelectronic device.

[0257] The system can further comprise an opaque external container, preferably containing a desiccant, in which one or more assay vessels containing the BRET biosensor can be stored at room temperature or colder until needed for use.

[0258] Solid compositions and precursor compositions

[0259] The biosensors used in the methods and other aspects and embodiments described herein are typically provided in the form of solid compositions. Accordingly, solid compositions suitable for the embodiments and aspects described herein are also provided. The solid compositions comprise a biosensor (e.g., a BRET biosensor). The solid compositions may optionally comprise other components such as (i) one or more uncaging agents, such as esterases, for uncaging caged substrate compounds in the assay composition; (ii) buffers; (iii) one or more salts; (iv) one or more metal ions (if required, e.g., for metalloprotease assays); (v) one or more carrier proteins, such as fish skin gelatin; and (vi) one or more cryoprotectants, such as trehalose, sucrose, and pullulan polysaccharide.

[0260] Solid compositions typically contain sufficient extracellular bioluminescence sensor molecules (or precursors) for only a single assay, the amount of which will depend on the particulars of the embodiment. In some embodiments, the assay vessel contains a solid composition comprising from about 10 ng to 1000 ng, or from about 10 ng to 900 ng, 10 ng to 800 ng, 10 ng to 700 ng, 50 ng to 1000 ng, 100 ng to 1000 ng, 200 ng to 1000 ng, 300 ng to 1000 ng, 400 ng to 1000 ng, 500 ng to 1000 ng, or 600 ng to 1000 ng of the biosensor. In some embodiments, the assay vessel contains a lyophilized solid composition comprising from about 400 ng to 700 ng of the biosensor. In some embodiments, the assay vessel contains a lyophilized solid composition comprising about 690 ng of the biosensor. In some embodiments, the assay vessel contains a lyophilized solid composition comprising about 500 ng of the biosensor. In one exemplary embodiment disclosed herein, for the analysis of AprX in 1 ml of milk sample, the solid composition comprises about 690 ng of BRET 2 biosensor, and other components including porcine liver esterase. In another exemplary embodiment disclosed herein, for the analysis of plasmin or plasminogen in 1 ml of milk sample, the solid composition comprises about 690 ng of BRET 2 biosensor, and other components including porcine liver esterase. In another exemplary embodiment disclosed herein, for the analysis of lactose in 1 ml of milk sample, the solid composition comprises about 500 ng of BRET 2 biosensor, and other components including porcine liver esterase.

[0261] The solid composition may comprise one or more uncaging agents as defined herein. In some embodiments, the amount of the uncaging agent (e.g., esterase) in the solid composition is from about 0.2 units to 20 units, 0.2 units to 18 units, 0.2 units to 16 units, 0.2 units to 14 units, 0.2 units to 12 units, 0.2 units to 10 units, 1 unit to 20 units, 1 unit to 10 units, or 1 unit to 5 units. In some embodiments, the concentration of the uncaging agent is about 1 unit, 5 units, or 10 units.

[0262] The solid composition may comprise one or more buffering agents as defined herein. In some embodiments, the amount of the buffering agent in the solid composition is between about 2 micromoles and 100 micromoles, such as 50 micromoles.

[0263] The solid composition may comprise one or more cryoprotectants as defined herein. In some embodiments, the amount of each cryoprotectant in the solid composition is between about 0.2 mg and 20 mg, such as 2.5 mg or 5 mg. In some embodiments, the total amount of the cryoprotectant in the solid composition before freezing is between about 0.4 mg and 40 mg, such as 5 mg.

[0264] In some embodiments, the solid composition may comprise an appropriate amount of a carrier protein, such as fish gelatin.

[0265] In some embodiments, the solid composition may comprise one or more salts, such as sodium chloride. In some embodiments, the amount of the one or more salts in the solid composition is between about 2 micromoles and 100 micromoles, such as 50 mM micromoles.

[0266] In some embodiments, the solid composition comprises one or more metal ions (if desired, e.g., for metalloprotease assays or for the activity of an enzyme). In some embodiments, the amount of the metal ion in the solid composition is between about 0.2 micromoles and 20 micromoles, such as 5 micromoles.

[0267] In some embodiments, the solid comprises one or more additional components as described herein. For example, in the case of a zymogen assay, the solid composition can optionally comprise an agent that activates the zymogen. For example, in the case of an assay for plasmin that has been disclosed to associate with casein micelles, the solid composition can optionally comprise an agent that dissociates plasmin from the casein micelles. For example, the solid composition can comprise 1 micromole of aminocaproic acid and 80 micromoles of trisodium acetate, which helps to dissociate plasmin from the casein micelles. For example, in the case of a plasmin assay, the solid composition can optionally comprise L-lysine that supports plasmin activity. In some embodiments, the amount of L-lysine in the solid composition is between 2 micromoles and 100 micromoles, such as 25 micromoles.

[0268] In some embodiments, the solid composition is prepared by filling an aqueous solution of the composition (referred to herein as the precursor composition) into a suitable assay vessel. The volume of the aqueous solution added to the assay vessel can vary, but typically provides only the extracellular bioluminescent sensor molecule (or precursor) for a single assay, and the amount will depend on the particulars of the embodiment. In some embodiments, the solid composition is prepared by filling 200 μL of the precursor composition into a suitable assay vessel. Unless otherwise noted, the following paragraphs describe the concentrations of certain components in the aqueous solution of the composition prior to freezing or drying (i.e., the concentrations in the precursor composition).

[0269] In some embodiments, the concentration of the BRET biosensor in the composition prior to freezing is from about 50 ng to 10,000 ng / mL, or from about 50 ng / mL to 8000 ng / mL, from about 50 ng / mL to 6000 ng / mL, from about 50 ng / mL to 5000 ng / mL, 50 ng / mL to 4500 ng / mL, 50 ng / mL to 4000 ng / mL, 50 ng / mL to 3500 ng / mL, 50 ng to 3000 ng / L, 500 ng / mL to 4000 ng / mL, 1000 ng / mL to 4000 ng / mL, 1500 ng / mL to 4000 ng / mL, 2000 4000 ng / mL, 2500 ng / mL to 4000 ng / mL, or 3000 4000 ng / mL. In some embodiments, the concentration of the BRET biosensor is from about 3000 ng / mL to 4000 ng / mL. In some embodiments, the concentration of the BRET biosensor is about 3450 ng / mL. In some embodiments, the concentration of the BRET biosensor is about 2500 ng / mL.

[0270] The precursor composition can comprise one or more uncaging agents as defined herein. In some embodiments, the concentration of the uncaging agent (e.g., esterase) in the pre-frozen composition is from about 1 U / mL to 100 U / mL, 1 U / mL to 90 U / mL, 1 U / mL to 80 U / mL, 1 U / mL to 70 U / mL, 1 U / mL to 60 U / mL, 1 U / mL to 50 U / mL, 5 U / mL to 100 U / mL, 5 U / mL to 50 U / mL, or 5 U / mL to 25 U / mL. In some embodiments, the concentration of the uncaging agent is about 5 U / mL, 25 U / mL, or 50 U / mL.

[0271] The precursor composition can comprise one or more buffers as defined herein. In some embodiments, the concentration of the buffer in the pre-frozen composition is between about 10 mM and 500 mM, such as 250 mM.

[0272] The precursor composition can comprise one or more cryoprotectants as defined herein. In some embodiments, the concentration of each cryoprotectant in the pre-frozen composition is between about 1 mg / mL and 100 mg / mL, such as 12.5 mg / mL or 25 mg / mL. In some embodiments, the total concentration of the cryoprotectants in the pre-frozen composition is between about 2 mg / mL and 200 mg / mL, such as 25 mg / mL.

[0273] In some embodiments, the precursor composition can comprise a carrier protein, such as fish gelatin. In some embodiments, the concentration of the carrier protein in the pre-frozen composition is between about 0.1% and 1% (v / v), such as about 0.1%, 0.2%, 0.3%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% (v / v). In some embodiments, the concentration of the carrier protein is about 0.45% (v / v). In some embodiments, %(v / v) refers to the volume of a fish gelatin stock solution containing 0.45 g / mL (45% w / v) of fish skin gelatin added to the precursor composition.

[0274] In some embodiments, the precursor composition can comprise one or more salts, such as sodium chloride. In some embodiments, the concentration of the one or more salts in the pre-frozen composition is between about 10 mM and 500 mM, such as 250 mM.

[0275] In some embodiments, the precursor composition comprises one or more metal ions (if desired, e.g., for metalloprotease assays or for enzyme activity). In some embodiments, the concentration of the metal ions in the pre-frozen composition is between about 1 mM and 100 mM, such as 25 mM.

[0276] In some embodiments, the precursor comprises one or more additional components. The additional components include, but are not limited to, activators, inhibitors, detergents, reducing agents, oxidizing agents, etc. For example, in the case of a zymogen assay, the assay composition may optionally comprise an agent that activates the zymogen. For example, in the case of an assay for plasmin that has been disclosed to associate with casein micelles, the precursor composition may optionally comprise an agent that dissociates plasmin from the casein micelles. For example, the precursor composition may comprise 5 mM aminocaproic acid and 400 mM trisodium acetate, which helps to dissociate plasmin from the casein micelles. For example, in the case of a plasmin assay, the precursor composition may optionally comprise L-lysine that supports plasmin activity. In some embodiments, the concentration of L-lysine in the pre-freezing composition is between 10 mM and 500 mM, such as 125 mM.

[0277] In one exemplary embodiment, a BRET for detecting AprX in milk 2 The freeze-dried solid composition of the biosensor system is prepared by filling an aqueous solution (200 μL) of the following composition into a suitable assay vessel (e.g., QSP TM natural screw cap microtube 520-GRD (Thermo Fisher Scientific)) and freeze-drying the aqueous solution:

[0278] Precursor Composition Assay Composition AprX Biosensor 3,450 ng / ml 690 ng / ml Tris (pH 8.4) 250 mM 50 mM NaCl 250 mM 50 mM <![CDATA[MgCl2.6H2O]]> 25 mM 5 mM <![CDATA[CaCl2.2H2O]]> 25 mM 5 mM Fish Skin Gelatin * 0.45% (v / v) 0.09% (v / v) Trehalose 12.5 mg / mL 2.5 mg / mL Pullulan 12.5 mg / mL 2.5 mg / mL Porcine Esterase 5 U / mL or 25 U / mL 1 U / mL or 5 U / mL

[0279] * The stock solution contains 45% (w / v) (0.45 g / mL) fish skin gelatin.

[0280] In one exemplary embodiment, the above formulation containing 25 U / mL esterase (final concentration of 5 U / mL) can also be used for the detection of subtilisin (alkaline protease).

[0281] In one exemplary embodiment, a BRET for detecting plasmin in milk 2 The freeze-dried solid composition of the biosensor system is prepared by filling an aqueous solution (e.g., 200 μL) of the following composition into a suitable assay vessel and freeze-drying the aqueous solution:

[0282] Precursor Composition Assay Composition <![CDATA[Plasmin BRET 2 Biosensor]]> 3,450 ng / ml 690 ng / ml Tris (pH 8.4) Buffer 250 mM 50 mM NaCl 250 mM 50 mM L-Lysine 125 mM 25 mM 6-Aminocaproic Acid 5 mM 1 mM Trisodium Acetate 400 mM 80 mM Fish Skin Gelatin * 0.45% (v / v) 0.09% (v / v) Trehalose 12.5 mg / mL 2.5 mg / mL Pullulan 12.5 mg / mL 2.5 mg / mL Porcine Esterase 25 U / mL 5 U / mL

[0283] * The stock solution contains 45% (w / v) (0.45 g / mL) fish skin gelatin.

[0284] In one exemplary embodiment, a BRET for detecting plasmin in milk 2The lyophilized solid composition of the biosensor system is prepared by filling an aqueous solution (e.g., 200 μL) of the following composition into a suitable assay vessel and lyophilizing the aqueous solution:

[0285] Precursor Composition Assay Composition <![CDATA[Plasmin BRET 2 Biosensor]]> 3,450 ng / ml 690 ng / ml Tris (pH 8.4) Buffer 250 mM 50 mM NaCl 250 mM 50 mM L-Lysine 125 mM 25 mM 6-Aminocaproic Acid - - Trisodium Acetate - - Fish Skin Gelatin * 0.45% (v / v) 0.09% (v / v) Trehalose 12.5 mg / mL 2.5 mg / mL Pullulan 12.5 mg / mL 2.5 mg / mL Porcine Esterase 5 U / mL 1 U / mL

[0286] * The stock solution contains 45% (w / v) (0.45 g / mL) fish skin gelatin.

[0287] In one exemplary embodiment, a BRET for detecting plasminogen in milk 2 The lyophilized solid composition of the biosensor system is prepared by filling an aqueous solution (200 μL) of the following composition into a suitable assay vessel and lyophilizing the aqueous solution:

[0288] * The stock solution contains 45% (w / v) (0.45 g / mL) fish skin gelatin.

[0289] In one exemplary embodiment, a BRET for detecting lactose in milk 2 The lyophilized solid composition of the biosensor system is prepared by filling an aqueous solution (200 μL) of the following composition into a suitable assay vessel and lyophilizing the aqueous solution:

[0290] Range Precursor Composition Assay Composition Lactose Biosensor 25 - 250 nM 25 nM 5 nM Tris (pH 7.5) 0 - 250 mM 50 mM 10 mM NaCl 0 - 250 mM 50 mM 10 mM Fish Skin Gelatin * 0 - 0.45% (v / v) 0.45% (v / v) 0.09% (v / v) Trehalose 0 - 25 mg / mL 0 0 Pullulan 0 - 25 mg / mL 25 mg / mL 5 mg / mL Porcine Esterase 50 U / mL 50 U / mL 10 U / mL

[0291] * The stock solution contains 45% (w / v) (0.45 g / mL) fish skin gelatin.

[0292] In each exemplary embodiment, an assay composition is formed by dispersing the lyophilized composition in the sample to be analyzed (1 mL).

[0293] Substrate storage vessel

[0294] The substrate storage vessel of the system contains a substrate solution that is stable at room temperature. Thus, the substrate solution can be stored until needed and then dispensed into the assay composition in the assay vessel. The substrate storage vessel can be configured to store the substrate solution for at least one week, or at least one month, or at least six months. Thus, the substrate storage vessel is sealable and preferably light - impermeable (or stored in a light - impermeable container), thereby minimizing air and light exposure during storage. The substrate storage vessel can contain a sufficient amount of the room - temperature - stable substrate solution for multiple assays, such as at least ten. Thus, caged substrate compounds can be conveniently provided for periodic in - field diagnostic BRET tests without the need to prepare fresh formulations each time.

[0295] In some embodiments, the substrate storage vessel is configured to dispense an aliquot of the substrate solution into the assay vessel, for example, by dispensing the substrate solution drop - by - drop. Thus, the substrate storage vessel can be equipped with a precision pipette capable of accurately and repeatedly dispensing droplets of a known volume. An example of a suitable substrate storage vessel for this purpose is a Nalgene pipette bottle with a controlled dispensing tip available from Thermo Fisher Scientific. TM As an example, a diluted DMSO solution can be accurately dispensed as 28 μL droplets from such a vessel. Thus, the substrate solution can be formulated such that the amount of caged substrate compound required for each assay is dispensed as one or more droplets. In some embodiments, each droplet of the substrate solution contains from 1 mM to 100 mM, 1 mM to 90 mM, 1 mM to 80 mM, 1 mM to 70 mM, 1 mM to 60 mM, 1 mM to 50 mM, 1 mM to 45 mM, 1 mM to 40 mM, 1 mM to 30 mM, 1 mM to 20 mM, 1 mM to 10 mM, or 1 mM to 5 mM, such as 43 mM, 12 mM, 6 mM, or 4.3 mM. In some embodiments, the concentration of each caged substrate in the substrate solution is about 1 mM to 50 mM, 1 mM to 40 mM, 1 mM to 30 mM, 1 mM to 20 mM, 1 mM to 15 mM, 1 mM to 12 mM, 1 mM to 10 mM, 1 mM to 9 mM, 1 mM to 8 mM, 1 mM to 7 mM, 1 mM to 6 mM, or 1 to 5 mM. In some instances, the concentration of the caged substrate compound is about 43 mM caged substrate compound. In some instances, the concentration of the caged substrate compound is about 12 mM caged substrate compound. In some instances, the concentration of the caged substrate compound is about 6 mM caged substrate compound. In some instances, the concentration of the caged substrate compound is about 4.3 mM caged substrate compound.

[0296] Optoelectronic device

[0297] The optoelectronic device of the system is configured to detect light emitted from the assay composition that passes through the wall of the assay vessel, including emissions associated with an acceptor fluorophore and a donor luminophore. To avoid interfering with the BRET signal, the optoelectronic device is typically configured to exclude all external light when detecting light emitted from the assay composition. In a preferred embodiment, the optoelectronic device is configured to house the assay vessel and incubate the assay composition contained therein for the required incubation period. Thus, the optoelectronic device can be configured to incubate the assay composition at a predetermined incubation temperature or within a narrow incubation temperature range (e.g., ±2 °C). Advantageously, this can allow the incubation and light detection steps of the methods disclosed herein to be performed in a single device and preferably without manual operator intervention after initiation.

[0298] The optoelectronic device of the system includes one or more photodetectors, such as two separate photodetectors, which are tuned to measure wavelength bands associated with a donor luminophore and an acceptor fluorophore. Each photodetector can include a micro-photon multiplier tube (e.g., Hamamatsu H12406 micro PMT photon counting head), which is equipped with a bandpass filter that is adapted to selectively transmit only the light wavelengths associated with the donor luminophore or the acceptor fluorophore. For BRET 2 systems, examples of suitable bandpass interference filters are 10 mm diameter chromaticity 515 nm / 30 nm (green) and 10 mm diameter chromaticity 410 nm / 80 nm (blue) filters. Conveniently, such filters can be replaced when it is desired to sample other wavelengths, for example, if a different BRET system is used.

[0299] Figure 1 and Figure 2 depicts an optoelectronic device according to some embodiments, particularly for use in point-of-care diagnostic testing deployed in production and other non-laboratory environments. As Figure 1 seen, an assay vessel (e.g., a capped 2 ml clear polypropylene sample tube) containing a luminescent assay composition (e.g., 1 ml milk sample + BRET 2 biosensor + caged substrate + uncaging agent) is housed in the optoelectronic device in a sample holder that positions the assay composition between two photodetectors. The assay composition can be incubated for the required incubation time in the device before light is detected. The two photodetectors include a blue bandpass optical filter (measuring emissions associated with the BRET 2 donor luminophore) or a green bandpass optical filter (measuring emissions associated with the BRET 2A microphotomultiplier tube associated with the emission of the acceptor fluorophore. In use, light is transmitted from the assay composition through the wall of the sample tube and detected by two photodetectors. Thus, the emission luminosities associated with the acceptor fluorophore and the donor luminophore are quantified, where the BRET 2 ratio provides an indication of any interaction of the extracellular analyte of interest (e.g., AprX protease) with the BRET 2 biosensor.

[0300] Figure 2 Depicts a complete optoelectronic device including Figure 1 the portions seen in. The optoelectronic device includes a sample holder for receiving the sample tube, a sliding light-tight cover for excluding extraneous light during light detection, a Peltier device (heater / cooler) and associated heat sink, a fan, a temperature monitor and control electronics for controlling the incubation temperature, and associated physical and electronic devices such as a drain tube and an overflow tray (in the event of sample leakage) and an air inlet. The optoelectronic device may include the depicted USB connection or other interfaces for wired or wireless connection to a computing device.

[0301] Software and quantification

[0302] The systems disclosed herein may include a computing device that includes one or more tangible, non-transitory computer-readable media having computer-executable instructions for performing a computer-implemented method to control the operation of the optoelectronic device and process data received therefrom. Typically, the computer-implemented method is automatically executed on a computer processor, which may be provided together with or separately from the optoelectronic device, such as in a desktop computer, a tablet computer, or a smartphone. The computer processor may include software applications installed thereon for performing one or more steps of the computer-implemented method. In alternative embodiments, the software application may be a cloud-based application accessible via a network such as the Internet. In some embodiments, the software application may be remotely accessible via a local network.

[0303] For each specific combination of sample type, extracellular analyte, and BRET biosensor, the optoelectronic device can be calibrated by measuring the BRET results of assays performed with a series of externally defined calibration standards. The calibration curve can then be fit to the calibration data, optionally using a non-linear curve fitting equation. A software application installed on a computer processor, such as GraphPad Prism software, can be used to achieve this. The curve fitting equation and parameters are input into the software and used by the software to calculate the analyte concentration (e.g., ligand concentration) or activity (e.g., protease activity) for each assay of a sample containing an unknown amount of analyte. This result is recorded and presented to the user, who interprets the value based on their actions. In some embodiments, the software is programmed to guide the user in their actions based on any particular result, such as accepting a shipment, retesting a shipment, rejecting a shipment, or seeking further advice.

[0304] The operation of the optoelectronic device can also be controlled by a software application on a computer processor, preferably via a user-friendly graphical interface that allows the user to input details of the sample and operator, assay format (time and temperature), etc. The software can capture and record data from the optoelectronic device during the assay, including, for example, temperature and photon counts in the blue and green channels sampled, for example, every 0.5 seconds during the assay. The software can also calculate and present the BRET ratio in real time.

[0305] Workflow

[0306] The systems disclosed herein can be used to perform methods for detecting extracellular analytes in a sample. Advantageously, the methods can be implemented as a simple workflow suitable for implementation in a production or other non-laboratory environment.

[0307] In some embodiments, the workflow comprises the following steps:

[0308] (a) Collect the sample to be analyzed in a suitable sample collection vessel. In some exemplary embodiments, the sample can be raw or processed milk, for example, collected in a sample cylinder, beaker, or cup from a sampling port of a tanker, buffer tank, silo, or sterile tank or from a finished milk container.

[0309] (b) Select the type of assay to be performed on a computing device connected to the optoelectronic device by entering relevant parameters or selecting a pre-programmed assay. In an exemplary embodiment, the assay type can be an AprX standard sensitivity assay, designed to determine the activity of AprX in milk using a combination of an AprX-sensitive BRET 2 biosensor with one or more caged coelenterazine compounds and one or more uncaging agents, as disclosed herein. Details of the operator and sample can also be entered.

[0310] (c) Dispense a desired volume of the sample into an assay vessel that already contains the biosensor and optional components (e.g., a de-caging agent). Optionally, the sample and the solid composition can be stirred to disperse the solid composition in the liquid sample. In an exemplary embodiment, 1 ml of a milk sample is dispensed into a 2 ml assay tube using an automatic dispenser or a pipette. The assay tube contains freeze-dried solid that includes a desired amount of AprX-sensitive BRET 2 biosensor (e.g., 690 ng), a predetermined amount of porcine liver esterase, and other ingredients as disclosed herein.

[0311] (d) Immediately thereafter (e.g., within one minute), dispense an aliquot of the substrate solution containing one or more caged substrate compounds from a substrate storage vessel into the assay composition and mix well to provide a well-mixed assay composition in which the solid composition is dispersed. In an exemplary embodiment, the assay tube is uncapped and a droplet (28 μL) of the substrate solution containing a 4.3 mM caged coelenterazine compound is dispensed from a dropper bottle equipped with a precision pipette into the assay composition. The assay tube is capped and inverted 10 or 20 times to mix the assay composition.

[0312] (e) Insert the assay vessel into a photoelectronic device and initiate a designated assay on a computing device connected to the photoelectronic device. Thus, the computing device commands the photoelectronic device to incubate the assay composition for a required incubation time at a designated incubation temperature and then detect the light emitted from the assay composition. In an exemplary embodiment, the assay tube is inserted into the Figure 1 and Figure 2 sample holder of the photoelectronic device depicted in, the sliding lid is closed to exclude ambient light, and an AprX standard sensitivity assay is initiated using a software application that controls the device. Thus, the photoelectronic device automatically incubates the assay composition for a required incubation period (e.g., 10 or 15 minutes) at the required incubation temperature (e.g., 37 °C) and detects the BRET ratio at the end of the incubation period.

[0313] (f) Receive the results presented on the computing device after the assay is completed. Typically, the results will be presented as the activity or concentration of the extracellular analyte of interest, obtained by comparing the observed BRET ratio to a pre-established calibration curve.

[0314] In some embodiments, all or part of the workflow can be automated. For example, steps (a), (c), and / or (d) can be performed using a preparation station as described herein. Automation of at least a portion of the workflow is believed to enable highly reproducible results by operators with minimal training and less developed fine motor skills. In some embodiments, the preparation station is configured to extract a defined volume of sample from a suitable sample collection vessel and transfer the sample to an open reagent tube (e.g., a capped 2 ml clear polypropylene sample tube containing a luminescence assay composition (e.g., 1 ml milk sample + BRET 2 biosensor + caged substrate + uncaging agent)). The preparation station can also be configured to facilitate placement of the open reagent tube and allow the reagent tube to move between different (e.g., two or more, or three) positions. In some embodiments, an orbital system (e.g., a linear orbital system) is used to facilitate positioning and movement of the open reagent tube. The preparation station can also be configured to accommodate a removable dropper bottle holder that holds a dropper bottle filled with a substrate solution. In some embodiments, the preparation station includes a cavity (e.g., a cylindrical cavity) capable of accommodating the removable dropper bottle holder. The preparation station can be configured to become airtight once the dropper bottle holder is inserted into the cavity. In some embodiments, the cavity can also be shaped and / or include means for fitting a desiccant pad. In some embodiments, the cavity is positioned to be able to dispense droplets of the substrate solution into the reagent tube. In some embodiments, the cavity can be positioned above a tube located within the orbital system such that droplets of the substrate solution can be dispensed into the reagent tube.

[0315] In an exemplary embodiment, as Figure 29 shown in A, the preparation station includes the following components:

[0316] ● A bottle-top dispenser designed to extract a sample from a sample collection vessel (e.g., a sample cup).

[0317] ● A linear orbital system that facilitates placement of the open reagent tube and allows the tube to move between three different positions.

[0318] ● A cylindrical cavity that houses a removable dropper bottle holder that holds a dropper bottle filled with a substrate solution. The bottle holder can be positioned above a tube located within the orbital system such that droplets of the substrate solution can be dispensed into the reagent tube. Once the dropper bottle holder is inserted, the cavity becomes airtight and includes means for fitting a desiccant pad.

[0319] In some embodiments, the preparation station can also be used to dilute a sample prior to analysis with a biosensor. This can be important, for example, when using a highly sensitive lactose sensor based on BRET to measure the lactose concentration in milk that has not been treated with lactase or is in the early stages of hydrolysis.

[0320] Advantageously, the preparation station provides a simplified assay preparation workflow for minimally trained operators without compromising assay performance. The storage stability of the substrate solution within the preparation station can further enhance the ease of use of the overall system.

[0321] BRET reagent system, solid biosensor composition, substrate dispensing system, and substrate composition

[0322] The present invention further relates to a BRET reagent system. The BRET reagent system comprises two separate formulations that are combined with a sample to produce a bioluminescence assay composition. According to the principles disclosed herein, after an appropriate incubation period, this assay composition has a BRET ratio indicative of an extracellular analyte in the sample. The BRET reagent system comprises (i) a solid composition that is dispersible in a liquid (e.g., a liquid sample) containing the sample to produce the assay composition, and (ii) a room temperature-stable substrate solution for dispensing into the assay composition. The solid composition comprises a biosensor system selected from (i) an extracellular bioluminescence sensor molecule comprising a donor luminophore, an analyte-sensitive domain that readily interacts with the extracellular analyte, and an acceptor fluorophore; and (ii) a precursor system thereof comprising a pair of molecules adapted to react in solution to form the extracellular bioluminescence sensor molecule. The room temperature-stable substrate solution comprises at least one caged substrate compound that is adapted to uncage in the assay composition to produce an uncaged substrate compound that excites the donor luminophore.

[0323] The solid composition can be contained within an assay vessel that is configured to receive the liquid containing the sample and to permit light from the assay composition to transmit through the assay vessel wall for detection. The assay vessel can be of any of the embodiments disclosed herein in the context of a system for detecting an extracellular analyte in a sample.

[0324] The solid composition can be a lyophilized composition prepared by lyophilizing an aqueous precursor composition in an assay vessel, as previously disclosed herein.

[0325] The extracellular bioluminescence sensor molecule and any optional components in the solid composition can be of any embodiment disclosed herein in the context of a method for detecting an extracellular analyte in a sample. According to some embodiments, as previously disclosed herein, the solid composition further comprises a caging agent, such as an esterase, for uncaging the at least one caged substrate compound in the assay composition.

[0326] The room temperature stable substrate solution can be contained in a substrate storage vessel configured to dispense an aliquot of the substrate solution into the assay composition. The substrate storage vessel can be of any embodiment disclosed herein in the context of a system for detecting an extracellular analyte in a sample. In some embodiments, the substrate storage vessel is configured to dispense the substrate solution dropwise into the assay composition.

[0327] The caged substrate compound and the solvent in the room temperature stable substrate solution can be of any embodiment disclosed herein in the context of a method for detecting an extracellular analyte in a sample. In some embodiments, the room temperature stable substrate solution comprises at least two different caged substrate compounds, the at least two different caged substrate compounds being adapted to uncage in the assay composition to produce the uncaged substrate compound at different reaction rates.

[0328] In some embodiments, the BRET reagent system is a kit comprising instructions for using the BRET reagent system in a method for detecting an extracellular analyte in a sample according to any embodiment disclosed herein.

[0329] The present invention further relates to a solid biosensor composition for performing BRET detection of extracellular analytes in a sample. The solid biosensor composition comprises: (a) a biosensor system selected from (i) an extracellular bioluminescent sensor molecule comprising a donor luminophore, an analyte-sensitive domain that is susceptible to interacting with the extracellular analyte, and an acceptor fluorophore; and (ii) a precursor system thereof, the precursor system comprising a pair of molecules adapted to react in solution to form the extracellular bioluminescent sensor molecule; and (b) a caged compound releasing agent. In use, the solid biosensor composition is capable of being dispersed in a liquid containing the sample (e.g., in a liquid sample) to produce an assay composition. According to the principles disclosed herein, the caged compound releasing agent is adapted to uncage at least one caged substrate compound in the assay composition, thereby producing an uncaged substrate compound that excites the donor luminophore. The solid biosensor composition may optionally comprise other components as described herein, such as (i) a buffer, (ii) one or more salts, (iii) one or more metal ions, (iv) one or more carrier proteins, and (v) one or more cryoprotectants.

[0330] It should be understood that, as disclosed herein, the solid biosensor composition is adapted for use in a method for detecting extracellular analytes in a sample. The extracellular bioluminescent sensor molecule, the caged compound releasing agent, and optionally other components can generally be any of the embodiments disclosed herein in the context of this method and other aspects and embodiments of the present invention.

[0331] The present invention further relates to a substrate dispensing system for performing BRET detection of extracellular analytes in a sample. The substrate dispensing system comprises a room-temperature stable substrate solution comprising at least one caged substrate compound. The substrate solution is contained in a substrate storage vessel configured to dispense a predetermined aliquot (e.g., one or more droplets) of the substrate solution into an assay composition comprising a sample and a BRET bioluminescent sensor molecule.

[0332] It should be understood that, as disclosed herein, the substrate dispensing system is adapted for use in a method for detecting extracellular analytes in a sample. The at least one caged substrate compound and the solvent in the room-temperature stable substrate solution can generally be any of the embodiments disclosed herein in the context of this method. In some embodiments, the room-temperature stable substrate solution comprises at least two different caged substrate compounds adapted to uncage at different reaction rates.

[0333] The present invention further relates to a substrate composition for performing BRET detection of extracellular analytes in a sample. The substrate composition comprises at least two different caged substrate compounds, and the at least two different caged substrate compounds are adapted to uncage in an assay composition comprising the sample and a BRET bioluminescence sensor molecule, thereby producing uncaged substrate compounds at different reaction rates. The substrate composition can generally be any embodiment as disclosed herein in the context of the methods and other aspects and embodiments of the present invention.

[0334] It should be understood that, as disclosed herein, the substrate composition is adapted to be used in a method for detecting extracellular analytes in a sample. The caged substrate compounds can generally be any embodiment as disclosed herein in the context of the methods.

[0335] Examples

[0336] The present invention is described with reference to the following examples. It should be understood that the examples are illustrative and do not limit the invention described herein.

[0337] Materials and methods.

[0338] Unless otherwise specified, all chemicals, reagents, and enzymes were obtained from Sigma-Aldrich.

[0339] The caged coelenterazine compounds 7, 8, and 10 were purchased from a custom organic synthesis company (Advanced Molecular Technologies, Australia) according to the previously reported synthetic method (Journal of the American Chemical Society (J Am Chem Soc) 2007 129(39) 11900-11901).

[0340]

[0341] Coelenterazine was obtained from Nanolight Technology, USA.

[0342] As described in the published US Patent Application No. US2019 / 0345535A1 (especially Example 1 and Sequence ID No. 105 of the application; see SEQ ID NO:1 of the present application), the AprX bioluminescence sensor molecule (hereinafter referred to as the AprX sensor) was constructed to generate the pRSET-Pflu expression plasmid. The expression and purification of the AprX sensor are as follows.

[0343] A tube of chemically competent Escherichia coli BL21(DE3) (New England Biolabs) was taken out from -80 °C and placed on ice for 10 minutes. The overexpression plasmid pRSET-Pflu prepared as described above was taken out from the -80 °C refrigerator and placed on ice. 1-2 μl of pRSET-Pflu plasmid was added to the thawed tube of BL21(DE3) competent cells, gently mixed and incubated on ice for 30 minutes. The transformation mixture was heat shocked by placing the tube at 42 °C for exactly 10 seconds. The transformation mixture was placed on ice for 3 minutes, and then 900 μl of sterile LB (Table 1) was added, and the transformation mixture was incubated at 37 °C for 1 hour.

[0344] Table 1. Buffered LB medium

[0345]

[0346] 100 μl and 20 μl aliquots of the reaction mixture were plated on agar supplemented with 100 μg / ml ampicillin and 1% (v / v) glucose and incubated at 37 °C for 24 hours.

[0347] To produce the AprX sensor protein, a transformed Escherichia coli BL21(DE3) colony transformed with pRSET-Pflu was picked into 10 mL of LB medium mixed with 1 ml of 20% (w / v) glucose and supplemented with 100 μg / mL ampicillin and incubated at 37 °C with orbital shaking (200 rpm) for 18 hours. The entire 10 mL overnight culture was added to 500 ml of buffered 2xYT medium (Table 2) supplemented with 100 μg / mL ampicillin in a 3 L sterile flask and incubated at 37 °C with orbital shaking (200 rpm) for 3 hours until the optical density of the culture at 600 nm reached approximately 0.5.

[0348] Table 2. Buffered 2xYT medium

[0349]

[0350] 250 μL of a 1 M isopropyl-β-D-1-thiogalactopyranoside (IPTG, final concentration 500 μM) stock solution was added to the flask, and the incubation temperature was adjusted to 25 °C for 18 - 24 hours. The bacterial cells were pelleted by centrifugation (20 minutes, 4,000 g) and the supernatant was discarded. The cell pellet was resuspended in 50 mL of phosphate buffered saline (PBS; Table 3) per 200 mL of the starting culture.

[0351] Table 3. Phosphate buffered saline (PBS)

[0352]

[0353]

[0354] Centrifuge the cells (20 min, 4,000 g), discard the supernatant, and resuspend the pellet in a total of 100 mL of Buffer HisA (Table 4).

[0355] Table 4. Buffer HisA

[0356]

[0357] Use lysozyme and sonication to lyse the cells. Transfer the resuspended bacterial cells to a 100 ml Schott bottle. Add a spatula load of egg lysozyme with a diameter of 3 mm to the cell suspension and mix well. Add 50 μL of an absolute ethanol solution of 200 mM phenylmethylsulfonyl fluoride (PMSF) to the suspension, mix it well and place it on ice. Place the probe of the Misonix 3000 sonicator in the suspension and sonicate the suspension as follows: (3 × 30 s at 51 W, during which the bottle is rotated ≈120°, followed by standing on ice for 5 minutes), three times. Add another 50 μl of 200 mM PMSF to the lysate and mix well, and centrifuge the lysate at 15,000 g for 20 minutes at 4 °C.

[0358] Purify the AprX sensor protein by metal ion chelation affinity chromatography. Pour the supernatant after lysis and centrifugation into a clean container and place it on ice. Prepare a TALON immobilized metal affinity chromatography column (Takara Bio) according to the manufacturer's instructions and load the clarified, lysed cell supernatant. Almost fill the column with the supernatant, seal it and invert it several times. Remove the top seal and allow the resin to settle by gravity for 10 minutes. Then remove the bottom seal of the column and drain the lysate. Repeat the loading and settling process for the remaining unprocessed supernatant until it is all applied. Wash the column with 20 mL of HisA buffer according to the same procedure for loading. Wash the column once with 3 mL of HisC buffer (Table 5) without disturbing the resin.

[0359] Table 5. Buffer HisC

[0360]

[0361] Elute the AprX sensor from the TALON column with 4 ml of HisB buffer (Table 6) and store it at 4 °C in the dark.

[0362] Table 6. Buffer HisB

[0363]

[0364] The purified AprX sensor protein was diluted to 100 nM in AprX storage buffer (Table 7), aliquoted into 1 mL portions, snap-frozen in liquid nitrogen and stored at -80 °C.

[0365] Table 7. AprX storage buffer

[0366]

[0367] UHT reference milk (Devondale full cream UHT milk, best before 10 December 2021, purchased from IGA supermarket in O'Connor; or Devondale full cream UHT milk, best before 17 September 2022, purchased from the ANU campus ACT general store) was frozen in aliquots and stored at -80 °C for up to 12 months before use.

[0368] Esterase from porcine liver was obtained from Sigma-Aldrich (Catalog No. E3019-20KU).

[0369] Alkaline protease (AprX) from Pseudomonas fluorescens was prepared as follows. An isolate of Pseudomonas fluorescens (strain 65) [P.D. Button, H. Roginski, H.C. Deeth, H.M. Craven, Improved shelf-life estimation UHT milk by prediction of proteolysis, Journal of Food Quality, 34 (2011) 229-35.] was obtained from the Dairy Culture Collection at the CSIRO Food Innovation Centre in Werribee and spread on an OXOID agar plate and incubated at 28 °C for 2 days. Three colonies from the plate were inoculated into 100 mL of sterile OXOID nutrient broth (NB) and incubated at 28 °C for 48 h. 10 mL of the culture was centrifuged at 6,000 g for 10 min. The supernatant was discarded and the pellet was resuspended in 1 mL of sterile isotonic saline solution (0.85% w / v) and added to 99 mL of skim UHT milk (skim milk from COLES Australian, 99% fat-free). The culture was incubated at 4 °C for 8 days with orbital stirring at 150 rpm. Thereafter, the culture was centrifuged at 27,000 g for 20 min at 4 °C. The supernatant was collected, filter-sterilized through a 0.22 μm filter and used as a source of AprX protease. 1 mL aliquots of the protease preparation were snap-frozen in liquid nitrogen and stored at -80 °C.

[0370] The activity of alkaline protease (AprX) was standardized based on measurements performed using the azocasein method as follows. Azocasein (A2765) was purchased from Sigma-Aldrich (lot number SLBR0712V, specific absorbance E1% at 440 nM was 35). A 3% (w / v) working stock solution was prepared by gradually dissolving 90 mg of azocasein in 750 μL of 0.1 M NaOH, followed by the addition of 750 μL of H2O and finally 1500 μL of 50 mM Na2HPO3 (pH 7.5). AprX serial dilutions were prepared in 400 μL of 50 mM Na2HPO3 (pH 7.5) in 1.5 ml Eppendorf tubes and mixed with 100 μl of 3% (w / v) azocasein solution. The tubes were incubated at 37 °C for 15 minutes to 24 hours. The reaction was terminated by adding 500 μL of 20% (v / v) trichloroacetic acid, followed by centrifugation at 14,000 rpm for 3 minutes to precipitate undigested azocasein. 500 μL of the supernatant was carefully removed and neutralized with 750 μl of 1 M NaOH in a microcuvette. The absorbance at 440 nm was measured using a SpectraMax Plus spectrophotometer (Molecular Devices, United States). Using the molecular weight and extinction coefficient of the azocasein batch and the incubation time, the average rate of change of Abs440nm at each incubation time was converted to enzyme activity. It was expressed as the number of micromoles of azocasein reacted per mL of enzyme solution per minute (units per mL).

[0371] The luminescence and BRET 2 ratio measurements in the plate reader were performed according to the following basic procedure, with modifications as described in the examples.

[0372] 50 μL of UHT milk standard was mixed with 29 μL of AprX buffer B3 (Table 8), 10 μL of standardized AprX protease of known activity, and 10 μL of 100 nM AprX biosensor protein in the wells of a 96-well plate.

[0373] Table 8. AprX buffer B3

[0374]

[0375] The plate was incubated in a preheated Inkubator 1000 (Heidolph Instruments, Germany) at a nominal temperature of 50 °C for 10 minutes. The plate was transferred to a CLARIOstar (BMG Labtech) plate reader, which was set to 30 °C with the following additional settings:

[0376]

[0377]

[0378] Immediately add 1 μL of substrate, such as CLZ400a (500 μM in dry ethanol) or a caged substrate (6 mM in dry DMSO solution). In the case of the caged substrate, also add 1 μL of an aqueous solution of porcine esterase. Mix the contents of the well by pipetting and start the reading protocol.

[0379] Human plasmin activity was normalized based on measurements performed using the following protocol with Chromozyme PL. Chromozyme PL is a chromogenic substrate that contains the proteolytic recognition site for plasmin. By measuring the absorbance at 410 nm, the colorimetric change due to proteolytic release of the plasmin-derived dye from the Chromozyme PL substrate can be detected. Human plasmin was purchased from Merck-Sigma-Aldrich (P1867), reconstituted in 50 mM Na2HPO4 (pH 7.0), filter sterilized and aliquoted into sterile microcentrifuge tubes. All aliquots were snap frozen in liquid nitrogen and stored at -80 °C. Chromozyme PL was purchased from Merck-Sigma-Aldrich (10378461001). Assays were prepared in a total volume of 100 μL in a clear 96-well plate containing serial dilutions of plasmin (in 50 mM Na2HPO3, pH 7.0). Chromozyme PL was added to a final concentration of 0.315 mM and the plate was incubated in a CLARIOstar plate reader set at 37 °C. The absorbance at 410 nm was measured every 2 minutes for 30 minutes. A control reaction without spiked plasmin was added in each set of tests. The difference in absorbance between 0 and 4 minutes (ΔA / min) was determined and used to calculate the plasmin activity in unit form, defined as

[0380] 1 U = ΔA / min × total assay volume / (sample volume × path length × extinction coefficient),

[0381] where the path length is 0.28 cm and the extinction coefficient is 9.7.

[0382] Example 1. Storage Stability of Caged Deoxycoelenterazine Compounds in Solution

[0383] For use in a reliable on-site BRET 2 test protocol, it was determined that the substrate compound solution should support an emission amplitude of not less than 10% of its original value when stored at 22 °C, while maintaining BRET 2The ratio is within ±5% of its original value. Here, the emission amplitude is based on the measurement of the emission of green fluorescent protein 2 (GFP 2 ), and the BRET ratio is calculated as green fluorescent protein 2 (green emission) ÷ Renilla luciferase 8 emission (RLuc8, blue emission). The substrate compound should be storable at room temperature for several weeks and preferably for a longer period, such as up to one year.

[0384] Stability tests of a series of substrates of Renilla luciferase were as follows. Coelenterazine (500 μM) (95%) was prepared in absolute ethanol, and coelenterazine-HCl (CLZ400a-HCl) (500 μM) was prepared in anhydrous DMSO. Separate solutions (each 6 mM) of compounds 7, 8, and 9 were prepared in dry dimethyl sulfoxide (DMSO). For the aging test, including accelerated aging under elevated temperature conditions, the substrate solutions were dried and stored in o-ring sealed 2 mL tubes (QSP Natural screw cap microtubes 520-GRD, Thermo Fisher Scientific) in re-closable plastic bags containing 5 g TM absorbent packs (Sigma-Aldrich, Z163635) in the dark and tested at different times using an AprX sensor in a BRET 2 assay.

[0385] To evaluate the storage stability of the substrates, the basic AprX sensor assay protocol (described above) was modified as follows to measure the brightness and BRET 2 ratio of the caged substrate or uncaged substrate after storage at different temperatures for different times. For CLZ400a itself, the standardized AprX protease was omitted from the reaction mixture and replaced with an additional 10 μL of AprX buffer B3. Before adding CLZ400a or CLZ400a-HCl, the assay mixture was incubated at 50 °C for 10 minutes and then measured immediately.

[0386] To evaluate the caged substrates, the following modifications were made to the procedure. For compound 7, AprX protease was replaced with 10 μL of AprX buffer B3. Compound 7 was added in 1 μL of anhydrous DMSO to a final concentration of 60 μM. The assay was then incubated at 50 °C for ten minutes. For compound 8 and 10 or their mixtures, the same procedure as for compound 7 was used, except that the AprX protease addition was replaced with 9 μL of AprX buffer B3. The caged substrate was added to 1 μL of anhydrous DMSO to a final total concentration of 60 μM, and then 1 μl of aqueous porcine esterase (2 U / μL) was immediately added to a final concentration of 20 U / mL. Then, the assay was incubated at 50 °C for ten minutes before reading the data on a Clariostar.

[0387] Figures 3 - 6 Results for uncaged coelenterazine (CLZ400a or CLZ400a-HCl) are shown. Coelenterazine produced the most intense light among all the substrates tested. However, at all temperatures, within 55 days, the coelenterazine solution in ethanol ( Figure 3 ) and the coelenterazine-HCl solution in DMSO ( Figure 5 ) lost more than 90% of their ability to excite the AprX biosensor within 31 days. Thus, uncaged coelenterazine does not meet the brightness stability requirements at any temperature, as a 90% loss in brightness results in an unstable BRET ratio and, due to the random variation in photon counting at low brightness, the coefficient of variation of the ratio is unacceptably high.

[0388] Figure 7 and Figure 8 Results for caged coelenterazine compound 7 are shown. Compound 7 is more stable in solution than uncaged coelenterazine and still meets the brightness and BRET ratio criteria when stored at 4 °C for up to 132 days and at 22 °C for up to 80 days. However, the loss of both brightness and BRET ratio experienced in the high-temperature samples indicates that the solution of compound 7 will no longer meet the acceptable criteria after being stored at temperatures above 4 °C for several months. However, if refrigerated for up to 132 days, compound 7 has potential utility in supporting rapid uncaging.

[0389] Figure 9 and Figure 10 Results for caged coelenterazine compound 8 are shown. Compound 8 was stable at 4 °C for up to 203 days, after which its activity decreased to approximately 13% of its initial capacity after 315 days. At all temperatures above 4 °C, the solutions of compound 8 were stable for approximately two weeks, but after 40 days of storage, they did not meet the requirements for both brightness and BRET ratio, indicating that they can be used for the timed release of uncaged substrates for up to one month.

[0390] Figure 11 and Figure 12 shows the results for the caged coelenterazine compound 10. Compound 10 maintains excellent stability in solution, meeting the brightness and BRET ratio criteria, maintaining for up to 300 days at 4 °C and at least 436 days at 22 °C, 37 °C or 45 °C.

[0391] The stability of DMSO solutions containing 1:2 mixtures of caged compounds 8 and 10 (6 mM and 12 mM, respectively) was also investigated ( Figure 13 and 14 ). It was found that in initial experiments, the 1:2 mixture provided a stable BRET ratio and a good delayed bioluminescence signal onset for 10-minute assays (data not shown). However, different mixtures of caged substrates can be used to achieve optimal timed light production for assays of different durations. After an apparent initial change within 14 days, the brightness and BRET ratio of the reactions excited by compounds 8 and 10 were stable at all temperatures (4 °C, 22 °C, 37 °C and 45 °C) after storing the solution for up to 315 days.

[0392] The results indicate that caged coelenterazine compounds (but not uncaged coelenterazine) can be formulated to meet the requirements of ready-to-use, medium to long shelf-life substrate solutions for use in on-site BRET 2 assays for detecting analytes in liquid samples.

[0393] Example 2. BRET 2 Timed activation of biosensors

[0394] The sensitivity of compounds 7, 8 and 10 to the uncaging reaction was then investigated by BRET 2 assays ( Figure 15 and 16 ). In the absence of esterase, compound 7 was consumed by the BRET 2 sensor in buffer, with a low and sustained bioluminescence signal. In UHT milk, compound 7 was activated without the addition of esterase, exhibiting a delayed flash-type bioluminescence. Compound 8 was also activated in milk, but to a lesser extent, while compound 10 was not activated in milk. In the absence of esterase, neither compound 8 nor 10 could be consumed by the BRET 2 sensor in buffer. In milk and buffer, both compounds 8 and 10 exhibited low but sustained bioluminescence signals upon addition of porcine liver esterase, with a longer activation delay compared to compound 7.

[0395] Preliminary experiments have shown that combining one to three caged substrate components with an esterase enables different timed activation windows, in which, in the presence of the AprX sensor, BRET is achieved at different times after mixing all components and depending on the incubation temperature. 2 Peak intensity. At 30 °C, the bioluminescence of 30 different assay compositions was tested over time (0 min to 20 min) on a CLARIOstar (Table 9). The assay compositions were as follows:

[0396] ● 50 μl UHT milk (r00014)

[0397] ● 0 - 4 μl porcine esterase

[0398] ● 10 μl AprX sensor (100 nM stock solution)

[0399] ● 33.5 - 37.5 μl buffer b0002

[0400] ● 2.5 μl substrate mixture (solution in DMSO).

[0401] The substrate mixture was added last, and then 63 cycles of BRET signal channels were run immediately within 20 minutes. The amounts of caged substrate compounds and exogenous uncaging agents in each assay composition are shown in Table 9. 2 The signal intensity based on the measurement of the emission of green fluorescent protein 2 (GFP2) and the BRET ratio, calculated as GFP (green emission) ÷ Renilla luciferase 2 emission (RLuc2, blue emission), are shown in

[0402] Table 9.

[0403] Assay Code Compound 7 [μM] Compound 8 [μM] Compound 10 [μM] Porcine Esterase (Units / 100 μl) 1 60 60 60 4 2 30 60 60 4 3 10 60 60 4 4 5 60 60 4 5 1 60 60 4 6 60 60 60 2 7 30 60 60 2 8 10 60 60 2 9 5 60 60 2 10 1 60 60 2 11 60 60 60 1 12 30 60 60 1 13 10 60 60 1 14 5 60 60 1 15 1 60 60 1 16 60 0 0 4 17 30 0 0 4 18 10 0 0 4 19 5 0 0 4 20 1 0 0 4 21 0 60 60 1 22 0 60 60 2 23 0 60 60 4 24 0 60 60 8 25 0 60 60 0 26 0 120 120 1 27 0 120 120 2 28 0 120 120 4 29 0 120 120 8 30 0 120 120 0

[0404] The signal intensity based on the measurement of the emission of green fluorescent protein 2 (GFP2) and the BRET ratio, calculated as GFP (green emission) ÷ Renilla luciferase 2 emission (RLuc2, blue emission), are shown in 2 (green emission) ÷ Renilla luciferase 2 emission (RLuc2, blue emission), respectively, in Figure 17 and 18 Three formulations (assay codes 20, 25, and 30) did not exhibit a signal peak intensity >50,000 and were discarded for subsequent analysis.

[0405] It was observed that the BRET ratio varied over time according to the formulation. However, the BRET ratio of each formulation was generally stable (i.e., low coefficient of variation) near its 80 - 100% signal peak. To highlight the assay times at which the formulations exhibited 80 - 100% of their signal peaks, Figure 19 only those normalized signals are shown. The normalized signal intensities clearly illustrate how different formulations result in different substrate activation windows ranging from 1 minute to 20 minutes.

[0406] Then, Figure 17and 18 is combined in Figure 20 to show the time at which the peak signal window (80 - 100% relative signal intensity) of each formulation appears and the peak intensity (normalized to the formulation with the highest signal peak intensity: 60 μM C - 7, 4U esterase (assay code 16)). The peak signal intensity time window follows a trend where lower signal intensities are associated with later and longer - lasting bioluminescence production.

[0407] The BRET ratio stability is analyzed by calculating the standard deviation of the BRET ratio for each formulation within its 80 - 100% signal peak window, and the results are shown in Figure 21 The standard deviation of the BRET ratio ranges from approximately 0.01 to 0.035. Most of the standard deviations are below 0.02, which is satisfactory for the BRET 2 assay sensitivity.

[0408] Therefore, for any given BRET 2 assay with a known or preferred analysis time (e.g., for the optimal interaction between a biosensor and an analyte), the results of this experiment can be used to select a suitable caged substrate formulation, i.e., one in which (1) the analysis time falls within the 80 - 100% signal peak window and (2) preferably, the signal intensity is also as high as possible.

[0409] Example 3. Storage - stable lyophilized biosensor formulations

[0410] It was found that the AprX biosensor is stable in solution at 4 °C in the presence of the antimicrobial agent thymol. However, when the solution of the AprX biosensor is stored at room temperature (22 °C), even in the presence of thymol, the signal intensity and the BRET 2 ratio steadily decline within 80 days. Therefore, storing the biosensor in solution at ambient temperature for on - site assay methods is considered infeasible. Thus, a study was conducted to determine whether the AprX biosensor and other components (including buffer, cryoprotectant, and uncaging esterase) could be lyophilized for long - term storage.

[0411] 200 μL of an aqueous solution containing the following components (referred to herein as FD - B4) was loaded into the required number of QSP TM natural screw - cap microtubes 520 - GRD (Thermo Fisher Scientific):

[0412] ● 250 mM Tris (pH 8.4)

[0413] ● 250 mM NaCl

[0414] ● 25 mM MgCl₂·6H₂O

[0415] ● 25 mM CaCl₂·2H₂O

[0416] ● 0.45% (v / v) fish skin gelatin (*stock solution contains 45% (w / v) (0.45 g / mL) fish skin gelatin)

[0417] ● 12.5 mg / ml trehalose

[0418] ● 12.5 mg / ml pullulan

[0419] ● 5 U / ml porcine esterase

[0420] ● 3,450 ng / ml AprX biosensor

[0421] Keep the uncapped assay tubes in a -80 °C freezer for at least 2 hours (although this initial step of rapid freezing can be omitted due to the gelatin / trehalose / pullulan components). Turn on and preheat the α1-2LDplus freeze dryer and the connected oil vacuum pump, and precool the backplate of the freeze dryer to -80 °C. Load the backplate and sample tubes into the freeze dryer and run it for 20 hours, then switch the freeze dryer to standby mode, slowly release the vacuum, and remove the sample tubes, cap them and store them in the dark in a heat-sealed plastic bag containing 5 g of absorbent packets.

[0422] Many other freeze-drying protocols using other equipment have proven successful. These include commercial protocols where the mixture is cooled to ≈0 °C before freeze-drying, but without pre-freezing.

[0423] Then subject the freeze-dried composition to accelerated aging testing by storing it in sealed assay tubes in the dark at 4 °C, 22 °C, 37 °C, and 45 °C for 9 months. The appearance of the samples stored at 4 °C did not change, and there were no signs of glass transition to rubber. The samples stored at temperatures above 4 °C turned yellow after 6 months (22 °C), 3 months (37 °C), and 2 months (45 °C).

[0424] Then evaluate the performance of the aged freeze-dried biosensor composition in a BRET 2 assay on a photoelectronic device. Reconstitute the freeze-dried assay tubes with 1 ml of raw milk supplemented with 28 μL of 4.3 mM compound 10 (in anhydrous DMSO), mix and insert into the photoelectronic device. Set the device to 37 °C and monitor the signal ( Figure 22 and 23 ) over 15 minutes.

[0425] The signal was monitored on the α device from 0 - 118 days and then analyzed on the β device (133 - 270 days). Due to hardware changes, the signal output between the two devices changed, so a correction factor of 0.19 for the signal intensity value and a correction factor of 1.39 for the BRET ratio for days 133 - 270 were applied to the data.

[0426] The lyophilized biosensor showed some change in overall intensity at 4 °C over more than 9 months, but the BRET 2 ratio did not decline, while for samples stored at 22 °C, the signal remained stable for up to 6 months. The lyophilized biosensor was stable at 37 °C for 2 months and at 45 °C for 1 month. The BRET ratio stability was related to the signal intensity and remained stable until the signal intensity decreased.

[0427] Example 4. Detection of AprX activity in milk samples using storage - resistant biosensor reagents

[0428] Examples 1 - 3 demonstrated a route to produce storage - resistant assay reagents that could be used for analysis (e.g., analysis of milk) within a 2 - 20 - minute incubation time. Next, it was tested whether these assay reagents could be applied to various milk samples on a photoelectronic device as described herein Figure 1 and Figure 2 and

[0429] A lyophilized AprX biosensor formulation was prepared in assay tubes using the same storage - resistant formulation (5 U / ml porcine esterase) and lyophilization procedure as determined in Example 3, except that pullulan was not added (pullulan has no effect on the assay other than stabilizing the reagent for long - term storage).

[0430] The lyophilized formulation was reconstituted with 1 mL of raw milk (providing 1 U / ml porcine esterase in the reconstituted formulation), supplemented with serial dilutions of standardized AprX protease, and then 28 μL of compound 10 solution (4.3 mM in anhydrous DMSO) was added to provide a assay concentration of 120 μM compound 10. The tube was capped and inverted 20 times and inserted into the photoelectronic device.

[0431] An initial assay (without adding AprX protease) was performed using the photoelectronic device incubated at 37 °C, and the BRET was recorded within 900 seconds (15 minutes). 2Bioluminescence signal. Measurements performed in raw milk showed a strong signal peak at approximately 250 seconds, followed by a slow decay of the signal, while fresh milk or UHT milk produced a continuous low signal( Figure 24 ).

[0432] An increase in bioluminescence in raw milk was also observed in raw milk samples heated at 95 °C for 5 minutes compared to fresh milk and UHT milk, but not in raw milk samples sonicated (QSonica, USA) for 1 minute at an amplitude of 40( Figure 25 ). Sonication homogenized the raw milk samples. Without wishing to be bound by any theory, it is believed that the caged substrate 10 is rapidly absorbed into the minute fat globules present in homogenized milk, reducing the uncaging rate. Raw milk may also contain factors that are heat-stable but susceptible to sonication / homogenization, which affect the bioluminescence production of the biosensor with the caged substrate.

[0433] Adjusting the esterase concentration from 1 U / mL to 5 U / mL in the final assay resolved the low signal generation problem in UHT milk( Figure 26 ). This indicates that different milk sample types require different formulations.

[0434] Different from the observations obtained earlier using a plate reader, the optoelectronic device exhibited a more stable BRET ratio after reaching the 100% peak luminescence window( Figure 27 ). The BRET ratio stabilized after 200 seconds and remained stable for at least 15 minutes. On this device, endpoint measurements could be reliably measured outside the 80 - 100% peak luminescence window.

[0435] Example 5. Calibration curve of AprX activity in raw milk samples using a storage-stable biosensor reagent.

[0436] Using the BRET 2 protocol developed in Example 4, raw milk samples spiked with known amounts of standardized AprX protease were tested using a storage-stable reagent containing 1 U / mL porcine esterase. Additionally, a reagent containing 5 U / mL porcine esterase was tested with UHT milk samples spiked with AprX. Bioluminescence values at 590 - 600 seconds were used to determine the BRET ratio for the 10-minute assay, and the bioluminescence signal values recorded from 890 - 900 seconds were used to determine the BRET ratio for the 15-minute assay. The observed BRET ratio decreased with an increase in the amount of AprX activity in the raw milk samples. The calibration plots for the 10-minute and 15-minute assays matched a non-linear calibration curve( Figure 28 and 29) It was found that the AprX activity detection depends on the incubation time, and the 15-minute assay is more sensitive than the 10-minute assay. For raw milk, the 10-minute assay achieved a limit of detection (LOD) of 83 μU / mL and an effective concentration at half-maximum (EC 50 ) of 1.82 mU / mL, while the 15-minute assay achieved an LOD of 67 μU / mL and an EC 50 of 1016 μU / mL. For UHT milk, the 10-minute assay achieved an LOD of 232 μU / mL and an EC 50 of 1.65 mU / mL, while the 15-minute assay achieved an LOD of 184 μU / mL and an EC 50 of 1084 μU / mL.

[0437] Before adding compound 10 ( Figure 26 ), the detection sensitivity can be further improved (referred to as the "high-sensitivity protocol") by incubating the reconstituted freeze-dried biosensor with the spiked raw milk sample for 1 hour on a heating block (Delvo MiniS incubator) set at 34 °C. Then the sample is analyzed on the optoelectronic device for 15 minutes, and the BRET ratio value of the high-sensitivity protocol is determined using the bioluminescence signal values recorded from 890 - 900 seconds. The LOD of the high-sensitivity protocol is 17 μU / mL, and the EC 50 is 148 μU / mL.

[0438] Example 6. Using a preparation station to prepare milk analysis samples

[0439] Next, an easy-to-use workflow was developed to prepare samples for the optoelectronic device used in the methods of Examples 3 - 5. The workflow includes a single pipetting step to add 1 mL of a liquid sample such as milk to a 2 mL screw-cap tube containing a freeze-dried biosensor system, followed by applying one drop of a solution containing the caged substrate from a dropper bottle to initiate the bioluminescence reaction.

[0440] To enable operators with minimal training and less-developed fine motor skills to produce highly reproducible results, a preparation station was created. This station was designed to eliminate the final pipetting step and improve user-friendliness, thereby simplifying the process of analyzing samples on the optoelectronic device.

[0441] As Figure 30 shown in A, the preparation station comprises the following components:

[0442] ● A bottle-top dispenser designed to extract milk samples from a cup.

[0443] ● A linear rail system that facilitates the placement of open reagent tubes and allows the tubes to move between three different positions.

[0444] ● A cylindrical cavity that houses a removable dropper bottle holder which in turn houses a dropper bottle filled with a substrate solution Figure 30 B). The bottle holder can be positioned above a tube within an orbital system such that droplets of the substrate solution can be dispensed into a reagent tube. Once the dropper bottle holder is inserted, the cavity becomes airtight and includes means for fitting a desiccant pad.

[0445] Next, the performance of specific components of the preparation stations and workflows described in Examples 3 - 5 was compared.

[0446] First, the performance of the dropper bottle holder was evaluated by quantifying the volume dispensed from a dropper bottle containing 2 mL of DMSO (where 1 mg equals 1 μL). This evaluation was performed 20 times, including both manually and operating within the dropper bottle holder Figure 30 C). The dropper bottle holder consistently dispensed 28.9 ± 0.7 μL of DMSO, while manually operating the dropper bottle resulted in a slightly lower dispensed volume of 27.8 ± 0.9 μL.

[0447] Similarly, the performance of the bottle - mouth dispenser was compared with that of a fixed - volume 1 - mL pipette for sucking milk from a sample cup Figure 30 D). The fixed - volume pipette dispensed slightly less milk, with a measured value of 975.4 ± 6.8 μL, compared to the bottle - mouth dispenser which consistently dispensed 1027.7 ± 2.6 μL.

[0448] Notably, two elements of the sample preparation station, namely the bottle - mouth dispenser and the dropper bottle holder, showed better reproducibility in dispensing performance compared to their manually - operated counterparts Figure 30 E).

[0449] Then, as described in Example 5, the assay performance (signal intensity and BRET ratio) of analyzing UHT whole - milk samples with FD - B4 (1 U / assay esterase, 5 U / mL esterase before freeze - drying) was compared. Samples were prepared using the preparation station or via a manual workflow (manual operation of a 1 - mL fixed - volume pipette and a dropper bottle) Figure 30 F and Figure 30 G). When using the preparation station, the signal intensity was slightly lower than the manual workflow Figure 30 F). This may be due to the higher dispensed milk volume affecting the amount of the attenuated bioluminescence signal. Importantly, no difference in the BRET ratio was observed between the two sample preparation methods Figure 30 G).

[0450] The dropper bottle holder enables the user to store the dropper bottle in a light - and moisture - free location. It was tested whether the substrate (compound 10, 4.3 mM, in anhydrous DMSO) remained stable within the holder cavity at room temperature. This was tested using a combination of the FD - B4 (5 U / assay esterase; 25 U / mL esterase before lyophilization) reagent and raw milk samples with droplets from the dropper bottle stored in the preparation station (as described in Example 5). Over 16 weeks, no significant decrease in signal intensity ( Figure 30 H) or change in BRET ratio ( Figure 30 I) was observed.

[0451] The preparation station provides a simplified assay preparation workflow for minimally trained operators without compromising assay performance. The storage stability of the substrate solution within the preparation station further enhances the ease of use of the entire system.

[0452] Before analysis with the biosensor, the preparation station can also be conveniently used to dilute samples, for example when using a highly sensitive BRET - based lactose sensor to measure the lactose concentration in milk that has not been treated with lactase or is in the early stages of hydrolysis.

[0453] Example 7. Formulation and operation of lactose detection tests on optoelectronic devices

[0454] Next, it was tested whether the optoelectronic device described herein was compatible with a storage - stable lactose assay. This example describes the development of this assay method and the development of a storage - stable two - component reagent system.

[0455] BRET has been previously described 2 lactose sensor molecules (see Australian Patent 2018315053; Caron, K. and S. C. Trowell (2018). “Highly Sensitive and Selective Biosensor for a Disaccharide Based on an AraC - Like Transcriptional Regulator Transduced with Bioluminescence Resonance Energy Transfer”. Analytical Chemistry 90(21):12986 - 12993; also see SEQ ID NO:2 herein). BRET has also been described 2Use of lactose sensor molecules in non-storage assays (Briggs, L. et al., (2023). “On-the-spot trace lactose test for flavored and unflavored lactose-free dairy beverages”. Talanta Open 8). The lactose biosensor comprises a native or modified lactose-binding domain from the BgaR transcription factor, sandwiched between RLuc2 and GFP 2 Therein. Binding of lactose causes a conformational change in the lactose-binding domain, which in turn alters the distance and / or orientation between the RLuc2 and GFP 2 components, thereby changing the energy transfer efficiency between RLuc2 and GFP 2 Therein. The sensor can be used to quantify the lactose content in dairy products.

[0456] In unpublished experiments, it has been observed that at 4 °C, the dynamic range of the change in the sensor BRET ratio for lactose binding (determined by comparing the ratio in lactose-free milk and milk supplemented with 5% (w / v) lactose-free lactose) decreases over time. It is hypothesized that the sensor dimerizes over time due to a dimerization domain located within the lactose-binding domain. Based on the dimerization surface deduced from the crystal structure (Newman, J. et al., (2019). “Structures of the transcriptional regulator BgaR, a lactose sensor”. Acta Crystallogr D Struct Biol 75(Pt7): 639-646), several variants containing mutations within the dimerization domain were constructed, and three were selected for further study (LacB6[GFP 2 -BgaR(D140A / I141A)-RLuc8], SEQ ID NO:3, LacB13[GFP 2 -BgaR(N12S / D140S / I141A)-RLuc8], SEQ ID NO:4 and LacB16[GFP 2 -BgaR(D140S / I141A / L142A / L144A)-RLuc8], SEQ ID NO:5). Wild-type and variant lactose sensors were expressed and purified as previously described.

[0457] The dynamic range of the BRET ratio of the 'wild-type' lactose biosensor and the three variants over time was measured using an improved plate reader assay. Briefly, 89 μL AprX buffer B3 (Table 8) was mixed with 10 μL 100 nM lactose biosensor protein in a 96-well plate. The plate was incubated for 5 minutes at 30 ° C in a preheated Inkubator 1000 (Heidolph Instruments, Germany). The plate was transferred to a CLARIOstar (BMG Labtech) plate reader set to 30 ° C, the settings of which are described in the method section. 1 μL CLZ400a substrate (500 μM in anhydrous ethanol) was added, mixed by pipetting, and the BRET ratio was measured.

[0458] The dynamic range of the wild-type, LacB6, and LacB16 biosensor variants varied over time. In contrast, the LacB13 biosensor had a stable dynamic range for 7 days, after which the dynamic range decreased ( Figure 31 A). Without wishing to be bound by theory, this relatively better stability may be due to a lower dimerization rate. It is believed that the improved stability allows for more reproducible measurements in lactose sensing assays performed on optoelectronic devices.

[0459] Using the protocol described above, a calibration curve was prepared by adding serial dilutions of lactose to PBS buffer ( Figure 31 B). The wild-type sensor exhibited the highest lactose detection sensitivity (LOD: 0.0000527%, EC50: 0.00032), followed by LacB13 (LOD: 0.00063%, EC50: 0.0074%), and finally LacB6 and LacB16 (LOD: 0.0053%, EC50 0.0202% and LOD: 0.0027%, EC50: 0.0208%). Previous reports (Briggs et al., 2023; Caron and Trowell, 2018) showed that the wild-type sensing ability was saturated in assays containing 50% (v / v) milk due to the presence of trace levels of lactose and interference from high levels of glucose and galactose. The reduction in LacB13 sensitivity is beneficial for lactose assays because it reduces the milk dilution step and simplifies the assay workflow. The LacB13 biosensor was used for the remainder of the assay development.

[0460] As described in Example 3, LacB13 biosensors were freeze-dried using the following formulation:

[0461] 250 mM Tris (pH 7.5)

[0462] 250 mM NaCl

[0463] ● 12.5 mg / mL pullulan

[0464] ● 12.5 mg / mL trehalose

[0465] ● 0.45% (w / v) fish skin gelatin (*stock solution contains 45% (w / v) (0.45 g / mL) fish skin gelatin.)

[0466] ● 250 nM LacB13 biosensor

[0467] ● 50 U / mL porcine esterase

[0468] The formulation includes 50 U / mL porcine esterase to achieve faster activation of Compound-10, thereby enabling shortening of the assay time. The lyophilized reagent was reconstituted in 1 mL of 10% lactose-free milk (Aldi Lidls), diluted in water, supplemented with one drop of C-10 (28 μL, 4.3 mM in anhydrous DMSO), and analyzed on a photoelectronic device for 4 minutes. The device was set at 37 °C.

[0469] The photoelectronic device detected the bioluminescence generated by the sensor molecule ( Figure 31 C). An increase in the BRET ratio was observed within the first 60 seconds, and the ratio decreased within the subsequent 3 minutes ( Figure 31 D). To determine the conditions for optimal BRET ratio stability within the 4-minute assay time range, a series of different lyophilized formulations (Table 10) were tested, where the concentrations of NaCl, pullulan, trehalose, and the biosensor were different.

[0470] Table 10. Lyophilized formulations of lactose biosensor

[0471]

[0472] *stock solution contains 45% (w / v) (0.45 g / mL) fish skin gelatin.

[0473] All tested formulations produced functional lactose sensors, which can be used to analyze milk samples on a photoelectronic device. For any tested formulation, the BRET ratio did not stabilize within 4 minutes ( Figure 31 E). Such stability would be beneficial but is not essential for assay utility. When analyzing lactose-free milk samples supplemented with 5% (w / v) lactose, the BRET ratio of the trehalose-containing formulation increased by up to 20%, while the ratio of the trehalose-free formulation decreased by up to 20% ( Figure 31F). The disaccharide trehalose was added as a cryoprotectant. However, it is known to bind to the biosensor (Caron and Trowell, 2018), although much less strongly than lactose. Thus, it is suspected that trehalose interferes with the lactose-sensing ability of the sensor as the BRET ratio decreases in the presence of lactose. The highest dynamic range was seen in formulation FD-L3-13 without any trehalose ( Figure 31 F). While all formulations were able to distinguish lactose-free and lactose-containing milk, comparison of end-point measurements at 1 minute, 2 minutes, 3 minutes, or 4 minutes showed that only FD-L3-13 exhibited a stable difference in BRET ratio.

[0474] Next, formulation FD-L3-13 was used in further experiments where serial dilutions of lactose were added to lactase-treated milk samples (Devondale full-fat milk was treated with 5000 NLU / L lactase at 4 °C for 6 days. 1 mL aliquots were incubated at 95 °C for 10 minutes to inactivate the lactase and the aliquots were stored at -80 °C). The lactose-sensing assay using the lyophilized reagents was able to distinguish lactose-free milk and lactose-free milk supplemented with 5% (w / v) lactose ( Figure 31 G), and showed a dose-dependent decrease in BRET ratio with increasing lactose levels ( Figure 31 H).

[0475] The method described herein is able to rapidly and accurately determine the lactose content in milk samples on a optoelectronic device within 4 minutes using a storable two-component formulation.

[0476] Example 8. Detection of plasmin activity in milk samples using storable biosensor reagents

[0477] Examples 1 to 5 demonstrated storable assay reagents that can be used to detect the protease AprX activity in milk samples on an optoelectronic device. The following example demonstrates that the methods and formulations described in Examples 1 to 5 can be used to detect other milk spoilage proteases in milk samples, such as plasmin. Unlike AprX, plasmin has been found to bind to its substrate casein. While it is possible to directly detect the activity in milk samples, its detection sensitivity can be enhanced using a dissociation protocol (e.g., as described in WO2018 / 085895).

[0478] Construct the plasmin BRET2 sensor molecule (hereinafter referred to as the plasmin sensor) to generate the pRSET-plasmin expression plasmid as described in WO2013 / 155553 (especially Example 10 and Figure 53 of said application, see also Dacres et al., (2019) A rapid and sensitive biosensor for measuring plasmin activity in milk, Sensors and Actuators B: Chemistry, Volume 301, 127141). Express and purify the plasmin sensor as described in the method section. Then use the purified plasmin sensor to prepare two separate lyophilized formulations as described below.

[0479] FD-plasmin-5 formulation: This formulation is used for the analysis of milk while improving the detection sensitivity for plasmin. It is designed to be used in combination with an ultrasonication step that homogenizes the milk and requires a higher concentration of esterase. The formulation contains:

[0480] ● 3,450 ng / ml plasmin BRET 2 biosensor

[0481] ● 250 mM Tris (pH 8.4) buffer

[0482] ● 250 mM NaCl

[0483] ● 125 mM L-lysine to support optimal plasmin activity

[0484] ● 5 mM aminocaproic acid for casein-plasmin dissociation

[0485] ● 400 mM trisodium acetate for casein-plasmin dissociation

[0486] ● 0.45% (v / v) fish skin gelatin as a carrier protein (*stock solution contains 45% (w / v) (0.45 g / mL) fish skin gelatin)

[0487] ● 12.5 mg / ml trehalose as a cryoprotectant

[0488] ● 12.5 mg / ml pullulan as a cryoprotectant

[0489] ● 25 U / ml porcine esterase as a caging agent.

[0490] FD-plasmin-6 formulation: The formulation is used for the analysis of non-homogenized milk. This formulation contains:

[0491] ● 3,450 ng / ml plasmin BRET2 Biosensor

[0492] ● 250 mM Tris (pH 8.4) buffer

[0493] ● 250 mM NaCl

[0494] ● 125 mM L-lysine to support optimal plasmin activity

[0495] ● 0.45% (v / v) fish skin gelatin as a carrier protein (*stock solution contains 45% (w / v) (0.45 g / mL) fish skin gelatin)

[0496] ● 12.5 mg / ml trehalose as a cryoprotectant

[0497] ● 12.5 mg / ml pullulan as a cryoprotectant

[0498] ● 5 U / ml porcine esterase as a de-caging agent

[0499] As described in Example 3, the formulation was prepared in assay tubes and lyophilized.

[0500] The lyophilized FD-plasmin-6 reagent was used for the protease assay described in Example 4. Briefly, the lyophilized formulation was reconstituted with 1 mL of raw milk supplemented with a known amount of standardized human plasmin protease, and then 28 μL of compound 10 solution (in anhydrous DMSO, 4.3 mM) was added to provide a final assay concentration of 120 μM compound 10. The tube was capped and inverted 20 times and inserted into a photoelectronic device. The activity of human plasmin was standardized as described in the Materials and Methods section above.

[0501] The protease assay was performed using a photoelectronic device incubated at 37 °C, and the BRET2 bioluminescence signal was recorded over 600 seconds (10 minutes) or 900 seconds (15 minutes). The bioluminescence value at 590 - 600 seconds was used to determine the BRET ratio for the 10-minute assay, and the bioluminescence signal value recorded from 890 - 900 seconds was used to determine the BRET ratio for the 15-minute assay.

[0502] The observed BRET ratio decreased with an increase in the amount of plasmin activity in the raw milk sample ( Figure 32)。In the 10-minute assay, the BRET ratio decreased from 0.572 ± 0.004 to 0.461 ± 0.050 in the presence of 10 nM human plasmin (450 mU / mL) and to 0.206 ± 0.018 in the presence of 100 nM human plasmin (4500 mU / mL). In the 15-minute assay, the BRET ratio decreased from 0.576 ± 0.005 to 0.382 ± 0.083 in the presence of 10 nM human plasmin (450 mU / mL) and to 0.157 ± 0.015 in the presence of 100 nM human plasmin (4500 mU / mL). The greater decrease in the BRET ratio indicates that the 15-minute assay is more sensitive than the 10-minute assay.

[0503] Example 9. Detection of plasminogen activity in milk samples using a storage-stable biosensor reagent

[0504] In milk or blood, plasmin is subject to a complex regulatory system that includes the inactive precursor, plasminogen, and a series of plasminogen activators and inhibitors. Plasminogen present in milk samples can be activated to plasmin by heat treatment, which inadvertently alters the balance of inhibitors and activators. High initial levels of plasminogen and / or malfunctioning heat treatment processes that activate plasminogen to plasmin can cause long-life or UHT milk to deteriorate during long-term storage. To test whether the protease sensing system developed herein can measure the potential plasmin activity present as plasminogen in milk, the plasminogen activator urokinase was added to the assay reagent formulation developed in Example 8.

[0505] The assay reagent formulation (referred to as FD-plasminogen-1) was prepared as follows:

[0506] ● 3,450 ng / ml plasmin BRET 2 Biosensor

[0507] ● 250 mM Tris (pH 8.4) buffer

[0508] ● 250 mM NaCl

[0509] ● 125 mM L-lysine to support optimal plasmin activity

[0510] ● 0.45% (v / v) fish skin gelatin as a carrier protein (*stock solution contains 45% (w / v) (0.45 g / mL) fish skin gelatin)

[0511] ● 12.5 mg / ml trehalose as a cryoprotectant

[0512] ● 12.5 mg / ml pullulan as a cryoprotectant

[0513] ● 1000 U / mL human urokinase for activating bovine plasminogen

[0514] ● 5 U / ml porcine esterase as a caging agent.

[0515] FD - plasminogen - 1 was lyophilized (FD) as described in Example 3, and the protease assay protocol developed in Example 4 was used to analyze plasminogen in milk samples.

[0516] Native plasminogen present in raw milk was inactivated by incubating 1 mL of the sample in a 1.5 mL Eppendorf tube at 90 °C for 20 minutes. The lyophilized formulation was reconstituted with 1 mL of raw milk (heat - treated or unheated raw milk). Subsequently, 28 μL of compound 10 solution (in anhydrous DMSO, 4.3 mM) was added to provide a assay concentration of 120 μM compound 10. The tube was capped and inverted 20 times and inserted into the optoelectronic device.

[0517] The protease assay was performed using an optoelectronic device incubated at 37 °C, and the BRET was recorded within 900 seconds (15 minutes) 2 Bioluminescence signal. The bioluminescence values at 890 - 900 seconds were used to determine the BRET ratio value.

[0518] After an initial change in the BRET ratio (< 100 seconds) due to the production of free coelenterazine in the system, the unheated raw milk samples showed a continuous decrease in the BRET ratio, while the BRET ratio of the heat - treated raw milk samples remained stable ( Figure 33 A). This indicates the presence of bovine plasminogen activated by plasmin generated by human urokinase. Then the plasmin sensor can detect plasmin protease. Using the 15 - minute assay, the BRET ratio showed a 20% decrease in the BRET ratio compared to raw milk and heat - treated raw milk samples ( Figure 33 B). Then protease assays were performed using heat - treated raw milk samples spiked with serial dilutions of human plasminogen. A dose - dependent response was observed where higher levels of plasminogen led to lower BRET ratios ( Figure 33 C). This example demonstrates that the methods and formulations described herein can be used to measure the potential activity levels of bovine plasminogen in raw milk samples.

[0519] Example 10. Detection of subtilisin (alkaline protease) activity and discrimination of AprX activity in milk samples using storage - stable biosensor reagents

[0520] Bacterial spores of the genus Bacillus, which form endospores, are present in milk powder and, after hydration and spore germination, secrete several different proteases in milk. The main protease class, subtilisin (also known as subtilisin protease or alkaline protease), is a serine protease that preferentially cleaves uncharged amino acids. It is hypothesized that the CT-AprX biosensor, which includes neutral amino acids, can be cleaved by subtilisin present in reconstituted milk powder and has the sensitivity required for practical applications in the dairy industry.

[0521] The lyophilized AprX biosensor formulation developed in Example 3 was prepared with a porcine esterase concentration of 25 U / mL (final working concentration 5 u / mL????), and the BRET 2 assay developed in Example 4 was used to analyze UHT milk samples spiked with active, calibrated subtilisin protease. The subtilisin protease was calibrated for activity according to the AprX protease activity calibration protocol described in the Methods section.

[0522] The protease assay was performed using a photoelectronic device incubated at 37 °C, and the BRET 2 bioluminescence signal was recorded over 600 seconds (10 minutes) or 900 seconds (15 minutes). The bioluminescence values from 590 - 600 seconds were used to determine the BRET ratio for the 10-minute assay, and the bioluminescence signal values recorded from 890 - 900 seconds were used to determine the BRET ratio for the 15-minute assay.

[0523] To distinguish the activity of AprX from that of subtilisin protease, the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) was added to milk samples spiked with AprX or subtilisin protease to a final concentration of 0.8 mM. After adding PMSF, the samples were incubated on ice for 30 minutes.

[0524] In both the 10-minute assay and the 15-minute assay, the UHT milk samples analyzed showed a protease activity-dependent decrease in the BRET ratio ( Figure 34 A). The 15-minute assay showed a greater decrease in the BRET ratio compared to the 10-minute assay. When milk samples spiked with 500 μU / mL subtilisin protease or 1000 μU / mL AprX were analyzed, the BRET ratio decreased over time ( Figure 34 B). Over time, samples spiked with 1000 μU / mL AprX and treated with PMSF showed a similar decrease in the BRET ratio, indicating that AprX is not inhibited by PMSF. In contrast, milk samples spiked with subtilisin protease and treated with PMSF produced a stable BRET ratio ( Figure 34 B).

[0525] Calibration Method for a Photodetector in a Photoelectronic Device Using a Gaseous Tritium Light Source

[0526] The optoelectronic device described herein contains two micro - photon multiplier tubes (μPMT). The photon detection sensitivities of these μPMTs vary from device to device, and the performance of an individual μPMT will decay over time. To ensure accurate and precise measurements, the μPMT signal output should be calibrated and adjusted over time.

[0527] To this end, it was tested whether a gaseous tritium light source (GTLS) could be used to calibrate the optoelectronic device. Various GTLS sources (all < 0.67 GBq, non - radioactive) were purchased from SP2 (Self Powered Safety Products Ltd, United Kingdom), and their luminescence characteristics in response to relevant external factors (temperature and light) were tested.

[0528] According to the supplier's specification sheet:

[0529] Each MiniGlow safety marker contains a gaseous tritium light source (GTLS), which provides a reliable, safe and self - powered continuous light output source. During the service life of the product, no battery or ambient light charging is required. The GTLS contains a sealed borosilicate glass capsule, which contains a trace amount of gaseous tritium to power the device. Beta particle radiation (i.e., the electrons released when gaseous tritium decays) is used to excite the phosphor coating on the inner surface of the capsule, causing it to emit light. As a sealed source, they are non - emissive and have a very low energy level. Each light source lamp is manufactured and tested in accordance with UK MOD Defence Standard 62 / 4. The GTLS contains no moving parts, and since the unit is continuously excited, no on / off switch is required. They will remain excited for a maintenance - free period of over 10 years, even during long periods of complete darkness. The device can be used in any environmental conditions (including underwater applications) and a temperature range of - 40° to 80°C. They are suitable for operation in Zone 0 hazardous areas....

[0530] Description

[0531] The MiniGlow marker is molded from clarity V0 polycarbonate, with stainless steel end caps and an opening ring [removed], providing a rugged protective housing and making it highly impact - resistant. Length: 18.0 mm Diameter: 9.0 mm Total weight: 3.0 g Gripping diameter: 20.0 mm Maximum activity: 0.67 GBq

[0532] First, to select the ideal 'color' for the filter set installed in the optoelectronic device, the emission spectra of three different colors (UV blue, green, and white) of GTLS tubes were analyzed. The tubes were molded as cylindrical tubes with a hexagonal transparent polycarbonate housing ("micro markers" UV blue, green, and white). By placing the GTLS source in a 96-well plate and subsequently measuring the emission at 320 - 610 nm, the emission spectrum was determined on a CLARIOstar plate reader (gain of 2000 at 30 °C).

[0533] Spectral scans showed that photons from the 'blue' and 'white' GTLS sources would pass through the green and blue filter windows of the optoelectronic device, while the 'green' source showed very little emission within the 'blue filter' window ( Figure 35 A). The 'white' GTLS source showed two emission peaks, which was considered an unnecessary complication. Therefore, GTLS with a "UV blue" phosphor was selected for all future experiments.

[0534] The GTLS source was temporarily adhered to the screw cap of a 2 mL tube using "Blu Tack" adhesive ( Figure 35 B), or the GTLS source was placed at the bottom of a capped 2 mL tube ( Figure 35 C) to test the positioning of the GTLS source within the optoelectronic device tube holder. The device temperature was set to 37 °C and the emission counts of two channels were recorded within 5 minutes.

[0535] The capped GTLS resulted in a lower amount of light reaching the PMT ( Figure 35 B; < 1000 counts), but the GTLS tube placed at the bottom of the tube holder gave 500,000 - 6,000,000 signal counts, depending on the type of GTLS and the version of the device hardware ( Figure 35 C). The counts from the latter experiment also showed high signal variation when the tube containing the GTLS source was repeatedly removed and reinserted into the tube holder, which may be due to the non-uniform diffusion of GTLS light emission. This indicates that the GTLS source must be placed at the bottom or in the lower half of the tube holder and at a fixed angle relative to the detection window of the μPMT.

[0536] Then, two different types of blue / UV blue were purchased and tested GTLS (“BivvyMarker” (TD302) or “MiniGlow Smooth” (TD912)). The blue of these is the color closest to the “UV blue” used in the original experiments. The advantage of these two types is that the GTLS source is encapsulated in a polycarbonate holder that is approximately cylindrical (rather than a polygonal prism). For ease of fabrication, only MiniGlow Smooth was selected because it has flat metal caps rejected on both ends, facilitating connection to the holder. The top end of the MiniGlow Smooth GTLS (the lateral hole for connection to the key ring) was glued into an embedded socket incorporated into the base of a lathe-turned replica of the upper half of a 2 mL screw-cap tube (Thermo Scientific TM QSPTM)( Figure 36 and Figure 37 ). Additionally, the calibration tool has a thickened area 2 mm below the cap to ensure a “transition” or push-fit of the trigalight in the tube holder of the device, thereby stabilizing the tube within the holder.

[0537] In a further refinement, and to ensure precise repeatable rotational alignment of the calibration tool in the device, positioning notches were machined at the top of the tube holder of the optoelectronic device, and complementary lugs were designed to fit into said notches of the cap of the calibration tool. To demonstrate the utility of such a method (prior to delivery of the handle with lugs), engraved and marked points were used to visually align the calibration tube with the notches. When assembling and gluing the GTLS into the holder, a jig was used that allowed the injection molding seam of the polycarbonate housing to be aligned with the axis of the lugs / points, thereby ensuring that the seam was at right angles to the axis of the PMT in the device and minimizing variations in light transmission to the PMT due to any non-uniformities in the optical properties of the polycarbonate at or near the seam.

[0538] Then, the calibration source (designated CT2) was tested on the optoelectronic device by monitoring the luminescence in the ‘blue’ and ‘green’ channels over 20 minutes at 37 °C.

[0539] Over time, the ‘green’ channel counts increased by approximately 9% until a stable plateau signal level was reached, while the ‘blue’ channel counts decreased by approximately 0.1% before reaching the stable plateau. When analyzing CT2 at other device temperatures (20 - 45 °C), the same effect was observed, although the plateau levels were different ( Figure 35 D). It is speculated that the temperature of the GTLS source itself affects the luminescence output, possibly due to a change in the efficiency of the phosphorescent coating on the inside of the tritium electron-excitation tube. Therefore, for precise measurements, CT2 should be equilibrated to the set device temperature before using the signal for calibration.

[0540] After each measurement was completed, the test of CT2 was repeated by removing and reinserting the tube. Three optoelectronic devices were tested. Each measurement was analyzed at 37 °C for 5 minutes, and the photon counts between 200 - 300 seconds were averaged. The first run was not included in the data analysis because it was used to equilibrate CT2 to the device temperature.

[0541] Comparing all three devices, the CT2 signal had a high degree of repeatability ( Figure 35 E), with the coefficient of variation in the 'green' channel in the range of 0.01% to 0.17%, and the coefficient of variation in the 'blue' channel being 0.13 - 0.56%.

[0542] Based on these measurements, setting reference data enabled the adjustment of the signal output of newly manufactured optoelectronic devices. Additionally, to correct for the brightness decline due to tritium decay (λ = 12.33 years), a reference data set was calculated to adjust for the brightness decline of the individual calibration tool over time, thereby isolating any changes in the photon sensitivity of the μPMT that might occur over time.

[0543] Those skilled in the art will understand that various changes and / or modifications can be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. Accordingly, this embodiment should be considered illustrative in all respects and not restrictive.

[0544] This application claims priority to AU2022903116, filed on October 21, 2022, the entire content of which is incorporated herein by reference.

[0545] This application is filed with a sequence listing in electronic form. The entire content of the sequence listing is hereby incorporated by reference.

[0546] All publications discussed and / or mentioned herein are incorporated herein in their entirety.

[0547] Any discussion of documents, statutes, materials, devices, articles, etc. that have been included in this specification is for the sole purpose of providing context for the present invention. This should not be construed as an admission that any or all of these matters form part of the prior art base or are common general knowledge in the field relevant to the present invention, as they existed prior to the priority date of each claim of this application.

[0548] Future patent applications may be filed in Australia or overseas based on this application or claiming priority from this application. It should be understood that the following provisional claims are provided by way of example only and are not intended to limit the scope of what may be claimed in any such future applications. Features may be added to or omitted from the provisional claims at a later date in order to further define or re-define one or more inventions.

Claims

1. A method for detecting an extracellular analyte in a sample, the method comprising: providing a biosensor system selected from (i) an extracellular bioluminescence sensor molecule comprising a donor luminophore, an analyte-sensitive domain, and a receptor fluorophore; and (ii) a precursor system thereof comprising a pair of molecules adapted to react in solution to form the extracellular bioluminescence sensor molecule; incubating a assay composition comprising the sample and the biosensor system for an incubation period sufficient for the extracellular analyte to interact with the analyte-sensitive domain, if the extracellular analyte is present in the sample; and detecting light emitted from the assay composition after the incubation period, wherein the ratio of the luminescence associated with the receptor fluorophore to the luminescence associated with the donor luminophore indicates the interaction of the extracellular analyte with the analyte-sensitive domain, wherein at least one caged substrate compound uncages in the assay composition during the incubation period to produce an uncaged substrate compound that excites the donor luminophore for the detection.

2. The method according to claim 1, wherein the at least one caged substrate compound is present in the assay composition from the start of the incubation period.

3. The method according to claim 1 or claim 2, wherein the at least one caged substrate compound is preferably uncaged by an uncaging agent that is present in the assay composition from the start of the incubation period.

4. The method according to claim 3, wherein the uncaging agent is independent of the sample, preferably wherein the uncaging agent is an esterase that is independent of the sample.

5. The method according to claim 3, wherein the uncaging agent is endogenous to the sample, preferably wherein the uncaging agent is an esterase that is endogenous to the sample.

6. The method according to any one of claims 1 to 5, wherein the incubation period is between 1 minute and 30 minutes.

7. The method according to any one of claims 1 to 6, wherein the incubation period is between 5 minutes and 15 minutes.

8. The method according to any one of claims 1 to 7, wherein the at least one caged substrate compound uncages to provide a luminescence associated with the receptor fluorophore that is greater than 80% of the maximum value when the light is detected in the absence of the extracellular analyte.

9. The method according to claim 8, wherein in the absence of the extracellular analyte, the luminescence associated with the receptor fluorophore is greater than 80% of the maximum value in a detection window of at least 3 minutes, preferably at least 5 minutes, wherein the light is detected during the detection window.

10. The method according to any one of claims 1 to 9, wherein at least two different caged substrate compounds present in the assay composition from the start of the incubation period uncage during the incubation period, thereby producing the uncaged substrate compounds at different reaction rates.

11. The method according to any one of claims 1 to 10, wherein each caged substrate compound is a caged coelenterazine compound comprising a carbonyl protecting group at the 3'-position.

12. The method according to any one of claims 1 to 11, wherein each caged substrate compound has a structure according to Formula 1 or Formula 2: Wherein: Each R 1 is independently selected from H, OH, OR alk and NH2, where R alk is a C1-C6 alkyl group, Each R 2 is independently selected from H, Ar and (CH2) n Ar, where each Ar is independently a carbocyclic aromatic group optionally substituted with OH, C1-C6 alkyl or halogen, and n is 1 or 2 Each L 1 is independently selected from CH2 or S, Each R 3 is independently selected from C6H5, CH2C6H5, C1-C6 alkyl, and C1-C6 cycloalkyl, R 4 independently selected from H and CH3, L 2 independently selected from (CH2) n and CH=CH, where n is 1-3, and Each X is independently a carbonyl protecting group.

13. The method according to claim 12, wherein each X has a structure according to Formula 3, Formula 4 or Formula 5: Wherein R 5 , R 6 and R 7 are each independently optionally substituted C1-C 12 alkyl, C1-C 12 cycloalkyl or C1-C 12 aryl.

14. The method according to any one of claims 1 to 13, wherein the uncaged substrate compound is a coelenterazine compound, preferably bisdeoxycoelenterazine.

15. The method according to any one of claims 1 to 14, wherein in a substrate solution stable at room temperature, the at least one caged substrate compound is introduced into the assay composition, preferably by dropwise dispensing the substrate solution into the assay composition.

16. The method according to claim 15, wherein the substrate solution is a non-aqueous solution comprising a water-miscible aprotic solvent, preferably DMSO.

17. The method according to claim 15 or claim 16, further comprising storing the substrate solution for at least one week, preferably at least one month, more preferably at least six months, before introducing the substrate solution into the assay composition.

18. The method according to any one of claims 1 to 17, which comprises preparing the assay composition by a method comprising dispersing a solid composition comprising the biosensor system in a liquid, preferably wherein the solid composition is a lyophilized solid composition, and preferably wherein the liquid comprises the sample.

19. The method according to claim 18, wherein the solid composition further comprises a caging agent for uncaging the at least one caged substrate compound in the assay composition.

20. The method according to claim 18 or claim 19, wherein the assay composition is prepared and incubated in an assay vessel in which the solid composition was previously lyophilized.

21. The method according to any one of claims 1 to 20, wherein the assay composition is prepared and incubated in an assay vessel, and the light emitted from the assay composition is detected passing through the wall of the assay vessel.

22. The method according to any one of claims 1 to 21, wherein the extracellular bioluminescence sensor molecule is a BRET 2 biosensor.

23. The method according to any one of claims 1 to 22, wherein the extracellular analyte is selected from the group consisting of enzymes, zymogens and carbohydrates, and is preferably a protease.

24. The method according to any one of claims 1 to 23, wherein the sample is a food or beverage composition, preferably a dairy composition.

25. The method according to any one of claims 1 to 24, which comprises: ● providing the sample as a liquid for analysis; ● providing an assay vessel containing a solid composition comprising an extracellular bioluminescent sensor molecule and optionally a caging agent; ● Provide a substrate storage container containing a substrate solution that can be stored at room temperature, wherein the substrate solution that can be stored at room temperature contains the at least one caged substrate compound; ● Add an aliquot of the sample to the assay vessel; ● Thereafter, transfer an aliquot of the substrate solution that can be stored at room temperature from the substrate storage container to the assay vessel and mix the assay composition contained therein; ● Incubate the assay composition in the assay vessel for the incubation period; And ● Detect the light emitted from the assay composition passing through the wall of the assay vessel with a photoelectronic device.

26. A system for detecting an extracellular analyte in a sample, the system comprising: A biosensor system selected from (i) an extracellular bioluminescence sensor molecule comprising a donor luminophore, an analyte-sensitive domain, and a receptor fluorophore; and (ii) a precursor system thereof, the precursor system comprising a pair of molecules adapted to react in solution to form the extracellular bioluminescence sensor molecule, the biosensor system being contained in an assay vessel configured to receive the sample to be combined with the biosensor system in an assay composition; A room-temperature stable substrate solution containing at least one caged substrate compound, the room-temperature stable substrate solution for dispensing into the assay composition in the assay vessel, the substrate solution being contained in a substrate storage vessel; and A photoelectronic device configured to detect light emitted from the assay composition passing through the wall of the assay vessel, wherein the ratio of the luminescence associated with the receptor fluorophore to the luminescence associated with the donor luminophore indicates the interaction of the extracellular analyte with the analyte-sensitive domain, wherein the at least one caged substrate compound is adapted to uncage in the assay composition, thereby producing an uncaged substrate compound that excites the donor luminophore.

27. The system according to claim 26, wherein the photoelectronic device is configured to receive the assay vessel and incubate the assay composition contained therein for an incubation period sufficient for the extracellular analyte to interact with the analyte-sensitive domain, if the extracellular analyte is present in the sample, preferably wherein the photoelectronic device is configured to incubate the assay composition at a predetermined incubation temperature.

28. The system according to claim 26 or claim 27, wherein the biosensor system is present in a solid composition, preferably a lyophilized solid composition.

29. The system according to claim 28, wherein the solid composition further comprises an uncaging agent for uncaging the at least one caged substrate compound in the assay composition.

30. The system according to any one of claims 26 to 29, wherein the substrate solution comprises at least two different caged substrate compounds, and the at least two different caged substrate compounds are adapted to uncage in the assay composition so as to produce the uncaged substrate compounds at different reaction rates.

31. The system according to any one of claims 26 to 30, wherein each caged substrate compound is a caged coelenterazine compound comprising a carbonyl protecting group at the 3'-position.

32. The system according to any one of claims 26 to 31, wherein each caged substrate compound has a structure according to Formula 1 or Formula 2: Wherein: Each R 1 is independently selected from H, OH, OR alk and NH2, wherein R alk is a C1-C6 alkyl group, Each R 2 is independently selected from H, Ar, and (CH2) n Ar, where each Ar is independently a carbocyclic aromatic group optionally substituted with OH, C1-C6 alkyl, or halogen, and n is 1 or 2 Each L 1 is independently selected from CH2 or S, Each R 3 is independently selected from C6H5, CH2C6H5, C1-C6 alkyl, and C1-C6 cycloalkyl, R 4 independently selected from H and CH3, L 2 independently selected from (CH2) n and CH=CH, where n is 1-3, and Each X is independently a carbonyl protecting group.

33. The system according to claim 32, wherein each X has a structure according to Formula 3, Formula 4 or Formula 5: wherein R 5 、R 6 and R 7 are each independently optionally substituted C1-C 12 alkyl, C1-C 12 cycloalkyl or C1-C 12 aryl.

34. The system according to any one of claims 26 to 33, wherein the uncaged substrate compound is a coelenterazine compound, preferably dideoxycoelenterazine.

35. The system according to any one of claims 26 to 34, wherein the substrate solution is a non-aqueous solution comprising a water-miscible aprotic solvent, preferably DMSO.

36. The system according to any one of claims 26 to 35, wherein the substrate storage vessel is configured to dispense an aliquot of the substrate solution into the assay vessel, and preferably is configured to dispense the substrate solution dropwise into the assay vessel.

37. The system according to any one of claims 26 to 36, wherein the extracellular bioluminescent sensor molecule is a BRET 2 biosensor.

38. The system according to any one of claims 26 to 37, wherein the optoelectronic device comprises a first photodetector and a second photodetector, the first photodetector comprising a micro-photon multiplier tube equipped with a band-pass filter to selectively permit transmission of the light wavelength associated with the acceptor fluorophore, and the second photodetector comprising a micro-photon multiplier tube equipped with a band-pass filter to selectively permit transmission of the light wavelength associated with the donor luminophore.

39. The system according to any one of claims 26 to 38, wherein the optoelectronic device is configured to incubate the assay composition at a predetermined incubation temperature.

40. A BRET reagent system for combination with a sample to produce a bioluminescence assay composition, which, after incubation of the bioluminescence assay composition, has a BRET ratio indicative of an extracellular analyte in the sample, the BRET reagent system comprising: (a) A solid composition comprising a biosensor system selected from (i) an extracellular bioluminescence sensor molecule comprising a donor luminophore, an analyte-sensitive domain adapted to interact with the extracellular analyte, and an acceptor fluorophore; and (ii) a precursor system thereof comprising a pair of molecules adapted to react in solution to form the extracellular bioluminescence sensor molecule, wherein the solid composition is capable of being dispersed in a liquid comprising the sample to produce the assay composition; and (b) A room-temperature stable substrate solution containing at least one caged substrate compound, the room-temperature stable substrate solution being for dispensing into the assay composition, wherein the at least one caged substrate compound is adapted to uncage in the assay composition to produce an uncaged substrate compound that excites the donor luminophore.

41. The BRET reagent system according to claim 40, wherein the solid composition is contained in an assay vessel configured to receive the liquid containing the sample and to permit light from the assay composition to transmit through the assay vessel wall for detection.

42. The BRET reagent system according to claim 41, wherein the solid composition is a lyophilized composition prepared by lyophilizing an aqueous precursor composition in the assay vessel.

43. The BRET reagent system according to any one of claims 40 to 42, wherein the solid composition further comprises an uncaging agent for uncaging the at least one caged substrate compound in the assay composition.

44. The BRET reagent system according to any one of claims 40 to 43, wherein the room-temperature stable substrate solution is contained in a substrate storage vessel configured to dispense an aliquot of the substrate solution into the assay composition and preferably configured to dispense the substrate solution dropwise into the assay composition.

45. The BRET reagent system according to any one of claims 40 to 44, wherein the room-temperature stable substrate solution contains at least two different caged substrate compounds, the at least two different caged substrate compounds being adapted to uncage in the assay composition to produce the uncaged substrate compounds at different reaction rates.

46. The BRET reagent system according to any one of claims 40 to 45, in the form of a kit, the kit comprising instructions for using the BRET reagent system in the method according to any one of claims 1 to 25.

47. A solid biosensor composition for BRET detection of an extracellular analyte in a sample, the solid biosensor composition comprising: (a) A biosensor system selected from (i) an extracellular bioluminescence sensor molecule comprising a donor luminophore, an analyte-sensitive domain that readily interacts with the extracellular analyte, and a receptor fluorophore; and (ii) a precursor system thereof comprising a pair of molecules adapted to react in solution to form the extracellular bioluminescence sensor molecule; and (b) An uncaging agent, wherein the solid biosensor composition is capable of being dispersed in a liquid containing the sample to produce an assay composition, and wherein the uncaging agent is adapted to uncage at least one caged substrate compound in the assay composition, thereby producing an uncaged substrate compound that excites the donor luminophore.

48. A substrate dispensing system for BRET detection of extracellular analytes in a sample, the substrate dispensing system comprising a room temperature-stable substrate solution, the room temperature-stable substrate solution comprising at least one caged substrate compound, wherein the substrate solution is contained in a substrate storage vessel configured to dispense a predetermined aliquot of the substrate solution into an assay composition comprising the sample and a BRET bioluminescent sensor molecule.

49. The substrate dispensing system according to claim 48, wherein the room temperature-stable substrate solution comprises at least two different caged substrate compounds, the at least two different caged substrate compounds being adapted to uncage at different reaction rates.

50. A substrate composition for BRET detection of extracellular analytes in a sample, the substrate composition comprising at least two different caged substrate compounds, the at least two different caged substrate compounds being adapted to uncage in an assay composition comprising the sample and a BRET bioluminescent sensor molecule, thereby producing uncaged substrate compounds at different reaction rates.

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