Reagents, devices, and accessories for kinetic fluorogenic chemical quantification

Abstract

A method for kinetic fluorogenic (FG) quantification of a target biomarker in a sample includes steps of exposing a sample to an extraction reagent or solvent comprising water and a water-miscible organic solvent to provide a sample extract, filtering to provide a sample supernatant, and admixing the supernatant with a reaction solvent to provide a reaction mixture. The reaction mixture is exposed to a fluorogenic probe specific to the target biomarker to provide a fluorescent probe-target biomarker complex. At predetermined time intervals, the probe-target biomarker complex is quantified by kinetic spectroscopic quantification by a two-step dual chromogenic (CG) and FG measurement.

Description

IN THE UNITED STATES PATENT AND TRADEMARK OFFICE A UTILITY PATENT APPLICATION for REAGENTS, DEVICES, AND ACCESSORIES FOR KINETIC FLUOROGENIC CHEMICAL QUANTIFICATION Inventors Dongmao Zhang Max Wamsley Assignee Mississippi State University, Starkville, MS Attorney Docket No. 18869N / 231234REAGENTS, DEVICES, AND ACCESSORIES FOR KINETIC FLUOROGENIC CHEMICAL QUANTIFICATION Related Applications

[0001] This utility patent application claims the benefit of priority in U.S. Provisional Application Serial No.63 / 541,245 filed on September 28, 2023, the entire disclosure of which is incorporated herein by reference. Technical Field

[0002] This disclosure relates in one aspect to a comprehensive system comprising devices, sampling accessories, and reagents designed for the swift kinetic fluorogenic quantification of chemicals. Another aspect of the disclosure relates to the quantification of biomarkers / chemical agents in samples, for example quantification of malondialdehyde (MDA), a widely utilized lipid peroxidation biomarker in the food industry and biological research. The disclosed technology can be readily adapted to encompass a wide spectrum of biomarkers and chemical agents, showcasing its versatility and broad applicability. Background

[0003] Chromogenic (CG-) and fluorogenic (FG)-reactions have been used extensively for chemical measurement and imaging. Numerous commercial and literature chromogenic / fluorogenic probes are available for chemical quantifications. However, the existing CG- / FG- based chemical quantifications use predominantly the time-consuming equilibrium quantification that is performed after the completion of the CG / FG reactions. The much more efficient kinetic spectroscopic quantification initially introduced in the 1970s has been limited only to a small number of CG / FG reactions and to research samples with known compositions. Embodiments of the invention relate to devices, reagents, accessories, and methodology for kinetic fluorogenic quantification of reactive species.

[0004] In this disclosure, reactive species refers to reagents that react with one or more chemicals and such reactions change the sample fluorescence properties such as spectral intensities, peak wavelengths, anisotropy, and / or lifetimes. Such reactive species include, butnot limited to reactive oxygen species (such as hydrogen peroxide, superoxide, ozone, malondialdehyde (MDA)), reactive sulfur / nitrogen / chlorine species, chemical warfare agents such as nerve agents, and radioactive materials.

[0005] In a prior study by the inventors, entitled “Kinetic spectroscopic quantification using two- step chromogenic and fluorogenic reactions: from theoretical modeling to experimental quantification of biomarkers in practical samples”1MDA was used as the model reactive species. This model reactive species in this disclosure is also MDA. However, it will be appreciated that the general designs of the device, accessory, reagents, and their usage is applicable to numerous other reactive species if there are fluorogenic probes currently available for their detection / quantification. Summary

[0006] The details of one or more embodiments of the presently-disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided in this document. The information provided in this document, and particularly the specific details of the described exemplary embodiments, is provided primarily for clarity of understanding and no unnecessary limitations are to be understood therefrom. In case of conflict, the specification of this document, including definitions, will control.

[0007] While the terms used herein are believed to be well understood by those of ordinary skill in the art, certain definitions are set forth to facilitate explanation of the presently-disclosed subject matter.

[0008] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong.

[0009] All patents, patent applications, published applications and publications, GenBank sequences, databases, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety.

[0010] Where reference is made to a URL or other such identifier or address, it understood that such identifiers can change and particular information on the internet can come and go, butequivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

[0011] Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently-disclosed subject matter, representative methods, devices, and materials are described herein.

[0012] As used herein, nomenclature for compounds, including organic compounds, can be given using common names, IUPAC, IUBMB, or CAS recommendations for nomenclature. When one or more stereochemical features are present, Cahn-Ingold-Prelog rules for stereochemistry can be employed to designate stereochemical priority, ElZ specification, and the like. One of skill in the art can readily ascertain the structure of a compound if given a name, either by systemic reduction of the compound structure using naming conventions, or by commercially available software, such as CHEMDRAW™ (Cambridgesoft Corporation, U.S.A.).

[0013] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the presently- disclosed subject matter belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently-disclosed subject matter, representative methods, devices, and materials are now described.

[0014] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently- disclosed subject matter.

[0015] As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a functional group," "an alkyl," or "a residue" includes mixtures of two or more such functional groups, alkyls, or residues, and the like.

[0016] Ranges can be expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0017] As used herein, the terms "optional" or "optionally" means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0018] As used herein, "kit" means a collection of at least two components constituting the kit. Together, the components constitute a functional unit for a given purpose. Individual member components may be physically packaged together or separately. For example, a kit comprising an instruction for using the kit may or may not physically include the instruction with other individual member components. Instead, the instruction can be supplied as a separate member component, either in a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation.

[0019] Certain materials, compounds, compositions, and components disclosed herein can be obtained commercially or readily synthesized using techniques generally known to those of skill in the art. For example, the starting materials and reagents used in preparing the disclosed compounds and compositions are either available from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), Acros Organics (Morris Plains, N.J.), Fisher Scientific (Pittsburgh, Pa.), or Sigma (St. Louis, Mo.) or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd's Chemistry of CarbonCompounds, Volumes 1-5 and Supplemental (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March's Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989).

[0020] There are currently numerous commercial MDA assay kits available in the market, most of which employ the well-established thiobarbituric acid (TBA) as chromogenic or fluorogenic probes. However, these TBA-based MDA quantification methods exclusively rely on the equilibrium quantification approach, which is time-consuming (typically taking 1 hour or longer), labor-intensive, and necessitates relatively large sample volumes. Moreover, certain techniques within this category require expensive instrumentation for sample handling and measurements.

[0021] The present invention offers many advantages over the prior art. For example, instead of relying on equilibrium fluorogenic quantification, which relies on measuring spectral intensity after the completion of fluorogenic reactions, the present invention utilizes kinetic fluorogenic MDA quantification. This novel approach is based on monitoring the rate of spectral intensity change during the fluorogenic reaction process.

[0022] Unlike current methods that use water as the reaction solvent, embodiments of the present invention employ water-miscible organic solvents such as methanol, ethanol, propanol, dimethyl sulfoxide, ethylene glycol, or their mixtures with water as the reaction solvent.

[0023] Embodiments of the present invention involve using a large excess of TBA (10 mM or higher) compared to the expected MDA concentration in the quantification process.

[0024] In certain embodiments, the invention incorporates a fluorescence internal reference to correct for measurement uncertainties arising from fluctuations in excitation power and emission detection intensity.

[0025] In other embodiments, a spectrofluorometer capable of simultaneously monitoring fluorescence from the internal reference and the TBA / MDA reaction products will be utilized.

[0026] For sampling cells, relatively low-cost optically transparent glass or polymer cuvettes can be used in specific implementations.

[0027] Furthermore, in certain scenarios, the TBA-reaction solutions can be conveniently pre- packaged for direct use prior to their expiration dates.Brief Description of the Drawings

[0028] Figure 1 shows embodiments of known MDA probes and their dual CG / FG reactions with MDA.

[0029] Figure 2 shows: (A) Photographs and UV-vis extinction spectra of an as-obtained beef extract. (B) Kinetic fluorescence of reaction solutions of TBA and beef extracts with different amount of added MDA standards. (C) As-acquired data and linear curve fitting of kinetic fluorescence in the linear time course regions. The kinetic data in (B) and (C) are offset for clarity. (D) Linear curve-fitting for standard-addition quantification of MDA concentration.

[0030] Figure 3 shows effects of solvents on assay rate and sensitivity. (A) Kinetic fluorescence spectra of TBA and 50 nM MDA reaction solution at 60 μC in water and in DMSO / H2O cosolvent (1:1, v:v) cosolvent. The TBA is at its room-temperature saturation concentration in water and in DMSO / H2O. (B) Kinetic UV-vis of the reaction solutions of 1 μM MDA and 205.2 mM TBA with DMSO / H2O cosolvents with volume fractions shown in the legends. (C) Linear region of the kinetic UV-vis extinction data shown in plot (B). (D) Fluorescence spectra of 1 ^M TBA-MDA in DMSO / H2O cosolvents of different DMSO volume fractions. Inset is the fluorescence peak intensity as a function of DMSO volume fractions.

[0031] Figure 4 shows a schematic of an embodiment of a presser used to extract biomarker- containing fluid from tissue samples. The presser handle can be operated manually or mechanically.

[0032] Figure 5 shows an embodiment of a miniaturized blender for extracting biomarker- containing fluid from tissue samples.

[0033] Figure 6 shows an embodiment of a cuvette holder for sample analysis.

[0034] Figure 7 shows: (Top left) Schematic of a reflective fluorescence cuvette embodiment. (Top right) Fluorescence cuvettes: one with and one without reflective metal coating. (Bottom) Comparative fluorescence spectra from the same solution using cuvettes with and without reflective coating.

[0035] Figure 8 shows an embodiment of a single sample kinetic FG analyzer including: BP: Bandpass filter; FC: Fiber coupler for separating light into two portions; LP: Linear polarizer; ND: Neutral density filter. Dashed lines refer to optional computational control or remote wireless control.

[0036] Figure 9 shows an alternative embodiment of a single sample kinetic FG analyzer including: BP: Bandpass filter; FC: Fiber coupler for separating light into two portions; LP: Linear polarizer; ND: Neutral density filter. Dashed lines refer to optional computational control or remote wireless control.

[0037] Figure 10 shows another alternative embodiment of a single sample kinetic FG analyzer including: BP: Bandpass filter; FC: Fiber coupler for separating light into two portions; LP: Linear polarizer; ND: Neutral density filter. Dashed lines refer to optional computational control or remote wireless control.

[0038] Figure 11 shows another alternative embodiment of a single sample kinetic FG analyzer including: BP: Bandpass filter; FC: Fiber coupler for separating light into two portions; LP: Linear polarizer; ND: Neutral density filter. Dashed lines refer to optional computational control or remote wireless control.

[0039] Figure 12 shows an embodiment of a multiplex kinetic fluorogenic analyzer design capable of simultaneously analyzing eight samples. Applications encompass quantification of various chemical and biological species within identical extracts, as well as the same species across different extracts.

[0040] Figure 13 shows embodiments of fluorescence well plates for use in the disclosure: (Top) Current fluorescence well plate with black walls surrounding the sample solutions. (Bottom) Schematic of the reflective fluorescence well plate, constructed by inserting externally coated reflective wells. This design improves fluorescence sensitivity by reflecting excitation and emission photons that would be absorbed by conventional plates, leading to enhanced fluorescence excitation and detection. Detailed Description

[0041] The details of one or more embodiments of the presently-disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided in this document. The information provided in this document, and particularly the specific details of the described exemplary embodiments, is provided primarily for clearness of understanding and no unnecessary limitations are to be understood therefrom. Further, while theterms used herein are believed to be well-understood by one of ordinary skill in the art, definitions are set forth to facilitate explanation of the presently-disclosed subject matter.

[0042] Reagents: Assays using commercial MDA assay kits use fluorogenic reactions performed in water. Examples of these reactions are illustrated in Figure 1. The most popular fluorogenic probe for MDA assay is 2-thiobarbituric acid (TBA) (reaction 1 in Figure 1). The kinetic fluorogenic MDA assay reported in our recent work is also performed in water.1In this disclosure, “reagents” refers to the chemicals or chemical solutions used for the reactive species assay processes. Such reagents can be classified as sample treatment / extraction reagents, fluorogenic reaction reagents, and post-reaction sample / cuvette treatment reagents.

[0043] Sample treatment / extraction reagents: Sample extraction refers to the process for separation of the reactive species from the materials of interest. One example is shown in Figure 2A where beef extract in the vials is obtained by blending the ground beef in water followed by paper filtration. In this particulate embodiment, the sample extraction reagent is water alone. Other embodiments of the sample extraction reagents include water mixed with organic solvents including, but not limiting to alcohol (methanol, ethanol, propanol, butanol), dimethyl sulfoxide (DMSO) acetone, acetonitrile, and dissolved electrolytes or any other reagents known to the skilled artisan for their abilities to promote macromolecule (such as protein, DNA, and fiber) aggregation / coagulation, thereby facilitate the separation of the fluid sample extracts from the sample matrix.

[0044] Fluorogenic reaction reagents include kinetic FG probes, solvents, catalysts, and any other chemicals known to knowledgeable person in the domain that facilitate the controls of reaction rates, photostability of the fluorogenic products, and / or the sensitivity and reliability of the assay.

[0045] The reaction solvent refers to the solvents in which the fluorogenic reaction occurs. Herein, the inventors have surprisingly discovered that fluorogenic MDA quantification in an organic solvent / water mixture is much more sensitive and efficient than that in water alone. Taking quantification of MDA in beef as a non-limiting example, the sample extracting reagent used for extracting MDA can be water or water-soluble organic solvent(s), or their mixture with mixing ratio of individual solvent compound varying from 0 to 100%. These organic solvents include but are not limited to dimethyl sulfoxide (DMSO), propanol, ethanol, methanol, and acetonitrile. The inclusion of organic solvents for the TBA based MDA quantification improvesboth assay efficiency and sensitivity. As an example, the MDA reaction with TBA dissolved in DMSO / H2O (1:1 volume ratio) is found to be 3 times faster than the reaction performed in 100% water, while the sensitivity of the MDA assay with the mixture solvent is five times better than in water alone.

[0046] Without intending any limitation, examples of FG probes, solvents, and catalysts suitable for use in the presently disclosed methods are set forth in the Table below. These fluorophore associated reagents have previously been used in equilibrium-based assay. However, the present inventors have expanded their use for the presently described kinetic fluorogenic quantification methods.

[0047]

[0048] Post-assay treatment reagents in this application refers to solvents and / or chemicals known to the skilled artisan that facilitate disinfection and cleaning of the sample handing devices including extraction and reaction containers before their disposal or reuse.

[0049] One aspect of this disclosure is the surprising ability to repurpose of known fluorogenic probes that included but not limited to that list in the Table above. These fluorophores have been used in equilibrium-based assay, while this invention expand their applications to kinetic fluorogenic assays.

[0050] Accessories: In this disclosure, “accessories” refers to reusable or disposable supplies needed during sample preparation, assay, and post-assay treatment processes.

[0051] Accessories for sample extractions: Besides its high assay efficiency and simple operation, another key appeal of kinetic FG quantification is its low sample consumption.However, current tools for obtaining the sample extracts (solutions containing the biomarker of interest) from sample size of real-world hard / soft solid samples are difficult. Current methods for getting the sample extracts from tissue include use of a blender, a probe sonicator, a sonication homogenizer, and others.

[0052] The sample preparation accessories described herein are designed to improve the efficiency, reliability, and convenience of the sample preparation for solid tissue sample and solution samples, and to reduce sample sizes and / or sample matrix interference for kinetic FG quantification.

[0053] One embodiment of the preparation accessory is a miniaturized presser (Figure 4), optionally made with plastic and / or metal. The presser 10 comprises a sample chamber 12, a plunger 14, and a screen 16. The presser 10 further includes a holder 18 configured to hold areusable or disposable pressor filter to separate solids from extruded liquid. In embodiments, filters having filter pore sizes varying from 100 nm to 4 mm are used.

[0054] Another embodiment of the sample preparation accessory is miniaturized blender 20 (Figure 5) comprising a motor 22, a threaded sample container 24 which may be disposable or replaceable, a cooperating threaded sample container receiver 26, and a disposable or replaceable mixing blade assembly 28. The blender and blade can be made of metal, plastics, or their combination. Part of the blender can be disposable or reusable.

[0055] Cuvette holders: One embodiment of a cuvette holder 30 according to the disclosure is depicted in Figure 6. It comprises a heating block 32, magnetic stirrer 34, and a port 36 for one or more of an optional bandpass filter (BP), linear polarizer (LP), and / or neutral density (ND) filter for conditioning the excitation and detection light. In an embodiment, the cuvette holder 30 also has additional ports 38 for fiber coupled excitation and detection. The cuvette holder 30 further includes a receiver 40 for receiving a sample cuvette 42.

[0056] Sampling cuvettes: An embodiment of a sample cuvette 42 according to the disclosure is shown in Figure 7. The cuvette 42 in the depicted embodiment is defined by side walls 44, 46, 48, and 50, and a floor 52. Two sides of the external surface of the cuvette (sides 44 and 46 in the depicted embodiment) are coated with silver, aluminum, or other reflective coating layers known to the skilled artisan. The two optically transparent sides (48 and 50 in the depicted embodiment) are used for the fluorescence excitation and detection as depicted in Figure 7 which showed the metal coating enhances fluorescence signal. A cap 54 may be provided, in embodiments also including a reflective coating in a sample-facing surface thereof. In still another embodiment, the bottom 52 of the cuvette 42 may also be coated with the reflective layer. Still another embodiment (not shown) is a cuvette 42’ which, except for discrete areas configured to allow passage of excitation and detection light therethrough, are partially or completely coated with reflective materials to control entry into / exit from the cuvette 42’ of excitation and / or detection light.

[0057] Single sample kinetic FG analyzer: Particular devices have been designed for acquiring and analyzing the kinetic FG assay data for the biomarker quantifications. In one embodiment, a single sample kinetic FG analyzer (SSKFA) 56 is described, configured as per the name for analysis of a single sample at a time.

[0058] One embodiment of the SSKFA 56 is depicted in Figure 8. The SSKFA 56 includes one sample fluorescence detector 58 and one reference detector 60. An excitation source 62 includes, but not limited to lamp(s) (not shown) in combination with a monochromator or bandpass filter 64 for providing monochromatic light for excitation. In alternative embodiments, the excitation source 62 may utilize laser, light emitting diode, and other light sources with or without bandpass filter 64 to control the bandwidth of the excitation light. Other optional optical elements include a linear polarizer and a neutral density filter, all depicted representatively as reference numeral 64. All or part of the components are manually controlled or computer-controlled by way of software associated with a computing device 68.

[0059] The SSKFA 56 is configured and adapted to implement an excitation and detection method. Excitation light emitted by excitation source 62 is passed through a beam splitter or optical fiber coupler (shown as reference numeral 70). One portion of the excitation light goes through the sample solution held in cuvette holder 30 for excitation of fluorescence that will be detected with sample fluorescence detector 58, the other portion goes directly to reference detector 60 for monitoring the excitation intensity changes and correcting such changes on the detected fluorescence signal. Suitable fluorescence / reference detectors contemplated for use in the disclosed method include but are not limited to photodiodes and photomultiplier tubes (PMT) include photon counting PMT. The sample fluorescence detector 58 and reference detector 60 are synchronized for detection and data output. The time intervals between the consecutive kinetic data acquisition can vary from 1 ms to 1 mins. Data recorded includes reference intensity (Ir), the sample detector (Is), and / or, the ratio between Is / Ir. In this embodiment, no polarizers are used for either the sample excitation or detection. The data can be directly stored in computing device 68 or transmitted via wired or wireless means to a centralized control center (not shown) for data analysis and storage.

[0060] The FG analyzer method also contemplates linear polarization of the excitation and / or detection by disposing a linear polarizer (generally, ref num 64) along the excitation and / or detection optical path.

[0061] Optionally, the excitation and detection wavelength are adjustable by changing the monochromator wavelength, band pass filter wavelength, laser excitation wavelength. The excitation and detection light intensity can also be optionally independently adjustable by changing the wavelength bandwidths and / or inclusion of a neutral density filter (generally, refnum 64), and additional means known to the skilled artisan. The excitation bandwidth may vary from sub 1 nm to 30 nm, while the detection bandwidth may vary from 1 nm to 600 nm.

[0062] Another embodiment of an SSKFA 56a is shown in Figure 9. The SSKFA 56a further includes a transmittance detector 72. The signal acquisition from each of the illustrated detectors are synchronized for displacement and analysis. Optionally, a solvent reference cell 72 is placed between the beam splitter (BS) and the reference detector for applications where kinetic UV-vis intensity is needed.

[0063] Another embodiment of an SSKFA 56b is shown in the general scheme in Figure 10. It has a transmittance detector 72 and fluorescence detector 58, but no reference detector. In this configuration, the transmittance detector 72 serves as the reference detector for monitoring excitation intensity fluctuation.

[0064] Still another embodiment of the SSKFA 56c is depicted in Figure 11 which comprises four single element detectors. Two fluorescence detectors 58 are used for the detection of fluorescence in the same wavelength region, or at different regions. One fluorescence detector 58 monitors the fluorescence signal of an internal reference, the other fluorescence detector 58 monitors a kinetic fluorescence signal that depends on the kinetic FG reactions. In this configuration, one or both of reference detector 60 or transmission detector 72 can be optionally removed.

[0065] Multiplex kinetic FG analyzer: Multiplex kinetic fluorogenic analyzers refer to kinetic fluorogenic analyzers that allow two or more samples be analyzed simultaneously. Figure 12 shows an example design of a multiplex kinetic FG analyzer which includes a fiber splitter 70 that splits emitted excitation light to a plurality of cuvette holders 30 placed on a multi-well stir plate 76. Fluorescence detected in cuvette holders 30 is transmitted via detection optical fibers to a spectrometer 78, and the data gathered by the spectrometer analyzed by computing device 68.

[0066] Reflective fluorescence well-plates: Alternatively, the excitation signal may be passed through samples held in a multi-well fluorescence well plate 80 (see Figure 13). In this embodiment, the side walls of the well plate are made non-reflective, for example by coating with a black paint (Figure 13) to avoid “cross talk” or signal contamination between samples held in adjoining wells. A reflective metal coated well plate is disclosed in this subsection.

[0067] It will be understood that various details of the presently disclosed subject matter can be changed without departing from the scope of the subject matter disclosed herein. Furthermore,the foregoing description is for the purpose of illustration only, and not for the purpose of limitation. Obvious modifications and variations are possible in light of the above teachings. All such modifications and variations are within the scope of the appended claims when interpreted in accordance with the breadth to which they are fairly, legally and equitably entitled.

[0068] Reference

[0069] 1. Peng, W.; Athukorale, S.; Hu, J.; Cui, X.; Zhang, D., Kinetic Spectroscopic Quantification Using Two-Step Chromogenic and Fluorogenic Reactions: From Theoretical Modeling to Experimental Quantification of Biomarkers in Practical Samples. Anal. Chim. Acta 2021, 1153, 338293.

Claims

WHAT IS CLAIMED:

1. A method for kinetic fluorogenic (FG) quantification of a target biomarker in a sample, comprising: exposing a sample to an extraction reagent or solvent comprising water and a water- miscible organic solvent to provide a sample extract; obtaining a sample supernatant; admixing at least a portion of the supernatant with a reaction solvent to provide a reaction mixture; admixing at least a portion of the reaction mixture with a fluorogenic probe specific to the target biomarker to provide a fluorescent probe-target biomarker complex; and at predetermined time intervals, quantifying the probe-target biomarker complex by kinetic spectroscopic quantification by a two-step dual chromogenic (CG) and FG measurement.

2. The method according to claim 1, wherein the predetermined time intervals are from about 1 ms to about 1 min.

3. The method according to any one of claims 1 or 2 wherein the target biomarker is a reactive species selected from the group consisting of: reactive oxygen species, reactive sulfur species, reactive nitrogen species, and reactive chlorine species.

4. The method according to claim 3, wherein the reactive oxygen species is selected from the group consisting of hydrogen peroxide, superoxide, ozone, and malondialdehyde.

5. The method according to any one of claims 1 or 2, wherein the water-miscible organic solvent is selected from the group consisting of alcohol, dimethyl sulfoxide (DMSO), acetone, acetonitrile, and dissolved electrolytes.

6. The method according to claim 5, wherein the alcohol is selected from the group consisting of methanol, ethanol, propanol, and butanol.

7. The method according to any one of claims 1or 2, wherein the reaction solvent is selected from the group consisting of DMSO, propanol, ethanol, methanol, and acetonitrile.

8. The method according to any one of claims 1 or 2, wherein the fluorogenic probe is selected from the group of probes set forth in Table 1.

9. The method according to claim 1, wherein the sample is processed by a miniature blender after the step of exposing to the extraction reagent or solvent to provide the sample extract.

10. The method according to claim 9, wherein the sample is processed by passing through a miniature presser holding a filter having a filter pore size of from about 100 nm to about 4 mm.

11. The method according to claim 1, wherein the reaction mixture including the fluorescent probe-target biomarker complex is placed in a sampling cuvette having at least two sides coated with a reflective coating.

12. The method according to any one of claims 1 or 2, further including providing a fluorescence internal reference control to allow correction for fluorescence detection artifacts.

13. The method according to claim 12, wherein the step of quantifying is performed by a spectrofluorometer adapted to simultaneously monitor fluorescence from the fluorescence internal monitor and fluorescence from the fluorescent probe-target biomarker complex.

14. The method according to claim 13, wherein the spectrofluorometer is configured to quantify the target biomarker in a single sample.

15. The method according to claim 13, wherein the spectrofluorometer is configured to simultaneously quantify the target biomarker in a plurality of samples.

16. The method according to any one of claims 13-15, wherein the spectrofluorometer comprises an excitation source, a fluorescence detector, and a reference fluorescence detector.

17. The method according to any one of claims 13-15, wherein the spectrofluorometer further comprises one or more of a bandpass filter, a linear polarizer, and a neutral density filter for varying a wavelength of a light emitted by the excitation source and / or a light detected by the fluorescence detector and / or the reference fluorescence detector.

18. The method according to any one of claims 13-15, wherein the spectrofluorometer further comprises a beam splitter adapted to redirect a portion of an excitation light emitted by the excitation source to the fluorescence detector and a portion of the excitation light to the reference fluorescence detector.

19. The method according to claim 17, wherein the wavelength of the light emitted by the excitation source can be varied from about 1 nm to about 30 nm.

20. The method according to claim 17, wherein the wavelength of the light detected by the fluorescence detector and reference fluorescence detector can be varied from about 1 nm to about 600 nm.

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