Reagents, apparatus and accessories for the quantification of kinematic fluorescence generation

Abstract

A kinetic fluorescence generation (FG) quantitative method for targeting biomarkers in a sample includes the following steps: exposing the sample to an extraction reagent or solvent composed of water and a water-soluble organic solvent to obtain a sample extract; filtering to obtain a sample supernatant; mixing the supernatant with a reaction solvent to obtain a reaction mixture; mixing the reaction mixture with a fluorescent probe specifically targeting the biomarker to generate a fluorescent probe-target biomarker complex; and quantifying the probe-target biomarker complex using kinetic spectroscopy at predetermined time intervals by a two-step dual-colorimetric method (CG) and FG measurements.

Description

Related applications

[0001] This patent application claims priority to U.S. Provisional Application 63 / 541,245, filed September 28, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This disclosure relates, in one aspect, to a comprehensive system comprising a device, sampling accessories, and reagents designed for rapid kinetic fluorogenic quantification of chemical substances. Another aspect relates to the quantification of biomarkers / chemical reagents in samples, such as the quantification of malondialdehyde (MDA), a lipid peroxidation biomarker widely used in the food industry and biological research. The disclosed techniques are readily adaptable to a wide range of biomarkers and chemical reagents, demonstrating their versatility and broad applicability. Background Technology

[0003] Colorimetric reactions (CG) and fluorescence generation reactions (FG) have been widely used in chemical measurements and imaging. Currently, various commercially available and literature-reported colorimetric / fluorescent probes are available for quantitative chemical analysis. However, existing CG / FG-based quantitative chemical analysis primarily employs time-consuming equilibrium quantification methods, which are performed after the CG / FG reaction is complete. More efficient kinematic spectroscopy-based quantitative methods were first proposed in the 1970s, but are currently limited to a few types of CG / FG reactions and are used for studying samples with known compositions. Embodiments of this invention relate to apparatus, reagents, accessories, and methods for quantifying active substances using kinematic fluorescence generation.

[0004] In this article, "reactive species" refers to reagents that react with one or more chemical substances and that such reactions alter the fluorescence properties of a sample (e.g., spectral intensity, peak wavelength, anisotropy, and / or shelf life). Such reactive species include, but are not limited to, reactive oxygen species (e.g., hydrogen peroxide, superoxide, ozone, malondialdehyde (MDA)), reactive sulfur / nitrogen / chlorine compounds, chemical weapon reagents (e.g., nerve agents), and radioactive substances.

[0005] In a 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" In prior studies, MDAs have been used as a class of model active substances. This class of model active substances is also an MDA in this disclosure. However, it should be understood that the general design of the apparatus, fittings, reagents, and methods of use also applies to a variety of other active substance classes, provided that fluorescent probes available for their detection / quantification are currently available. Invention Overview

[0006] Details of one or more embodiments of the subject matter disclosed herein are given below. Modifications to the embodiments described herein, as well as other embodiments, will be apparent to those skilled in the art upon reading the information provided herein. The information provided herein, particularly the specific details of the exemplary embodiments described, is primarily for ease of understanding and should not be construed as limiting in any way. In case of any conflict, the description (including definitions) herein shall prevail.

[0007] While the terminology used herein should be readily understood by one of ordinary skill in the art, some definitions are provided for the purpose of explaining the subject matter disclosed herein.

[0008] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0009] All patents, patent applications, published applications and publications, GenBank sequences, databases, websites and other published materials are incorporated herein by reference in their entirety.

[0010] Whenever a URL or other similar identifier or address is mentioned, it should be understood that such identifiers are subject to change, and specific information on the Internet may be updated or disappear at any time, but equivalent information can be found through Internet searches. Mentioning such information indicates that it is available and publicly disseminated.

[0011] Although any methods, apparatuses and materials similar to or equivalent to those described herein may be used to practice or test the subject matter disclosed herein, representative methods, apparatuses and materials have been described herein.

[0012] In this document, the naming of compounds (including organic compounds) may follow common names, IUPAC, IUBMB, or CAS nomenclature guidelines. When one or more stereochemical features are present, the Cahn-Ingold-PreloG stereochemical rule can be used to specify stereochemical priority, E / Z nomenclature, etc. Those skilled in the art, knowing the compound name, can easily determine its structure—either through systematic deduction using nomenclature guidelines or with the aid of commercially available software (such as CHEMDRAW™, produced by Cambridgesoft).

[0013] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art. While any methods, apparatuses, and materials similar to or equivalent to those described herein may be used to practice or test the subject matter disclosed herein, representative methods, apparatuses, and materials are currently described herein.

[0014] Unless otherwise stated, all figures used in this specification and claims indicating quantities of components, reaction conditions, and other properties should be understood to include the word "about" in all cases. Therefore, unless otherwise stated, the numerical parameters listed in this specification and claims are approximate values, which may vary depending on the properties desired by the invention.

[0015] As described in the specification and appended claims, the singular forms “a” and “the” include plural references unless the context clearly specifies otherwise. For example, “functional group,” “alkyl”, or “residue” all include mixtures of two or more such functional groups, alkyl groups, or residues.

[0016] A range may be expressed herein as starting “about” of a particular value and / or ending “about” of another particular value. When such a range is expressed, the other aspect includes starting from and / or ending at that particular value. Similarly, when “about” is used as an antecedent to represent a value as an approximation, it should be understood that the particular value constitutes another aspect. It should also be understood that the two endpoints of each range are significant, whether related to or independent of the other endpoint. It should also be understood that several values ​​are disclosed herein, and each value is disclosed not only as itself but also as “about” that particular value. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It should also be understood that every unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0017] In this document, the terms “optional” or “optionally” mean that an event or situation described below may or may not occur, and the description includes examples of the event or situation occurring and examples of it not occurring.

[0018] As used herein, the term "kit" refers to a collection of at least two components that constitute the kit. These components together form a functional unit for a specified purpose. The components may be packaged together or separately. For example, a kit containing instructions for use may include the instructions for use as well as other individual components. Alternatively, the instructions for use may be provided as a separate component in paper or electronic form (e.g., stored on a computer-readable storage device, downloaded from an internet website, or as a recorded presentation).

[0019] Some of the materials, compounds, compositions, and components disclosed herein are available from commercial sources or can be easily synthesized using techniques generally known to those skilled in the art. For example, the starting materials and reagents used to prepare the compounds and compositions described herein may be obtained from suppliers such as Aldrich Chemical Co. (Milwaukee, Wis.), AcrosOrganics (Morris Plains, NJ), Fisher Scientific (Pittsburgh, Pa.), or Sigma (St. Louis, Mo.), or may be prepared by methods known to those skilled in the art according to the steps described in the literature, such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1–17 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1–5 and Supplements (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] Currently, there are many commercially available MDA detection kits, most of which use the well-established thiobarbituric acid (TBA) as a chromogenic or fluorescent probe. However, these TBA-based MDA quantification methods rely on balanced quantification, which is time-consuming (usually requiring one hour or more), labor-intensive, and requires a relatively large sample volume. Furthermore, some techniques in this type of method require expensive equipment for sample processing and measurement.

[0021] This invention offers numerous advantages over existing technologies. For instance, unlike conventional methods which rely on equilibrium fluorescence quantification (which requires measuring spectral intensity after the fluorescence reaction is complete), this invention employs kinematic fluorescence-based MDA quantification. This novel method is based on monitoring the rate of change in spectral intensity during the fluorescence reaction.

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

[0023] Embodiments of the present invention involve using TBA (10 mM or higher) in far greater amounts than the MDA concentration expected during the quantification process.

[0024] In some embodiments, the present invention incorporates an internal fluorescence reference to correct for measurement uncertainties caused by fluctuations in excitation power and emission detection intensity.

[0025] In other embodiments, the fluorescence spectrometer used is capable of simultaneously monitoring the fluorescence of the internal reference and the TBA / MDA reaction product.

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

[0027] In addition, in some cases, TBA reaction solutions can be conveniently pre-packaged for direct use before the expiration date. Attached Figure Description

[0028] Figure 1 A known MDA probe and its implementation of a dual CG / FG reaction with MDA are shown.

[0029] Figure 2Display: (A) Photograph and UV-Vis absorption spectrum of the obtained beef extract. (B) Kinematic fluorescence generation curves of reaction solutions containing TBA and beef extract after adding different amounts of MDA standards. (C) Raw acquisition data and linear curve fitting of kinematic fluorescence generation in the linear time progression region. The kinematic data in (B) and (C) have been shifted for clarity. (D) Linear curve fitting of quantitative analysis using standards added for MDA concentrations.

[0030] Figure 3 The effects of solvent on detection rate and sensitivity are shown. (A) Kinematic fluorescence generation spectra of TBA and 50 nM MDA reaction solution at 60°C in water and in a DMSO / H2O (1:1, v:v) mixed solvent. TBA is present at a saturated concentration at room temperature in water and in the DMSO / H2O mixed solvent. (B) Kinematic UV-Vis extinction spectra of 1 μM MDA and 205.2 mM TBA in different DMSO / H2O mixed solvents (volume fractions are shown in the figure). (C) Linear region of the kinematic UV-Vis extinction data in Figure (B). (D) Fluorescence spectra of 1 μM TBA-MDA in DMSO / H2O mixed solvents with different DMSO volume fractions. The inset shows the fluorescence peak intensity as a function of DMSO volume fraction.

[0031] Figure 4 A schematic diagram of a press for extracting liquid containing biomarkers from tissue samples is shown. The press handle can be operated manually or mechanically.

[0032] Figure 5 A miniature mixer for extracting liquid containing biomarkers from tissue samples is shown.

[0033] Figure 6 An example of a cuvette holder used for sample analysis is shown.

[0034] Figure 7 Showing: (Top left) A schematic diagram of a reflective fluorescent cuvette. (Top right) Fluorescent cuvettes: one with a reflective metallic coating, one without. (Bottom) Comparison of fluorescence spectra of the same solution using cuvettes with and without reflective coatings.

[0035] Figure 8 This image shows an example of a single-sample kinematics FG analyzer, including: BP: bandpass filter; FC: fiber optic coupler for splitting light into two parts; LP: linear polarizer; ND: neutral density filter. Dashed lines indicate optional computational control or remote wireless control.

[0036] Figure 9Another example of a single-sample kinematic FG analyzer is shown, including: BP: bandpass filter; FC: fiber optic coupler for splitting light into two parts; LP: linear polarizer; ND: neutral density filter. Dashed lines indicate optional computer control or remote wireless control.

[0037] Figure 10 Another example of a single-sample kinematic FG analyzer is shown, including: BP: bandpass filter; FC: fiber optic coupler for splitting light into two parts; LP: linear polarizer; ND: neutral density filter. Dashed lines indicate optional computational control or remote wireless control.

[0038] Figure 11 Another example of a single-sample kinematic FG analyzer is shown, including: BP: bandpass filter; FC: fiber optic coupler for splitting light into two parts; LP: linear polarizer; ND: neutral density filter. Dashed lines indicate optional computer control or remote wireless control.

[0039] Figure 12 This paper demonstrates the design of a multiplex kinematic fluorescence generation analyzer capable of simultaneously analyzing eight samples. Applications include the quantitative analysis of various chemical and biological substances in the same extract, as well as the quantitative analysis of the same substances in different extracts.

[0040] Figure 13 Examples of the fluorescent multiwell plates used in this paper are shown: (Top) A conventional fluorescent multiwell plate with black walls surrounding the sample solution. (Bottom) A schematic diagram of a reflective fluorescent multiwell plate, wherein the microplate is constructed by inserting reflective holes with an external coating. This design enhances fluorescence excitation and detection by reflecting excitation and emission photons that would be absorbed by conventional multiwell plates, thereby improving fluorescence sensitivity. Invention Details

[0041] Details of one or more embodiments of the subject matter disclosed herein are shown in this document. Modifications to the embodiments described herein, as well as other embodiments, will be apparent to those skilled in the art upon reading the information provided herein. The information provided herein, particularly the specific details of the exemplary embodiments described, is primarily for ease of understanding and should not be construed as limiting in any way. Furthermore, while we believe that those skilled in the art will fully understand the terminology used herein, definitions are provided to facilitate the interpretation of the subject matter disclosed herein.

[0042] Reagents: The detection using a commercial MDA detection kit involves a fluorescent reaction in water. Figure 1 Examples of these reactions are shown. The most commonly used fluorescent probe for MDA detection is 2-thiobarbituric acid (TBA). Figure 1The reaction 1 in the text. Our recently reported kinematic fluorescence-generated MDA detection was also performed in water. 1 In this disclosure, "reagent" refers to chemicals or chemical solutions used in the detection of active substances. These reagents can be categorized into sample preparation / extraction reagents, fluorescent reaction reagents, and post-reaction sample / cuvette preparation reagents.

[0043] Sample processing / extraction reagents: Sample extraction refers to the process of separating active substances from target materials. Figure 2 Example A illustrates a case where the beef extract in the vial is obtained by mixing minced beef with water and then filtering it through paper. In this specific embodiment, the sample extraction reagent consists only of water. Other examples of sample extraction reagents include mixtures of water with organic solvents (e.g., but not limited to alcohols (methanol, ethanol, propanol, butanol), dimethyl sulfoxide (DMSO), acetone, acetonitrile, and dissolved electrolytes), or any other agent known to those skilled in the art capable of promoting the aggregation / coagulation of macromolecules (e.g., proteins, DNA, and fibers), thereby facilitating the separation of the liquid sample extract from the sample matrix.

[0044] Fluorescent reaction reagents include kinematic FG probes, solvents, catalysts, and any other chemicals known to those skilled in the art that contribute to the control of reaction rate, photostability of fluorescent products, and / or sensitivity and reliability of detection.

[0045] The reaction solvent refers to the solvent in which the fluorescence reaction occurs. In this paper, the inventors unexpectedly discovered that quantitative analysis of fluorescent MDA in an organic solvent / water mixture is more sensitive and efficient than in water alone. Taking the quantitative analysis of MDA in beef as a non-limiting example, the sample extraction reagent used to extract MDA can be water or a water-soluble organic solvent, or a mixture thereof (wherein the solvents are mixed in a 0%-100% ratio). These organic solvents include, but are not limited to, dimethyl sulfoxide (DMSO), propanol, ethanol, methanol, and acetonitrile. The addition of an organic solvent to TBA-based quantitative analysis of MDA improves both the efficiency and sensitivity of the analysis. For example, the reaction of MDA with TBA dissolved in a DMSO / H2O (volume ratio 1:1) solvent is 3 times faster than the reaction in 100% water, while the sensitivity of MDA analysis using said mixed solvent is 5 times higher than that in water alone.

[0046] The table below lists examples of FG probes, solvents, and catalysts suitable for, but not limited to, the methods disclosed herein. These fluorophore-associated reagents have previously been used in equilibrium-based analytical methods. However, the inventors have extended their use to the quantification of kinematic fluorescence generation described herein.

[0047]

[0048]

[0049]

[0050]

[0051]

[0052] The post-processing reagents used in this application refer to solvents and / or chemicals known to those skilled in the art that help to disinfect and clean sample processing equipment (including extraction and reaction vessels) before disposal or reuse.

[0053] One aspect of this disclosure is its surprising ability to reuse known fluorescent probes (including, but not limited to, those listed in the table above). These fluorophores have been used in balance-based detection, and this invention extends their application to kinematic fluorescence generation detection.

[0054] Accessories: In this disclosure, “accessories” refers to reusable or disposable supplies required for sample preparation, testing, and post-testing processing.

[0055] Sample extraction accessories: Besides high detection efficiency and ease of operation, another key advantage of kinematic FG quantification is its low sample consumption. However, currently, obtaining sample extracts (i.e., solutions containing target biomarkers) from real-world hard or soft solid samples (with a certain volume) still faces many challenges. Current methods for obtaining sample extracts from tissues include using blenders, probe-type ultrasonic instruments, and ultrasonic homogenizers.

[0056] The sample preparation accessories described herein are designed to improve the efficiency, reliability, and convenience of sample preparation from solid tissue and solution samples, and to reduce sample size and / or sample matrix interference in kinematic FG quantification.

[0057] An example of component manufacturing is a micro-press ( Figure 4 The filter membrane, optionally made of plastic and / or metal, includes a sample chamber 12, a piston 14, and a screen 16. The press 10 also includes a support 18 for securing a reusable or disposable press filter membrane to separate solids from the extruded liquid. In some embodiments, the filter membrane pore size ranges from 100 nm to 4 mm.

[0058] Another example of a sample preparation accessory is a micro stirrer 20 ( Figure 5The mixer includes a motor 22, a disposable or replaceable threaded sample cup 24, a matching threaded sample cup receiver 26, and a disposable or replaceable mixing blade assembly 28. The mixer and blades may be made of metal, plastic, or a combination of both. Some parts of the mixer may be disposable or reusable.

[0059] cuvette holder: Figure 6 An example of a cuvette holder 30 according to this disclosure is shown. It includes a temperature control module 32, a magnetic stirrer 34, and a port 36 for connecting one or more optional bandpass filters (BP), linear polarizers (LP), and / or neutral density filters (ND) to modulate the excitation and detection light. In one embodiment, the cuvette holder 30 also has an additional port 38 for fiber-optic coupled excitation and detection. The cuvette holder 30 also includes a receiver 40 for receiving a sample cuvette 42.

[0060] Sampling cuvette: Figure 7 An example of a sampling cuvette 42 according to this disclosure is shown. The cuvette 42 in the illustrated example comprises sidewalls 44, 46, 48, and 50 and a bottom surface 52. The two sides of the outer surface of the cuvette (sidewalls 44 and 46 in the illustrated example) are coated with silver, aluminum, or other reflective coatings known to those skilled in the art. Figure 7 As shown, two optically transparent sidewalls (sidewalls 48 and 50 in the illustrated example) are used for fluorescence excitation and detection, and the figure shows that the metallic coating enhances the fluorescence signal. A lid 54 may be provided, and in some embodiments, the sample-facing surface of the lid is also coated with a reflective coating. In another embodiment, the bottom surface 52 of the cuvette 42 may also be coated with a reflective layer. Another example (not shown) is a cuvette 42', which is partially or entirely coated with a reflective material, except for discrete areas that allow excitation and detection light to pass through, to control the entry and / or exit of excitation and / or detection light into and out of the cuvette 42'.

[0061] Single-sample kinematic FG analyzer: Devices have been designed specifically for acquiring and analyzing kinematic FG detection data for quantitative analysis of biomarkers. In one embodiment, a single-sample kinematic FG analyzer (SSKFA) 56 is described, which, as the name suggests, analyzes one sample at a time.

[0062] Figure 8An example of an SSKFA 56 is shown. The SSKFA 56 includes a sample fluorescence detector 58 and a reference detector 60. The excitation source 62 includes, but is not limited to, a lamp (not shown) and a monochromator or bandpass filter 64 for providing monochromatic excitation light. In other embodiments, the excitation source 62 may utilize a laser, a light-emitting diode, or other light sources, and the bandwidth of the excitation light may or may not be controlled using a bandpass filter 64. Other optional optical elements include linear polarizers and neutral density filters, all of which are indicated by reference numeral 64. All or some components may be manually controlled or computer-controlled via software associated with a computing device 68.

[0063] The SSKFA 56, after configuration and adaptation, enables an excitation detection method. Excitation light emitted from excitation source 62 passes through a beam splitter or fiber coupler (indicated by reference numeral 70 in the figure). A portion of the excitation light passes through the sample solution contained in cuvette holder 30 to excite fluorescence, which is detected by sample fluorescence detector 58; another portion of the excitation light directly enters reference detector 60 to monitor changes in excitation light intensity and correct for such changes in the detected fluorescence signal. Fluorescence / reference detectors suitable for the method described herein include, but are not limited to, photodiodes and photomultiplier tubes (PMTs), including photon-counting PMTs. Sample fluorescence detector 58 and reference detector 60 perform detection and data output synchronously. The time interval between two consecutive kinematic data acquisitions can vary between 1 millisecond and 1 minute. Recorded data includes the reference intensity (I0). r ), Sample detector intensity (I) s ), and / or I s / I r Ratio. In this embodiment, no polarizer is used for sample excitation and detection. Data can be stored directly in computing device 68, or transmitted via wired or wireless means to a central control center (not shown) for data analysis and storage.

[0064] The FG analyzer method also considers achieving linear polarization of excitation and / or detection by placing a linear polarizer (typically, reference 64) in the excitation and / or detection optical path.

[0065] Optionally, the excitation and detection wavelengths can be adjusted by changing the monochromator wavelength, the bandpass filter wavelength, and the laser excitation wavelength. The excitation and detection light intensities can be independently adjusted by changing the wavelength bandwidth and / or adding a neutral density filter (typically, reference 64), as well as other methods known to those skilled in the art. The excitation bandwidth can vary from less than 1 nm to 30 nm, while the detection bandwidth can vary from 1 nm to 600 nm.

[0066] Another example, SSKFA 56a, is shown in Figure 9 The SSKFA 56a also includes a transmission detector 72. Signal acquisition from each detector shown in the figure is synchronized for displacement measurement and analysis. Optionally, a solvent reference cell 72 is placed between the beam splitter (BS) and the reference detector for situations requiring kinematic UV-Vis intensity.

[0067] Another example, the overall schematic diagram of SSKFA 56b, can be found here. Figure 10 It has a transmission detector 72 and a fluorescence detector 58, but no reference detector. In this configuration, the transmission detector 72 is used as a reference detector to monitor fluctuations in the excitation light intensity.

[0068] Another example is SSKFA 56c. Figure 11 It contains four single-factor detectors. Two fluorescence detectors 58 are used to detect fluorescence in the same wavelength region or different regions. One fluorescence detector 58 monitors the fluorescence signal of an internal reference, and the other fluorescence detector 58 monitors the kinematic fluorescence generation signal (depending on the kinematic FG response). In this configuration, one or both of the reference detector 60 or the transmission detector 72 can be selectively removed.

[0069] Multiple kinematic fluorescence (FG) analyzer: A multiple kinematic fluorescence generator is a kinematic fluorescence generator that can simultaneously analyze two or more samples. Figure 12 The design of a multi-kinematic fluorescence (FG) analyzer is shown, which includes an optical fiber splitter 70 that splits the emitted excitation light into a plurality of cuvette holders 30 placed on a porous stirring plate 76. Fluorescence signals detected in the cuvette holders 30 are transmitted via detection optical fibers to a spectrometer 78, and the data acquired by the spectrometer is analyzed by a computing device 68.

[0070] Reflective fluorescent multi-well plate: Alternatively, the excitation signal can pass through the sample placed in the multi-well fluorescent plate 80 (see...). Figure 13 In this embodiment, the sidewalls of the perforated plate are made non-reflective, for example, coated with a black paint. Figure 13 To avoid crosstalk or signal contamination between samples in adjacent wells, this subsection discloses a reflective metal-coated porous plate.

[0071] It should be understood that various details of the subject matter disclosed herein may be modified without departing from the scope of the subject matter disclosed herein. Furthermore, the above description is for illustrative purposes only and not for limitation. Obvious modifications and variations can be made under the guidance of the foregoing teachings. All such modifications and variations, when interpreted to their reasonable, legal, and equitable extent, fall within the scope of the appended claims.

[0072] References

[0073] 1. Peng, W.; Athukorale, S.; Hu, J.; Cui, X.; Zhang, D., KineticSpectroscopic Quantification Using Two-Step Chromogenic and FluorogenicReactions: From Theoretical Modeling to Experimental Quantification ofBiomarkers in Practical Samples. Anal. Chim. Acta 2021, 1153, 338293.

Claims

1. A method for quantifying the kinematic fluorescence (FG) generation of target biomarkers in a sample, including: Expose the sample to an extraction reagent or solvent containing water and water-soluble organic solvents to provide a sample extract; Obtain the sample supernatant; At least a portion of the supernatant is mixed with the reaction solvent to provide the reaction mixture; At least a portion of the reaction mixture is mixed with a fluorescent probe specific to the target biomarker to provide a fluorescent probe-target biomarker complex; and The probe-target biomarker complex was quantified by kinematic spectroscopy quantification at predetermined time intervals using two-step dual-colorimetric (CG) and fG measurements.

2. The method of claim 1, wherein the predetermined time interval is from 1 millisecond to 1 minute.

3. The method of claim 1 or 2, wherein the target biomarker is an active substance selected from the group consisting of reactive oxygen species, reactive sulfur species, reactive nitrogen species, and reactive chlorine species.

4. The method of claim 3, wherein the reactive oxygen species are selected from hydrogen peroxide, superoxide, ozone and malondialdehyde.

5. The method of claim 1 or 2, wherein the water-soluble organic solvent is selected from alcohols, dimethyl sulfoxide (DMSO), acetone, acetonitrile, and dissolved electrolytes.

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

7. The method of claim 1 or 2, wherein the reaction solvent is selected from DMSO, propanol, ethanol, methanol and acetonitrile.

8. The method of any one of claims 1 or 2, wherein the fluorescent probe is selected from the probe group listed in Table 1.

9. The method of claim 1, wherein, After exposing the sample to the extraction reagent or solvent, the sample is processed with a micro-stirrer to provide the sample extract.

10. The method of claim 9, wherein, The samples were processed by passing them through a micropressor equipped with filter membranes with pore sizes ranging from 100 nanometers to 4 millimeters.

11. The method of claim 1, wherein, The reaction mixture containing the fluorescent probe-target biomarker complex was placed in a sampling cuvette with a reflective coating on at least two sides.

12. The method of any one of claims 1 or 2, further comprising providing an internal fluorescence reference control to allow correction of fluorescence detection artifacts.

13. The method of claim 12, wherein, The quantitative steps are performed by a fluorescence spectrometer adapted to simultaneously monitor fluorescence from both the fluorescent internal monitor and the fluorescent probe-target biomarker complex.

14. The method of claim 13, wherein, The fluorescence spectrometer is configured to quantify target biomarkers in a single sample.

15. The method of claim 13, wherein, The fluorescence spectrometer is configured to simultaneously quantify target biomarkers in multiple samples.

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

17. The method according to any one of claims 13-15, wherein, The fluorescence spectrometer also includes one or more bandpass filters, linear polarizers, and neutral density filters for changing the wavelength of light emitted from the excitation source and / or changing the wavelength of light detected by the fluorescence detector and / or reference fluorescence detector.

18. The method according to any one of claims 13-15, wherein, The fluorescence spectrometer also includes a beam splitter adapted to redirect a portion of the excitation light emitted from the excitation source to the fluorescence detector and a portion of the excitation light to the reference fluorescence detector.

19. The method of claim 17, wherein the wavelength of the light emitted by the excitation source can vary between 1 nm and 30 nm.

20. The method of claim 17, wherein the wavelength of the light detected by the fluorescence detector and the reference fluorescence detector can vary between 1 nm and 600 nm.

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