Transducers, nanoparticle transducer devices and systems, and related methods of use

Abstract

This invention describes transducers, kits, systems, and methods for determining the concentration of an analyte. In one embodiment, the transducer comprises a chromophore and an enzyme physically associated with the chromophore. In one embodiment, the transducer is configured to catalyze a reaction comprising a plurality of reaction elements. In one embodiment, the plurality of reaction elements comprises one or more reactants and one or more products, the one or more reactants comprising the analyte. In one embodiment, the amount of fluorescence emitted from the chromophore is determined by the concentration of one of the plurality of reaction elements.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of co-pending U.S. Provisional Patent Application No. 63 / 012,002, filed April 17, 2020, which is hereby incorporated herein by reference in its entirety. Background Technology

[0003] Monitoring fluid metabolite levels is a crucial part of managing disease and injury. Examples include phenylalanine for assessing phenylketonuria (PKU), glucose for managing diabetes, leucine for monitoring maple syrup urine disease, lactate for assessing tissue oxygenation, tyrosine for detecting hypertyrosinemia, glutamate for assessing ischemic stroke, and alpha-ketoglutarate for monitoring non-alcoholic fatty liver disease (NAFLD). Biosensors for measuring metabolites of interest have significantly improved the quality of life for both patients and caregivers. In recent years, there has been a growing demand in the medical diagnostics and healthcare management fields for reliable, easy-to-use, and cost-effective metabolite monitors suitable for the point of care (POC). However, technical hurdles and challenges still hinder the commercialization of some disease-related metabolite biosensors. These challenges may include multiplexing monitoring, specificity, portability, operability, and long-term stability.

[0004] Recently, semiconductor polymer dots (P-dots) have attracted considerable attention in interdisciplinary research across materials science, biology, and medicine. Compared to miniature fluorescent dyes and inorganic semiconductor quantum dots (Q-dots), P-dots exhibit highly desirable properties, including high brightness, fast emission rates, large absorption cross-sections, excellent photostability, non-toxicity, and a variety of surface modification options. These outstanding properties make them widely applicable in cell labeling, in vivo imaging, single-particle tracking, drug / gene delivery, and cancer therapy. Furthermore, small-scale biosensors based on P-dots have been developed, including biosensors for pH, temperature, metal ions, oxygen, and glucose.

[0005] However, to date, routine monitoring of reactions using the P-point requires a covalent bond between the enzyme and the P-point. This covalent bond limits how the P-point is prepared and deployed when determining analyte concentrations. Summary of the Invention

[0006] To address these and related challenges, this disclosure provides transducers, such as nanoparticle transducers, nanoparticle transducer devices and systems, and related methods of use. In some embodiments, and as discussed further in detail herein, enzymes are physically associated with P-point and / or chromophore polymers, such as when the enzyme and P-point and / or enzyme and chromophore polymers are dispersed in a common solvent, coupled to a common substrate, coupled together, encapsulated together in hydrogel beads, etc.

[0007] Therefore, in one aspect, this disclosure provides a nanoparticle transducer for measuring analyte concentration, the nanoparticle transducer comprising: nanoparticles including chromophores; and an NADH-dependent or NADPH-dependent enzyme coupled to the nanoparticles and configured to catalyze a reaction comprising a plurality of reaction elements; wherein the plurality of reaction elements comprises one or more reactants and one or more products, the one or more reactants comprising an analyte, and wherein the amount of fluorescence emitted from the chromophore is determined by the concentration of the reaction element among the plurality of reaction elements.

[0008] On the other hand, this disclosure provides a transducer substrate for measuring analyte concentration, the transducer substrate comprising: nanoparticles including a chromophore coupled to the substrate; and an enzyme coupled to the substrate and configured to catalyze a reaction comprising a plurality of reaction elements; wherein the plurality of reaction elements comprises one or more reactants and one or more products, the one or more reactants comprising an analyte, and wherein the amount of fluorescence emitted from the chromophore is determined by the concentration of the reaction element among the plurality of reaction elements.

[0009] On the other hand, this disclosure provides a kit for measuring analyte concentration, the kit comprising: nanoparticles including chromophores; and an enzyme physically associated with the nanoparticles and configured to catalyze a reaction comprising a plurality of reaction elements; wherein the plurality of reaction elements comprises one or more reactants and one or more products, the one or more reactants comprising an analyte, and wherein the amount of fluorescence emitted from the chromophore is determined by the concentration of the reaction element among the plurality of reaction elements.

[0010] On the other hand, this disclosure provides a transducer for measuring analyte concentration, the transducer comprising: a chromophore comprising a semiconductor chromophore polymer; and an enzyme physically associated with the semiconductor chromophore polymer and configured to catalyze a reaction comprising a plurality of reaction elements; wherein the plurality of reaction elements comprises one or more reactants and one or more products, the one or more reactants comprising an analyte, and wherein the amount of fluorescence emitted from the chromophore is determined by the concentration of the reaction element among the plurality of reaction elements.

[0011] On one hand, this disclosure provides a system for measuring analyte concentration, the system comprising: a nanoparticle transducer according to any embodiment described herein, a transducer substrate according to any embodiment described herein, a kit according to any embodiment described herein, or a transducer according to any embodiment described herein; an irradiation source configured to irradiate the chromophore of the nanoparticle transducer, the transducer substrate, the kit, or the transducer to induce fluorescence from the chromophore; a photodetector configured to generate a signal based on the fluorescence from the chromophore; and a controller operatively coupled to the irradiation source and the photodetector, and including logic that, when executed by the controller, causes the system to perform operations including: irradiating the chromophore with the irradiation source; and determining the concentration of the analyte based on the signal from the photodetector.

[0012] On one hand, this disclosure provides a method for measuring the concentration of an analyte in a fluid, the method comprising: contacting the fluid with a P-point and an NADH-dependent or NADPH-dependent enzyme, the P-point comprising a chromophore, the NADH-dependent or NADPH-dependent enzyme being coupled to the P-point, the NADH-dependent or NADPH-dependent enzyme being configured to catalyze a reaction comprising a plurality of reaction elements, wherein the plurality of reaction elements comprises one or more reactants and one or more products, the one or more reactants comprising the analyte, and wherein the amount of fluorescence emitted from the chromophore is determined by the concentration of the reaction elements among the plurality of reaction elements; irradiating the P-point to induce fluorescence from the P-point; measuring the fluorescence from the P-point; and determining the concentration of the analyte based on the measured fluorescence.

[0013] On one hand, this disclosure provides a method for measuring the concentration of an analyte in a fluid, the method comprising: contacting the fluid with a point P and an enzyme, the point P comprising a chromophore, the enzyme being physically associated with the point P, the enzyme being configured to catalyze a reaction comprising a plurality of reaction elements, wherein the plurality of reaction elements comprises one or more reactants and one or more products, the one or more reactants comprising the analyte, and wherein the amount of fluorescence emitted from the chromophore is determined by the concentration of the reaction element among the plurality of reaction elements; irradiating the point P to induce fluorescence from the point P; measuring the fluorescence from the point P; and determining the concentration of the analyte based on the measured fluorescence. In one embodiment, the fluorescence emitted from the chromophore defines a fluorescence ratio equal to the ratio of the amount of fluorescence emitted at a signal fluorescence wavelength to the amount of fluorescence emitted at a control fluorescence wavelength. In one embodiment, the fluorescence emitted from one or more chromophores defines a fluorescence ratio equal to the ratio of the amount of fluorescence emitted at a signal fluorescence wavelength to the amount of fluorescence emitted at a control fluorescence wavelength. In one embodiment, the fluorescence ratio is determined by the concentration of a fluid component or fluid element.

[0014] On one hand, this disclosure provides a method for measuring the concentration of an analyte in a fluid, the method comprising: contacting the fluid with a chromophore and an enzyme, the chromophore comprising a semiconductor chromophore polymer, the enzyme being physically associated with the chromophore, the enzyme being configured to catalyze a reaction comprising a plurality of reaction elements, wherein the plurality of reaction elements comprises one or more reactants and one or more products, the one or more reactants comprising the analyte, and wherein the amount of fluorescence emitted from the chromophore is determined by the concentration of the reaction elements among the plurality of reaction elements; irradiating the chromophore to induce fluorescence from the chromophore; measuring the fluorescence from the chromophore; and determining the concentration of the analyte based on the measured fluorescence.

[0015] This summary provides a simplified description of selected concepts, which will be further described in the detailed description below. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to help determine the scope of the claimed subject matter. Attached Figure Description

[0016] The foregoing aspects and many accompanying advantages of the invention will become more readily and better understood when taken in conjunction with the accompanying drawings and the following detailed description, wherein:

[0017] Figure 1A This is a transmission electron microscope (TEM) image of the PFBT P point according to an embodiment of the present disclosure;

[0018] Figure 1BThe figure shows, graphically, measurements obtained by dynamic light scattering (DLS) according to embodiments of the present disclosure. Figure 1A The size distribution of point P;

[0019] Figure 1C The zeta potentials of six different P points according to embodiments of the present disclosure are illustrated graphically, from left to right: PFO, PDHF, PFBT, PFBTTBT, PFTBT, and DPA-CNPPV P points;

[0020] Figure 1D Photographs of the P-point solution according to embodiments of the present disclosure under white light (top) and 365nm ultraviolet light irradiation (bottom);

[0021] Figure 1E and 1F The absorption (1E) and emission (1F) spectra of PFO, PDHF, PFBT, PFBTTBT, PFTBT and DPA-CNPPV P points according to embodiments of the present disclosure are shown graphically.

[0022] Figure 2A The embodiments of the present disclosure are illustrated graphically using λ. ex Fluorescence at the P-point of PFO obtained by excitation at 380 nm with increased NADH concentration;

[0023] Figure 2B The embodiments of the present disclosure are illustrated graphically using λ. ex Fluorescence at the P-point of PDHF obtained by excitation at 380 nm with increased NADH concentration;

[0024] Figure 2C The embodiments of the present disclosure are illustrated graphically using λ. ex Fluorescence at the P-point of PFBT with increased NADH concentration obtained by excitation at 330 nm;

[0025] Figure 2D The embodiments of the present disclosure are illustrated graphically using λ. ex Fluorescence at the PFO P-point obtained by excitation at 380 nm with increased PFBTTBT concentration;

[0026] Figure 2E The embodiments of the present disclosure are illustrated graphically using λ. ex Fluorescence at the P-point of PFTBT with increased NADH concentration obtained by excitation at 380 nm;

[0027] Figure 3AThe DPA-CNPPV P point (λ) is based on an embodiment of this disclosure in the presence of physiologically relevant NADH in the range (0-2 mM). ex The emission spectrum of (385nm) is shown.

[0028] Figure 3B The ratio calibration curves (R / R0; R=1) according to embodiments of the present disclosure are shown graphically. 458 nm / I 627 nm R0 indication Figure 3A (The ratio of fluorescence intensity at point P);

[0029] Figure 3C The following is a graphical illustration of embodiments according to the present disclosure in the presence of high NADH concentrations (2-10 mM). Figure 3A DPA-CNPPV P point (λ) ex The emission spectrum of (385nm) is shown.

[0030] Figure 3D The ratio calibration curves (R / R0; R=1) according to embodiments of the present disclosure are shown graphically. 458 nm / I 627 nm R0 indication Figure 3C (The ratio of fluorescence intensity at point P);

[0031] Figure 3E An embodiment according to the present disclosure is illustrated graphically. Figure 3A The photostability of the DPA-CNPPV P-point under 385nm light excitation for 30 minutes;

[0032] Figure 3F An embodiment according to the present disclosure is illustrated graphically. Figure 3A The response curve of DPA-CNPPV P point to NADH (1mM) in aqueous suspension;

[0033] Figure 3G The selectivity of the DPA-CNPPV P point according to embodiments of the present disclosure in the presence of various potentially interfering biologically relevant analytes (1 mM): (1) water; (2) NADH; (3) NAD + (4) Glucose; (5) H2O2; (6) Lactate; (7) Citrate; (8) Na + (9)K + (10)Ca 2+ (11)Mg 2+ (12)Cl - ;

[0034] Figure 3H The emission spectra of the DPA-CNPPV P point according to embodiments of the present disclosure are shown graphically in the presence of various potentially interfering biologically relevant analytes (1 mM): (1) water; (2) NADH; (3) NAD. + (4) Glucose; (5) H2O2; (6) Lactate; (7) Citrate; (8) Na + (9)K + (10)Ca 2+ (11)Mg 2+ (12)Cl - ;

[0035] Figure 3I The reversibility of the response of the DPA-CNPPV P point to NADH (1mM) according to an embodiment of the present disclosure is illustrated graphically.

[0036] Figure 3J The emission spectra of DPA-CNPPV P point and NADH excited at 385 nm under 0 mM and 2 mM NADH according to embodiments of the present disclosure are shown.

[0037] Figure 3K According to embodiments of this disclosure Figure 3J A photograph of point P in a solution irradiated with 365nm UV light;

[0038] Figure 3L An embodiment according to the present disclosure is illustrated graphically. Figure 3J The fluorescence response of point P to NADH and NADPH, wherein the DPA-CNPPV / PSMA titrated with NADH, NADPH, NAD+ and NADP+ showed a fluorescence response of point P to NADH and NADPH only, indicating that NAD+ and NADP+ do not quench the emission of point P and that it does not emit at 458 nm under UV irradiation.

[0039] Figure 4A These are combined bright-field and fluorescence microscopy images of PFBT P-point labeled MCF-7 cells treated with phosphate-buffered saline (PBS) according to embodiments of the present disclosure.

[0040] Figure 4B An embodiment according to the present disclosure is illustrated graphically. Figure 4A The three-dimensional fluorescence intensity of the image;

[0041] Figure 4C These are combined bright-field and fluorescence microscopy images of PFBT P-point labeled MCF-7 cells treated with NADH according to embodiments of the present disclosure;

[0042] Figure 4D An embodiment according to the present disclosure is illustrated graphically. Figure 4C The three-dimensional fluorescence intensity of the image;

[0043] Figure 5A These are photographs of DPA-CNPPV P points in solutions within the physiologically relevant NADH range (0-2 mM) taken under 365 nm UV light irradiation, according to embodiments of this disclosure.

[0044] Figure 5B The following are examples of embodiments according to the present disclosure. Figure 5A The original region of interest (ROI) image is split into its RGB channels;

[0045] Figure 5C According to embodiments of this disclosure Figure 5A The ratio calibration curve (R / R0) of point P within the physiologically relevant NADH range (0-2mM);

[0046] Figure 5D The following illustration shows an embodiment of the present disclosure in the absence of NADH (0mM). Figure 5A The three-dimensional distribution of fluorescence intensity at point P;

[0047] Figure 5E The following illustration shows an embodiment of the present disclosure in the presence of NADH (2mM). Figure 5A The three-dimensional distribution of fluorescence intensity at point P;

[0048] Figure 5F The embodiments of the present disclosure are illustrated graphically in the presence and absence of NADH. Figure 5A The average R / R0 fluorescence intensity ratio at point P;

[0049] Figure 5G The illustration schematically depicts in vivo ratioographic imaging of NADH with DPA-CNPPV P-points and a smartphone according to an embodiment of the present disclosure, wherein when the p-points are injected into two sites in a mouse, the emission changes from red to blue with and without NADH (0.1 mmol), as the NADH concentration increases. The inset: the right side shows a thermal image of the ratio (B / R ratio) of the blue and red channel intensities from the two injection sites, where a high B / R ratio (red) indicates a high NADH concentration.

[0050] Figure 5HConcentration-dependent ratio imaging of NADH in live mice using a smartphone camera according to embodiments of the present disclosure is shown, wherein the region of interest (the square-marked area) corresponds to a subcutaneous injection site of DPA-CNPPV P point (0.1 mg / mL, 100 μL; 0 mM NADH) only or a subcutaneous injection site of DPA-CNPPV P point and NADH (0.25 mM, 0.5 mM and 1.0 mM);

[0051] Figure 5I Embodiments according to this disclosure are illustrated. Figure 5G The fluorescence intensity of the B and R channels;

[0052] Figure 5J Showing Figure 5H The average R / R0 of the region of interest;

[0053] Figure 6A The fluorescence spectra at point P are shown graphically in the presence of various concentrations of phenylalanine (0-2400 μM) according to embodiments of the present disclosure.

[0054] Figure 6B According to embodiments of this disclosure Figure 6A The ratio calibration plot (R / R0) at point P changes with phenylalanine concentration;

[0055] Figure 6C According to embodiments of this disclosure, at phenylalanine concentrations of 0-120 μM... Figure 6A The ratio calibration plot (R / R0) of point P;

[0056] Figure 6D According to embodiments of this disclosure, at phenylalanine concentrations of 120-360 μM corresponding to mild benign HPA... Figure 6A The ratio calibration plot (R / R0) of point P;

[0057] Figure 6E According to embodiments of this disclosure, at phenylalanine concentrations of 360-600 μM corresponding to mild HPA... Figure 6A The ratio calibration plot (R / R0) of point P;

[0058] Figure 6F According to embodiments of this disclosure, at phenylalanine concentrations of 600-900 μM corresponding to mild PKU... Figure 6A The ratio calibration plot (R / R0) of point P;

[0059] Figure 6G According to embodiments of this disclosure, at phenylalanine concentrations of 900-1200 μM corresponding to moderate PKU... Figure 6A The ratio calibration plot (R / R0) of point P;

[0060] Figure 6H According to embodiments of this disclosure, at phenylalanine concentrations corresponding to classical PKU of 1200-1800 μM... Figure 6A The ratio calibration plot (R / R0) of point P;

[0061] Figure 6I According to embodiments of this disclosure, at phenylalanine concentrations corresponding to classical PKU of 1800-2400 μM... Figure 6A The ratio calibration plot (R / R0) of point P;

[0062] Figure 7A A 96-well microplate with P-points, according to an embodiment of this disclosure, is used in various phenylalanine concentrations (100 μL wells). -1 The image shows the concentration of phenylalanine in the solution of phenylalanine.

[0063] Figure 7B It is configured to be according to embodiments of this disclosure. Figure 7A Images from a digital camera used to image a 96-well microplate.

[0064] Figure 7C According to embodiments of this disclosure Figure 7B The P point is calibrated based on the ratio of phenylalanine concentration (R / R0);

[0065] Figure 7D Images are from a smartphone camera of the system of the present disclosure for reading microplates according to embodiments of the present disclosure;

[0066] Figure 7E According to embodiments of this disclosure Figure 7D The P-point sensor is calibrated based on the ratio of phenylalanine concentration (R / R0).

[0067] Figure 7F Images are of transducer substrates according to embodiments of the present disclosure;

[0068] Figure 7G It is according to the embodiments of this disclosure for use in... Figure 7F Images of transducer substrates measured by a fluorescence plate reader;

[0069] Figure 7H According to embodiments of this disclosure Figure 7A Point P according to Figure 7F Ratio calibration plot (R / R0) of phenylalanine concentration in fluorescence measured on the transducer substrate;

[0070] Figure 7IThe diagram schematically illustrates a phenylalanine sensing mechanism according to embodiments of the present disclosure, wherein phenylalanine dehydrogenase senses NAD through NAD. + Catalyzing the oxidation of L-phenylalanine leads to the formation of the NADH stoichiometry. NADH quenches the 627 nm P-point fluorescence emission and the 458 nm fluorescence. As the concentration of phenylalanine increases, the fluorescence emission changes from red (P-point emission) to blue (NADH emission). Metabolite concentrations are measured proportionally using a digital camera or plate reader, based on the ratio of blue channel to red channel emission intensity, in the form of solution- or paper-based determinations.

[0071] Figure 7J The pixel intensity distribution in a single aperture in the blue and red channels of a 60 μM Phe (healthy) image according to an embodiment of the present disclosure is shown.

[0072] Figure 7K The pixel intensity distribution in a single hole in the blue and red channels of a 1200 μM Phe (classical PKU threshold) according to an embodiment of the present disclosure is shown.

[0073] Figure 7L Demonstrated as Figure 7J and 7K The figure shows a significant increase in the average ratio of blue and red channel emission between 60 μM and 1200 μM Phe;

[0074] Figure 8 Examples of molecular structures of polymers PFO, PDHF, PFBT, PFBTTBT, PFTBT, DPA-CNPPV and DPA-CNPF suitable for P-point conjugation according to embodiments of the present disclosure are shown.

[0075] Figure 9 The hydrodynamic diameter of the PFBT P point in PBS solution according to the storage time at room temperature is graphically shown according to an embodiment of the present disclosure, wherein the error bars represent the standard deviation of the three measurements;

[0076] Figure 10 The fluorescence emission (λ) of the PFO P point according to embodiments of the present disclosure is illustrated graphically. ex =380nm);

[0077] Figure 11 The ratio of fluorescence emission at point PFO in the absence of (F0) and presence of (F) NADH, according to embodiments of the present disclosure, is illustrated graphically as F0 / F.

[0078] Figure 12AThe F0 / F and λ of the PDHF P point according to embodiments of the present disclosure are illustrated graphically. em =428nm;

[0079] Figure 12B The F0 / F and λ of the PFBT P point according to embodiments of the present disclosure are illustrated graphically at different NADH concentrations (0-2 mM). em =546nm;

[0080] Figure 12C The F0 / F and λ of the PFBTTBT P point according to embodiments of the present disclosure are illustrated graphically at different NADH concentrations (0-2 mM). em =626nm;

[0081] Figure 12D The F0 / F and λ of the PFTBT P point according to embodiments of the present disclosure are illustrated graphically at different NADH concentrations (0-2 mM). em =638nm;

[0082] Figure 13 The figure illustrates, graphically, the addition of λ according to an embodiment of the present disclosure. ex Fluorescence emission of the P-point of PFBTTBT before and after NADH (10mM) excitation at 380nm;

[0083] Figure 14 The fluorescence (λ) of the DPA-CNPFP point is graphically shown according to embodiments of the present disclosure as NADH concentration increases. ex =385nm);

[0084] Figure 15 This is a schematic diagram of PFBT P-point bioconjugation for specific cell targeting according to embodiments of the present disclosure;

[0085] Figure 16 This is a schematic diagram illustrating the quantification of metabolites by NAD(P)H levels according to embodiments of the present disclosure, wherein a specific enzymatic reaction is performed using an NAD(P)H-dependent enzyme to quantify metabolites by NAD(P)H levels. + The analyte of interest is oxidized, and the level of NAD(P)H corresponds to the level of the analyte in the sample;

[0086] Figure 17A The fluorescence spectra of a P-point sensor with lactate dehydrogenase in the presence of various concentrations of lactate according to embodiments of the present disclosure are shown graphically.

[0087] Figure 17B According to embodiments of this disclosure Figure 17A The P-point sensor is calibrated based on the ratio chart (R / R0) of lactate concentration;

[0088] Figure 17C The fluorescence spectra of a P-point sensor according to an embodiment of the present disclosure, which contains glutamate dehydrogenase in the presence of various concentrations of glutamate, are shown graphically.

[0089] Figure 17D According to embodiments of this disclosure Figure 17C The P-point sensor is calibrated based on the ratio of glutamate concentration (R / R0).

[0090] Figure 17E The fluorescence spectra of a P-point sensor with glutamate dehydrogenase in the presence of various concentrations of glutamate according to embodiments of the present disclosure are shown graphically.

[0091] Figure 17F According to embodiments of this disclosure Figure 17E The P-point sensor is calibrated based on the ratio chart (R / R0) of glucose concentration;

[0092] Figure 17G The fluorescence spectra of a P-point sensor with BHB dehydrogenase in the presence of various concentrations of β-hydroxybutyrate (BHB) according to embodiments of the present disclosure are shown graphically.

[0093] Figure 17H According to embodiments of this disclosure Figure 17G The P-point sensor is calibrated based on the ratio (R / R0) of BHB concentration.

[0094] Figure 18A Fluorescence emission of a phenylalanine biosensor according to embodiments of the present disclosure is demonstrated in the presence or absence of phenylalanine dehydrogenase (PheDH).

[0095] Figure 18B Background correction for endogenous NADH levels according to embodiments of the present disclosure is demonstrated, wherein Figure 18A A sensor without PheDH measures endogenous NADH and subtracts the endogenous NADH from the value containing PheDH to obtain the phenylalanine concentration; and

[0096] Figure 18C and 18D The use of a fluorescence plate reader according to embodiments of the present disclosure is illustrated. Figure 18C ) and digital camera ( Figure 18D The fluorescence of phenylalanine incorporated into a plasma sample containing a phenylalanine biosensor was measured. Detailed Implementation

[0097] This disclosure generally relates to apparatus, compositions, kits, systems, and methods for monitoring, determining, and / or measuring the concentration of an analyte in a fluid using a transducer. In one embodiment, the analyte is a molecule in the fluid. In one embodiment, the fluid is blood; for example, the compositions, systems, and methods disclosed herein can be used to monitor the concentration of one or more selected molecules in the blood of a subject. In one embodiment, the fluid is tears; for example, the compositions, systems, and methods disclosed herein can be used to monitor the concentration of one or more selected molecules in the tears of a subject. In one embodiment, the fluid is sweat; for example, the compositions, systems, and methods disclosed herein can be used to monitor the concentration of one or more selected molecules in the sweat of a subject. In one embodiment, the fluid is saliva; for example, the compositions, systems, and methods disclosed herein can be used to monitor the concentration of one or more selected molecules in the saliva of a subject. In one embodiment, the fluid is lymph; for example, the compositions, systems, and methods disclosed herein can be used to monitor the concentration of one or more selected molecules in the lymph of a subject. In one embodiment, the fluid is cerebrospinal fluid; for example, the compositions, systems, and methods disclosed herein can be used to monitor the concentration of one or more selected molecules in the cerebrospinal fluid of a subject. In one embodiment, the fluid is urine; for example, the compositions, systems, and methods disclosed herein can be used to monitor the concentration of one or more selected molecules in the urine of a subject.

[0098] In the following description, numerous specific details are set forth in order to provide a thorough understanding of the embodiments. However, those skilled in the art will recognize that the techniques described herein can be practiced without one or more of the specific details stated herein, or can be practiced using other methods, components, materials, etc. In other instances, well-known structures, materials, or operations have not been shown or described in detail to avoid obscuring certain aspects.

[0099] Throughout this specification, references to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the invention. Therefore, the phrases "in one embodiment" or "in an embodiment" appearing in different places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, specific features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0100] transducer

[0101] On one hand, this disclosure provides a transducer for monitoring the concentration of an analyte in a fluid. As further described herein, in one embodiment, such a transducer is adapted to emit a signal, such as an optical signal, based on or proportional to the concentration of an analyte adjacent to an element of the transducer. In such embodiments, the transducer is configured to generate an optical signal based on the presence or absence of the analyte or its concentration.

[0102] In one embodiment, the transducer is a nanoparticle transducer, such as a nanoparticle transducer for analyte concentration measurement, said nanoparticle transducer comprising nanoparticles containing one or more chromophores (such as one or more semiconductor chromophore polymers); and an enzyme physically associated with, such as coupled to, the nanoparticles. In one embodiment, the enzyme is a nicotinamide adenine dinucleotide (NADH)-dependent or nicotinamide adenine dinucleotide dinucleotide (NADPH)-dependent enzyme. In one embodiment, the activity or stoichiometry (between the analyte and one or more reaction elements), such as the rate at which it acts on the substrate, depends on the concentration of NADH or NADPH and / or the NAD adjacent to the enzyme. + or NADP + The concentration. In one embodiment, the analyte includes NADH or NADPH, or NAD... + or NADP + As further discussed herein with reference to examples of this disclosure, it has been surprisingly found that the transducers described herein are suitable for monitoring or determining analytes and / or reaction elements (including NADH and / or NADPH or NAD). + and / or NADP + The concentration of ).

[0103] In one embodiment, the enzyme is configured to catalyze a reaction comprising a plurality of reaction elements. In one embodiment, the plurality of reaction elements comprises one or more reactants and one or more products, the one or more reactants comprising an analyte, and wherein the amount of fluorescence emitted from the chromophore is determined by and / or related to the concentration of the reaction element among the plurality of reaction elements. In one embodiment, the reaction element among the plurality of reaction elements comprises NADH, and wherein the amount of fluorescence emitted from the chromophore is determined by the concentration of the NADH.

[0104] In another embodiment, the transducer is a transducer substrate for analyte concentration measurement, the transducer substrate comprising: nanoparticles including chromophores coupled to the substrate; and an enzyme coupled to the substrate and configured to catalyze a reaction comprising multiple reaction elements. As further discussed herein with respect to Example 7, it has been surprisingly found that transducers comprising chromophores (such as nanoparticles) and enzymes coupled to a common substrate are suitable for measuring and / or monitoring analyte concentrations.

[0105] As described above, in one embodiment of the transducer substrate, the nanoparticles and enzyme are coupled to the substrate. The substrate can comprise any substrate suitable for coupling with the nanoparticles and enzyme. Such substrates can include, but are not limited to, polymer substrates, glass substrates, silica-based substrates, silicone-based substrates, metal substrates, woven substrates, non-woven substrates, etc. In one embodiment, the substrate is a fiber and / or paper-based substrate. In one embodiment, the substrate is a porous membrane. In one embodiment, the porous membrane comprises paper, nitrocellulose, nylon, and many other materials that those skilled in the art consider capable of serving as a core. In one embodiment, the substrate is a paper substrate.

[0106] In one embodiment, the enzyme is covalently bound to the substrate. In one embodiment, the nanoparticles are covalently bound to the substrate. In one embodiment, the enzyme and / or nanoparticles are physically associated with the substrate. Such physical association may include covalent binding, but may also include other non-covalent associations between the enzyme, nanoparticles, and the substrate. In this regard, the enzyme and / or nanoparticles may associate with the substrate via ionic bonding, van der Waals forces, hydrogen bonding, etc. In one embodiment, the enzyme and / or nanoparticles are deposited on the substrate, such as by liquid deposition or imprinting. In one embodiment, the enzyme and nanoparticles are lyophilized onto the substrate.

[0107] In one embodiment, the nanoparticles are coupled to the substrate at a point or other spatially confined region (e.g., including fewer than all of the substrate). Such confined placement or positioning of the nanoparticles on the substrate, as further discussed herein, allows for the testing and analysis of a variety of samples and reaction conditions. In one embodiment, an enzyme is coupled to the substrate at a point adjacent to the nanoparticles. In another embodiment, the enzyme is coupled to the substrate, and the nanoparticles are coupled to the substrate at a point.

[0108] In one embodiment, the substrate comprises a plurality of nanoparticles coupled to the substrate at spatially different portions or points. In another embodiment, the substrate comprises a plurality of enzymes also coupled to points and corresponding to the number of nanoparticles also coupled thereto. In this respect, in one embodiment, the substrate comprises pairs of nanoparticles and enzymes, or nanoparticle types and enzyme types, coupled to the substrate at various points.

[0109] In one embodiment, the substrate further comprises one or more nanoparticles coupled to regions or sites on the substrate that do not contain an enzyme coupled thereto. Such one or more nanoparticles coupled to the substrate in the absence of an enzyme are suitable for use as controls, such as control points for calibration.

[0110] In one embodiment, the enzyme is a first enzyme, the nanoparticle is a first nanoparticle including a first chromophore, and the reaction is a first reaction. In such embodiments, the transducer substrate may further include: a second nanoparticle including a second chromophore coupled to the substrate; and a second enzyme, different from the first enzyme coupled to the substrate, the second enzyme being configured to catalyze a second reaction including a second plurality of reaction elements. In one embodiment, the second plurality of reaction elements includes one or more second reactants, the one or more second reactants comprising the analyte and one or more second products, and wherein the amount of fluorescence emitted from the second chromophore is determined by the concentration of the second reaction element among the second plurality of reaction elements.

[0111] In one embodiment, the point is a first point, and the second nanoparticle is coupled to the substrate at a second point separate from the first point. In this regard, a sample can be applied to the first and second points to determine the sample with different enzymes, for example, if the first and second enzymes are different. In one embodiment, the second reaction is different from the first reaction. In one embodiment, a single sample is applied to different points to use spatial multiplexing to determine the concentration of different analytes through different points. In one embodiment, a sample can be applied to the first and second points to perform multiple iterations of the same reaction, as if the first and second enzymes were the same. Therefore, in one embodiment, the second reaction is the same as the first reaction. In one embodiment, a sample is applied to the first and second points to perform the same reaction, but in different concentration ranges or dynamic ranges, in which case the first and second enzymes are also the same. Therefore, in one embodiment, the second reaction is the same as the first reaction.

[0112] In one embodiment, the second nanoparticle is coupled in proximity to the first nanoparticle on the substrate. Therefore, in one embodiment, the second nanoparticle is coupled to the substrate at said point. Such embodiments can be adapted to measure a single sample using nanoparticles containing optically distinct chromophores, to use spectral multiplexing to determine the concentrations of different analytes on different nanoparticles with different chromophores.

[0113] In one embodiment, the first chromophore is configured to absorb light within a first absorption wavelength range, and the second chromophore is configured to absorb light within a second absorption wavelength range different from the first absorption wavelength range. This configuration is suitable for, for example, absorption or excitation multiplexing, where different nanoparticles are excited by light of different wavelengths and / or different light sources. Similarly, in one embodiment, the fluorescence emitted from the first chromophore is within a first emission wavelength range, and the fluorescence emitted from the second chromophore is within a second emission wavelength range different from the first emission wavelength range. This configuration is suitable for emission multiplexing, where fluorescence from different nanoparticles is detected in different wavelength ranges and / or by different sensors. In one embodiment, the first and second chromophores are configured to absorb light within the same or similar absorption wavelength ranges but emit light within different wavelength ranges. This configuration utilizes emission multiplexing instead of excitation multiplexing. In one embodiment, the first and second chromophores are configured to emit light within the same or similar emission wavelength ranges but absorb light within different wavelength ranges. This configuration utilizes excitation multiplexing instead of emission multiplexing. In one embodiment, the first chromophore and the second chromophore are configured to absorb light in different absorption wavelength ranges and emit light in different emission wavelength ranges. This configuration utilizes both excitation multiplexing and emission multiplexing.

[0114] As described above, in some embodiments, nanoparticles and enzymes are coupled to the substrate in multiple spatially separated portions or points, such as for multiplexing different reactions, performing the same reaction at different concentrations, or repeating the reaction. In one embodiment, the number of points on the substrate coupled with nanoparticles and enzymes is selected from 2, 4, 6, 8, 24, 96, 384, and 1536. Such points can be configured to spatially correspond to the wells of a standard multiwell plate, and thus to a reader or other sensor for measuring, for example, the fluorescence of a sample contained in the wells of such a standard multiwell plate. In one embodiment, the number of points on the substrate coupled with nanoparticles and enzymes is in the range of 2 to 10, 2 to 50, 2 to 100, 2 to 500, or 2 to 1000 or more.

[0115] Enzymes and nanoparticles can be applied to substrates, such as paper-based or other porous substrates, to achieve high spatial resolution and small dot sizes. Such configurations are suitable for determining a large number of different nanoparticle / enzyme pairs on a single substrate. In one embodiment, the dot sizes are in the range of about 1 μM to about 10 μM, about 1 μM to about 25 μM, about 1 μM to about 50 μM, about 1 μM to about 100 μM, about 1 μM to about 250 μM, about 1 μM to about 400 μM, or about 1 μm to about 500 μm.

[0116] In one embodiment, the substrate is configured to wick a fluid sample to the point. Such wicking substrates are suitable for reactions and assays in which a fluid sample is applied to a first portion of the substrate and wicked to another portion of the substrate (e.g., nanoparticles and enzymes coupled thereto) via capillary action. In one embodiment, the substrate includes one or more fluid-isolated pathways that extend from the application area and individually to spatially distinct points where the enzyme and nanoparticle pairs are deposited.

[0117] In one embodiment, the wicking substrate is configured to filter or separate cells or other particles, such as blood cells, from a fluid sample, such that the fluid reaching one or more points on the substrate is free of or substantially free of cells or other particles that could interfere with the measurement. In one embodiment, the substrate includes a sample application portion and a filter disposed between the sample application portion and one or more points in fluid communication with the sample application portion.

[0118] In one embodiment, the nanoparticles comprise polymer dots (P-dots). As used herein, the terms "polymer dot" or "P-dot" refer to a particle structure comprising one or more semiconductor polymers that collapse to form stable submicron-sized particles, such as nanoparticles. In one embodiment, the polymer dot is a highly fluorescent nanoparticle with tunable emission, for example, from the visible region to the near-IR region. The polymer dot may comprise a chromophore polymer that can, for example, absorb light and then emit light via fluorescence. In some embodiments, the polymer dot comprises at least one condensed polymer, such as a semiconductor polymer. For polymer dots having more than one condensed polymer (e.g., more than one semiconductor polymer), the condensed polymers may be the same or different types of polymers. For example, a P-dot may comprise both a semiconductor polymer and a non-semiconductor polymer.

[0119] Nanoparticle transducers for monitoring selected analytes can be assembled based on the appropriate selection of enzymes, nanoparticles, and chromophores. As further discussed herein, the enzyme does not need to be coupled to the nanoparticles, such as through covalent bonding. The enzyme can be selected as the one catalyzing the reaction involving the analyte, such that the concentration of the analyte can affect the rate of the reaction or the amount of reaction elements produced or consumed. The reaction can involve multiple reaction elements, including reactants and products. The enzyme can be selected such that each reactant of its catalyzed reaction is present in the fluid to be analyzed. The chromophore can be selected such that the fluorescence of the chromophore is determined by the concentration of reactants or products of the enzyme-catalyzed reaction or the rate of reactant consumption or product formation. The nanoparticles can be selected to allow both the enzyme and the chromophore to be incorporated into or conjugated to the nanoparticles. For example, the nanoparticle can be a P-point, allowing the enzyme to covalently bind to the P-point, and the chromophore to be incorporated into and / or covalently bound to the P-point. In some cases, the chromophore can comprise all or substantially all of the nanoparticles; for example, in some cases, the P-point can be entirely or substantially entirely composed of one or more chromophores.

[0120] Although transducers incorporating nanoparticles (such as P-points) have been described, in some embodiments, the transducers of this disclosure comprise chromophores in an uncondensed state. For example, in one embodiment, the transducer comprises: a chromophore comprising a semiconductor chromophore polymer; and an enzyme physically associated with the semiconductor chromophore polymer and configured to catalyze a reaction comprising a plurality of reaction elements, wherein such chromophores do not comprise condensed semiconductor chromophore polymers.

[0121] In one embodiment, the uncondensed semiconductor chromophore polymer and the enzyme are coupled to a substrate. In another embodiment, the uncondensed semiconductor chromophore polymer and the enzyme are in the form of a lyophilized powder. In yet another embodiment, the semiconductor chromophore polymer and the enzyme are dispersed in a common solvent.

[0122] In one embodiment, the enzyme, chromophore, and nanoparticles may be selected from a potential set of enzymes, chromophores, and nanoparticles to generate a nanoparticle transducer for detecting a given analyte as follows: An enzyme catalyzing a reaction is selected from a set of enzymes, where the analyte is the reactant. For each such reaction, other reaction elements whose concentrations will change as the reaction occurs are identified—for example, each time the reaction occurs, the reactant concentration decreases and the product concentration increases (for reversible reactions, the reverse reaction produces the opposite effect). From these reaction elements, for each enzyme, a corresponding chromophore is identified from a set of chromophores whose fluorescence intensity changes in response to a change in the concentration of one of the reaction elements. If no chromophore matches, the enzyme is eliminated. One such pair is selected from the remaining enzyme / chromophore pairs, and nanoparticles, such as P-points, are selected, each of which can be physically associated or coupled and / or incorporated, thereby selecting elements to construct the nanoparticle transducer. A second chromophore, emitting at a different wavelength and whose intensity does not change in response to any reaction element, may be selected from the list of chromophores to serve as a control chromophore. Alternatively, if the initially selected chromophore emits fluorescence at wavelengths where the intensity changes in response to reactant or product concentrations and at different wavelengths where the intensity does not change, then this single chromophore can serve as a control for itself.

[0123] In one embodiment, the transducer described herein includes an enzyme that catalyzes a reaction involving an analyte. The reaction has reaction elements comprising reactants and products, one of which is the analyte. The nanoparticles include chromophores that emit fluorescence at one or more wavelengths in response to illumination with a light beam. The amount of fluorescence at at least one wavelength depends on the concentration of molecules of either the reactant or the product, excluding the analyte. The enzyme and the chromophore of the nanoparticles are closely or physically associated; therefore, as the enzyme-catalyzed reaction consumes reactants and produces products, the respective concentrations of the reactants and products change, with the reactant concentration decreasing and the product concentration increasing. The presence of an increased concentration of the analyte in the presence of the analyte causes the reaction to proceed more rapidly compared to a low concentration, thus resulting in a relatively high product concentration and a relatively low reactant concentration. Therefore, the enzyme and the chromophore of the nanoparticles together act as a transducer, thereby translating changes in analyte concentration into changes in fluorescence. In one embodiment, the fluorescence intensity emitted at one wavelength of the transducer is used to determine the analyte concentration. In one embodiment, the ratio of the fluorescence intensities emitted at two wavelengths of the transducer is used to determine the analyte concentration. This fluorescence can be easily measured in a wavelength-selective manner, thereby determining the concentration of the analyte based on the signal from the optical sensor.

[0124] In one embodiment, the nanoparticles comprise a semiconductor polymer that emits fluorescence at one or more wavelengths in response to illumination with a light beam. The amount of fluorescence at at least one of these wavelengths depends on the concentration of molecules of the reactant or product, rather than the concentration of the analyte. In some cases, the nanoparticles comprise a semiconductor polymer and a dye that emits fluorescence at one or more wavelengths. The dye may be physically doped or chemically linked to the semiconductor polymer to form the nanoparticles. The semiconductor polymer may transfer energy to the dye to enhance or amplify the fluorescence intensity of the dye.

[0125] In one embodiment, the fluid described herein is a bodily fluid, such as bodily fluids present in or expelled from the subject, such as blood, plasma, serum, sweat, tears, lymph, cerebrospinal fluid, urine, saliva, or other fluids present in, derived from, or secreted by body tissues. The subject may be an animal, and in one embodiment, the subject is a human.

[0126] Various embodiments of this disclosure provide chromophores with properties that facilitate efficient and accurate measurement of analyte concentrations using the transducers provided herein. Examples of such properties include, but are not limited to: (1) high brightness, thus allowing easy detection and recovery of the transducer signal; (2) high sensitivity to reaction elements of enzyme-catalyzed reactions; (3) high absorption cross-section, thus allowing easy induction of fluorescence in the nanoparticle transducer without strong energy application; (4) good stability (e.g., thermal stability), thus allowing the transducer to remain active over long periods in vivo; (5) detectable and distinguishable wavelengths, including, in some cases, wavelengths detectable and distinguishable transdermally; and / or (6) good fatigue resistance to reduce degradation when used for continuous analyte monitoring. In one embodiment, the chromophore of the nanoparticle transducer described herein comprises some or all of these properties.

[0127] For example, in one embodiment, this disclosure provides a transducer exhibiting signal fluorescence emission intensity at a peak emission wavelength that varies with the concentration of the fluid component. The nanoparticle transducer may also include chromophores with different control emission intensities at the peak emission wavelength, the emission intensities being substantially independent of the concentration of the fluid component. In one embodiment, the peak emission wavelength is in the range of about 200 nm to about 300 nm, about 250 nm to about 350 nm, about 300 nm to about 400 nm, about 350 nm to about 450 nm, about 400 nm to about 500 nm, about 450 nm to about 550 nm, about 500 nm to about 600 nm, about 550 nm to about 650 nm, about 600 nm to about 700 nm, about 650 nm to about 750 nm, about 700 nm to about 800 nm, about 750 nm to about 850 nm, about 800 nm to about 900 nm, about 850 nm to about 950 nm, about 900 nm to about 1000 nm, about 950 nm to about 1050 nm, about 1000 nm to about 1100 nm, about 1150 nm to about 1250 nm, or about 1200 nm to about 1300 nm.

[0128] As another example, some embodiments of this disclosure provide transducers that exhibit sufficient stability for long-term in vivo analyte concentration monitoring; for example, the transducer is capable of stably detecting analyte concentrations over extended periods without significant degradation. In various embodiments, the stability of the nanoparticle transducers helps ensure that the transducers can be used in vivo for extended periods without replacement. In one embodiment, a population of transducers is considered "stable" if at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 99.95% of the transducers maintain their ability to modulate fluorescence in response to changes in analyte concentration over a specified period. In one embodiment, a transducer is considered "stable" if its emission intensity maintains its ability to measure changes in analyte concentration over a specified period. In one embodiment, a transducer is considered "stable" if the intensity ratio of two emission peaks maintains its ability to measure changes in analyte concentration over a specified period, even if the absolute emission intensity may decrease significantly. In one embodiment, a transducer is considered "stable" if the time constant (e.g., the time to decay to 1 / e of the fluorescence signal intensity) is at least about 3 hours, about 6 hours, about 12 hours, about 24 hours, about 1 day, about 2 days, about 4 days, about 10 days, about 20 days, about 30 days, about 1 month, about 2 months, about 4 months, about 6 months, about 1 year, or longer. In one embodiment, the nanoparticle transducer maintains sufficient signal intensity so that analyte detection can be reliably performed throughout the specified time period.

[0129] In embodiments of this disclosure, the emission spectrum of the chromophore is selected or designed to exhibit narrow-band emission properties at the peak emission wavelength, thereby reducing or minimizing overlap with other emission sources. For example, in one embodiment, the peak emission bandwidth of the chromophore (e.g., the full width at half maximum (FWHM) of the emission peak) does not exceed about 5 nm, about 10 nm, about 15 nm, about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, or about 100 nm.

[0130] Chromophore composition

[0131] Various types of chromophores are applicable to the transducers, compositions, methods, kits, and systems disclosed herein, including but not limited to dyes, staining agents, proteins, polymers, beads, particles, or combinations thereof. In one embodiment, the transducer comprises one or more chromophores (e.g., fluorophores). The chromophores described herein can be used to produce transducers according to various mechanisms. In one embodiment, the chromophore comprises a semiconductor polymer that emits fluorescence at one or more wavelengths in response to illumination with a light beam. In one embodiment, the chromophore comprises a semiconductor polymer P-point that emits fluorescence at one or more wavelengths in response to illumination with a light beam. The amount of fluorescence of the semiconductor polymer can depend on the concentration of reactant or product molecules. The amount of fluorescence of the semiconductor polymer in one wavelength range can depend on the concentration of reactant or product molecules, while the amount of fluorescence of the semiconductor polymer in another different wavelength range can be relatively independent of the concentration of reactant or product molecules, and thus can serve as a control wavelength range for ratio measurements.

[0132] In one embodiment, the transducer of this disclosure comprises one or more, such as two or more, chromophores. In one embodiment, the one or more chromophores are configured to emit fluorescence in two or more different wavelength ranges, as applicable to ratiometric fluorescence measurements as discussed further herein. In one embodiment, the chromophore comprises a semiconductor polymer and a dye that emits fluorescence at one or more wavelengths. The amount of fluorescence from the dye depends on the concentration of molecules of the reactant or product. The dye may be physically doped or chemically linked to the semiconductor polymer to form nanoparticles. The chromophore polymer may transfer energy to the dye to enhance or amplify the fluorescence intensity of the dye.

[0133] In one embodiment, the fluorescence emitted from the chromophore includes a signal fluorescence wavelength and a control fluorescence wavelength. In one embodiment, the fluorescence emitted from the chromophore defines a signal fluorescence ratio, which is equal to the ratio of the amount of fluorescence emitted at the signal wavelength to the amount of fluorescence emitted at the control fluorescence wavelength, and wherein the signal fluorescence ratio is determined by the concentration of the reaction element among the plurality of reaction elements. In one embodiment, the fluorescence ratio varies proportionally with the concentration of the analyte. In one embodiment, the fluorescence emitted from the chromophore varies proportionally with the concentration of the analyte within a certain analyte concentration range. In one embodiment, the fluorescence at a control wavelength remains constant with different concentrations of the reaction element. In one embodiment, the fluorescence at a control wavelength varies with different concentrations of the reaction element.

[0134] As described above, in one embodiment, the chromophore emits fluorescence in two or more different wavelength ranges. In one embodiment, the chromophore emits fluorescence in one wavelength range (e.g., the control fluorescence wavelength) of the two or more wavelength ranges, the fluorescence remaining constant or relatively constant with changes in the concentration of the reactant, and emits fluorescence in another wavelength range (e.g., the control fluorescence wavelength) of the two or more wavelength ranges, the fluorescence varying with changes in the concentration of the reactant. In one embodiment, the chromophore is configured to emit fluorescence in two or more different wavelength ranges, wherein the emitted fluorescence varies with the concentration of the reactant at at least two of the two or more different wavelength ranges.

[0135] In one embodiment, the transducer comprises at least one chromophore semiconductor polymer particle (also referred to as a "polymer dot" or "P-dot") comprising one or more polymers (e.g., semiconductor polymers, non-semiconductor polymers, or combinations thereof) that have collapsed into stable submicron-sized particles. In one embodiment, semiconductor polymer particles are advantageous compared to other types of chromophores for several reasons: (1) the semiconductor polymer particles are very bright, up to 30 times brighter than quantum dots, and are photostable; (2) the semiconductor polymer particles have a fast photon emission rate, typically with a sub-nanosecond lifetime, making them ideal for rapid optical detection; (3) the semiconductor polymer particles are biocompatible and, unlike quantum dots, are not composed of cytotoxic heavy metals; and (4) the semiconductor polymer particles exhibit amplified energy transfer, thus their fluorescence emission can be well modulated by dyes, for example, through energy transfer.

[0136] Various structures and compositions of chromophore polymer particles apply to all aspects presented herein. The chromophore polymer particles provided herein are made from a single polymer, or alternatively, comprise blends of polymers. In one embodiment, one or more polymers collapse, precipitate, and / or condense to form a polymer matrix. In one embodiment, the properties of the chromophore polymer particles depend on the structure and / or properties of the constituent polymers. Therefore, in one embodiment, the polymer backbone, side chains, terminal units, and substituents are modified to obtain specific properties. In one embodiment, the optical properties of the chromophore polymer particles are tuned by modifying the structure of the polymer backbone.

[0137] In one embodiment, the chromophore polymer particles provided herein comprise one or more chromophores, also referred to herein as chromophore units. In one embodiment, the chromophore absorbs light of certain wavelengths, for example, from the UV region to the near-infrared region, and may or may not be emissive. In one embodiment, the chromophore unit comprises, but is not limited to, structural units having delocalized π electrons, small organic dye molecule units, and / or metal complex units. In various embodiments, the chromophore is part of the polymer matrix or, for example, incorporated into the polymer matrix through blending, crosslinking, etc. In one embodiment, the chromophore polymer is a semiconductor polymer.

[0138] In one embodiment, the chromophore polymer particles of this disclosure comprise one or more chromophore polymers. In one embodiment, the chromophore polymer comprises light that absorbs certain wavelengths, for example, at least a portion of the spectrum ranging from UV to near-infrared. The chromophore polymer according to this disclosure may or may not be radioactive. In one embodiment, the chromophore polymer comprises one or more chromophore units. Examples of chromophore polymers include, but are not limited to, polymers comprising structural units having delocalized π electrons (e.g., semiconductor polymers), polymers comprising small organic dye molecule units, polymers comprising metal complex units, and polymers comprising any combination thereof. In one embodiment, the chromophore unit is incorporated into the polymer backbone. In one embodiment, the chromophore unit is covalently linked to a side chain or terminal unit of the polymer. In one embodiment, the chromophore polymer is prepared using standard synthetic methods generally known in the art.

[0139] Various types of chromophore polymer particles are suitable for use as a platform for the optical sensing methods of this disclosure. Chromophore polymer particles can take on a variety of configurations, including, but not limited to, monolithic polymer particles having a uniform, homogeneous composition or polymer particles with different core and cap structures. The chromophore polymer particles provided herein can be formed by any method known in the art, including, but not limited to, precipitation-dependent methods, emulsion-dependent (e.g., mini or microemulsion) formation methods, and condensation-dependent methods. Figure 8 Examples of chemical structures for repeating units suitable for chromophore polymer particles are shown herein. Examples of chromophore polymer particles suitable for use with the techniques described herein can be found, for example, in PCT applications PCT / US2010 / 056079, PCT / US2012 / 071767, PCT / US2011 / 056768, PCT / US2013 / 024300, and PCT / US2013 / 063917, and U.S. Patent Publication 2013 / 0266957, each of which is incorporated herein by reference.

[0140] In one embodiment, the chromophore polymer particles are nanoparticles. In one embodiment, the size of the nanoparticles provided herein is defined according to a “critical size,” which refers to the minimum size of the nanoparticle. Some nanoparticles are approximately spherical in shape, resulting in a critical size that is the diameter of the spherical particle. In one embodiment, some nanoparticles (such as nanospheres and nanocubes) are entirely nanoscale in size. In one embodiment, not every dimension of the nanoparticle is at the nanoscale. For example, a nanocylinder may have a nanoscale diameter but a micrometer-scale length. A variety of nanoparticle shapes are applicable to the aspects described herein, including but not limited to spheres, cylinders, ellipsoids, polyhedra, prisms, rods, lines, or combinations thereof. In one embodiment, as those skilled in the art will understand, the shape of the nanoparticle contributes to optical properties (e.g., nanorods may have different optical properties than nanospheres).

[0141] In one embodiment, the typical size of the chromophore polymer particles is less than 100 nanometers. In one embodiment, the colloidal polymer nanoparticles are composed of a hydrophobic polymer interior. In one embodiment, the chromophore polymer particles comprise at least one chromophore polymer that has formed stable particles. For example, the particle size can vary between 5 nanometers and 500 nanometers. In one embodiment, the critical size (e.g., diameter) of the particles is less than 1,000 nanometers, less than 700 nanometers, less than 500 nanometers, less than 400 nanometers, less than 300 nanometers, less than 200 nanometers, less than 100 nanometers, less than 50 nanometers, or less than 40 nanometers. In one embodiment, the critical size of the particles is less than 30 nanometers, less than 20 nanometers, or less than 10 nanometers.

[0142] In one embodiment, the chromophore polymer particles described herein comprise a polymer matrix formed of one or more chromophore polymers. Any suitable number and combination of chromophore polymer types may be incorporated into the chromophore polymer particles described herein, such as one or more chromophore polymers, two or more chromophore polymers, three or more chromophore polymers, four or more chromophore polymers, five or more chromophore polymers, six or more chromophore polymers, seven or more chromophore polymers, eight or more chromophore polymers, nine or more chromophore polymers, ten or more chromophore polymers, fifty or more chromophore polymers, or one hundred or more chromophore polymers. The mass concentration or mass ratio of the chromophore polymer relative to the total mass of the chromophore polymer particles may vary between 1% and 99%, 10% and 99%, 20% and 99%, 30% and 99%, 40% and 99%, or 50% and 99%.

[0143] Chromophore polymers of various types and compositions are suitable for use in various aspects of this disclosure. Chromophore polymers can be homopolymers or hybrids. In various embodiments, the chromophore polymer is a semiconductor polymer, a non-semiconductor polymer, or a combination thereof. For example, many semiconductor polymers are suitable for chromophore polymer particles according to this disclosure. Examples of semiconductor polymers include, but are not limited to: polyfluorene-based polymers, including, but not limited to, poly(9,9-dihexylfluorenyl-2,7-diyl) (PDHF) and poly(9,9-dioctylfluorenyl-2,7-diyl) (PFO); fluorene-based copolymers, including, but not limited to: poly[{9,9-dioctyl-2,7-divinylfluoreneyl}-alternating-copolymer-{2-methoxy-5- (2-Ethylhexyloxy)-1,4-phenylene] (PFPV), based on poly[(9,9-dioctylfluorenyl-2,7-diyl)-copolymer-(1,4-benzo-{2,1,3}-thiadiazole)] (PFBT), based on poly[(9,9-dioctylfluorenyl-2,7-diyl)-copolymer-(4,7-di-2-thienyl-2,1,3-benzothiadiazole)] (PFTBT), and based on poly[( 9,9-Dioctylfluorenyl-2,7-diyl)-copolymer-(4,7-di-2-thienyl-2,1,3-benzothiadiazole)](PF-0.1TBT); phenylene vinylidene polymers, including but not limited to: semiconductor polymers based on poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene vinylidene](MEH-PPV) and semiconductor polymers based on poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-(1-cyanovinylidene-1,4-phenylene)](CN-PPV); polymers based on phenylene ethynyl, including but not limited to semiconductor polymers based on poly(2,5-di(3',7'-dimethyloctyl)phenylene-1,4-ethynyl (PPE); semiconductor polymers based on BODIPY; semiconductor polymers based on squaric acid; or combinations thereof.

[0144] Various chromophore polymer structures are suitable for use according to various embodiments of the present disclosure. In one embodiment, the chromophore polymer is a linear polymer. In other aspects, the chromophore polymer is a branched polymer. In one embodiment, the chromophore polymer is a dendritic polymer. In one embodiment, the chromophore polymer is a brush polymer. In one embodiment, the chromophore polymer is a star polymer.

[0145] In one embodiment, the chromophore polymer particles described herein contain a comb-like polymer based on polystyrene. Non-limiting examples of polystyrene-based comb-like polymers include polystyrene grafted with acrylic acid, polystyrene grafted with ethylene oxide, and polystyrene grafted with butanol. In one embodiment, the chromophore polymer particles described herein contain a comb-like polymer based on poly(methyl methacrylate). Non-limiting examples of poly(methyl methacrylate)-based comb-like polymers include poly(methyl methacrylate) grafted with acrylic acid and poly(methyl methacrylate) grafted with ethylene oxide. In one embodiment, the chromophore polymer particles described herein contain a comb-like polymer comprising carboxyl, amine, thiol, ester, succinimidyl ester, azide, alkyne, cyclooctylene, or phosphine groups.

[0146] In one embodiment, the chromophore polymer particles described herein contain polymers functionalized at the terminal repeating units, for example with carboxyl, amine, thiol, ester, succinimide ester, azide, alkyne, cyclooctyne, phosphine, or similar functional groups. Examples of such polymers include, but are not limited to, poly(meth)acrylate polymers, polyacrylamide polymers, polyisobutylene, polydiene, polyphenylene, polyethylene, poly(ethylene glycol), polylactide, polystyrene, polysiloxane, poly(vinylpyridine), poly(vinylpyrrolidone), polyurethane, block copolymers thereof, random or alternating copolymers thereof, etc.

[0147] In one embodiment, the chromophore polymer particles described herein contain copolymers having one or more functionalized repeating units, such as amphiphilic polymers, including, but not limited to, copolymers based on poly((meth)acrylic acid), such as: poly(acrylic acid-b-acrylamide), poly(acrylic acid-b-methyl methacrylate), poly(acrylic acid-bN-isopropylacrylamide), poly(n-butyl acrylate-b-acrylic acid), poly(sodium acrylate-b-methyl methacrylate), poly(methyl methacrylate-b-neopentyl acrylate), poly(methyl methacrylate-b-acrylic acid), poly(methyl methacrylate-b-methacrylic acid), poly(methyl methacrylate-bN,N-dimethyl methacrylate). Poly(methyl methacrylate-b-acrylate), poly(methyl methacrylate-b-methacrylate), poly(neopentyl methacrylate-b-methacrylic acid), poly(t-butyl methacrylate-b-ethylene oxide), poly(2-acrylamido-2-methylpropanesulfonic acid-b-acrylic acid); copolymers based on polydiene, such as: poly(butadiene(1,2-addition)-b-ethylene oxide), poly(butadiene(1,2-addition)-b-methacrylic acid), poly(butadiene(1,4-addition)-b-acrylic acid), poly(butadiene(1,4-addition)-b-ethylene oxide), poly(butadiene(1,4-addition)-b-acrylate), poly(butadiene(1,4-addition)-b-acrylate), poly(butadiene(1,4-addition)-b-acrylate), poly(butadiene(1,4-addition)-b-acrylate), poly(butadiene(1,4-addition)-b-acrylate), poly(butadiene(1,4-addition)-b-acrylate), poly(butadiene(1,4-addition)-b-acrylate). (Addition)-bN-methyl4-vinylpyridine iodide), poly(isoprene-b-ethylene oxide), poly(isoprene-b-ethylene oxide), and poly(isoprene-bN-methyl2-vinylpyridine iodide); copolymers based on poly(ethylene oxide), such as: poly(ethylene oxide-b-acrylic acid), poly(ethylene oxide-b-acrylamide), poly(ethylene oxide-b-epoxybutane), poly(ethylene oxide-bc-caprolactone), poly(ethylene oxide-b-lactide), poly(ethylene oxide-b-lactide), poly(ethylene oxide-b-methacrylic acid), poly(ethylene oxide-b-methyl acrylate), poly(ethylene oxide-bN-isopropylacrylamide), poly(ethylene oxide-b- Methyl methacrylate, poly(ethylene oxide-b-nitrobenzyl methacrylate), poly(ethylene oxide-bN,N-dimethylaminoethyl methacrylate), poly(ethylene oxide-b-propylene oxide), poly(ethylene oxide-bt-butyl acrylate), poly(ethylene oxide-b-tert-butyl methacrylate), poly(ethylene oxide-b-tetrahydrofurfuryl methacrylate), poly(ethylene oxide-b-2-ethyloxazoline), poly(ethylene oxide-b-2-hydroxyethyl methacrylate), poly(ethylene oxide-b-2-methyloxazoline); copolymers based on polyisobutylene, such as poly(isobutylene-b-acrylic acid), poly(isobutylene-b-ethylene oxide), poly(isobutylene-b-methacrylic acid);Copolymers based on polystyrene, such as poly(styrene-b-acrylamide), poly(styrene-b-acrylic acid), poly(styrene-b-acrylic acid cesium), poly(styrene-b-ethylene oxide), poly(styrene-b-ethylene oxide) acid that can be cut at block junctions, poly(styrene-b-methacrylic acid), poly(4-styrenesulfonate-b-ethylene oxide), poly(styrenesulfonate-b-methylbutene), poly(styrene-bN,N-dimethylacrylamide), poly(styrene-bN-isopropylacrylamide), poly(styrene-bN-methyl2-vinyl iodide pyridine), poly(styrene-bN-methyl-4-vinyl iodide pyridine), poly(styrene-b-propylacrylic acid), poly(sodium styrene-b-acrylate), poly(sodium styrene-b-methacrylate), poly(p-chloromethylstyrene-b-acrylamide), poly(styrene-copolymer-p-chloromethylstyrene-b-acrylamide), poly(styrene-copolymer-p-chloromethylstyrene-b-acrylic acid), poly(styrene-ethylhexylene- ... Poly(dimethylsiloxane-b-methylbutene-copolymer-isoprene sulfonate); copolymers based on polysiloxanes, such as poly(dimethylsiloxane-b-acrylic acid), poly(dimethylsiloxane-b-ethylene oxide), poly(dimethylsiloxane-b-methacrylic acid); copolymers based on poly(ferrocene dimethylsilane), such as poly(ferrocene dimethylsilane-b-ethylene oxide); copolymers based on poly(2-vinylnaphthalene), such as poly(2-vinylnaphthalene-b-acrylic acid); copolymers based on poly(vinylpyridine and N-methyl... Copolymers of vinylpyridine (2-vinylpyridine-b-ethylene oxide), such as poly(2-vinylpyridine-b-methacrylic acid), poly(N-methyl-2-vinylpyridine-b-ethylene oxide), poly(N-methyl-4-vinylpyridine-b-methacrylate), poly(4-vinylpyridine-b-ethylene oxide)PEO-terminated OH; and copolymers based on poly(vinylpyrrolidone), such as poly(vinylpyrrolidone-bD / L-lactide); etc.

[0148] In embodiments of this disclosure, the chromophore polymer particles provided herein comprise the polymer CN-PPV, also known as poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-(1-cyanovinylene-1,4-phenylene)], which is a bright, compact, orange-emitting semiconductor polymer particle. In one embodiment, CN-PPV exhibits excellent fluorescence properties, such as a large absorption cross-section, high quantum yield, and fast emission rate. In one embodiment, the chromophore polymer particles comprise a polymer substantially composed of CN-PPV. In one embodiment, the particles comprise CN-PPV and at least one other material. For example, CN-PPV may be blended with copolymers or other materials that provide additional functionality.

[0149] In one embodiment, the chromophore polymer particles of this disclosure comprise a semiconductor copolymer having at least two different chromophore units. For example, the conjugated copolymer may contain fluorene and benzothiazole chromophore units present in a given ratio. Typical chromophore units used to synthesize semiconductor copolymers include, but are not limited to, fluorene units, phenylene vinylidene units, phenylene units, phenylene ethynylidene units, benzothiazole units, thiophene units, carbazole fluorene units, boron-dipyrrole methylene units, and derivatives thereof. Different chromophore units may be separated, such as in block copolymers, or doped. In one embodiment, the chromophore copolymer is indicated by writing the identity of the primary chromophore species. For example, PFBT is a chromophore polymer containing fluorene and benzothiazole units in a certain ratio. In some cases, a dash is used to indicate the percentage of the secondary chromophore species, followed by the identity of the secondary chromophore species. For example, PF-0.1BT is a chromophore copolymer containing 90% polyfluorene (PF) and 10% benzothiazole (BT).

[0150] In one embodiment, the chromophore polymer particles comprise a blend of semiconductor polymers. The blend can comprise any combination of homopolymers, copolymers, and oligomers. The polymer blends used to form the chromophore polymer particles can be selected to tune the properties of the resulting polymer particles, for example, to achieve a desired excitation or emission spectrum of the polymer particles.

[0151] In various embodiments of this disclosure, the semiconductor chromophore polymer particles provide improved detection sensitivity, in part because they exhibit a higher quantum yield than other fluorescent reporter materials. In one embodiment, the quantum yield of the chromophore polymer particles used is greater than 5%, greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%. In various embodiments, the semiconductor chromophore polymer particles provide improved detection sensitivity, in part because they exhibit a large absorption cross-section. In various embodiments, the semiconductor chromophore polymer particles provide improved detection sensitivity, in part because they exhibit a faster emission rate than other fluorescent reporter materials. In one embodiment, the emission rate of the chromophore polymer particles used is between about 100 picoseconds and about 50 nanoseconds.

[0152] In one embodiment, the chromophore polymer particles described herein comprise polymers carrying units of small organic dye molecules, metal complexes, environmentally conductive dyes, photochromic dyes, and any combination thereof, such as optically inactive polymers, like polystyrene covalently linked or grafted with small organic dyes, metal complexes, environmentally conductive dyes, photochromic dyes, or any combination thereof. In one embodiment, the chromophore polymer particles comprise semiconductor polymers covalently linked as emission units to small organic dye molecules, metal complexes, environmentally conductive dyes, photochromic dyes, or any combination thereof. Such emission units can tune emission colors, increase quantum yield, or improve the photophysical properties of the chromophore polymer particles. In one embodiment, the small organic dye or metal complex has sensing capabilities, and thus adds additional functionality to the chromophore polymer particles, such as ion sensing capabilities.

[0153] In one embodiment, the nanoparticle transducer comprises one or more chromophores (e.g., fluorophores). Chromophore emission depends on the fluorescence of a fluid component. In one embodiment, the fluid component is a reaction element of an enzyme-catalyzed reaction involving an analyte within the nanoparticle transducer. In some cases, the fluid component is a product of the reaction; in others, it is a reactant. In one embodiment, the reaction rate varies according to the analyte concentration, thereby altering the concentration of the fluid component and causing a corresponding change in transducer fluorescence.

[0154] In one embodiment, the chromophore comprises a dye. In another embodiment, the dye is sensitive to one or more fluid components. Examples of dyes that can be used with the nanoparticle transducers disclosed herein include Pt(II)-porphyrins and Pd(II)-porphyrins, phosphorescent Ru(II) complexes, and Ir(III) complexes. Examples of dyes include, but are not limited to, Pt(II) octaethylporphyrin (PtOEP), Pt(II) meso-tetra(pentafluorophenyl)porphyrin (PtTFPP), Pt(II) octaethylporphyrinone (PtOEPK), Pd(II) octaethylporphyrin (PdOEP), Pd(II) meso-tetra(pentafluorophenyl)porphyrin (PdTFPP), Pd(II)-meso-tetra-(4-carboxyphenyl)porphyrin (PdTPCPP), and Pd(II)-meso-tetra-(4-carboxyphenyl)tetrabenzoporphyrin dendritic (P dTCPTBP), Pt(II)-coprophyll (PtCP), Pt(II)-meta-tetrabenzoporphyrin butyl octaester (PtTBP), Pt(II)-coprophyll-one (PtCPK), cyclometalated Ir(III)1-chlorobridged dimer coumarin complex (Ir(III)(Cx)2(acac)) and [Ru(bpy)2(2-(4-carboxyphenyl)imidazo-[4,5-f][1,10]o-phenanthroline)H2)]2+([Ru(bpy)2(picH2)]2+).

[0155] In one embodiment, the chromophore includes a chromophore unit sensitive to ions, pH, reactive oxygen species, reactive nitrogen species, and temperature. In another embodiment, the chromophore includes a chromophore unit or dye sensitive to ions, pH, reactive oxygen species, reactive nitrogen species, and temperature. Examples of chromophore units or dyes used to construct nanoparticle transducers include sodium-sensitive, potassium-sensitive, calcium-sensitive, magnesium-sensitive, iron-sensitive, zinc-sensitive, copper-sensitive, manganese-sensitive, pH-sensitive, reactive oxygen species-sensitive, reactive nitrogen species-sensitive, or temperature-sensitive dyes or chromophore units. Nanoparticles including chromophores sensitive to ions, pH, reactive oxygen species, reactive nitrogen species, and temperature include, for example, those nanoparticles described in PCT / US 2010 / 056079.

[0156] In one embodiment, the chromophore comprises a semiconductor chromophore polymer that is sensitive to one or more fluid components. The semiconductor polymer can be designed and synthesized to have fluorescence that is sensitive to one or more fluid components.

[0157] In one embodiment, chromophore emission depends on NADH and / or NADPH or NAD. + and / or NADP + Fluorescence at concentrations as further discussed herein with respect to examples of this disclosure.

[0158] In one embodiment, the chromophore emits fluorescence dependent on the concentration of hydrogen peroxide (H₂O₂). Hydrogen peroxide may be a product reaction element. In one embodiment, the nanoparticles comprise a chromophore polymer that emits fluorescence dependent on the concentration of hydrogen peroxide. In one embodiment, the nanoparticles comprise a chromophore polymer that emits fluorescence at one or more wavelengths and a dye. The amount of fluorescence of the dye may depend on the concentration of hydrogen peroxide. For example, the dye may be physically doped or chemically linked to the chromophore polymer to form nanoparticles. Energy transfer may occur between the chromophore polymer and the dye to enhance or amplify the fluorescence intensity of the dye. Examples of hydrogen peroxide-sensitive dyes that can be used with the nanoparticle transducers disclosed herein include coumarin derivatives, fluorescein derivatives, rhodamine derivatives, anthocyanin derivatives, and boron-dipyrrole methylene (BODIPY) derivatives.

[0159] In one embodiment, the chromophore emits fluorescence dependent on the oxygen concentration. Oxygen can be a reactant element. In one embodiment, the nanoparticles comprise a chromophore polymer that emits fluorescence dependent on the oxygen concentration. In another embodiment, the nanoparticles comprise a chromophore polymer that emits fluorescence at one or more wavelengths and a dye. The amount of fluorescence of the dye can depend on the oxygen concentration. For example, the dye can be physically doped or chemically linked to the chromophore polymer to form nanoparticles. Energy transfer can occur between the chromophore polymer and the dye to enhance or amplify the fluorescence intensity of the dye. Examples of oxygen-sensitive dyes that can be used with the nanoparticle transducers disclosed herein include Pt(II)-porphyrins and Pd(II)-porphyrins, phosphorescent Ru(II) complexes and Ir(III) complexes and their derivatives.

[0160] In one embodiment, the chromophore comprises a dye and a semiconductor chromophore polymer, and the dye and semiconductor polymer interact to produce enhanced fluorescence. In one embodiment, the semiconductor polymer is insensitive to fluid components; fluorescence from such polymers can provide a stable internal standard, thereby serving as a control for variable fluorescence signals at other wavelengths. The semiconductor chromophore polymer can transfer energy to the dye to amplify and enhance the dye's fluorescence. In one embodiment, the semiconductor polymer is sensitive to fluid components; fluorescence from such polymers can indicate the presence and / or concentration of an analyte. Examples of semiconductor chromophore polymers that can be used with the nanoparticle transducers disclosed herein include: poly(9,9-dihexylfluorene) (PDHF), poly(9,9-dioctylfluorene) (PFO), and poly{[9,9-di-(3-(3-methyloxacyclobut-3-yl)methoxy)hexylfluorene-2,7-diyl-copolymer-[9,9-dioctylfluorene-2,7-diyl]} (do-PFO); poly[{9,9-dioctyl-2,7-divinylfluorene}-alternating-copolymer-{2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene}] (PFPV); and poly[(9,9-dioctylfluorene-2,7-diyl)-copolymer-(1,4-benzo-{2,1,3}-thiadiazole)] (PFPV). The dye comprises, but is not limited to, poly((9,9-dioctylfluorenyl-2,7-diyl)-copolymer-(4,7-di-2-thienyl-2,1,3-benzothiadiazole)] (PFTBT); phenylene vinylidene polymers, including but not limited to, poly(2-methoxy-5-(2-ethylhexoxy)-1,4-phenylene vinylidene) (MEH-PPV); poly(2-methoxy-5-(2-ethylhexoxy)-1,4-(1-cyanovinylene-1,4-phenylene)] (CN-PPV); poly(2,5-di(3',7'-dimethyloctyl)phenylene-1,4-ethynyl (PPE); BODIPY; and squaric acid-based semiconductor polymers; and derivatives thereof. In one embodiment, the dye is sensitive to one or more fluid components.

[0161] In one embodiment, the chromophore comprises multiple dyes. A first dye is sensitive to one or more fluid components, and a second dye can interact with the sensitive dye to produce enhanced fluorescence. In one embodiment, at least one dye is insensitive to the fluid component, thus providing stable fluorescence as an internal standard. The multiple dyes can emit fluorescence at different wavelengths, allowing for independent measurement of the fluorescence of each dye. Sensitive and insensitive dyes can interact with each other to amplify and enhance the fluorescence of the dye sensitive to one or more fluid components.

[0162] In one embodiment, the chromophore comprises a plurality of semiconductor polymers. In one embodiment, a first semiconductor polymer among the plurality of semiconductor polymers is sensitive to one or more fluid components, and a second semiconductor polymer among the plurality of semiconductor polymers is insensitive to fluid components, thus providing stable fluorescence as an internal standard. The plurality of semiconductor polymers can emit fluorescence at different wavelengths, thereby allowing independent measurement of the fluorescence of each semiconductor polymer. In one embodiment, the chromophore comprises a semiconductor polymer. In one embodiment, a first monomer unit of the semiconductor polymer among the plurality of monomer units of the semiconductor polymer is sensitive to one or more fluid components, and a second monomer unit of the semiconductor polymer among the plurality of monomer units of the semiconductor polymer is insensitive to fluid components, thus providing stable fluorescence as an internal standard. The plurality of monomer units of the semiconductor polymer can emit fluorescence at different wavelengths, thereby allowing measurement of the fluorescence of different semiconductor polymer monomer units.

[0163] In one embodiment, the chromophore polymer particles include semiconductor polymers that are physically mixed or chemically crosslinked with other chromophore polymers, such as inactive polymers covalently linked or grafted with small organic dyes, metal complexes, photochromic dyes or any combination thereof, to have additional functions such as ion sensing or metabolite sensing.

[0164] In one embodiment, the chromophore polymer particles comprise semiconductor polymers that are physically mixed or chemically crosslinked with other components such as fluorescent dyes, inorganic luminescent materials, magnetic materials, and metallic materials, to tune emission colors, improve quantum yield and / or photostability, and / or provide additional functions such as magnetic functions and plasmon resonance functions.

[0165] Given the optical properties of chromophore polymer particles, such as absorption wavelength, they can be tuned by modifying their composition and / or structure. Semiconductor polymers have been developed to absorb wavelengths in the UV to infrared range, encompassing the entire visible spectrum. In one embodiment, chromophore polymer particles with peak absorption wavelengths between about 200 nm and about 300 nm, about 250 nm and about 350 nm, about 300 nm and about 400 nm, about 350 nm and about 450 nm, about 400 nm and about 500 nm, about 450 nm and about 550 nm, about 500 nm and about 600 nm, about 550 nm and about 650 nm, about 600 nm and about 700 nm, about 650 nm and about 750 nm, about 700 nm and about 800 nm, about 750 nm and about 850 nm, about 800 nm and about 900 nm, about 850 nm and about 950 nm, or about 900 nm and about 1000 nm are used.

[0166] Semiconductor polymers have been developed to emit wavelengths in the UV to infrared range, encompassing the entire visible spectrum. In one embodiment, peak emission wavelengths between about 200 nm and about 300 nm, about 250 nm and about 350 nm, about 300 nm and about 400 nm, about 350 nm and about 450 nm, about 400 nm and about 500 nm, about 450 nm and about 550 nm, about 500 nm and about 600 nm, about 550 nm and about 650 nm, about 600 nm and about 700 nm, and about 650 nm and about 700 nm are used. Chromophore polymer particles between approximately 750 nm, approximately 700 nm and approximately 800 nm, approximately 750 nm and approximately 850 nm, approximately 800 nm and approximately 900 nm, approximately 850 nm and approximately 950 nm, approximately 900 nm and approximately 1000 nm, approximately 950 nm and approximately 1050 nm, approximately 1000 nm and approximately 1100 nm, approximately 1150 nm and approximately 1250 nm, or approximately 1200 nm and approximately 1300 nm.

[0167] In one embodiment, this disclosure provides one or more chromophores having narrow-band emission. Narrow-band emission is advantageous for certain applications, including but not limited to resolution of multiple fluorescence signals. The emission wavelength of one or more chromophores can vary between the ultraviolet and near-infrared regions. In one embodiment, the FWHM of the emission band is less than about 100 nm, about 70 nm, about 65 nm, about 60 nm, about 55 nm, about 50 nm, about 45 nm, about 40 nm, about 35 nm, about 30 nm, about 25 nm, about 20 nm, or about 10 nm. In one embodiment, the FWHM of the polymer particles described herein can be in the range of about 5 nm to about 100 nm, about 10 nm to about 70 nm, about 20 nm to about 60 nm, or about 30 nm to about 50 nm.

[0168] In one embodiment, the various chromophores of this disclosure comprise polymers having narrow-band emission units (e.g., narrow-band repeating units and / or narrow-band units). For example, this disclosure may comprise homopolymers or hybrids containing narrow-band repeating units such as BODIPY and / or BODIPY derivative repeating units, squaric acid and / or squaric acid derivative repeating units, metal complexes and / or metal complex derivative repeating units, porphyrins and / or porphyrin derivative repeating units, metalloporphyrins and / or metalloporphyrin derivative repeating units, phthalocyanines and / or phthalocyanine derivative repeating units, lanthanide complexes and / or lanthanide complex derivative repeating units, perylene and / or perylene derivative repeating units, anthocyanins and / or anthocyanin derivative repeating units, rhodamine and / or rhodamine derivative repeating units, coumarin and / or coumarin derivative repeating units, and / or xabutane and / or xabutane derivative repeating units. In one embodiment, the narrowband unit is, for example, a narrowband repeating unit or a fluorescent nanoparticle embedded in or connected to the polymer particles. One or more chromophores may comprise, for example, quantum dots. Optionally, the narrowband unit comprises a polymer or fluorescent dye molecule that produces narrow emission within the polymer particles of this disclosure.

[0169] In some embodiments, the chemical composition and structure of one or more chromophores can affect the absorption spectrum of one or more chromophores. The absorption peak can shift from the ultraviolet region to the infrared region. In some embodiments, the absorption peak of one or more chromophores can be tuned to a laser wavelength. In some embodiments, for example, the absorption peak can be tuned to 405 nm. In some embodiments, the absorption peak can be tuned to about 450 nm. In some embodiments, the absorption peak can be tuned to about 488 nm. In some embodiments, the absorption peak can be tuned to about 532 nm. In some embodiments, the absorption peak can be tuned to about 561 nm. In some embodiments, the absorption peak can be tuned to about 633 nm. In some embodiments, the absorption peak can be tuned to about 635 nm. In some embodiments, the absorption peak can be tuned to about 640 nm. In some embodiments, the absorption peak can be tuned to about 655 nm. In some embodiments, the absorption peak can be tuned to about 700 nm. In some embodiments, the absorption peak can be tuned to about 750 nm. In some embodiments, the absorption peak can be tuned to about 800 nm. In some embodiments, the absorption peak can be tuned to about 850 nm. In some embodiments, the absorption peak can be tuned to about 900 nm. In some embodiments, the absorption peak can be tuned to approximately 980 nm. In some embodiments, the absorption peak can be tuned to the near-infrared region of the wavelength spectrum (e.g., from 750 nm to 1200 nm). In some embodiments, the absorption peak can be tuned to approximately 1064 nm. In some embodiments, for example, the absorption peak can be tuned to between 380 nm and 420 nm. In some embodiments, the absorption peak can be tuned to between 440 nm and 460 nm. In some embodiments, the absorption peak can be tuned to between 478 nm and 498 nm. In some embodiments, the absorption peak can be tuned to between 522 nm and 542 nm. In some embodiments, the absorption peak can be tuned to between 550 nm and 570 nm. In some embodiments, the absorption peak can be tuned to between 625 nm and 645 nm. In some embodiments, the absorption peak can be tuned to between 645 nm and 665 nm. In some embodiments, the absorption peak can be tuned to between 690 nm and 710 nm. In some embodiments, the absorption peak can be tuned to between 740 nm and 760 nm. In some embodiments, the absorption peak can be tuned to between 790 nm and 810 nm. In some embodiments, the absorption peak can be tuned to between 890 nm and 910 nm. In some embodiments, the absorption peak can be tuned to between 970 nm and 990 nm. In some embodiments, the absorption peak can be tuned to between 1054 nm and 1074 nm.

[0170] In some embodiments, the absorbance width of the chromophore is measured at 20% to 16% of the maximum absorbance. In some embodiments, the absorbance width of the chromophore at 20% of the maximum absorbance is less than 200 nm, less than 190 nm, less than 180 nm, less than 170 nm, less than 160 nm, less than 150 nm, less than 140 nm, less than 130 nm, less than 120 nm, less than 110 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, or less than 40 nm. In some embodiments, the absorbance width of the nanoparticles at 19% of the maximum absorbance is less than 200 nm, less than 190 nm, less than 180 nm, less than 170 nm, less than 160 nm, less than 150 nm, less than 140 nm, less than 130 nm, less than 120 nm, less than 110 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, or less than 40 nm. In some embodiments, the absorbance width of the chromophore at 18% of the maximum absorbance is less than 200 nm, less than 190 nm, less than 180 nm, less than 170 nm, less than 160 nm, less than 150 nm, less than 140 nm, less than 130 nm, less than 120 nm, less than 110 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, or less than 40 nm. In some embodiments, the absorbance width of the chromophore at 17% of its maximum absorbance is less than 200 nm, less than 190 nm, less than 180 nm, less than 170 nm, less than 160 nm, less than 150 nm, less than 140 nm, less than 130 nm, less than 120 nm, less than 110 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, or less than 40 nm. In some embodiments, the absorbance width of the chromophore at 16% of its maximum absorbance is less than 200 nm, less than 190 nm, less than 180 nm, less than 170 nm, less than 160 nm, less than 150 nm, less than 140 nm, less than 130 nm, less than 120 nm, less than 110 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, or less than 40 nm. In some embodiments, the absorbance width of the chromophore at 15% of its maximum absorbance is less than 200 nm, less than 190 nm, less than 180 nm, less than 170 nm, less than 160 nm, less than 150 nm, less than 140 nm, less than 130 nm, less than 120 nm, less than 110 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, or less than 40 nm.In some embodiments, the absorbance width of the chromophore at 14% of its maximum absorbance is less than 200 nm, less than 190 nm, less than 180 nm, less than 170 nm, less than 160 nm, less than 150 nm, less than 140 nm, less than 130 nm, less than 120 nm, less than 110 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, or less than 40 nm. In some embodiments, the absorbance width of the chromophore at 13% of its maximum absorbance is less than 200 nm, less than 190 nm, less than 180 nm, less than 170 nm, less than 160 nm, less than 150 nm, less than 140 nm, less than 130 nm, less than 120 nm, less than 110 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, or less than 40 nm. In some embodiments, the absorbance width of the chromophore at 12% of its maximum absorbance is less than 200 nm, less than 190 nm, less than 180 nm, less than 170 nm, less than 160 nm, less than 150 nm, less than 140 nm, less than 130 nm, less than 120 nm, less than 110 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, or less than 40 nm. In some embodiments, the absorbance width of the chromophore at 11% of its maximum absorbance is less than 200 nm, less than 190 nm, less than 180 nm, less than 170 nm, less than 160 nm, less than 150 nm, less than 140 nm, less than 130 nm, less than 120 nm, less than 110 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, or less than 40 nm. In some embodiments, the absorbance width of the chromophore at 10% of its maximum absorbance is less than 200 nm, less than 190 nm, less than 180 nm, less than 170 nm, less than 160 nm, less than 150 nm, less than 140 nm, less than 130 nm, less than 120 nm, less than 110 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, or less than 40 nm.

[0171] In embodiments of this disclosure, the devices, compositions, systems, and methods provided herein utilize one or more chromophores (e.g., dyes or semiconductor chromophore polymers) capable of generating fluorescence at one or more wavelengths, for example, in response to incident radiation such as UV light, visible light, far-red light, near-infrared light, or other light. In some cases, the amount of fluorescence from the chromophore at a given wavelength varies according to the local concentration of the fluid component (signal chromophore); in others, the amount of fluorescence from the chromophore does not vary in response to the local concentration (control chromophore). In one embodiment, the nanoparticles provided herein may incorporate both a signal chromophore and a control chromophore that emit fluorescence at signal and control wavelengths, respectively. Although the various embodiments herein are described in the context of nanoparticles having one or two different emission wavelengths, it should be understood that the methods presented herein are also applicable to nanoparticles emitting more than two wavelengths. For example, nanoparticles emitting at two signal wavelengths and one or two control wavelengths can be provided, and these nanoparticles can be used for measuring signals of multiple analytes. Multiple different nanoparticles with different signal / control wavelength pairs can be provided, each nanoparticle responding to a different analyte (or optionally responding to the same analyte, for example, for redundant signal transduction).

[0172] In one embodiment, the chromophore that generates fluorescence at the signal wavelength exhibits different optical properties (e.g., emission spectrum, absorption spectrum, peak emission wavelength, peak excitation wavelength, emission intensity, emission lifetime, emission rate) when present in different concentrations of the fluid component. For example, the chromophore may exhibit increased (or decreased) fluorescence in response to an increase in the concentration of the fluid component (e.g., molecules). In one embodiment, the change in fluorescence may be proportional to the concentration of the fluid component. For example, the molecule may be oxygen, NADH, NADPH, or NAD. + or NADP + Alternatively, hydrogen peroxide can be used, which can be a reactant or product involved in a reaction of an analyte to be measured and catalyzed by an enzyme. The enzyme can physically associate or couple with nanoparticles including chromophores, such that the enzyme-catalyzed reaction alters the local concentration of the molecule, thereby changing the fluorescence of the chromophore in response to changes in the concentration of the analyte. In one embodiment, such changes can be proportional. For example, fluorescence at a control wavelength can be generated, such that the fluorescence ratio of the control to the signal can serve as a signal of the analyte concentration, thereby eliminating or reducing certain noise sources and uncertainties in fluorescence intensity measurements.

[0173] Enzyme composition

[0174] In the embodiments disclosed herein, small molecule detection is provided based on the integration of an enzyme, such as a NADH-dependent or NADPH-dependent enzyme, with a chromophore. The enzyme catalyzes a small molecule reaction and comprises multiple reaction elements, and the chromophore is configured to emit fluorescence based on the concentration of one of the reaction elements (e.g., NADH or NADPH). In the embodiments disclosed herein, molecule detection (e.g., lipids, carbohydrates, proteins, nucleic acids, metabolites, peptides, drugs, enzyme substrates) is provided based on the integration of an enzyme, such as a NADH-dependent or NADPH-dependent enzyme, with a chromophore. The enzyme catalyzes a molecular reaction and comprises multiple reaction elements, and the chromophore is configured to emit fluorescence based on the concentration of one of the reaction elements (e.g., NADH or NADPH). In the embodiments disclosed herein, small molecule detection is provided based on the integration of a transducer with an enzyme, such as a NADH-dependent or NADPH-dependent enzyme catalyzing a small molecule reaction.

[0175] In some cases, chromophores can be directly mixed with enzymes for measurement. In others, covalent conjugation is provided to link nanoparticles to enzymes, thereby creating compact probes that can be used, for example, for intracellular sensing. In one embodiment, the enzyme and chromophore are physically associated. As discussed further herein, such physical association can include the enzyme and chromophore dispersed in a common solvent, the enzyme and chromophore coupled to a common substrate, the enzyme and chromophore lyophilized in a common powder, the enzyme and chromophore encapsulated in hydrogel beads, or the enzyme and chromophore otherwise physically / chemically contacted. In one embodiment, the enzyme physically associated with the chromophore is coupled to the chromophore. In this respect, the enzyme is physically linked to the chromophore directly or indirectly. Such coupling can contain covalent bonds. In another embodiment, the coupling between the enzyme and chromophore is through one or more non-covalent bonds or interactions, such as ionic bonds, van der Waals forces, hydrogen bonding, etc. Therefore, in one embodiment, the coupling does not contain covalent bonds.

[0176] As discussed elsewhere herein, chromophores can take many different forms and include many different types of chromophores. In one embodiment, the chromophore is in the form of nanoparticles. In one embodiment, the chromophore is a semiconductor polymer, such as in the form of semiconductor polymer nanoparticles. As described elsewhere herein, in one embodiment, the chromophore is part of a polymer dot or p-dot, such as where the chromophore and other polymer components exist in a condensed, stable, submicron state. In one embodiment, the chromophore may comprise a chromophore polymer in an uncondensed state, such as a semiconductor chromophore polymer.

[0177] When enzyme crowns form on the surface of nanoparticles, nanoparticle-enzyme bioconjugates exhibit depletion or generation in the presence of small molecule analytes to which the enzyme is sensitive, such as NAD+. + or NADP +Alternatively, nanoreactors for NADH or NADPH. Therefore, the concentration of small molecules is monitored by the optical signal from the transducer when the analyte is depleted or generated. The performance of this sensing scheme depends on factors including: (1) whether the presence of the analyte causes a significant change in the distribution curve of the reaction elements; and (2) whether the transducer can convert changes in analyte concentration into optical signals. Furthermore, in vivo detection is also closely related to issues such as local microvascular perfusion, the effectiveness of the analyte in tissues, and enzyme activity. In the following examples, NAD... + / NADH is provided as an example, through which the effectiveness of the transducer described herein in sensing analyte concentrations can be demonstrated by both theoretical analysis and experimental evidence for in vitro and in vivo applications. Based on the example described herein, transducers can be fabricated to generate fluorescence signals for the detection of a variety of analytes, including small molecules, macromolecules, and other fluid components, by selecting appropriate reactive enzymes and corresponding chromophores sensitive to the reaction elements catalyzed by these enzymes.

[0178] In one embodiment, the nanoparticle transducer provided herein includes an enzyme, and the enzyme catalyzes a reaction. The reaction involves an analyte to be measured and produces products and consumes reactants, which are collectively referred to as reaction elements. In one embodiment, the reaction elements include a fluid component, and the concentration of the fluid component is changed by the reaction. For example, the fluid component may be a reaction product, and the reaction may increase the concentration of the fluid component. Alternatively, the fluid component may be a reactant, and the reaction may decrease the concentration of the reactant.

[0179] In one embodiment, the fluid component is NADH, and NADH is the product. In one embodiment, the fluid component is NADH, and NADH is a reactant. In one embodiment, the fluid component is NAD. + And NAD + It is a reactant. In one embodiment, the fluid component is NAD. + And NAD + It is a product. In one embodiment, the fluid component is NADPH, and NADPH is a product. In one embodiment, the fluid component is NADPH, and NADPH reactants. In one embodiment, the fluid component is NADP. + And NADP + It is a reactant. In one embodiment, the fluid component is NADP. + And NADP + It is a product. In one embodiment, the NADH-dependent or NADPH-dependent enzyme and analyte each comprise one or more of the following pairs: phenylalanine and phenylalanine dehydrogenase (see...). Figure 6A and 6B ), lactate and lactate dehydrogenase (see) Figure 17A and 17B ); glutamate and glutamate dehydrogenase (see Figure 17C and 17D ); glucose and glucose dehydrogenase (see Figure 17E and 17F ); and β-hydroxybutyrate (BHB) and BHB dehydrogenase (see Figure 17G and 17H ).

[0180] In one embodiment, the fluid component is not NADH; for example, the fluid component may be ions, where an enzyme catalyzes a reaction that changes the ion concentration, and the chromophore produces fluorescence regulated by the ion concentration; the fluid component may be an acid or base, where an enzyme catalyzes a reaction that changes the pH, and the chromophore produces fluorescence regulated by the pH; or the fluid component may be heat energy, where an enzyme catalyzes a reaction that changes the temperature, and the chromophore produces fluorescence regulated by the temperature. In one embodiment, the fluid component may be hydrogen peroxide, where an enzyme catalyzes a reaction that changes the hydrogen peroxide concentration, and the chromophore produces fluorescence regulated by the hydrogen peroxide concentration. For example, hydrogen peroxide may be a product of the reaction. In one embodiment, the fluid component is oxygen, where an enzyme catalyzes a reaction that changes the oxygen concentration, and the chromophore produces fluorescence regulated by the oxygen concentration. For example, oxygen may be a reactant of the reaction.

[0181] In one embodiment, multiple enzymes are coupled to a nanoparticle transducer to catalyze corresponding multiple reactions. The multiple reactions form a reaction chain, where one or more products of one reaction are reactants of another reaction. For example, the enzyme cascade can be provided by multiple enzymes, each performing the steps of the cascade. At least one of the multiple reactions involves an analyte as a reactant, and at least one of the reactions has a fluid component as a reaction element for modulating the emission intensity of the chromophore.

[0182] In one embodiment, the enzyme is an NADH-dependent or NADPH-dependent enzyme. In one embodiment, the NADH-dependent or NADPH-dependent enzyme is selected from the group consisting of: dehydrogenases, reductases, oxygenases, synthases, hydroxylases, and combinations thereof. In one embodiment, the NADH-dependent or NADPH-dependent enzyme is a dehydrogenase. In one embodiment, the dehydrogenase is selected from the group consisting of: (-)-menthol dehydrogenase; (+)-neomenthol dehydrogenase; (+)-juniperol dehydrogenase; (+)-trans-resveratrol dehydrogenase; (3S,4R)-3,4-dihydroxycyclohexyl-1,5-diene-1,4-dicarboxylic acid dehydrogenase; (R)-2-hydroxy fatty acid dehydrogenase; (R)-2-hydroxy acid dehydrogenase; (R)-4-hydroxyphenyl lactate dehydrogenase; (R)-aminopropanol dehydrogenase; (R)-dehydroubilizing acid dehydrogenase; (S)-2-hydroxy-fatty acid dehydrogenase; (S)-carnitine 3-dehydrogenase; 1,2-dihydroxy-6-methylcyclohexane-3,5- Dienecarboxylic acid dehydrogenase; 1,3-propanediol dehydrogenase; 1,6-dihydroxycyclohexane-2,4-diene-1-carboxylic acid dehydrogenase; 2-alkynyl-1-ol dehydrogenase; 2-dehydro-3-deoxy-D-gluconic acid 5-dehydrogenase; 2-deoxy-D-gluconic acid 3-dehydrogenase; 2-hydroxymethylglutarate dehydrogenase; 2-hydroxypropyl-CoM dehydrogenase; 2-oxoaldehyde dehydrogenase; 2-oxoisovalerate dehydrogenase; 2,3-dihydro-2,3-dihydroxybenzoic acid dehydrogenase; 2,3-dihydroxy-2,3-dihydro-p-cumate dehydrogenase; 2,4-diaminovalerate dehydrogenase; 2,5-dioxovalerate dehydrogenase; 3-(imidazol-5-yl) Lactate dehydrogenase; 3-α-hydroxy-5β-androstan-17-one 3α-dehydrogenase; 3-α-hydroxycholate dehydrogenase; 3-α-hydroxyglycyrrhetinic acid dehydrogenase; 3-α-hydroxysteroid dehydrogenase; 3-α(17β)-hydroxysteroid dehydrogenase; 3-α(or 20β)-hydroxysteroid dehydrogenase; 3-β-hydroxy-5α-steroid dehydrogenase; 3-β-hydroxy-5β-steroid dehydrogenase; 3-β-hydroxysteroid dehydrogenase; 3-β(or 20α)-hydroxysteroid dehydrogenase; 3-dehydro-L-gulonate 2-dehydrogenase; 3-hydroxy-2-methylbutyryl-CoA dehydrogenase; 3-hydroxy ester dehydrogenase; 3 3-Hydroxyacyl-CoA dehydrogenase; 3-Hydroxybenzyl alcohol dehydrogenase; 3-Hydroxyisobutyrate dehydrogenase; 3-Hydroxypimehryl-CoA dehydrogenase; 3-Hydroxypropionate dehydrogenase; 3-Isopropylmalate dehydrogenase; 3(or 17)α-hydroxysteroid dehydrogenase; 4-(hydroxymethyl)benzenesulfonic acid dehydrogenase; 4-Carboxy-2-hydroxymuconic acid-6-semialdehyde dehydrogenase; 4-Formylbenzenesulfonic acid dehydrogenase; 4-Hydroxybenzaldehyde dehydrogenase; 4-Hydroxybutyrate dehydrogenase; 4-Hydroxycyclohexanecarboxylic acid dehydrogenase; 4-Hydroxymuconic acid-semialdehyde dehydrogenase; 4-Hydroxyphenylacetaldehyde dehydrogenase; 4-Hydroxythreonine-4-phosphate dehydrogenase; 4-Phosphorelate dehydrogenase;4-Trimethylammonium butyraldehyde dehydrogenase; 5-Carboxymethyl-2-hydroxymucin-semialdehyde dehydrogenase; 5,6-Dihydroxy-3-methyl-2-oxo-1,2,5,6-tetrahydroquinoline dehydrogenase; 5-Hydroxyarachidate dehydrogenase; 6-N-hydroxyeucalyptol dehydrogenase; 6-Hydroxyhexanoate dehydrogenase; 6-O-eucalyptol dehydrogenase; 6-O-hexanoate dehydrogenase; 6-Phosphoglucose dehydrogenase; 7-α-Hydroxysteroid dehydrogenase; 7-β-Hydroxysteroid dehydrogenase ; 11-β-hydroxysteroid dehydrogenase; 12-α-hydroxysteroid dehydrogenase; 12-β-hydroxysteroid dehydrogenase; 15-hydroxyeicosatetraenoic acid dehydrogenase; 15-hydroxyprostaglandin dehydrogenase; 16-α-hydroxysteroid dehydrogenase; 17-β-hydroxysteroid dehydrogenase; 20-α-hydroxysteroid dehydrogenase; 21-hydroxysteroid dehydrogenase; acyl-CoA dehydrogenase; alanine dehydrogenase; alanine dehydrogenase; α-ketoglutarate dehydrogenase; α-glycerol phosphate dehydrogenase; alcohol dehydrogenase; aldehyde dehydrogenase; aldose 1-dehydrogenase; allyl alcohol dehydrogenase; aminobutyraldehyde dehydrogenase; aminomucosamide-semialdehyde dehydrogenase; aromate dehydrogenase (dehydrogenase); aryl alcohol dehydrogenase; aryl aldehyde dehydrogenase; aspartate dehydrogenase; aspartate-semialdehyde dehydrogenase; benzaldehyde dehydrogenase; benzyl-2-methyl-hydroxybutyrate dehydrogenase; β-alanine dehydrogenase; β-hydroxybutyrate dehydrogenase; betaine-aldehyde dehydrogenase; borneol dehydrogenase; n-butyraldehyde dehydrogenase; butanediol dehydrogenase; carnitine 3-dehydrogenase; carnauba alcohol dehydrogenase; cholesterol-5-en-3β,7α-diol 3β-dehydrogenase; cholesterol 26-dehydrogenase; cholesterol dehydrogenase; cinnamyl alcohol dehydrogenase; cis-1,2-dihydro-1,2-dihydroxynaphthalene dehydrogenase; cis-1,2-dihydrophenyl-1,2-diol dehydrogenase; cis-1,2-dihydro-4-methylcyclohexane-3,5-diene-1-carboxylic acid dehydrogenase; cis-2 3-Dihydrobiphenyl-2,3-diol dehydrogenase; cis-3,4-dihydrophenanthrene-3,4-diol dehydrogenase; cis-dihydroethylcatechol dehydrogenase; coniferyl alcohol dehydrogenase; coniferyl aldehyde dehydrogenase; cyclohexane-1,2-diol dehydrogenase; cyclohexyl alcohol dehydrogenase; cyclopentanol dehydrogenase; D-arabinol 2-dehydrogenase; D-arabinol 4-dehydrogenase; D-arabinose 1-dehydrogenase; D-arabinose 1-dehydrogenase; D-lysine dehydrogenase; D-iduroyl alcohol 2-dehydrogenase; D-malate dehydrogenase; D-caryolipoic acid dehydrogenase; D-pineol dehydrogenase; D-threuronic acid 1-dehydrogenase; D-xylose 1-dehydrogenase; diaminopimelic acid dehydrogenase; dibenzothiophene dihydrogenyl alcohol dehydrogenase; dihydrobunolol dehydrogenase dehydrogenase; dihydropyrimidine dehydrogenase; dihydrouracil dehydrogenase; dimethyl malate dehydrogenase;DTDP-6-deoxy-L-taloose 4-dehydrogenase; DTDP-galactose 6-dehydrogenase; ephedrine dehydrogenase; erythrose 4-phosphate dehydrogenase; estradiol 17-α-dehydrogenase; estradiol 17-β-dehydrogenase; farnesol dehydrogenase; fluorene-9-ol dehydrogenase; fluoroacetaldehyde dehydrogenase; formaldehyde dehydrogenase; formate dehydrogenase; formyltetrahydrofolate dehydrogenase; fructose dehydrogenase; euonymus 2-dehydrogenase; euonymus 5-phosphate dehydrogenase; galactose dehydrogenase; γ-guanidinylbutyraldehyde dehydrogenase; GDP-6-deoxy-D-taloose 4-dehydrogenase; GDP-mannose 6-dehydrogenase; geissoschizine dehydrogenase; geraniol dehydrogenase; gluconate 2-dehydrogenase; gluconate 5-dehydrogenase; glucose dehydrogenase; glucose-6-phosphate dehydrogenase; glutamate (glutamate) dehydrogenase; glutamate-5-semialdehyde dehydrogenase; glutamate-semialdehyde dehydrogenase; glyceraldehyde-3-phosphate dehydrogenase; glycerate dehydrogenase; glycerol dehydrogenase; glycerol 2-dehydrogenase; glycerol-3-phosphate dehydrogenase; glycine dehydrogenase; glycolaldehyde dehydrogenase; glyoxylate dehydrogenase; hexadecyl alcohol dehydrogenase; histidine dehydrogenase; homoisocitrate dehydrogenase; homoserine dehydrogenase; hydrogen dehydrogenase Hydrogenases; Hydroxycyclohexanecarboxylic acid dehydrogenase; Hydroxymalonic acid dehydrogenase; Taurine dehydrogenase; Indanol dehydrogenase; Indolelactate dehydrogenase; Inosine-5'-monophosphate dehydrogenase; Inositol 2-dehydrogenase; Isocitrate dehydrogenase; Isomentelenol dehydrogenase; Isopropanol dehydrogenase; Kynurenate-7,8-dihydrodiol dehydrogenase L-amino acid dehydrogenase; L-aminooxalic acid-semialdehyde dehydrogenase; L-arabinol 2-dehydrogenase; L-arabinol 4-dehydrogenase; L-arabinose 1-dehydrogenase; L-arginine dehydrogenase; L-erythro-3,5-diaminohexanoic acid dehydrogenase; L-ethylene glycol dehydrogenase; L-gulonic acid 3-dehydrogenase; L-idonol 2-dehydrogenase; L-idonol 5-dehydrogenase (L-idonate) 5-Dehydrogenase; L-Rhamnose 1-Dehydrogenase; L-Threonate 3-Dehydrogenase; L-Threonine 3-Dehydrogenase; L-Xylose 1-Dehydrogenase; Lactaldehyde Dehydrogenase; Lactate (Lactate) Dehydrogenase; Leucine Dehydrogenase; Long-chain Alcohol Dehydrogenase; Lysine Dehydrogenase; Malate (Malate) Dehydrogenase; Malonic Acid-Semialdehyde Dehydrogenase; Mannitol 2-Dehydrogenase; Mannitol Dehydrogenase; Mannitol-1-phosphate 5-Dehydrogenase; Mesotartrate Dehydrogenase; Methylene Tetrahydrofolate Dehydrogenase; Methyl Malonate-Semialdehyde Dehydrogenase; Morphine 6-Dehydrogenase; Mycothiol-dependent Formaldehyde Dehydrogenase; N-Acetylhexosamine 1-Dehydrogenase;N-Acylmannosamine 1-dehydrogenase; N-methylalanine dehydrogenase; NADH dehydrogenase; NADPH dehydrogenase; Nicotinate dehydrogenase; Octyl alcohol dehydrogenase; ω-hydroxydecanoate dehydrogenase; Opine dehydrogenase; Oxyglutarate dehydrogenase; Pantoate 4-dehydrogenase; Perillyl alcohol dehydrogenase; Phenylacetaldehyde dehydrogenase; Phenylalanine dehydrogenase; Phenylacetate dehydrogenase; Phosphoglucate dehydrogenase; Phosphoglycerate dehydrogenase; Phosphonate dehydrogenase; Phthalate 4,5-cis-dihydrodiol dehydrogenase; Pimecroyl-CoA dehydrogenase; Precorrin-2 dehydrogenase; Prephenate dehydrogenase (dehydrogenase); Propylene glycol-phosphate dehydrogenase; Pyridoxal 4-dehydrogenase; Pyridoxine 4-dehydrogenase; Pyruvate dehydrogenase; Quinic acid dehydrogenase; Retinaldehyde dehydrogenase; Retinol dehydrogenase; Ribitol 2-dehydrogenase; Ribitol-5-phosphate 2-dehydrogenase; Ribose 1-dehydrogenase; S-(hydroxymethyl)glutathione dehydrogenase; Yeast amino acid dehydrogenase; Salicylate aldehyde dehydrogenase; Taxol dehydrogenase; Serine 2-dehydrogenase; Serine 3-dehydrogenase; Shikimate dehydrogenase; Sn-glycerol-1-phosphate dehydrogenase; Sorbitol 6-phosphate 2-dehydrogenase; Sorbitol 5-dehydrogenase; Sterol-4α-carboxylic acid 3-dehydrogenase; strombine dehydrogenase; Succinate-semialdehyde dehydrogenase; Succinylglutamate-semialdehyde dehydrogenase; Tartrate dehydrogenase; Tauropine (dehydrogenase); terephthalic acid 1,2-cis-dihydrodiol dehydrogenase; testosterone 17β-dehydrogenase; thiomorpholine-carboxylic acid dehydrogenase; trans-1,2-dihydrophenyl-1,2-diol dehydrogenase; trans-acenaphthene-1,2-diol dehydrogenase; tryptophan dehydrogenase; UDP-glucose 6-dehydrogenase; UDP-N-acetylglucosamine 6-dehydrogenase; UDP-N-acetylmuracetate dehydrogenase; ureoglycolate dehydrogenase; uronic acid dehydrogenase; valine dehydrogenase; vanillin dehydrogenase; vellosimine dehydrogenase; vomifoliol dehydrogenase; xanthine dehydrogenase; and xanthine aldehyde dehydrogenase.

[0183] In one embodiment, the NADH-dependent or NADPH-dependent enzyme is a reductase. In one embodiment, the reductase is selected from the group consisting of: (S)-lichenate reductase; 1,2-dehydroreticulinium reductase; 1,2-dihydrovomilenine reductase. (reductase); 1,5-dehydro-D-fructose reductase; 2-enal reductase; 2-coumaric acid reductase; 2-dehydropantolytic acid 2-reductase; 2-dehydropantolytic acid lactone reductase; 2-enoic acid reductase; 2-hexadecenal reductase; 2-hydroxy-1,4-benzoquinone reductase; 2-hydroxy-3-oxopropionic acid reductase; 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid reductase; 2-oxoadipic acid reductase; 2-oxopropyl-CoM reductase; 2,4-dichlorobenzoyl-CoA reductase; 2,5-didehydrogluconic acid reductase; 2'-hydroxydaidzein reductase; 2'-hydroxyisoflavones reductase; 3"-deamin-3"-oxozincamine reductase; 3-dehydrodihydrosphingomyelin Amino alcohol reductase; 3-ketosteroid reductase; 3-methylbutyraldehyde reductase; 3-methylene hydroxyindole reductase; 3-oxoacyl-(acyl-carrier-protein) reductase; 4-(dimethylamino)phenyloxyazobenzene reductase; 4-hydroxy-tetrahydropyridine dicarboxylic acid reductase; 4-oxoproline reductase; 5-amino-6-(5-phosphoribosylamino)uracil reductase; 6-pyruvyltetrahydropterin 2'-reductase; 6,7-dihydropterin reductase; 8-oxo-meta-mycin reductase; 12-oxophytedienoic acid reductase; acetylacetyl-CoA reductase; acylglycerol ketone-phosphate reductase; aldose reductase; aldose-6-phosphate reductase; α-artemisinin 1,2-reductase (α-santonin) 1,2-reductase; anthocyanin reductase; apigenin 1-reductase; cobalamin reductase; asparagus acid reductase; azobenzene reductase; berberine reductase; β-nitroacrylate reductase; biliverdin reductase; biochanin-A reductase; bis-γ-glutamylcysteine ​​reductase; carbonyl reductase; CDP-4-dehydro-6-deoxyglucose reductase; decachloroone reductase; cholesterol 5α-reductase ; Cinnamyl-CoA reductase; cis-2-enoyl-CoA reductase; CoA-glutathione reductase; CoA-disulfide reductase; cobalamin(II) reductase; codeine reductase; cortisone α-reductase; cucurbitacin δ23-reductase; cyanocobalamin reductase; cystine reductase; D-xylulose reductase; δ1-piperidine-2-carboxylic acid reductase; δ14-sterol reductase; δ24-sterol reductase;δ24(241)-sterol reductase; diethyl 2-methyl-3-oxosuccinate reductase; di-iron-transferrin reductase; dihydrokaempferol 4-reductase; diiodophenylpyruvate reductase; divinyl chlorophyll a 8-vinyl-reductase; DTDP-4-dehydro-6-deoxyglucose reductase; DTDP-4-dehydro-rhamnose reductase; enoyl-(acyl-carrier-protein) reductase; erythrulose reductase; ferredoxin—NAD; + reductase; ferrochelatase reductase; flavanone 4-reductase; flavin reductase; FMN reductase; fructonic acid reductase; fumarate reductase; GDP-4-dehydro-6-deoxy-D-mannose reductase; GDP-4-dehydro-D-rhamnose reductase; glucuronate reductase; glucuronolactone reductase; glutamyl-tRNA reductase; glyoxylate reductase; hydroxylamine reductase; hydroxymethylglutaryl-CoA reductase; hydroxyphenylpyruvate reductase; hydroxypyruvate reductase; hyponitrite reductase; indole-3-acetaldehyde reductase; L-xylulose reductase; lactaldehyde reductase; leghemoglobin reductase; leucoanthocyanidin reductase; long-chain fatty acyl-CoA reductase; maleylacetic acid reductase; mannose 6-phosphate 6-reductase; mannuronate reductase; mercury(II) reductase; (methionine synthase) reductase; methylglyoxal reductase; 3-hydroxy-3-methylvaleraldehyde reductase; monodehydroascorbate reductase; mycothione reductase; N-hydroxy-2-acetylaminofluorene reductase; N-acetyl-γ-glutamyl-phosphate reductase; NADPH-cytochrome-c2 reductase; NADPH-heme protein reductase; NADPH:quinone reductase; nitrite reductase; nitroquinoline-N-oxide reductase; orotate reductase; oxaloacetolactate reductase; p-benzoquinone reductase; phloroglucinol reductase; pre-corrin-6A reductase; progesterone 5α-reductase; prostaglandin-E2 9-reductase; protein-disulfide reductase; protochlorophyllide reductase; pteridine reductase; pyrroline-2-carboxylic acid reductase; pyrroline-5-carboxylic acid reductase; rubredoxin-NAD + reductase; rubredoxin-NAD(P) +Reductases; salutaridine reductase; sepiapterin reductase; sorbitol reductase; sulcatone reductase; sagaturonate reductase; tetrahydroxynaphthalene reductase; trans-2-enoyl-CoA reductase; trimethylamine-N-oxide reductase; sropinone reductase; srypanothione-disulfide reductase; vomilenine reductase; xanthommatin reductase; and zeatin reductase.

[0184] In one embodiment, the NADH-dependent or NADPH-dependent enzyme is an oxygenase. In one embodiment, the oxygenase is selected from the group consisting of: (S)-limonene 3-monooxygenase; (S)-limonene 7-monooxygenase; 2-hydroxybiphenyl 3-monooxygenase; 2-hydroxycyclohexanone 2-monooxygenase; 2-hydroxyquinoline 8-monooxygenase; 2-nitrophenol 2-monooxygenase; 2,4-dichlorophenol 6-monooxygenase; 2,6-dihydroxypyridine 3-monooxygenase; 3-hydroxybenzoic acid 4-monooxygenase; 3-hydroxybenzoic acid 6-monooxygenase; 3,9-dihydroxy... Pterocarboxylic acid 6α-monooxygenase; 4-aminobenzoic acid 1-monooxygenase; 4-hydroxyacetophenone monooxygenase; 4-hydroxybenzoic acid 3-monooxygenase; 4-hydroxyphenylacetaldehyde oxime monooxygenase; 4-hydroxyphenylacetic acid 1-monooxygenase; 4-hydroxyquinoline 3-monooxygenase; 4-nitrophenol 2-monooxygenase; 5-O-(4-coumaryl)-D-quinic acid 3'-monooxygenase; 27-hydroxycholesterol 7α-monooxygenase; albendazole monooxygenase Monooxygenase; olefin monooxygenase; dehydrotetracycline monooxygenase; an-aminobenzoic acid 3-monooxygenase; an-aminobenzoyl-CoA monooxygenase; benzoic acid 4-monooxygenase; benzoyl-CoA 3-monooxygenase; cholesterol 26-monooxygenase; cholesterol 7α-monooxygenase; cyclopentanone monooxygenase; dihydrochelirubine 12-monooxygenase; dihydrosanguinarine 10-monooxygenase; flavonoid 3'-monooxygenase; hydroxyphenylacetonitrile 2-monooxygenase; imidazoleacetic acid 4-monooxygenase; kynurenine 3-monooxygenase; L-lysine 6-monooxygenase; leukotriene-B4 20-monooxygenase; leukotriene-E4 20-Monooxygenase; Limonene 6-Monooxygenase; Melilotate 3-Monooxygenase; Methyltetrahydroproberberine 14-Monooxygenase; N-Methylcodonine 3'-Monooxygenase; Orcinol 2-Monooxygenase; Phenol 2-Monooxygenase; Phenylacetone Monooxygenase; Phosphatidylcholine 12-Monooxygenase; Chelidonine 6-Monooxygenase 6-monooxygenase; monomethyl ether monooxygenase; quinine 3-monooxygenase; salicylic acid 1-monooxygenase; taurine 8-monooxygenase; trans-cinnamic acid 2-monooxygenase; trans-cinnamic acid 4-monooxygenase; vanillic acid monooxygenase; 2-aminobenzenesulfonic acid 2,3-dioxygenase; 2-chlorobenzoic acid 1,2-dioxygenase; 2-hydroxyquinoline 5,6-dioxygenase; 3-hydroxy-2-methylpyridinecarboxylic acid dioxygenase; 3-phenylpropionic acid dioxygenase;4-Chlorophenylacetic acid 3,4-dioxygenase; 4-sulfobenzoic acid 3,4-dioxygenase; 5-pyridoxine dioxygenase; anthranilic acid 1,2-dioxygenase; benzene 1,2-dioxygenase; benzoic acid 1,2-dioxygenase; biphenyl 2,3-dioxygenase; naphthalene 1,2-dioxygenase; nitric oxide dioxygenase; phthalic acid 4,5-dioxygenase; senecionine N-oxygenase; terephthalic acid 1,2-dioxygenase; and toluene dioxygenase.

[0185] In one embodiment, the NADH-dependent or NADPH-dependent enzyme is a synthase. In one embodiment, the synthase is selected from the group consisting of: (S)-berberine synthase; (S)-cheilanthifoline synthase; (S)-stylopine synthase; 6-methylsalicylic acid synthase; 6'-deoxychalcone synthase; berbamunine synthase; corydaline synthase; fatty acid synthase; fatty acyl-CoA synthase; GDP-L-fucose synthase; glutamate synthase; soybean antitoxin synthase; glyceollin synthase; glycine cleavage system (glycine synthase); icosanoyl-CoA synthase; licodione synthase; lovastatin nonaketide synthase. Synthesizers include: mycocerosate synthase, N5-(carboxyethyl)ornithine synthase, precorline-3B synthase, PreQ1 synthase, prostaglandin-F synthase, psoralen synthase, salidomide synthase, and secologanin synthase.

[0186] In one embodiment, the NADH-dependent or NADPH-dependent enzyme is a hydroxylase. In one embodiment, the hydroxylase is selected from the group consisting of: 3-hydroxyphenylacetic acid 6-hydroxylase; 4-hydroxybenzoic acid 1-hydroxylase; 4'-methoxyisoflavones 2'-hydroxylase; 5-β-cholestan-3α,7α-diol 12α-hydroxylase; 7-deoxyloganin 7-hydroxylase; 7-α-hydroxycholestan-4-en-3-one 12α-hydroxylase; 8-dimethylallylnaringenin 2'-hydroxylase; 24-hydroxycholesterol 7α-hydroxylase; cholesterol 24-hydroxylase; deoxysarpagine hydroxylase. hydroxylase); isoflavone 2'-hydroxylase; isoflavone 3'-hydroxylase; lithochondyl salt 6β-hydroxylase; tabersonine 16-hydroxylase; taxane 10-β-hydroxylase; taxane 13-α-hydroxylase; vinorine hydroxylase.

[0187] In one embodiment, the NADH-dependent or NADPH-dependent enzyme is selected from the group consisting of: 15-oxoprostaglandin 13-oxidase; cholesterol oxidase; NADH peroxidase; NADPH peroxidase; 3α,7α,12α-trihydroxycholestan-26-aldehyde 26-oxidoreductase; myristoyl-CoA11 desaturase; phosphatidylcholine desaturase; ATP-dependent NAD(P)H-hydrate dehydratase; GDP-mannose 4,6-dehydratase; ketol acid reductase; monoisoprene isoflavone cyclooxygenase; and sterol 14-demethylase.

[0188] Reagent test kit

[0189] On the other hand, this disclosure provides a kit for measuring analyte concentration. In one embodiment, the kit includes: a transducer, such as a nanoparticle transducer, the transducer including a chromophore; and an enzyme physically associated with the nanoparticles and configured to catalyze a reaction comprising multiple reaction elements. In one embodiment, the enzyme is physically associated with the transducer and / or the chromophore, such as when the enzyme and the transducer and / or the enzyme and the chromophore are dispersed in a common solvent, coupled to a common substrate, coupled together, or encapsulated together in hydrogel beads, etc. In one embodiment, the transducer is any transducer according to the transducers described herein. In one embodiment, the nanoparticles include P-points. In one embodiment, the enzyme is an enzyme as described herein. In one embodiment, the kit contains components suitable for carrying out one or more reactions according to the methods of this disclosure.

[0190] In one embodiment, the plurality of reaction elements includes one or more reactants and one or more products, the one or more reactants comprising an analyte, and wherein the amount of fluorescence emitted from the chromophore is determined by the concentration of the reaction element among the plurality of reaction elements. In one embodiment, the enzyme is an NADH-dependent or NADPH-dependent enzyme, as further discussed herein with respect to the transducers of this disclosure. In one embodiment, the NADH-dependent or NADPH-dependent enzyme is selected from the group consisting of dehydrogenases, reductases, oxygenases, synthases, hydroxylases, and combinations thereof. In one embodiment, the reaction element among the plurality of reaction elements includes NADH and / or NAD. + And wherein the amount of fluorescence emitted from the chromophore is determined by the NADH and / or NAD. + The concentration is determined by the analyte. In one embodiment, the analyte includes NADH and / or NAD. + In one embodiment, the reaction elements among the plurality of reaction elements include NADPH and / or NADP. + And wherein the amount of fluorescence emitted from the chromophore is determined by the NADPH and / or NADP. + The concentration is determined by the analyte. In one embodiment, the analyte includes NADPH and / or NADP. + .

[0191] Although NADH and NADH-dependent or NADPH and NADPH-dependent enzymes have been described, it should be understood that other analytes and enzyme pairs are also within the scope of this disclosure. In this regard, in one embodiment, the analyte is glucose, and the enzyme is glucose oxidase. In one embodiment, the reaction element among the plurality of reaction elements includes oxygen, and wherein the amount of fluorescence emitted from the chromophore is determined by the concentration of the oxygen.

[0192] As described above, enzymes and nanoparticles are physically associated. In some cases, chromophores can be directly mixed with enzymes for measurement. In others, covalent conjugation is provided to link nanoparticles to enzymes, thereby creating compact probes that can be used, for example, for intracellular sensing. In one embodiment, the enzyme and chromophore are physically associated. As discussed further herein, such physical association can include the enzyme and chromophore dispersed in a common solvent, the enzyme and chromophore coupled to a common substrate, the enzyme and chromophore lyophilized in a common powder, the enzyme and chromophore encapsulated in hydrogel beads, or the enzyme and chromophore otherwise physically / chemically contacted. In one embodiment, the enzyme physically associated with the chromophore is coupled to the chromophore. In this respect, the enzyme is physically linked to the chromophore directly or indirectly. Such coupling can contain covalent bonds. In another embodiment, the coupling between the enzyme and chromophore is through one or more non-covalent bonds or interactions, such as ionic bonds, van der Waals forces, hydrogen bonding, etc. Therefore, in one embodiment, the coupling does not contain covalent bonds.

[0193] As discussed elsewhere herein, chromophores can take many different forms and include many different types of chromophores. In one embodiment, the chromophore is in the form of nanoparticles. In one embodiment, the chromophore is a semiconductor polymer, such as in the form of semiconductor polymer nanoparticles. As described elsewhere herein, in one embodiment, the chromophore is part of a polymer dot or p-dot, such as where the chromophore and other polymer components exist in a condensed, stable, submicron state. In one embodiment, the chromophore may comprise a chromophore polymer in an uncondensed state, such as a semiconductor chromophore polymer.

[0194] As discussed further herein, it has been unexpectedly discovered that the transducer of this disclosure can be operated to detect or monitor analyte concentrations via physical association rather than solely via covalent bonding. Therefore, such physical association can include covalent bonding, wherein the enzyme is covalently bonded to the nanoparticle. Additionally, such physical association includes other forms of association, such as non-covalent bonding between the enzyme and the nanoparticle. Thus, in one embodiment, the enzyme is not covalently bonded to the nanoparticle. In one embodiment, the enzyme and nanoparticle associate via ionic bonding, van der Waals forces, hydrogen bonding, etc.

[0195] In one embodiment, the enzyme and nanoparticles are mixed together. In one embodiment, the enzyme and nanoparticles are encapsulated together in hydrogel beads. In one embodiment, the enzyme and nanoparticles are in the form of a lyophilized powder. Such lyophilized powders can, for example, be rehydrated and / or reconstituted into a common solvent. In one embodiment, the enzyme and nanoparticles are dispersed in a common solvent. In one embodiment, the enzyme and nanoparticles are covalently or non-covalently linked to a substrate or surface. In one embodiment, the enzyme and nanoparticles are covalently or non-covalently linked to a common substrate or surface.

[0196] method

[0197] On the other hand, this disclosure provides a method for measuring the concentration of an analyte in a fluid. In one embodiment, the method is performed in part or in whole using the system of this disclosure.

[0198] In one embodiment, the method includes contacting a fluid with a transducer according to the present disclosure. In one embodiment, the method includes contacting a fluid with a P-point and an NADH- or NADPH-dependent enzyme, the P-point comprising a chromophore, the NADH- or NADPH-dependent enzyme being coupled to the P-point and configured to catalyze a reaction. In one embodiment, the method includes contacting a fluid with a P-point and an enzyme, the P-point comprising a chromophore, the enzyme being physically associated with the P-point, and the enzyme being configured to catalyze a reaction. In one embodiment, the method includes contacting a fluid with a chromophore comprising a semiconductor chromophore polymer and an enzyme physically associated with the chromophore configured to catalyze a reaction. In one embodiment, the fluid is a liquid. In one embodiment, the fluid is a gas. In one embodiment, the fluid is a combination of liquid and gas. Although methods comprising contacting a fluid with, for example, a transducer have been described, it should be understood that in some embodiments, the methods of the present disclosure include contacting the transducer or the present disclosure with a solid or slurry. In one embodiment, the method includes contacting a transducer as described herein with the biological samples in Table 1.

[0199] As further described herein with respect to the transducer of this disclosure, the chromophore and enzyme can be coupled and / or physically associated. Thus, in one embodiment, the chromophore and enzyme are coupled to a substrate. In one embodiment, the chromophore and enzyme are coupled to a common substrate or to the same surface. Similarly, in one embodiment, the chromophore and enzyme are dispersed in a common solvent. In one embodiment, the chromophore and enzyme are encapsulated together in hydrogel beads. In one embodiment, the enzyme is not coupled to point P, as if the enzyme is not covalently bound to point P. In one embodiment, point P is covalently bound to the enzyme.

[0200] In some cases, chromophores can be directly mixed with enzymes for measurement. In others, covalent conjugation is provided to link nanoparticles to enzymes, thereby creating compact probes that can be used, for example, for intracellular sensing. In one embodiment, the enzyme and chromophore are physically associated. As discussed further herein, such physical association can include the enzyme and chromophore dispersed in a common solvent, the enzyme and chromophore coupled to a common substrate, the enzyme and chromophore lyophilized in a common powder, the enzyme and chromophore encapsulated in hydrogel beads, or the enzyme and chromophore otherwise physically / chemically contacted. In one embodiment, the enzyme physically associated with the chromophore is coupled to the chromophore. In this respect, the enzyme is physically linked to the chromophore directly or indirectly. Such coupling can contain covalent bonds. In another embodiment, the coupling between the enzyme and chromophore is through one or more non-covalent bonds or interactions, such as ionic bonds, van der Waals forces, hydrogen bonding, etc. Therefore, in one embodiment, the coupling does not contain covalent bonds.

[0201] As discussed elsewhere herein, chromophores can take many different forms and include many different types of chromophores. In one embodiment, the chromophore is in the form of nanoparticles. In one embodiment, the chromophore is a semiconductor polymer, such as in the form of semiconductor polymer nanoparticles. As described elsewhere herein, in one embodiment, the chromophore is part of a polymer dot or p-dot, such as where the chromophore and other polymer components exist in a condensed, stable, submicron state. In one embodiment, the chromophore may comprise a chromophore polymer in an uncondensed state, such as a semiconductor chromophore polymer.

[0202] As further discussed herein, in one embodiment, the reaction includes multiple reaction elements, such that the multiple reaction elements include one or more reactants and one or more products, the reactants comprising an analyte. As further discussed herein, in one embodiment, the amount of fluorescence emitted from the chromophore is determined by the concentration of the reaction element among the multiple reaction elements.

[0203] In one embodiment, the method further includes irradiating a chromophore to induce fluorescence from the chromophore. In one embodiment, such irradiation includes irradiating the chromophore with a wavelength absorbed by the chromophore. In one embodiment, the method further includes irradiating a second chromophore, such as in a wavelength range different from that used to irradiate the chromophore, for example, for exciting multiplexing. In one embodiment, the irradiation is provided by an irradiation source of a system according to embodiments of the present disclosure.

[0204] In one embodiment, the method further includes measuring fluorescence from the chromophore. In one embodiment, the fluorescence emitted from the chromophore defines a fluorescence ratio equal to the ratio of the amount of fluorescence emitted at a signal fluorescence wavelength to the amount of fluorescence emitted at a control fluorescence wavelength. In one embodiment, the fluorescence ratio is determined by the concentration of a fluid component or fluid element.

[0205] In one embodiment, the method further includes determining the concentration of the analyte based on measured fluorescence. In one embodiment, determining the concentration of the analyte includes: measuring fluorescence at the signal fluorescence wavelength and fluorescence at the control fluorescence wavelength; determining a measured fluorescence ratio based on the measurement; and determining the concentration of the analyte based on the measured fluorescence ratio.

[0206] As described above, the method involves contacting a fluid with a transducer according to this disclosure. Such a fluid may contain any fluid suitable for determining an analyte. In one embodiment, the fluid is a biological fluid that is believed or suspected of containing an analyte. In one embodiment, the fluid is selected from blood, plasma, serum, lymph, saliva, tears, interstitial fluid, cerebrospinal fluid, urine, sweat, and combinations thereof.

[0207] The methods disclosed herein are applicable to the determination or monitoring of the concentration of various analytes in fluids. Such analytes may be those consumed or altered by NADH-dependent or NADPH-dependent enzymes or other enzymes described herein. In one embodiment, the analyte is an amino acid. In one embodiment, the analyte is NADH. In one embodiment, the analyte is selected from the group consisting of ascorbic acid, glutamate, dopamine, cholesterol, and alcohol. In one embodiment, the analyte is a drug. In one embodiment, the analyte is a drug metabolite. In one embodiment, the analyte is a protein, nucleic acid molecule, or neurotransmitter molecule. In one embodiment, the analyte is a carbohydrate, lipid, or metabolite. In one embodiment, the analyte is a sugar. In one embodiment, the analyte is a metabolite. In one embodiment, the metabolite is selected from the group consisting of lactate, glutamate, glucose, and β-hydroxybutyrate. In one embodiment, the metabolite is a metabolite of any one or more metabolites according to Table 1.

[0208] Table 1: List of possible metabolites

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217] In one embodiment, the metabolite is a metabolite of any one or more metabolites according to Table 2.

[0218] Table 2: Medically relevant metabolites compatible with the P-point biosensor.

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225] In some embodiments, the order in which some or all steps are described in each process should not be considered limiting. Rather, those skilled in the art who benefit from this disclosure will understand that some steps in the process may be performed in various orders not shown, or even in parallel.

[0226] system

[0227] On the other hand, this disclosure provides a system for measuring analyte concentration. In one embodiment, the system includes a transducer, such as a nanoparticle transducer and / or a transducer substrate, or a kit, as further described herein. In one embodiment, the system is configured and adapted to perform the methods of this disclosure.

[0228] As discussed elsewhere herein, the transducers and kits of this disclosure include chromophores, such as chromophores physically associated with enzymes. In some cases, the chromophore can be directly mixed with the enzyme for measurement. In others, covalent conjugation is provided to link nanoparticles to the enzyme, thereby creating compact probes that can be used, for example, for intracellular sensing. In one embodiment, the enzyme is physically associated with the chromophore. As further discussed herein, such physical association can include the enzyme and chromophore dispersed in a common solvent, the enzyme and chromophore coupled to a common substrate, the enzyme and chromophore lyophilized in a common powder, the enzyme and chromophore encapsulated in hydrogel beads, or the enzyme and chromophore otherwise physically / chemically contacted. In one embodiment, the enzyme physically associated with the chromophore is coupled to the chromophore. In this respect, the enzyme is physically linked to the chromophore directly or indirectly. Such coupling can include covalent bonds. In another embodiment, the coupling between the enzyme and the chromophore is achieved through one or more non-covalent bonds or interactions, such as ionic bonds, van der Waals forces, hydrogen bonding, etc. Therefore, in one embodiment, the coupling does not involve covalent bonds.

[0229] As discussed elsewhere herein, chromophores can take many different forms and include many different types of chromophores. In one embodiment, the chromophore is in the form of nanoparticles. In one embodiment, the chromophore is a semiconductor polymer, such as in the form of semiconductor polymer nanoparticles. As described elsewhere herein, in one embodiment, the chromophore is part of a polymer dot or p-dot, such as where the chromophore and other polymer components exist in a condensed, stable, submicron state. In one embodiment, the chromophore may comprise a chromophore polymer in an uncondensed state, such as a semiconductor chromophore polymer.

[0230] In one embodiment, the system includes an illumination source configured to illuminate the transducer, the transducer substrate, the reagent kit, or the chromophore of the transducer to induce fluorescence from the chromophore. In one embodiment, the illumination source is a laser. In one embodiment, the illumination source is a laser diode. In one embodiment, the illumination source is an LED (light-emitting diode). In one embodiment, the illumination source is a lamp.

[0231] In one embodiment, the irradiation source is configured to emit electromagnetic radiation, which is configured to excite chromophores, such as by emitting fluorescence from the electromagnetic radiation.

[0232] In one embodiment, the irradiation source is a first irradiation source, and the system includes a second irradiation source configured to emit a second electromagnetic radiation, such as electromagnetic radiation having a wavelength range different from that emitted from the first irradiation source. Such first and second irradiation sources can be adapted to excite transducers with different chromophores, such as chromophores configured to absorb electromagnetic radiation with different wavelength ranges and be excited by electromagnetic radiation with different wavelength ranges. In this respect, such systems are suitable for excitation multiplexing, as further described herein.

[0233] In one embodiment, the system includes a photodetector configured to generate a signal based on the fluorescence from the chromophore. In one embodiment, the photodetector is a first photodetector, and the signal is a first signal, and the system includes a second photodetector configured to generate a second signal. In one embodiment, the second photodetector is configured to generate a second signal based on light having a wavelength range different from the first light. In this respect, the system can be configured to generate the first and second signals based on fluorescence, for example, from different chromophores (such as chromophores belonging to a portion of different transducers configured to react with different enzymes). In this respect, the system can be configured to perform emission multiplexing. In this respect, the system can also be configured to generate, for example, a first signal and a second signal at a signal wavelength and a control wavelength. In this respect, the system can be configured to perform ratio fluorescence measurement, as further described herein with respect to the methods of this disclosure.

[0234] In one embodiment, the system includes a controller operatively coupled to an irradiation source and a photodetector. In another embodiment, the controller includes logic that, when executed by the controller, causes the system to perform operations. Such operations can be configured to perform one or more methods of this disclosure. In one embodiment, the operations include: irradiating the chromophore with the irradiation source; and determining the concentration of the analyte based on the signal from the photodetector. As further discussed herein with reference to examples of this disclosure, the amount or intensity of fluorescence emitted from the transducer of this disclosure can be based on the concentration of the analyte. In this respect, the amount or intensity of fluorescence detected / measured by the transducer's chromophore can be used to infer and / or calculate the concentration of the analyte.

[0235] In one embodiment, the photodetector is configured to detect the amount of signal fluorescence at the signal fluorescence wavelength and the amount of control fluorescence at the control fluorescence wavelength. In one embodiment, the controller includes additional logic that, when executed by the controller, causes the system to perform the operation of determining a measured fluorescence ratio based on the measured amounts of signal fluorescence and control fluorescence. In one embodiment, the determination of the concentration of the analyte is based on the measured fluorescence ratio. In one embodiment, the system is configured to generate a signal indicating the concentration of the analyte.

[0236] In one embodiment, the system is shaped to house a transducer substrate as further described herein. In another embodiment, the transducer substrate is configured to house a sample containing or potentially containing an analyte, such as a fluid sample, for system analysis.

[0237] In some embodiments, the processes or operations explained above are described based on computer software and hardware. The described techniques may constitute machine-executable instructions embodied in a tangible or non-transitory machine-readable storage medium, which, when executed by a machine, will cause the machine to perform the described operations.

[0238] In one embodiment, a non-transitory machine-readable storage medium has instructions stored thereon that, when executed by a processing system, cause the processing system to perform operations including steps or portions of methods such as those disclosed herein.

[0239] Tangible machine-readable storage media include any means that provides (i.e., stores) information in a non-transitory form accessible to a machine (e.g., a computer, network device, personal digital assistant, manufacturing tool, any device having one or more processors, etc.). For example, machine-readable storage media include recordable / non-recordable media (e.g., read-only memory (ROM), random access memory (RAM), magnetic disk storage media, optical storage media, flash memory devices, etc.). Additionally, the process may be embodied in hardware such as application-specific integrated circuits (“ASICs”) or other hardware.

[0240] The above description of the embodiments of the present invention (including those described in the abstract) is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Although specific embodiments and examples of the invention have been described herein for illustrative purposes, various modifications can be made within the scope of the invention, as will be recognized by those skilled in the art.

[0241] Based on the above detailed description, these modifications can be made to the present invention. The terminology used in the following claims should not be construed as limiting the invention to the specific embodiments disclosed in the specification. Rather, the scope of the invention will be fully defined by the following claims, which will be interpreted in accordance with the established principles of claim interpretation.

[0242] Example

[0243] Example 1: Materials.

[0244] Poly[9,9-dioctylfluorenyl-2,7-diyl]-terminated with dimethylphenyl (DMP) (PFO, ADS129BE, Mw: 40,000–150,000), poly[9,9-dihexylfluorenyl-2,7-diyl]-terminated with DMP (PDHF, ADS130BE, Mw: 40,000–150,000), poly[(9,9-dioctylfluorenyl-2,7-diyl) - Alternating-copolymer-(1,4-benzo-{2,1',3}-thiadiazole)](PFBT, ADS133YE, Mw: 15,000–200,000), Poly[{2-methoxy-5-(2-ethylhexyloxy)-1,4-(1-cyanovinylphenylene)}-copolymer-{2,5-bis(N,N'-diphenylamino)-1,4-phenylene}](DPA-CNPPV, ADS) 113RE, Mw: 15,000–50,000), poly[{9,9-dihexyl-2,7-bis(1-cyanovinylene)fluorene}-alternating-copolymer-{2,5-bis(N,N'-diphenylamino)-1,4-phenylene}](DPA-CNPF, ADS111RE, Mw: 25,000–250,000), poly[2-methoxy-5-(2-ethylhexyloxy)- 1,4-Phenylidene-vinylene]-terminated with polysilsesquioxane (POSS) (MEHPPV, ADS200RE, Mw: >100,000), poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-(1-cyanovinylene-1,4-phenylene)] (CNPPV, ADS110RE, Mw: ~15,000) was obtained from America Dye Source Inc. (Montreal, Canada). L-phenylalanine dehydrogenase (PheDH, 1.4.1.20) from Sporosarcina sp., poly(styrene-maleic anhydride) (PSMA, average Mw: approx. 1,700), and anhydrous tetrahydrofuran (THF, ≥99.9%) were obtained from Sigma-Aldrich (St. Louis, USA); oxidized form (NAD) + ) and reduced form (NADH) of β-nicotinamide adenine dinucleotide hydrate, oxidized form (NADP) +The β-nicotinamide adenine dinucleotide phosphate (NADPH) and its reduced form were obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan) and were not further purified unless otherwise stated. Poly[(9,9-dioctylfluorenyl-2,7-diyl)-copolymer-(1,4-benzo-{2,1',3}-thiadiazole)]-co-4,7-bis(thiophene-2-yl)benzo-2,1,3-thiadiazole] (PFBTTBT) and poly[2,7-(9,9-dioctylfluorenyl)-alternating-4,7-bis(thiophene-2-yl)benzo-2,1,3-thiadiazole] (PFTBT) were synthesized in this group. Milli-Q water (18.2 MΩ·cm at 25 °C) was used throughout the experiments. -2 All other chemical reagents should be used as is.

[0245] Example 2: Synthesis of NADH-sensitive P-points.

[0246] P-points were prepared using a nanoprecipitation method. In a typical preparation, the fluorescently conjugated polymer was dissolved in anhydrous THF to prepare a stock solution (1.0 mg / mL). -1 The stock solution was further diluted in THF to produce a solution containing the fluorescent polymer (0.1 mg / mL). -1 ) and functional polymer PSMA (0.02 mg / mL) -1 A mixture of the above solution was rapidly dispersed in 10 mL of Milli-Q water under intense sonication. THF was removed by blowing nitrogen at 90°C for approximately 60 minutes. A small fraction of the aggregates was removed by filtration through a 0.2 μm membrane filter.

[0247] The P-point has been developed using band theory spanning the entire visible spectrum. Figure 8 The chemical structures of the semiconductor polymers used in this work (including PFO, PDHF, PFBT, PFBTTBT, PFTBT, DPA-CNPPV, and DPA-CNPF) are presented. NADH-sensitive P-points were prepared via a simple nanoprecipitation method that folds and twists the polymer through hydrophobic interactions with the amphiphilic polymer PSMA.

[0248] Example 3: Characterization of NADH-sensitive P-points

[0249] The morphology of the obtained point P was characterized by transmission electron microscopy (TEM), which showed that point P is monodisperse and approximately spherical in shape. Figure 1A The average diameter of point P was determined by dynamic light scattering (DLS), where the hydrodynamic diameter was approximately 19 nm. Figure 1BThe zeta potential measurements showed that point P has a negatively charged surface at neutral pH, and the initial zeta potential is approximately -37 mV. Figure 1C These transparent P-points remained stable for several weeks at room temperature in phosphate-buffered saline (PBS) solution. Figure 9 ), and there were no obvious signs of further aggregation or decomposition. Figure 1D The images show the suspension at point P under white light and 365nm ultraviolet (UV) light (from left to right: PFO, PDHF, PFBT, DPA-CNPPV, PFBTTBT, and PFTBTP points). Figure 1E As indicated, the changes in absorption and fluorescence spectra vary depending on the polymer structure. Upon excitation, the aqueous P-point suspension exhibits strong fluorescence and emits almost the entire spectrum (400-750 nm). Figure 1F ).

[0250] UV-Vis absorption spectra were recorded on a DU 720 scanning spectrophotometer (Beckman Coulter, Inc., CA, USA). Fluorescence spectra were acquired and calibrated using an LS-55 fluorescence spectrometer (LS55, PerkinElmer Life and Analytical Sciences, Shelton, CT, USA). Fluorescence quantum yield was measured using a Hamamatsu C10027 photon multichannel analyzer equipped with a CCD camera and integrating sphere. Solvent was used as a reference for quantum yield calibration. The size distribution and zeta potential of the P-points in aqueous solutions were determined by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS instrument. The morphology of the P-points was recorded on a FEI Tecnai F20 TEM operating at 200 kV.

[0251] Table 3 summarizes the size and photophysical properties of point P.

[0252] Table 3. Summary of the size and photophysical properties of point P.

[0253] Point P <![CDATA[λ max abs (nm) a ]]> <![CDATA[λ max em (nm) b ]]> Size (nm) <![CDATA[ζ(mV) c ]]> <![CDATA[LOD(μM) d ]]> <![CDATA[K SV (M –1 ) e ]]> PFO 380 436 20.2 -36.2 25 <![CDATA[1.04×10 3 ]]> PDHF 374 428 22.1 -37.2 36 <![CDATA[0.94×10 3 ]]> PFBT 322，458 546 19.3 -36.4 14 <![CDATA[4.89×10 3 ]]> PFBTTBT 380 626 19.7 -38.2 27 <![CDATA[1.17×10 3 <!-- 46 -->]]> PFTBT 374，528 638 17.5 -36.9 28 <![CDATA[0.97×10 3 ]]> DPA-CNPPV 294 627 18.8 -38.5 3.1 <![CDATA[2.89×10 5f ]]>

[0254] a Maximum absorption.

[0255] b Maximum fluorescence.

[0256] c ζ potential.

[0257] d Detection limit.

[0258] e Quenching constant (0-2mM).

[0259] f Sensitivity (0-2mM).

[0260] Example 4: Fluorescence response of point P to NADH.

[0261] First, the fluorescence response of point P to NADH was studied in aqueous solution to identify the best candidate. This example demonstrates point P dispersed in a co-solvent containing NADH.

[0262] Figure 2A The representative spectral change at the P-point of PFO is shown when NADH is added (a similar spectral evolution was shown in the determination of NADPH, as...). Figure 10 (Provided in [the source]). Increased NADH concentration strongly quenches emission. Figure 11 A Stern-Volmer plot of fluorescence intensity at the P-point of PFO versus dissolved NADH was plotted. Within the physiologically relevant range of 0–2 mM, the data showed a good linear fit with NADH concentration. The limit of detection (LOD) was 25 μM, and the quenching constant (K0) was also satisfactory. SV The value is 1.04 × 10 3 M –1 The luminescence quenching at other P-points under the same conditions was also investigated. The LOD of the PDHF P-point was 36 μM, and K... SV It is 0.94×10 3 M –1 ( Figure 2B , Figure 12A ); The LOD of PFBT point P is 14 μM, and K SV It is 4.89×10 3 M –1 ( Figure 2C , Figure 12B ); PFBTTBT has a LOD of 27 μM, and K SV It is 1.17 × 10 3 M –1 ( Figure 2D , Figure 12C ); and the LOD of PFTBT is 28 μM, and K SV It is 0.97×10 3 M –1 ( Figure 2E , Figure 12D Of these P points, the PFBT P point exhibits the smallest LOD and the largest K. SV This indicates that it has excellent sensitivity in detecting NADH. Figure 13 The figure illustrates, graphically, the addition of λ according to an embodiment of the present disclosure. ex Fluorescence emission of the P-point of PFBTTBT before and after NADH (10 mM) excitation at 380 nm was obtained. These results suggest that the P-point may be a promising "turn-off" fluorescent probe for NADH detection.

[0263] Ratiometric fluorescent probes rely on analyte-induced variations in emission intensity at two or more different wavelengths, which significantly increase the signal-to-noise ratio and improve quantization. The luminescence response of the P-point of the DPA-CNPPV probe to NADH was investigated, revealing significant changes in the emission band, with a sharp decrease in red emission at 627 nm and a subsequent increase in blue emission at 458 nm, thus generating a ratiometric sensor for NADH.

[0264] In the presence of 100 mM NADH, the fluorescence quantum yield (QY) of the DPA-CNPPV P-points recorded in the 500–800 nm region decreased from 10.8% to 3.4%, while the corresponding QY recorded in the 400–500 nm region increased from 0.2% to 1.3% (Table 4). Fluorescence intensity ratio (R = I) 458nm / I 627nm Relative variation: R / R0; where R0 indicates the fluorescence intensity ratio of pure P point in the absence of NADH, and R is the fluorescence intensity ratio at different NADH concentrations) changes with 0-2mM ( Figure 3A , 3B ) and 2-10mM ( Figure 3C , 3D The NADH concentration exhibited excellent linearity over a wide range. The LOD of NADH at the DPA-CNPPV P point was determined to be as low as 3.1 μM.

[0265] Table 4: Characterization of quantum yield (QY) at the P point of DPA-CNPPV (2MG ML) -1 )

[0266] Point P + [NADH] 0μM 10μM 20μM 30μM 40μM 60μM 100μM QY (500-800nm) 10.8 7.6 6.2 5.4 4.7 4.0 3.4 QY (400-500nm) 0.2 0.7 0.9 1.0 1.1 1.2 1.3

[0267] The photostability of fluorescent probes is a key issue for long-term monitoring of analytes. Figure 3E It can be seen that the fluorescence intensity of the DPA-CNPPV P spot remained almost constant after 30 minutes of continuous irradiation with 385nm light. Furthermore, the reaction kinetics results indicate that the reaction of the DPA-CNPPV P spot with NADH caused a time-dependent change in the free radical fluorescence intensity ratio, which was completed within 5 seconds. Figure 3FThis demonstrates the rapid response between the DPA-CNPPV P-point probe and NADH. Selectivity is another important sensor parameter for biosensing. The DPA-CNPPV P-point sensor exhibits high selectivity in the presence of various potentially interfering substrates, including active oxidizing and reducing species, different carbohydrate derivatives, and abundant cellular cations. NADH-triggered I 458 nm / I 627 A significant enhancement in the emission ratio of nm ( Figure 3G ), while the spectral changes of at most 1 mM in other potentially interfering substrates are not discernible. Figure 3H The reversibility of the ratio P-point sensor was also investigated. The DPA-CNPPV P-point sensor was repeatedly separated from NADH (1 mM) by ultrafiltration and gel filtration. For each cycle of measurement, the sensor response remained constant. Figure 3I This indicates that the DPA-CNPPV P-point sensor has good reversibility, which is consistent with the electron transfer mechanism without chemical reaction. Figure 3J and 3K The results show that NADH quenches P-point emission at 627 nm and emits fluorescence at 458 nm. Figure 3J The emission spectra of DPA-CNPPV P-point and NADH excited at 385 nm are shown. Figure 3K A photograph of the solution at point P irradiated with 365nm UV light is shown. Figure 3L The fluorescence response of the sensor to NADH and NADPH is shown. Figure 3L The fluorescence response of the sensor to NADH and NADPH is shown. Using NADH, NADPH, and NAD... + and NADP + The titration of DPA-CNPPV / PSMA at point P showed only a fluorescence response to NADH and NADPH, indicating that NAD... + and NADP + It will not quench P-point emission, and it will not emit 458nm light under UV irradiation.

[0268] The luminescence properties of DPA-based P-point (DPA-CNPF) in other NADH-containing cases were also investigated, yielding results similar to those of DPA-CNPPV. Figure 14 The rapid, sensitive, selective, and reversible response to NADH gives P-point sensors great potential for monitoring metabolites.

[0269] Example 5: Detection of NADH in living cells.

[0270] After confirming the sensitivity, selectivity, and stability of the P-point probe, the potential applications of P-point imaging for NADH in live cells were explored. This example demonstrates the p-point in a co-solvent containing NADH.

[0271] P-dots with ultra-bright fluorescence have been successfully applied for specific cell labeling. Here, the PFBT P-dot is selected as an example of its in vitro application. Streptavidin-functionalized P-dots were used to label the specific cellular target protein EpCAM to detect circulating tumor cells MCF-7 (…). Figure 15 ).

[0272] P-point labeled MCF-7 cells were incubated with PBS and NADH, respectively. The breast cancer cell line MCF-7 was purchased from the American Type Culture Collection (Manassas, Virginia, USA). Primary cultured MCF-7 cells were cultured at 37°C in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum and 1% penicillin / streptomycin under a humidified environment of 5% CO2. The medium was changed every two days. Cells were isolated at 80% confluence using 0.25% trypsin-EDTA and then centrifuged at 800 rpm for 5 minutes. The pellet was resuspended in medium and passaged in culture flasks.

[0273] Bioconjugation was performed using an EDC-catalyzed reaction between the carboxyl group at the P-point and the amino group on the biomolecule streptavidin. In a typical bioconjugation reaction, 80 μL of polyethylene glycol (PEG, 5% w / v) and an equal volume of concentrated HEPES buffer (1M) were added to 4 mL of PFBT P-point solution (50 μg / mL, in Milli-Q water) to produce a P-point solution in 20 mM HEPES buffer at pH 7.3. Then, 240 μL of streptavidin (5 mg / mL) was added to the solution and vortexed. Next, 80 μL of freshly prepared EDC solution (5 mg / mL, in deionized water) was added, and the resulting mixture was incubated on a rotary shaker at room temperature for 4 hours. The resulting P-point-streptavidin bioconjugation was separated from the free biomolecule by gel filtration using Sephacryl HR-300 gel media. To label the surface marker EpCAM, MCF-7 cells were collected from culture flasks, washed, centrifuged, and resuspended in labeling buffer (1×PBS, 1% BSA). MCF-7 cells were dispersed in 100 μL of labeling buffer in 5 mL round-bottom tubes and sequentially incubated with biotinylated primary anti-EpCAM (0.5 mg / mL) and P-point-streptavidin. The P-point-labeled MCF-7 cells were then incubated at 37°C in PBS solution (10 mM, pH 7.4) for 30 min in the absence or presence of NADH (1 mM). Fluorescence imaging was performed using a fluorescence microscope with a 20× objective. Excitation light was provided by a xenon lamp and filtered by a bandpass filter (Semrock FF01-350 / 52). The fluorescence signal was filtered by a bandpass filter (Semrock FF01-525 / 20). Image processing and analysis were performed using ImageJ and Matlab software.

[0274] Figure 4A Combined bright-field and fluorescence images of PFBT P-point labeled MCF-7 cells in PBS without NADH are shown. P-point labeled MCF-7 cells exhibit strong fluorescence. Cells receiving NADH showed significantly reduced fluorescence compared to the control group. Figure 4C The differences in fluorescence intensity are presented more vividly through their 3D interactive intensity. Figure 4B and 4D The fluorescence intensity at point P is inversely proportional to the NADH concentration, indicating that the sensor at point P has successfully detected NADH in living cells.

[0275] Example 6: Using ratio NAD(P)H sensing from a digital camera or smartphone for point-of-care (POC) testing and in vivo imaging.

[0276] This example demonstrates the p-point dispersion in a co-solvent containing NADH.

[0277] Significant innovations in biosensors for point-of-care (POC) applications have emerged in recent years, allowing individuals to perform simple diagnostic or prognostic tests without visiting medical laboratories or hospitals, thereby improving convenience. This also increases the likelihood that physicians will receive results faster, enabling better immediate clinical management decisions. Due to economic considerations and device availability, digital and smartphone camera-based POC testing is rapidly becoming a potential technology for generating mobile diagnostic and monitoring systems for POC testing. Figure 5A As shown, after incubation with NADH within a physiologically relevant range, the red to blue fluorescence color change of the DPA-CN-PPV P point can be directly observed via a smartphone camera. In the absence of NADH, the solution fluorescence color is red; as the NADH concentration increases, the emission color eventually turns blue. The large ratio change also allows for clear visualization of the fluorescence color change of the solution. Each primary color true-color image can be divided into red (R), green (G), and blue (B) channels. After digitization using image processing algorithms... Figure 5B The intensity ratio of the B / R channels is used to quantify NADH concentration. Figure 5C The linear response of the intensity ratio (R = B / R) is shown within the physiologically relevant range of 0–2 mM. The maximum ratio enhancement can reach more than 100-fold as the NADH concentration increases from 0 mM to 2 mM. Figure 5D-5F These results indicate that the ratio DPA-CNPPV P-point sensor, combined with a smartphone camera or digital camera, provides a viable method for NADH monitoring.

[0278] Furthermore, the feasibility of using DPA-CNPPV P-points for in vivo NADH imaging will be evaluated by processing the fluorescence of P-points imaged by a smartphone under UV lamp excitation. Figure 5G-5J ).

[0279] Here, female Balb / c nude mice were used in accordance with the guidelines for the care and use of research animals. Anesthetized nude mice were subcutaneously injected with PBS containing DPA-CNPPV P-points and different concentrations of NADH (0 mM, i.e., no NADH, and 0.25 mM, 0.5 mM, and 1.0 mM NADH) into the dorsal region. UV lamps were used for irradiation, and a smartphone camera was used for imaging. Figure 5G ). Figure 5H Concentration-dependent sensing at various NADH concentrations (0.25 mM, 0.5 mM, and 1.0 mM) is illustrated. True-color images of the region of interest were split into blue and red channel images to calculate the B / R ratio. Figure 5I , 5JThe B / R ratio exhibits excellent linearity in the in vivo NADH detection response of the P-point sensor.

[0280] Example 7: PKU's P-point sensor

[0281] This example demonstrates NADH-dependent enzymes coupled to p-points and NADH-dependent enzymes coupled to a common substrate having p-points.

[0282] Metabolites play a vital role in all aspects of living organisms because they have a variety of functions, such as energy conversion, structure, signal transduction, epigenetic effects, cofactor activity, and interactions with other organisms. NAD + / NADH and NADP + NADPH is a crucial cofactor for metabolism. + / NADH and NADP + There are over 500 reactions involving NADPH. Most of these reactions involve dehydrogenases. Dehydrogenases are enzymes belonging to the oxidoreductase group, which typically work by reducing NADPH. + The NADH produced is oxidized to the substrate of interest. The stoichiometric NADH production can be quantified using an NADH-sensitive P-point sensor. The NADH level corresponds to the substrate level in the sample. Figure 16 Here, the metabolite detection strategy is based on the integration of an NADH-sensitive P-point sensor with an NADH-dependent enzyme that catalyzes the oxidation of the analyte of interest. The metabolite detection strategy can also be based on the integration of an NADPH-sensitive P-point sensor with an NADPH-dependent enzyme that catalyzes the oxidation of the analyte of interest. Figure 16 ).

[0283] As the first application of a NADH-sensitive P-point sensor, a smartphone-based assay for phenylketonuria (PKU) was developed. PKU is a hereditary disorder of phenylalanine metabolism caused by a deficiency in phenylalanine hydroxylase (PAH). Infants and children with PKU typically exhibit signs of progressive neurological disorders. To quantify phenylalanine, a DPA-CNPPV P-point sensor with phenylalanine dehydrogenase (PheDH) was used for measurement, which has high specific activity for phenylalanine (…). Figure 6A A predetermined concentration of phenylalanine was incorporated into the P-point sensor (containing 0.05 mg / mL LDA-CNPPV P-point, 3 mM NAD). + The mixture was incubated in a 200 mM glycine buffer (pH 10.5, 1 μM PheDH). After incubation for 10 minutes, the emission ratio of the sensor in the reaction mixture was measured using a fluorescence spectrometer.

[0284] When titrated with phenylalanine, the resulting luminescent sensor showed an 18.9-fold change in emission ratio. The LOD of phenylalanine at point P was measured to be 3.5 μM. The sensor's c 50 (The phenylalanine concentration that produces 50% of the maximum sensor response) was measured at 279.7 μM. Figure 6B ).

[0285] Patients with PKU should be managed with all available options from birth to lifelong control of their blood phenylalanine levels. This is important because toxic levels of phenylalanine in the blood can lead to irreversible brain damage and neurological complications. PKU patients are classified according to their pre-treatment blood, serum, or plasma phenylalanine concentrations: newly diagnosed newborns with levels between 120 μM and 360 μM indicate benign mild hyperphenylalaninemia (HPA); 360 μM to 600 μM, mild HPA; 600 μM to 900 μM, mild PKU; 900 μM to 1200 μM, moderate PKU; and levels above 1200 μM, classic PKU. Figure 6C-6I As shown, within different clinical symptom-related ranges of blood phenylalanine levels, the emission ratio exhibits a linear relationship with phenylalanine levels. For healthy levels, the P-point sensor has a resolution of 2.6 μM and a sensitivity of 4.89 × 10⁻⁶. 4 M -1 Table 5 summarizes the performance parameters of the PKU sensor in other dynamic ranges, including maximum signal variation, sensitivity, and resolution.

[0286] Table 5. Summary of performance parameters of PKU sensors

[0287]

[0288] a The concentration of phenylalanine in blood plasma.

[0289] b The linear range of biosensors.

[0290] c Maximum signal change within the dynamic range.

[0291] d Sensitivity (S = Δs / Δc; s = R / R0, c = [Phe]).

[0292] e Resolution (R = σ / S).

[0293] Frequent blood tests measure phenylalanine levels in children's blood in efforts to prevent health problems. Many methods and reagents have been developed for phenylalanine quantification, including the Guthrie bacterial inhibition assay, ferric chloride test, dye-based fluorescence assay, and PCR methods. However, these methods often involve complex preparation or lengthy testing times. Therefore, it is important to develop new materials and methods that enable simple, rapid, and accurate detection of phenylalanine at home. For self-testing phenylalanine measurement, a assay using a 96-well microplate and a digital camera was designed (…). Figure 7A This enzyme assay requires a 10-minute incubation. The phenylalanine concentration can then be calculated based on the emission ratio (R / R0) of the ROI image. Figure 7B Due to different color temperature settings, photos taken by digital cameras and smartphones look different. Figure 7D , 7E For the 96-well microplate measurement, photographs were taken using a Sony a7 camera (file format: RAW; WB: 3000, ISO: 2000; shutter speed: 1 / 20). A smartphone camera (iPhone iOS 13.1, Auto mode) was used as an alternative for photographic capture. The ratio was used to calculate the phenylalanine concentration. Furthermore, its great potential in paper-based sensors has been demonstrated.

[0294] Figure 7I This is an example demonstration of the described scheme. Here, a metabolite biosensor is designed that combines the NAD(P)H-sensitive P-site with a metabolite-specific NAD(P)H-dependent enzyme in solution-based or paper-based assays. Figure 7I Enzymatic oxidation of metabolites produces NAD(P)H; under ultraviolet (UV) irradiation, NAD(P)H quenches the red emission of the P-point while fluorescing in the blue region. The NAD(P)H-sensitive P-point is composed of the luminescent conjugated polymer poly[{2-methoxy-5-(2-ethylhexyloxy)-1,4-(1-cyanovinylphenylene)}-copolymer-{2,5-bis(N,N'-diphenylamino)-1,4-phenylene}] (DPA-CNPPV) and the amphiphilic polymer poly(styrene-copolymer-maleic anhydride) (PSMA). Excitation of the P-point with UV irradiation produces a 627 nm emission that is quenched by NAD(P)H, while NAD(P)H emits at 458 nm. The ratio of the emission intensities at 458 nm and 627 nm—or the ratio of the emission intensities from the blue to the red channel—is used to accurately measure the concentration of oxidized metabolites when using a digital camera or cellular phone and RGB image processing.

[0295] Using an HP Pro400 M401 dne printer, grade 1 Chr cellulose chromatography paper (GE Healthcare) was patterned using a 96-well plate template. The pattern was created by inserting 4 μL of 4 μL of paper containing 0.05 mg / mL DPA-CNPPV P-point and 3 mM NAD-1. + To prepare the paper-based assay, a buffer solution of 2 μM PheDH and 200 mM glycine (pH 10.5) was lyophilized onto a paper tray. A liquid nitrogen cooling bath was used during the first 2 hours of lyophilization. For measurement, 4 μL of the analyte (in pH 10.5 buffer) was added directly to the test strip. After incubation for 10 minutes, the emissivity of the sensor on the test strip was measured using a fluorescence plate reader, digital camera, or cellular phone camera. A blank paper was used as a control to eliminate background fluorescence interference from the paper.

[0296] The dehydrogenase and NADH-sensitive P-spot were lyophilized onto 96-well test strips. Simply add 0.4 μL of analyte to the test strip containing the buffer and the lyophilized sensor. Figure 7F Then a fluorescence plate reader can be used. Figure 7G or digital camera or cellular phone ( Figure 7C and Figure 7E )Analytical results. An enzymatic reaction is initiated by adding a small amount of sample containing the analyte to the test strip. Figure 7F The image was analyzed using RGB image processing algorithms to calculate the average blue and red channel intensities within each hole based on pixel intensity distribution. Figures 7J to 7L The ratio of blue to red channel intensities was significantly lower at 60 μM Phe (healthy) than at 1200 μM Phe (classical PKU threshold). Figure 7L ).like Figure 7H As shown, the phenylalanine measurement results of the paper-based system clearly reflect PKU levels. The simple and rapid testing procedure allows patients to perform self-testing at designated care locations. Table 6 illustrates the performance of the P-point sensor in paper measurements read using a digital camera (e.g., a digital camera from a cellular phone or a standalone camera).

[0297] Table 6. Performance of the phenylalanine P-point biosensor in paper measurements read using a digital camera.

[0298]

[0299]

[0300] a The concentration of phenylalanine found in blood plasma. b s = R / R0 (blue to red channel emission ratio).

[0301] Example 8: Using a P-point sensor to measure human plasma samples for PKU.

[0302] This example demonstrates the coupling of a NADH-dependent enzyme with a common substrate as a p-point.

[0303] In this regard, the performance of the paper-based assay in measuring human plasma samples was next evaluated. The biosensor was calibrated by analyzing samples with and without PheDH. Figure 18A To correct for inter-patient differences in endogenous NADH concentrations in the blood, values ​​obtained without PheDH were subtracted from values ​​obtained using PheDH to obtain phenylalanine concentrations (in...). Figure 18B (Marked as "difference"). As a proof of principle for PKU screening applications, paper assays were used to analyze plasma samples mixed with different concentrations of phenylalanine, and the results were compared using a plate reader. Figure 18C ) and digital camera ( Figure 18D The result obtained when reading it out.

[0304] To correct for endogenous NADH in the blood, whole blood (containing EDTA) from healthy human donors was obtained from PlasmaLab International (Everett, USA). Plasma was separated from the whole blood by centrifugation. Correction for endogenous NADH was achieved using a P-point sensor containing and without PheDH enzyme. Plasma (0.2 mL) was added to a P-point sensor containing 0.0625 mg / mL DPA-CNPPV / PSMAP and 3.75 mM NAD. + Add 0.8 mL of 0 M or 2.5 μM PheDH and 250 mM glycine buffer (pH 10.5) and incubate the mixture for 10 minutes. Measure endogenous NADH using the reaction without PheDH, and measure NADH produced by the conversion of endogenous NADH to phenylalanine using the reaction with PheDH; subtract both values ​​to obtain the phenylalanine concentration.

[0305] To prepare a paper-based assay for measuring phenylalanine in plasma, grade 1 Chr cellulose chromatographic paper was patterned as described above. This was achieved by using 4 μL of paper containing 0.05 mg / mL DPA-CNPPV / PSMA at the P point and 3 mM NAD. + Test strips were prepared by lyophilizing a buffer solution of 2 μM HeDH and 200 mM glycine (pH 10.5) onto a paper tray. A liquid nitrogen cooling bath was used during the first 2 hours of lyophilization.

[0306] Example 9: P-point sensor for other metabolic diseases, drug metabolism, or metabolites

[0307] This example demonstrates point p dispersed in a co-solvent containing an NADH-dependent enzyme.

[0308] NAD can be used + or NADP + Any oxidized metabolite can be analyzed or measured using the sensors described herein. As non-limiting examples, NADH-dependent enzymatic reactions are used to accurately analyze samples with various analyte concentrations for lactate, glutamate, glucose, and β-hydroxybutyrate (BHB). Figures 17A-17H Here, different amounts of lactate, glucose, glutamate, or β-hydroxybutyrate analytes were incorporated into the corresponding P-point sensor solutions. For lactate, the solution contained 0.05 mg / mL P-point and 3 mM NAD. + 1 μM lactate dehydrogenase, 200 mM glycine buffer (pH 9.8); for glucose: 0.05 mg / mL at point P, 3 mM NAD. + 1 μM glucose dehydrogenase, 50 mM HEPES buffer (pH 8.0); for glutamate: 0.05 mg / mL at point P, 3 mM NAD. + 1 μM glutamate dehydrogenase, 50 mM HEPES buffer (pH 7.3); for β-hydroxybutyrate: 0.05 mg / mL at point P, 3 mM NAD + 1 μM β-hydroxybutyrate dehydrogenase, 50 mM HEPES buffer (pH 7.8). Lactate detection is important for medical conditions including bleeding, respiratory failure, liver disease, and sepsis; glucose monitoring is important for managing diabetes; glutamate monitoring can be used for the diagnosis and monitoring of neurodegenerative diseases; and β-hydroxybutyrate sensing is used to detect hyperketosis. More than 500 NAD(P)H-dependent enzymes and more than 300 related metabolites are compatible with this system (see, for example, Table 2), including more than 100 medically relevant metabolites (see, for example, Tables 1 and 2).

[0309] Although illustrative embodiments have been described and illustrated, it should be understood that various changes may be made therein without departing from the spirit and scope of the invention.

Claims

1. A nanoparticle transducer for measuring analyte concentration, the nanoparticle transducer comprising: Nanoparticles, wherein the nanoparticles include chromophores; as well as Nicotinamide adenine dinucleotide NADH-dependent enzyme or nicotinamide adenine dinucleotide phosphate NADPH-dependent enzyme, wherein the NADH-dependent enzyme or NADPH-dependent enzyme is physically associated with the nanoparticles and configured to catalyze a reaction comprising multiple reaction elements; The plurality of reaction elements includes one or more reactants and one or more products, the one or more reactants comprising an analyte, and wherein the amount of fluorescence emitted from the chromophore is determined by the concentration of the reaction element among the plurality of reaction elements. The enzymes and nanoparticles are dispersed in a common solvent, coupled with a common substrate, encapsulated together in hydrogel beads, or in the form of lyophilized powder. The enzyme is not directly connected to the nanoparticles via covalent bonds.

2. The nanoparticle transducer of claim 1, wherein the reaction element among the plurality of reaction elements includes NADH, and wherein the amount of fluorescence emitted from the chromophore is determined by the concentration of the NADH.

3. The nanoparticle transducer according to claim 1, wherein the analyte comprises NADH.

4. The nanoparticle transducer according to any one of the preceding claims, wherein the NADH-dependent or NADPH-dependent enzyme is selected from the group consisting of: dehydrogenases, reductases, oxygenases, synthases, and combinations thereof.

5. The nanoparticle transducer according to any one of claims 1-3, wherein the NADH-dependent or NADPH-dependent enzyme is a hydroxylase.

6. The nanoparticle transducer according to any one of claims 1-3, wherein the nanoparticles comprise polymer dots P-dots.

7. The nanoparticle transducer according to any one of claims 1-3, wherein the chromophore comprises a semiconductor polymer.

8. The nanoparticle transducer according to any one of claims 1-3, wherein the chromophore comprises a blend of two or more semiconductor polymers.

9. The nanoparticle transducer according to any one of claims 1-3, wherein the chromophore comprises a dye, and wherein the dye is contained within the nanoparticle.

10. The nanoparticle transducer according to any one of claims 1-3, wherein the chromophore comprises a semiconductor polymer and a dye, and wherein the dye and the semiconductor polymer interact upon irradiation to generate enhanced fluorescence.

11. The nanoparticle transducer according to any one of claims 1-3, wherein the fluorescence emitted from the chromophore comprises a signal fluorescence wavelength and a control fluorescence wavelength.

12. The nanoparticle transducer of claim 11, wherein the fluorescence emitted from the chromophore defines a signal fluorescence ratio, the signal fluorescence ratio being equal to the ratio of the amount of fluorescence emitted at the signal fluorescence wavelength to the amount of fluorescence emitted at the control fluorescence wavelength, and wherein the signal fluorescence ratio is determined by the concentration of the reaction element among the plurality of reaction elements.

13. The nanoparticle transducer of claim 12, wherein the fluorescence ratio varies proportionally with the concentration of the analyte.

14. The nanoparticle transducer according to any one of claims 1-3, wherein the fluorescence emitted from the chromophore varies proportionally with the concentration of the analyte within a certain analyte concentration range.

15. A transducer substrate for measuring analyte concentration, the transducer substrate comprising: Nanoparticles, said nanoparticles including chromophores coupled to a substrate; as well as An enzyme, coupled to the substrate and configured to catalyze a reaction comprising multiple reaction elements, The plurality of reaction elements includes one or more reactants and one or more products, the one or more reactants comprising an analyte, and wherein the amount of fluorescence emitted from the chromophore is determined by the concentration of the reaction element among the plurality of reaction elements. The enzyme is not directly physically linked to the chromophore via covalent bonds.

16. The transducer substrate of claim 15, wherein the reactive element of the plurality of reactive elements comprises NADH, and wherein the amount of fluorescence emitted from the chromophore is determined by the concentration of the NADH.

17. The transducer substrate of claim 15, wherein the analyte comprises NADH.

18. The transducer substrate according to any one of claims 15 to 17, wherein the enzyme is an NADH-dependent or NADPH-dependent enzyme.

19. The transducer substrate according to claim 18, wherein the NADH-dependent enzyme or NADPH-dependent enzyme is selected from the group consisting of: dehydrogenases, reductases, oxygenases, synthases, and combinations thereof.

20. The transducer substrate of claim 18, wherein the NADH-dependent enzyme or NADPH-dependent enzyme is a hydroxylase.

21. The transducer substrate according to claim 15, wherein the enzyme is glucose oxidase.

22. The transducer substrate of claim 15, wherein the reactive element of the plurality of reactive elements includes oxygen, and wherein the amount of fluorescence emitted from the chromophore is determined by the concentration of the oxygen.

23. The transducer substrate according to any one of claims 15 to 17, wherein the enzyme is not coupled to the nanoparticles.

24. The transducer substrate according to any one of claims 15 to 17, wherein the substrate is a paper substrate.

25. The transducer substrate according to any one of claims 15 to 17, wherein the enzyme is covalently bound to the substrate.

26. The transducer substrate according to any one of claims 15 to 17, wherein the nanoparticles are covalently bonded to the substrate.

27. The transducer substrate according to any one of claims 15 to 17, wherein the enzyme is physically associated with the substrate.

28. The transducer substrate of claim 27, wherein the enzyme and the nanoparticles are freeze-dried onto the substrate.

29. The transducer substrate according to any one of claims 15 to 17, wherein the enzyme is coupled to the substrate at a point adjacent to the nanoparticle.

30. The transducer substrate according to claim 29, wherein the enzyme is a first enzyme, the nanoparticles are first nanoparticles, the chromophore is a first chromophore, and the reaction is a first reaction; and The transducer substrate further comprises: The second nanoparticle includes a second chromophore coupled to the substrate; as well as A second enzyme, which is different from the first enzyme coupled to the substrate, is configured to catalyze a second reaction comprising a second plurality of reaction elements; The second plurality of reaction elements includes one or more second reactants and one or more second products, the one or more second reactants comprising the analyte, and wherein the amount of fluorescence emitted from the second chromophore is determined by the concentration of the second reaction element among the second plurality of reaction elements.

31. The transducer substrate of claim 30, wherein the point is a first point, and wherein the second nanoparticle is coupled to the substrate at a second point separate from the first point.

32. The transducer substrate of claim 30, wherein the second nanoparticle is coupled to the substrate at the point described.

33. The transducer substrate of claim 30, wherein the first chromophore is configured to absorb light within a first absorption wavelength range, and the second chromophore is configured to absorb light within a second absorption wavelength range different from the first absorption wavelength range.

34. The transducer substrate of claim 30, wherein the fluorescence emitted from the first chromophore is in a first emission wavelength range, and wherein the fluorescence emitted from the second chromophore is in a second emission wavelength range different from the first emission wavelength range.

35. The transducer substrate of claim 30, wherein the second reaction is different from the first reaction.

36. The transducer substrate of claim 30, wherein the second reaction is the same as the first reaction.

37. The transducer substrate of claim 29, wherein the number of dots on the substrate coupled with nanoparticles and enzymes is in the range of 2 to 10, 2 to 50, 2 to 100, 2 to 500, or 2 to 1,000.

38. The transducer substrate of claim 29, wherein the size of the spot is in the range of 1 µM to 500 µM.

39. The transducer substrate of any one of claims 29, wherein the substrate is configured to draw a fluid sample core to the point.

40. The transducer substrate according to any one of claims 15 to 17, wherein the nanoparticles comprise P-points.

41. The transducer substrate according to any one of claims 15 to 17, wherein the chromophore comprises a semiconductor polymer.

42. The transducer substrate according to any one of claims 15 to 17, wherein the chromophore comprises a blend of two or more semiconductor polymers.

43. The transducer substrate according to any one of claims 15 to 17, wherein the chromophore comprises a dye, and wherein the dye is contained within the nanoparticles.

44. The transducer substrate according to any one of claims 15 to 17, wherein the chromophore comprises a semiconductor polymer and a dye, and wherein the dye and the semiconductor polymer interact upon irradiation to produce enhanced fluorescence.

45. The transducer substrate according to any one of claims 15 to 17, wherein the fluorescence emitted from the chromophore comprises a signal fluorescence wavelength and a control fluorescence wavelength.

46. ​​The transducer substrate of claim 44, wherein the fluorescence emitted from the chromophore defines a signal fluorescence ratio, the signal fluorescence ratio being equal to the ratio of the amount of fluorescence emitted at the signal fluorescence wavelength to the amount of fluorescence emitted at the control fluorescence wavelength, and wherein the signal fluorescence ratio is determined by the concentration of the reactive elements among the plurality of reactive elements.

47. The transducer substrate of claim 46, wherein the fluorescence ratio varies proportionally with the concentration of the analyte.

48. A kit for measuring analyte concentration, the kit comprising: Nanoparticles, wherein the nanoparticles include chromophores; as well as An enzyme that is physically associated with the nanoparticles and configured to catalyze a reaction comprising multiple reaction elements; The plurality of reaction elements includes one or more reactants and one or more products, the one or more reactants comprising an analyte, and wherein the amount of fluorescence emitted from the chromophore is determined by the concentration of the reaction element among the plurality of reaction elements. The enzymes and nanoparticles are dispersed in a common solvent, coupled with a common substrate, encapsulated together in hydrogel beads, or in the form of lyophilized powder. The enzyme is not directly connected to the nanoparticles via covalent bonds.

49. The kit according to claim 48, wherein the enzyme is an NADH-dependent or NADPH-dependent enzyme.

50. The kit according to claim 49, wherein the NADH-dependent or NADPH-dependent enzyme is selected from the group consisting of: dehydrogenases, reductases, oxygenases, synthases, and combinations thereof.

51. The kit according to claim 49, wherein the NADH-dependent or NADPH-dependent enzyme is a hydroxylase.

52. The kit according to any one of claims 48 to 50, wherein the reaction element among the plurality of reaction elements comprises NADH, and wherein the amount of fluorescence emitted from the chromophore is determined by the concentration of the NADH.

53. The kit according to any one of claims 48 to 50, wherein the analyte comprises NADH.

54. The kit according to claim 48, wherein the enzyme is glucose oxidase.

55. The kit of claim 48, wherein the reaction element of the plurality of reaction elements includes oxygen, and wherein the amount of fluorescence emitted from the chromophore is determined by the concentration of the oxygen.

56. The kit according to any one of claims 48 to 50, wherein the amount of fluorescence emitted from the chromophore is determined by the concentration of the reactant in one or more of the reactants.

57. The kit according to any one of claims 48 to 50, wherein the nanoparticles comprise P-points.

58. The kit according to any one of claims 48 to 50, wherein the chromophore comprises a semiconductor polymer.

59. The kit according to any one of claims 48 to 50, wherein the chromophore comprises a blend of two or more semiconductor polymers.

60. The kit according to any one of claims 48 to 50, wherein the chromophore comprises a dye, and wherein the dye is contained within the nanoparticles.

61. The kit according to any one of claims 48 to 50, wherein the chromophore comprises a semiconductor polymer and a dye, and wherein the dye and the semiconductor polymer interact upon irradiation to produce enhanced fluorescence.

62. The kit according to any one of claims 48 to 50, wherein the fluorescence emitted from the chromophore comprises a signal fluorescence wavelength and a control fluorescence wavelength.

63. The kit of claim 62, wherein the fluorescence emitted from the chromophore defines a signal fluorescence ratio, the signal fluorescence ratio being equal to the ratio of the amount of fluorescence emitted at the signal fluorescence wavelength to the amount of fluorescence emitted at the control fluorescence wavelength, and wherein the signal fluorescence ratio is determined by the concentration of the reaction element among the plurality of reaction elements.

64. The kit of claim 63, wherein the fluorescence ratio varies proportionally with the concentration of the analyte.

65. A transducer for measuring analyte concentration, the transducer comprising: Chromophores, wherein the chromophores comprise semiconductor chromophore polymers; as well as An enzyme that is physically associated with the semiconductor chromophore polymer and configured to catalyze a reaction comprising multiple reaction elements; The plurality of reaction elements includes one or more reactants and one or more products, the one or more reactants comprising an analyte, and wherein the amount of fluorescence emitted from the chromophore is determined by the concentration of the reaction element among the plurality of reaction elements. The semiconductor chromophore polymer and the enzyme are dispersed in a common solvent, coupled with a common substrate, encapsulated together in hydrogel beads, or in the form of lyophilized powder. The enzyme is not directly connected to the semiconductor chromophore polymer via covalent bonds.

66. The transducer of claim 65, wherein the semiconductor chromophore polymer is not in a condensed state.

67. The transducer according to any one of claims 65 to 66, wherein the reactive element of the plurality of reactive elements comprises NADH, and wherein the amount of fluorescence emitted from the chromophore is determined by the concentration of the NADH.

68. The transducer according to any one of claims 65 to 66, wherein the analyte comprises NADH.

69. The transducer according to any one of claims 65 to 66, wherein the enzyme is an NADH-dependent or NADPH-dependent enzyme.

70. The transducer of claim 69, wherein the NADH-dependent or NADPH-dependent enzyme is selected from the group consisting of: dehydrogenases, reductases, oxygenases, synthases, and combinations thereof.

71. The transducer of claim 69, wherein the NADH-dependent or NADPH-dependent enzyme is a hydroxylase.

72. The transducer according to any one of claims 65 to 66, wherein the chromophore comprises a blend of two or more semiconductor chromophore polymers.

73. The transducer according to any one of claims 65 to 66, wherein the chromophore comprises the semiconductor chromophore polymer and a dye, and wherein the dye and the semiconductor chromophore polymer interact upon irradiation to produce enhanced fluorescence.

74. The transducer according to any one of claims 65 to 66, wherein the fluorescence emitted from the chromophore comprises a signal fluorescence wavelength and a control fluorescence wavelength.

75. The transducer of claim 74, wherein the fluorescence emitted from the chromophore defines a signal fluorescence ratio, the signal fluorescence ratio being equal to the ratio of the amount of fluorescence emitted at the signal fluorescence wavelength to the amount of fluorescence emitted at the control fluorescence wavelength, and wherein the signal fluorescence ratio is determined by the concentration of the reaction element among the plurality of reaction elements.

76. The transducer of claim 75, wherein the fluorescence ratio varies proportionally with the concentration of the analyte.

77. A system for measuring analyte concentration, the system comprising: The nanoparticle transducer according to claim 1, the transducer substrate according to claim 15, the kit according to claim 48, or the transducer according to claim 65; An irradiation source configured to irradiate the nanoparticle transducer, the transducer substrate, the reagent kit, or the chromophore of the transducer to induce fluorescence from the chromophore; A photodetector configured to generate a fluorescence signal based on the chromophore; as well as A controller, operatively coupled to the irradiation source and the photodetector, and including logic that, when executed by the controller, causes the system to perform operations including: Irradiate the chromophore with the irradiation source; and The concentration of the analyte is determined based on the signal from the photodetector.

78. The system of claim 77, wherein the fluorescence emitted from the chromophore comprises a signal fluorescence wavelength and a control fluorescence wavelength.

79. The system of claim 78, wherein the fluorescence emitted from the chromophore defines a signal fluorescence ratio, the signal fluorescence ratio being equal to the ratio of the amount of fluorescence emitted at the signal fluorescence wavelength to the amount of fluorescence emitted at the control fluorescence wavelength, and wherein the signal fluorescence ratio is determined by the concentration of the reaction element among the plurality of reaction elements.

80. The system of claim 79, wherein the photodetector is configured to detect the amount of signal fluorescence at the signal fluorescence wavelength and the amount of control fluorescence at the control fluorescence wavelength, and wherein the controller includes another logic that, when executed by the controller, causes the system to perform an operation comprising: determining a measured fluorescence ratio based on the measured amount of signal fluorescence and the measured amount of control fluorescence.

81. The system of claim 80, wherein the determination of the concentration of the analyte is based on the measured fluorescence ratio.

82. The system according to any one of claims 77 to 81, wherein the system is shaped to house the transducer substrate according to claim 15.

83. A method for measuring the concentration of an analyte in a fluid, the method comprising: The fluid is contacted with a P-point and an NADH-dependent or NADPH-dependent enzyme, the P-point containing a chromophore, the NADH-dependent or NADPH-dependent enzyme being coupled to the P-point, the NADH-dependent or NADPH-dependent enzyme being configured to catalyze a reaction comprising multiple reaction elements, wherein the multiple reaction elements include one or more reactants and one or more products, the one or more reactants containing the analyte, and wherein the amount of fluorescence emitted from the chromophore is determined by the concentration of the reaction elements among the multiple reaction elements; Irradiate point P to induce fluorescence from point P; Measure the fluorescence from point P; as well as The concentration of the analyte is determined based on the measured fluorescence. The enzyme and chromophore are dispersed in a common solvent, coupled with a common substrate, or encapsulated together in hydrogel beads. The enzyme is not directly linked to the chromophore via covalent bonds.

84. A method for measuring the concentration of an analyte in a fluid, the method comprising: The fluid is brought into contact with point P and an enzyme, point P containing a chromophore, the enzyme being physically associated with point P, the enzyme being configured to catalyze a reaction comprising a plurality of reaction elements, wherein the plurality of reaction elements comprises one or more reactants and one or more products, the one or more reactants comprising the analyte, and wherein the amount of fluorescence emitted from the chromophore is determined by the concentration of the reaction elements among the plurality of reaction elements. Irradiate the chromophore to induce fluorescence from the chromophore; Measure the fluorescence from the chromophore; as well as The concentration of the analyte is determined based on the measured fluorescence. The enzyme and chromophore are dispersed in a common solvent, coupled with a common substrate, or encapsulated together in hydrogel beads. The enzyme is not directly linked to the chromophore via covalent bonds.

85. A method for measuring the concentration of an analyte in a fluid, the method comprising: The fluid is brought into contact with a chromophore and an enzyme, the chromophore comprising a semiconductor chromophore polymer, the enzyme being physically associated with the chromophore, the enzyme being configured to catalyze a reaction comprising a plurality of reaction elements, wherein the plurality of reaction elements comprises one or more reactants and one or more products, the one or more reactants comprising the analyte, and wherein the amount of fluorescence emitted from the chromophore is determined by the concentration of the reaction elements among the plurality of reaction elements; Irradiate the chromophore to induce fluorescence from the chromophore; Measure the fluorescence from the chromophore; as well as The concentration of the analyte is determined based on the measured fluorescence. The enzyme and chromophore are dispersed in a common solvent, coupled with a common substrate, or encapsulated together in hydrogel beads. The enzyme is not directly linked to the chromophore via covalent bonds.

86. The method according to any one of claims 83 to 85, wherein the fluorescence emitted from the chromophore defines a fluorescence ratio equal to the ratio of the amount of fluorescence emitted at a signal fluorescence wavelength to the amount of fluorescence emitted at a control fluorescence wavelength, and wherein the fluorescence ratio is determined by the concentration of the fluid component.

87. The method of claim 86, wherein the determination of the concentration of the analyte comprises: Measure the fluorescence at the signal fluorescence wavelength and the fluorescence at the control fluorescence wavelength; The fluorescence ratio was determined based on the aforementioned measurements; The concentration of the analyte is determined based on the measured fluorescence ratio.

88. The method according to any one of claims 83 to 85, wherein the fluid is selected from blood, plasma, serum, lymph, saliva, tears, interstitial fluid, cerebrospinal fluid, urine, sweat, and combinations thereof.

89. The method according to any one of claims 83 to 85, wherein the analyte is an amino acid.

90. The method according to any one of claims 83 to 85, wherein the analyte is NADH or NADPH.

91. The method according to any one of claims 83 to 85, wherein the analyte is selected from the group consisting of ascorbic acid, glutamate, dopamine, cholesterol, and alcohol.

92. The method according to any one of claims 83 to 85, wherein the analyte is a drug or a drug metabolite.

93. The method according to any one of claims 83 to 85, wherein the analyte is a protein, nucleic acid molecule, or neurotransmitter molecule.

94. The method according to any one of claims 83 to 85, wherein the analyte is a carbohydrate, lipid, or metabolite.

95. The method according to any one of claims 83 to 85, wherein the analyte is a metabolite.

96. The method of claim 95, wherein the metabolite is selected from the group consisting of lactate, glutamate, glucose, and β-hydroxybutyrate.

97. The method according to any one of claims 83 to 85, wherein the analyte is sugar.

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