Nanoparticle transducer sensors and methods of use thereof

Abstract

To provide nanoparticle transducers and methods of use thereof for detection of analyte concentrations in a fluid.SOLUTION: Nanoparticle transducers can comprise a nanoparticle, such as a Pdot, coupled to an enzyme that catalyzes a reaction with the analyte. The nanoparticle transducers further comprise chromophores that emit fluorescence that varies as a function of the concentration of one of the elements of the reaction. The nanoparticle transducer thus changes fluorescence as the analyte concentration changes, transforming analyte concentration values into fluorescence intensity values. Measurement of these intensity values provides measurement of the analyte concentration. The nanoparticle transducers are biocompatible, allowing use in vivo for monitoring analyte blood concentrations such as blood glucose concentrations.SELECTED DRAWING: None

Description

[Technical field]

[0001] This application claims the benefit of International Patent Application No. PCT / CN2016 / 084986, filed Jun. 6, 2016, the entire contents of which are incorporated by reference. [Background technology]

[0002] Measuring the concentration of fluid components is important in medicine, biomedical research, and biotechnology. For example, it is desirable to monitor the concentration of various molecules, including small molecules, in a patient's blood. Indeed, small molecules play a vital role in all aspects of life sciences, as they are widely involved in various cellular processes, such as cell signaling, enzyme activity, and molecular transport. Advances in biology and medicine have led to the identification of a vast number of small molecules that are related to human diseases. In drug discovery, small molecules constitute a large but fast-growing library of active drugs. Small molecules are also widely used as research tools to probe biological functions and assess emerging therapeutic agents.

[0003] Treatment of chronic diseases such as diabetes may require continuous monitoring of one or more blood constituents, such as glucose, the concentrations of which can be dangerous if too high or too low. Rapid and accurate measurement of blood contents allows for the detection of undesirable fluctuations in such contents as well as more efficient and responsive treatment of the underlying condition.

[0004] However, specific and sensitive detection of small molecules in biological systems is extremely challenging. Many of the previous blood concentration monitoring methods require drawing blood from the patient for external analysis, resulting in poor response speed that depends on patient compliance, poor biocompatibility of device implantation, and insufficient accuracy and sensitivity to blood concentration changes. For example, electrochemical transducers have difficult problems such as response failure and unpredictable signal drift, which require frequent replacement of the implanted electrodes of such transducers. Optical techniques such as Raman technology have also been applied. However, although some unique Raman tags can generate signals in typical Raman silent regions of cells, the Raman signals of small molecules tend to be weak and can be easily masked by strong overlap by dominant biological species. Therefore, it is extremely difficult to detect intact small molecules, such as amino acids, sugars, lipids, neurotransmitters, metabolites, and drug molecules, in vivo in real time.

[0005] The detection of larger molecules, such as proteins or nucleic acid chains, poses even greater challenges due to their large numbers and the need for higher measurement specificity. Real-time in vivo detection of such fluid constituents has so far proven to be largely difficult to achieve. Summary of the Invention [Means for solving the problem]

[0006] Thus, there is a need for improved sensors and methods for monitoring the concentration of fluid constituents with high response speed, specificity, and sensitivity. The present disclosure addresses this need and beyond.

[0007] The present disclosure provides transducer sensors and methods for using such sensors to monitor the concentration of fluid constituents.

[0008] In various aspects, the present disclosure provides a nanoparticle transducer for particle-specific concentration measurement. The nanoparticle transducer includes a nanoparticle including a chromophore and an enzyme. The enzyme is bound to the nanoparticle. The enzyme is configured to catalyze a reaction. The reaction includes a plurality of reaction elements including one or more reactants and one or more products. The chromophore emits an amount of fluorescence determined by the concentration of a reaction element of the plurality of reaction elements.

[0009] Semiconducting polymer dots (Pdots) have been developed as a new class of fluorescent nanoparticles. Compared to organic dyes and fluorescent proteins, Pdots can have several orders of magnitude greater brightness and are more resistant to photobleaching. Compared to quantum dots, for example, Pdots can have an order of magnitude greater brightness. Furthermore, the dimensions of Pdots can be tuned from a few nanometers to tens of nanometers without affecting their spectral properties. Small size Pdots are desirable in situations where labeling with large nanoparticles may perturb the natural behavior of tagged biomolecules. Small Pdots may also be useful in crowded cellular or intracellular spaces, where small Pdots can better penetrate and distribute. Various schemes for controlling the surface properties and bioconjugation of Pdots have been developed, which provide for the use of Pdots for cell surface and subcellular labeling. In addition, Pdot-based ratiometric sensors have been developed, including those for pH, temperature, small molecules such as oxygen and hydrogen peroxide, and ions such as iron and copper.

[0010] In some embodiments, the present disclosure provides a Pdot transducer for measuring glucose concentration. The Pdot transducer includes Pdots that include a chromophore that emits a fluorescence determined by the concentration of oxygen. Glucose oxidase is bound to the Pdots. The glucose oxidase is configured to catalyze a reaction involving a reaction element. The reaction element includes glucose and oxygen as reactants. The chromophore emits fluorescence at a first fluorescent wavelength and a second fluorescent wavelength; the amount of fluorescence at the first fluorescent wavelength varies as a function of the oxygen concentration. Thus, the Pdot transducer includes a fluorescence ratio that is equal to the ratio of the amount of fluorescence at the first fluorescent wavelength to the amount of fluorescence at the second fluorescent wavelength, and the fluorescence ratio is determined by the oxygen concentration. The oxygen concentration is affected by the reaction with glucose catalyzed by the enzyme; thus, the fluorescence ratio varies as a function of the glucose concentration, thereby measuring the glucose concentration.

[0011] In various aspects, the present disclosure provides a method of determining a concentration of an analyte in a fluid, the method comprising contacting the fluid with a nanoparticle transducer as described herein and measuring the fluorescence of the nanoparticle transducer, the method further comprising determining the concentration of the analyte in the fluid based on the measured fluorescence.

[0012] In various aspects, a device is provided for measuring a concentration of a target reactant in blood. The device comprises a plurality of fluorescent nanoparticle transducers. The device further comprises an optical sensor coupled to a processor and a memory. The memory includes instructions that, when executed, configure the processor to measure fluorescence emitted by the plurality of fluorescent nanoparticle transducers using the optical sensor. The fluorescent nanoparticle transducers can be placed in a subdermal location and in contact with blood-borne fluid of the subject. The optical sensor can be adapted to detect fluorescence transmitted through the skin by the plurality of fluorescent nanoparticle transducers when the sensor faces the skin.

[0013] In various aspects, the present disclosure provides a method for measuring the concentration of an analyte in a fluid. Pdots are provided in the fluid. The Pdots include a chromophore that emits an amount of fluorescence determined by the concentration of a fluid component. The analyte undergoes a reaction in the fluid, which reaction changes the concentration of the fluid component. The fluorescence emitted by the chromophore is measured, and the concentration of the analyte in the fluid is based on the measured fluorescence.

[0014] In various aspects, an artificial pancreas is provided with a glucose sensor using nanoparticle transducers that provides a feedback loop to trigger insulin dispensing to maintain blood glucose concentrations within a predetermined range. In some aspects, the artificial pancreas is an implantable device, and in some aspects, the artificial pancreas is configured for transcutaneous optical sensing of glucose concentrations and for insulin injection. The artificial pancreas comprises a glucose-sensitive nanoparticle transducer, an illumination source, and a detector adapted to detect fluorescence at a nanoparticle emission wavelength. The device further comprises a processor for determining blood glucose concentrations from the measured fluorescence and for controlling insulin dispensing from the storage chamber to the patient via an insulin pump. The detector, processor, and pump provide a feedback loop for maintaining blood glucose levels within a predetermined concentration range that may be optionally user adjustable. In some aspects, the device also comprises a memory that stores a log of measured glucose levels as a function of time. In some aspects, the device comprises a transmitter that allows wireless communication with a mobile device and / or over a computer network. In some embodiments, the nanoparticle transducers, optical sensor, and processor are integrated together and placed in contact with a blood-borne fluid of a subject at a subdermal location to detect fluorescence from the nanoparticle transducers. In some embodiments, an integrated device comprising a nanoparticle transducer, a detector, and a processor at a subdermal location can have wireless communication through the skin with a mobile device and / or over a computer network.

[0015] The present disclosure provides sensitive detection and in vivo dynamic monitoring of analytes using optically bright transducers based on nanoparticles such as semiconducting polymer dots (Pdots). Fluorescent Pdots are a bright and versatile nanoparticle platform for biological imaging and sensing applications. In certain embodiments, oxygen-responsive Pdots are conjugated with oxygen-consuming enzymes on the surface for sensitive detection of analytes in the form of small molecule substrates in biological environments. In certain embodiments, the analyte is glucose. Analytical modeling and simulation of certain glucose-sensitive transducers disclosed herein, based on enzyme kinetic constants and Fick's law of oxygen diffusion, shows that different concentrations of small molecules can be well distinguished at typical tissue oxygen concentrations. Experimental results are also described that demonstrate intracellular glucose detection in a mouse model and long-term in vivo dynamic monitoring of blood glucose. Given the large library of oxygen-consuming enzymes, as well as other enzymes known to consume or produce suitable fluid components for which a suitably sensitive fluorescent chromophore can be used, this approach can be generalized for the in vivo detection of a wide range of small molecules, including, for example, amino acids, transmitters (e.g., neurotransmitters), metabolites, and pharmaceuticals. The present invention provides, for example, the following items. (Item 1) 1. A nanoparticle transducer for measuring an analyte concentration, comprising: A nanoparticle comprising a chromophore; an enzyme bound to the nanoparticle and configured to catalyze a reaction comprising a plurality of reaction components; Including, the reaction elements include one or more reactants including the analyte and one or more products, and the amount of fluorescence emitted from the chromophore is determined by the concentration of a reaction element among the plurality of reaction elements; Nanoparticle transducers. (Item 2) 2. The nanoparticle transducer of claim 1, wherein one of the plurality of reactive elements comprises oxygen, and the amount of fluorescence emitted from the chromophore is determined by the concentration of the oxygen. (Item 3) 2. The nanoparticle transducer of any preceding claim, wherein the analyte comprises glucose. (Item 4) 2. The nanoparticle transducer of any preceding item, wherein the enzyme comprises glucose oxidase. (Item 5) The nanoparticle transducer of any of the preceding items, wherein the one or more reactants include glucose and oxygen, and the one or more products include hydrogen peroxide and D-glucono-1,5-lactone. (Item 6) 2. The nanoparticle transducer of any of the preceding items, further comprising catalase. (Item 7) 2. The nanoparticle transducer of any of the preceding items, wherein the amount of fluorescence emitted from the chromophore is determined by the concentration of reactants. (Item 8) 2. The nanoparticle transducer of any of the preceding items, wherein the amount of fluorescence emitted from the chromophore is determined by the concentration of a product. (Item 9) 2. The nanoparticle transducer of any of the preceding items, wherein the nanoparticles comprise Pdots. (Item 10) 2. The nanoparticle transducer of any of the preceding items, wherein the enzyme is covalently bound to the nanoparticle. (Item 11) 2. The nanoparticle transducer of any of the preceding items, wherein the chromophore comprises a semiconducting polymer. (Item 12) 2. The nanoparticle transducer of any of the preceding items, wherein the chromophore comprises a dye, the dye being contained in the nanoparticle. (Item 13) 2. The nanoparticle transducer of any of the preceding items, wherein the chromophore comprises a semiconducting polymer and a dye, the dye and the semiconducting polymer interacting to produce enhanced fluorescence. (Item 14) 2. The nanoparticle transducer of any of the preceding items, wherein the chromophore comprises a blend of two or more semiconducting polymers. (Item 15) The nanoparticle transducer of any of the preceding items, wherein the fluorescence emitted from the chromophore comprises a signal fluorescence wavelength and a control fluorescence wavelength. (Item 16) 16. The nanoparticle transducer of claim 15, wherein the fluorescence emitted from the chromophore comprises a fluorescence ratio 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 the signal fluorescence ratio is determined by the concentration of the reaction element among the plurality of reaction elements. (Item 17) 17. The nanoparticle transducer of claim 16, wherein the fluorescence ratio varies ratiometrically with the concentration of the analyte. (Item 18) A nanoparticle transducer according to any of the preceding items, wherein the fluorescence emitted from the chromophore varies ratiometrically with the concentration of the analyte within a range of analyte concentrations. (Item 19) 2. The nanoparticle transducer of any of the preceding items, wherein the analyte is glucose and the analyte concentration range is within the range of between 0 and about 20 mM of glucose. (Item 20) 20. The nanoparticle transducer of claim 19, wherein the analyte concentration range is within the range of about 3 mM glucose to about 15 mM glucose. (Item 21) 2. The nanoparticle transducer of any of the preceding items, wherein the nanoparticles contain at least 20 percent by weight of chromophore. (Item 22) 2. The nanoparticle transducer of any of the preceding items, wherein the nanoparticles contain at least 50 percent by weight of chromophore. (Item 23) 2. The nanoparticle transducer of any of the preceding items, wherein the nanoparticles contain at least 90 percent by weight of chromophore. (Item 24) 2. The nanoparticle transducer of any preceding item, wherein the nanoparticles comprise about 100 percent by weight of the chromophore. (Item 25) the nanoparticles comprise a second chromophore for fluorescent detection of a second analyte; a second enzyme is bound to the nanoparticle and configured to catalyze a second reaction involving a second plurality of reaction elements; the second plurality of reaction elements includes a second reactant or reactants including the second analyte and a second product or products, and an amount of fluorescence emitted from the second chromophore is determined by a concentration of a second reaction element of the second plurality of reaction elements; the fluorescence of the second chromophore comprises a wavelength that is different from the fluorescence of other chromophores; 2. A nanoparticle transducer according to any of the preceding items. (Item 26) a second nanoparticle comprising a second chromophore; and a second enzyme bound to the nanoparticle and configured to catalyze a second reaction involving a second plurality of reaction elements; Further comprising: the second plurality of reaction elements includes a second reactant or reactants including a second analyte and a second product or products, and an amount of fluorescence emitted from the second chromophore is determined by a concentration of a second reaction element of the second plurality of reaction elements. 2. A nanoparticle transducer according to any of the preceding items. (Item 27) The nanoparticle transducer of any of the preceding items, wherein the enzyme is selected from the group consisting of ascorbic acid oxidase, glutamate oxidase, dopamine beta-hydroxylase, cholesterol oxidase, alcohol oxidase, amine oxidase, and cytochrome P450. (Item 28) 2. The nanoparticle transducer of any of the preceding items, wherein the analyte is selected from the group consisting of ascorbic acid, glutamate, dopamine, cholesterol, and alcohol. (Item 29) The nanoparticle transducer of any of the preceding items, wherein the analyte is an amino acid. (Item 30) The nanoparticle transducer of any of the preceding items, wherein the analyte is a drug. (Item 31) 2. The nanoparticle transducer of any of the preceding items, wherein the analyte is a protein, a nucleic acid molecule, or a transmitter molecule. (Item 32) The nanoparticle transducer of any of the preceding items, wherein the analyte is a carbohydrate, lipid, or metabolite. (Item 33) 2. The nanoparticle transducer of any of the preceding items, wherein the analyte is a sugar. (Item 34) The nanoparticle transducer of any of the preceding items, wherein the analyte concentration is a blood concentration. (Item 35) 36. The nanoparticle transducer according to any one of the preceding items, comprising a plurality of enzymes, each of which catalyzes a respective plurality of reactions, each of which includes a respective plurality of reaction components, the analyte being a reactant in one of the reactions, and the fluorescence emitted from the chromophore being determined by the concentration of at least one of the reaction components in the reactions. 2. The nanoparticle transducer of any of the preceding items, wherein the critical dimension of the nanoparticle transducer is less than about 1000 nm. (Item 37) 2. The nanoparticle transducer of any of the preceding items, wherein the critical dimension of the nanoparticle transducer is less than about 100 nm. (Item 38) 2. The nanoparticle transducer according to any of the preceding items, wherein the critical dimension of the nanoparticle transducer is in the range of about 15 to about 45 nm. (Item 39) An apparatus for measuring the concentration of an analyte in a biological fluid, comprising a plurality of fluorescent nanoparticle transducers according to any of the preceding items, an optical sensor coupled to a processor and a memory; an illumination source configured to irradiate the plurality of fluorescent nanoparticles with light thereby inducing fluorescence therefrom; Further equipped with the memory includes instructions that, when executed, cause the processor to use the optical sensor to measure fluorescence emitted by the plurality of fluorescent nanoparticle transducers. Device. (Item 40) 40. The apparatus of claim 39, wherein the plurality of fluorescent nanoparticle transducers are adapted to be positioned under the patient's skin, and the optical sensor is adapted to detect fluorescence transmitted through the skin by the plurality of fluorescent nanoparticle transducers when facing the skin. (Item 41) 41. The apparatus of claim 39 or 40, wherein the optical sensor is configured to detect an amount of signal fluorescence at a signal fluorescence wavelength and an amount of control fluorescence at a control fluorescence wavelength, and the memory comprises instructions for causing the processor to determine a measured fluorescence ratio based on the measured amounts of signal fluorescence and control fluorescence. (Item 42) 42. The apparatus of claim 41, wherein the memory comprises instructions that cause the processor to determine a concentration of the analyte based on the measured fluorescence ratio. (Item 43) 43. The apparatus according to any of items 39 to 42, wherein the biological fluid is blood, lymph, saliva, tears, interstitial fluid, cerebrospinal fluid, or urine. (Item 44) A contact lens for detecting glucose concentration, comprising a permeable transparent membrane wearable on an eye and containing a plurality of nanoparticle transducers according to any of the preceding items, the nanoparticle transducers being configured to generate fluorescence in response to illumination by a scanner, the amount of fluorescence providing a signal detectable by the scanner for determining the concentration of glucose in a fluid at the surface of the eye. (Item 45) Item 45. The contact lens of item 44, wherein the membrane of the lens is shaped to correct human vision when worn. (Item 46) 1. A device for measuring glucose concentration from sweat of a subject, comprising: A plurality of fluorescent nanoparticle transducers according to any of the preceding items positioned by the device so as to contact the skin of the subject when worn; and an illumination source configured to irradiate the plurality of fluorescent nanoparticles with light to induce fluorescence therefrom; an optical sensor disposed within the device and oriented to detect fluorescence from the plurality of fluorescent nanoparticle transducers; a processor coupled to the optical sensor and configured to determine a concentration of glucose in the sweat based on the fluorescence detected by the optical sensor; A device comprising: (Item 47) Item 47. The device of item 46, wherein the processor is further configured to determine a concentration of glucose in blood based on the concentration of glucose in the sweat. (Item 48) 1. A method for determining an analyte concentration in a fluid, comprising: contacting the fluid with a nanoparticle transducer according to any of the preceding items; measuring the fluorescence of the nanoparticle transducer; determining a concentration of the analyte in the fluid based on the measured fluorescence; A method comprising: (Item 49) 49. The method of claim 48, wherein the determining step comprises comparing the measured fluorescence to a calibration curve of the nanoparticle transducer to determine the analyte concentration in the fluid. (Item 50) 50. The method of claim 48 or 49, wherein the fluid is blood, sweat, or tears. (Item 51) 51. The method of any of items 48 to 50, wherein the measuring step comprises measuring a plurality of fluorescence wavelengths, and the determining step is based on a ratio of the measured wavelengths. (Item 52) 1. A method for determining a concentration of an analyte in a fluid, comprising: contacting the fluid with Pdots, the Pdots comprising chromophores that fluoresce in an amount determined by the concentration of fluid components; measuring the fluorescence emitted by the chromophore; determining a concentration of the analyte in the fluid based on the fluorescence; Including, The method, wherein the analyte undergoes a reaction in the fluid, the reaction changing the concentration of the fluid component. (Item 53) 53. The method of claim 52, wherein the fluorescence emitted from the chromophore comprises 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, the fluorescence ratio being determined by the concentration of the fluid component. (Item 54) 54. The method of claim 53, wherein the determining of the concentration comprises measuring fluorescence at the signal fluorescence wavelength and a control fluorescence wavelength, determining a measured fluorescence ratio based on the measurements, and determining the concentration of the analyte based on the measured fluorescence ratio. (Item 55) 55. The method of any of items 52 to 54, wherein the analyte is ascorbic acid and the fluid constituent is oxygen. (Item 56) 56. The method of claim 55, wherein the determined concentration is between 1 mM and 20 mM ascorbic acid. (Item 57) 57. The method of any of items 52 to 56, wherein the fluid is blood, sweat, or tears.

[0016] This Summary is provided to introduce selected concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. Incorporation by Reference

[0017] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0018] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [Brief description of the drawings]

[0019] [Figure 1-1]1A-1H illustrate simulated O2 depletion kinetics and distribution profiles for an exemplary nanoparticle transducer for glucose detection using an O2-modulated signal from a glucose oxidase-catalyzed reaction. FIG. 1A and FIG. 1B illustrate glucose-induced O2 depletion kinetics in closed and open cuvettes, respectively. FIG. 1C illustrates a 3-D plot of temporal and spatial oxygen distribution in a system containing 5 nM GOx and 20 mM glucose along the Z-axis in an open cuvette configuration. FIG. 1D illustrates oxygen distribution profiles in open cuvettes with different glucose concentrations at 500 seconds. FIG. 1E illustrates O2 depletion kinetics in a closed tissue oxygen environment. FIG. 1F illustrates O2 depletion kinetics in tissue with oxygen diffusion. FIG. 1G illustrates 2-D mapping of the circular configuration with oxygen diffusion at 20 seconds. FIG. 1H illustrates the sensitivity of oxygen depletion for quantification of glucose with and without oxygen diffusion at the 20 second time point. [Figure 1-2] Same as above.

[0020] [Figure 2-1] Figures 2A-D illustrate the preparation and characterization of nanoparticle transducers containing Pdot-GOx assemblies. Figure 2A is a schematic representation of the formation of Pdot-GOx bioconjugates for in vivo glucose monitoring. Figure 2B illustrates the hydrodynamic diameters of bare Pdots and Pdot-GOx. Figure 2C illustrates the zeta potentials of carboxyl Pdots and Pdot-GOx. Figure 2D illustrates representative TEM images of carboxyl Pdots (left) and Pdot-GOx (right). [Figure 2-2] Same as above.

[0021] [Diagram 3]Figures 3A-3D illustrate further properties of nanoparticle transducers such as Pdot transducers. Figure 3A illustrates the colloidal stability of Pdot-GOx transducers over 30 days. Figure 3B shows the UV-visible (vis) absorption and photoluminescence spectra of Pdot-GOx sensors. Figure 3C illustrates the spectral overlap between the fluorescence emission of polyfluorene nanoparticles (PDHF) and the absorption of a phosphorescent dye (PtOEP). Figure 3D illustrates the emission spectra of undoped PDHF Pdots and PtOEP-doped Pdots with an excitation wavelength of 380 nm.

[0022] [Figure 4] Figures 4A-D illustrate the spectroscopic properties of the Pdot-GOx nanoparticle transducer. Figure 4A shows the emission spectrum of the Pdot-GOx transducer at different glucose concentrations. Figure 4B shows the ratiometric calibration plot (I648 / I428) of the Pdot-GOx transducer as a function of glucose concentration. Figure 4C illustrates the response curve of Pdot-GOx to glucose in aqueous suspension. Figure 4D illustrates the selectivity of the Pdot-GOx transducer for glucose over potentially interfering carbohydrates.

[0023] [Diagram 5] Figures 5A and 5B illustrate the use of enzyme-densely coated nanoparticles for sensing at low concentrations. Figure 5A illustrates the emission spectra of Pdots densely coated with GOx at various glucose concentrations. Figure 5B shows the ratiometric calibration plot (I648 / I428) of Pdot-GOx in the low analytical range.

[0024] [Figure 6]Figures 6A-6D illustrate the cell viability of HeLa cells treated with various materials, including the Pdot-GOx transducer. Figures 6A-6C show the cell viability at 24 hours of cells treated with varying concentrations of the Pdot-GOx transducer, GOx, and catalase, respectively. Figure 6D shows cells treated with both the Pdot-GOx transducer and catalase at a ratio of about 1:6.

[0025] [Figure 7] Figures 7A-C illustrate intracellular glucose sensing in HeLa cells: Figure 7A shows HeLa cells as a control without Pdot-GOx incubation, Figure 7B shows cells incubated in sugar-free medium with Pdot-GOx nanoparticles for 24 h, and Figure 7C shows cells incubated with Pdot-GOx for 24 h and supplemented with glucose for 4 h.

[0026] [Figure 8] Figure 8A illustrates the fluorescence imaging of a mouse with a Pdot-GOx transducer subcutaneously. Figure 8B illustrates the fluorescence imaging of a mouse injected with Pdot-GOx transducer at different concentrations at three sites. Figure 8C shows the fluorescence intensity of the three different sites injected with Pdot-GOx.

[0027] [Figure 9] FIG. 9 shows images of a mouse subcutaneously injected with a nanoparticle transducer under room light (top) and UV (bottom).

[0028] [Figure 10-1]Figures 10A-D illustrate continuous glucose monitoring in vivo in live mice. Figure 10A shows in vivo fluorescence imaging of fluctuating glucose concentrations in live mice injected with Pdot-GOx. Figure 10B shows in vivo fluorescence imaging of Pdot-GOx in a live mouse from a glucose- and insulin-free control group. Figure 10C shows the fluorescence intensity of Pdot-GOx transducers injected into live mice and glucose concentrations measured from cut tail blood samples. Figure 10D shows the fluorescence response of Pdot-GOx to blood glucose concentrations in a glucose- and insulin-free control group. [Figure 10-2] Same as above.

[0029] [Figure 11-1] Figures 11A-11F illustrate long-term glucose monitoring and in vivo distribution. Figure 11A shows fluorescence images of organs and skin tissues excised from mice subcutaneously injected with Pdot-GOx transducers (bottom) or sterile phosphate-buffered saline (top). Figure 11B shows quantification of the fluorescence intensity of Pdot-GOx distributed in organs and tissues taken from injected and control mice (*P<0.05). Figures 11C-11E show the fluorescence intensity of injected Pdot-GOx in response to blood glucose concentration 7, 15, and 30 days after subcutaneous administration, respectively. Figure 11F illustrates hematoxylin and eosin staining of organ sections excised from Pdot-GOx-injected mice (right) and PBS-injected control group (left). [Figure 11-2] Same as above.

[0030] [Figure 12] FIG. 12 shows histochemical analysis of tissue sections from mice 30 days after nanoparticle transducer injection.

[0031] [Figure 13]13A-C show fluorescent imaging of live mice at 7 days (FIG. 13A), 15 days (FIG. 13B), and 30 days (FIG. 13C) after injection of the nanoparticle transducer.

[0032] [Figure 14] Figure 14A shows in vivo glucose measurement using a miniature fluorometer. Figure 14A shows a photograph of a mouse subcutaneously injected with Pdot-GOx under UV light (385 nm). Figure 14B illustrates the kinetic changes in the fluorescence emission spectrum of a Pdot-GOx transducer implanted in a live mouse, excited at 385 nm. Figure 14C shows the change in the intensity ratio (480 nm to 650 nm) of a Pdot-GOx transducer implanted in a live mouse after glucose and insulin injection.

[0033] [Figure 15-1] Figures 15A and 15B show the fluorescence emission from a nanoparticle transducer with chromophores including do-PFO, 10% PdOEP, and 10% PSMA in a Pdot-GOx transducer for the detection of glucose. Figure 15A shows the emission spectrum for multiple glucose concentrations. Figure 15B shows a calibration plot of the transducer detecting glucose for a concentration range of 0 to approximately 20 mM, showing a ratiometric response curve across the range. Figure 15C shows in vivo mouse response data showing emission curves that substantially overlap the measured glucose concentrations. Figure 15D shows images with time data for glucose-fed mice (top image) and a control group not fed glucose (bottom image). [Figure 15-2] Same as above.

[0034] [Figure 16]Figures 16A and 16B show the fluorescence emission from a nanoparticle transducer with chromophores including PSMA, 1% PdOEP, and 0.1% coumarin 1 in a Pdot-GOx transducer for the detection of glucose. Figure 16A shows the emission spectrum for multiple glucose concentrations. Figure 16B shows a calibration plot of the transducer for detecting glucose for a concentration range of 0 to about 20 mM, showing the ratiometric response curve.

[0035] [Figure 17] Figures 17A and 17B show the fluorescence emission from a nanoparticle transducer with chromophores including PSMA, 1% PtOEPK, and 0.1% Nile Red in a Pdot-GOx transducer for the detection of glucose. Figure 17A shows the emission spectrum for multiple glucose concentrations. Figure 17B shows a calibration plot of the transducer for detecting glucose for a concentration range of 0 to about 20 mM, showing the ratiometric response curve.

[0036] [Figure 18] 18A and 18B show the fluorescence emission from a nanoparticle transducer having a chromophore containing PDHF, 10% PtOEP, and 10% PSMA in a nanoparticle sensor for the detection of ascorbic acid. FIG. 18A shows the emission spectrum for multiple concentrations of ascorbic acid. FIG. 18B shows a calibration plot of the transducer detecting ascorbic acid for a concentration range of about 2 to about 20 mM, showing the ratiometric response curve over that range.

[0037] [Figure 19]Figures 19A and 19B illustrate continuous in vivo monitoring of ascorbic acid in live mice using nanoparticle sensors. Figure 19A illustrates the fluorescence intensity of nanoparticle sensors injected into live mice administered different concentrations of ascorbic acid. Figure 19B shows in vivo fluorescence imaging of varying ascorbic acid concentrations in live mice injected with Pdot sensors.

[0038] [Figure 20] Figures 20A and 20B illustrate the monitoring of ascorbic acid blood levels by a miniaturized optical detection system. Figure 20A shows the kinetic changes in the fluorescence emission spectrum of Pdots injected into a live mouse with respect to the blood concentration of ascorbic acid, excited at 385 nm. Figure 20B shows the response of fluorescence intensity to the blood concentration of ascorbic acid as a function of time after intravenous administration of ascorbic acid.

[0039] [Figure 21] FIG. 21 shows the spectral response of the H2O2-based nanoparticle transducer containing nanoparticle-GOx assemblies. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] The present disclosure generally relates to devices, compositions, systems, and methods for monitoring the concentration of an analyte in a fluid using nanoparticle transducers. In many embodiments, the analyte is a molecule in a fluid. In many embodiments, the fluid is blood; for example, the compositions, systems, and methods disclosed herein are useful for monitoring the concentration of one or more selected molecules in the blood of a subject. In many embodiments, the fluid is tears; for example, the compositions, systems, and methods disclosed herein are useful for monitoring the concentration of one or more selected molecules in the tears of a subject. In many embodiments, the fluid is sweat; for example, the compositions, systems, and methods disclosed herein are useful for monitoring the concentration of one or more selected molecules in the sweat of a subject. In many embodiments, the fluid is saliva; for example, the compositions, systems, and methods disclosed herein are useful for monitoring the concentration of one or more selected molecules in the saliva of a subject. In many embodiments, the fluid is lymph; for example, the compositions, systems, and methods disclosed herein are useful for monitoring the concentration of one or more selected molecules in the lymph of a subject. In many embodiments, the fluid is cerebrospinal fluid; for example, the compositions, systems, and methods disclosed herein are useful for monitoring the concentration of one or more selected molecules in the cerebrospinal fluid of a subject. In many embodiments, the fluid is urine; for example, the compositions, systems, and methods disclosed herein are useful for monitoring the concentration of one or more selected molecules in the urine of a subject.

[0041] As used herein, the term "polymer dot" or "Pdot" refers to a particle structure comprising one or more semiconducting polymers that are crushed to form stable submicron-sized particles, e.g., nanoparticles. In some aspects, the polymer dots are highly fluorescent nanoparticles with tunable emission, e.g., from the visible to the near-IR range. The polymer dots can comprise, for example, chromophore polymers that can absorb light and then emit light by fluorescence. In some embodiments, the polymer dots comprise at least one condensation polymer, e.g., a semiconducting polymer. In the case of polymer dots having more than one condensation polymer (e.g., more than one semiconducting polymer), the condensation polymers can be the same type of polymer or different types of polymers. For example, the Pdots can comprise both semiconducting and non-semiconducting polymers.

[0042] A nanoparticle transducer for monitoring a selected analyte may be assembled from an appropriate selected enzyme, nanoparticle, and chromophore. The enzyme may be selected to catalyze a reaction involving the analyte such that the concentration of the analyte may affect the rate of the reaction. The reaction may involve multiple reaction components, including reactants and products. The enzyme may be selected such that each reactant of the enzyme-catalyzed reaction is present in the fluid being analyzed. The chromophore may be selected such that the fluorescence of the chromophore is determined by the concentration of a reactant or product of the enzyme-catalyzed reaction. The nanoparticle may be selected such that both the enzyme and the chromophore are incorporated into the nanoparticle or are conjugated to the nanoparticle. For example, the nanoparticle may be a Pdot such that the enzyme can be covalently attached to the Pdot and the chromophore can be incorporated into and / or covalently attached to the Pdot. In some cases, the chromophore may constitute all or substantially all of the nanoparticle; for example, in some cases, the Pdot may be entirely or substantially entirely composed of one or more chromophores.

[0043] In many embodiments, a nanoparticle transducer can be created to detect a given analyte by selecting enzymes, chromophores, and nanoparticles from a set of possible enzymes, chromophores, and nanoparticles as follows: From the set of enzymes, select one that catalyzes a reaction in which the analyte is a reactant. For each such reaction, identify other reaction elements whose concentration changes as a result of the reaction occurring (e.g., each time the reaction occurs, the concentration of the reactant decreases and the concentration of the product increases (for a reversible reaction, reversal of the reaction causes the opposite effect)). From these reaction elements, for each enzyme, identify a corresponding chromophore from the set of chromophores whose fluorescence changes in response to a change in the concentration of one of the reaction products. If none of the chromophores match, eliminate the enzyme. From these remaining enzyme / chromophore pairs, select one that applies and select nanoparticles, such as Pdots, to which each can be bound and / or incorporated to select elements to construct the nanoparticle transducer. A second chromophore that emits light at a different wavelength and does not change its intensity in response to any of the reaction elements can be selected from the list of chromophores to serve as a control chromophore. Alternatively, if the initially selected chromophore fluoresces both at a wavelength that changes intensity in response to reactant or product concentration and at a different wavelength that does not change intensity, that single chromophore can serve as its own control.

[0044] In many embodiments, the nanoparticle transducers described herein include an enzyme that catalyzes a reaction involving an analyte. The reaction has reaction components including reactants and products, one of which is the analyte. The nanoparticle includes a chromophore that emits fluorescence at one or more wavelengths in response to irradiation with a beam of light. The amount of fluorescence at at least one of the wavelengths depends on the concentration of the reactant or product molecules other than the analyte. The enzyme and the chromophore of the nanoparticle are in close proximity; thus, as the reaction catalyzed by the enzyme consumes reactants and produces products, the respective concentrations of the reactants and products change, the reactant concentration decreasing and the product concentration increasing. The presence of the analyte at a high concentration causes the reaction to proceed more quickly than if it were at a low concentration, so the presence of the analyte causes a relatively high concentration of the product and a relatively low concentration of the reactant. Since the amount of fluorescence of the chromophore depends on one of the reactants or products, the amount of fluorescence from the chromophore affects the amount of fluorescence of the chromophore due to the change in the concentration of the other reaction component as the reaction occurs. Thus, the enzyme and nanoparticle chromophore together act as a transducer to convert variations in analyte concentration into variations in fluorescence. In some embodiments, the fluorescence intensity of a single wavelength emission of the transducer is used to determine the analyte concentration. In some embodiments, the fluorescence intensity ratio of a dual wavelength emission of the transducer is used to determine the analyte concentration. This fluorescence can be readily measured in a wavelength-selective manner to determine the analyte concentration from the optical sensor signal.

[0045] In some embodiments, the nanoparticles include a semiconducting polymer that fluoresces at one or more wavelengths in response to irradiation with light. The amount of fluorescence at at least one of the wavelengths depends on the concentration of reactant or product molecules other than the analyte. In some cases, the nanoparticles include a semiconducting polymer and a dye that fluoresces at one or more wavelengths. The dye can be physically doped or chemically attached to the semiconducting polymer to form the nanoparticles. The semiconducting polymer can have an energy transfer to the dye to enhance or amplify the fluorescence intensity of the dye.

[0046] In many embodiments, the fluids described herein are bodily fluids of a subject, such as blood, sweat, tears, lymphatic fluid, cerebrospinal fluid, urine, saliva, or other fluids within or secreted by bodily tissues. The subject may be an animal, and in many embodiments, the subject is a human.

[0047] Various aspects of the present disclosure provide chromophores with advantageous characteristics for efficient and accurate measurement of analyte concentrations using the nanoparticle transducers provided herein. Examples of such characteristics include, but are not limited to, (1) high brightness allowing easy detection and recovery of transducer signals, (2) high sensitivity to reaction components of enzyme-catalyzed reactions, (3) high absorption cross-sections such that nanoparticle transducer fluorescence can be easily induced without the need for application of intense energy, (4) good stability (e.g., thermal stability) such that the nanoparticle transducers can remain active in vivo for extended periods of time, (5) wavelengths that can be detected and distinguished, for example, transdermally, in some cases, and / or (6) good fatigue resistance to reduce degradation when used for continuous analyte monitoring. In certain aspects, the chromophores of the nanoparticle transducers described in the present disclosure include some or all of these characteristics.

[0048] For example, the present disclosure provides, in some embodiments, nanoparticle transducers that exhibit a signal fluorescent emission intensity at a peak emission wavelength that varies as a function of the concentration of a fluid component. The nanoparticle transducers may also include chromophores that have different control emission intensities at the peak emission wavelength that do not vary substantially in response to the concentration of a fluid component. In certain embodiments, the peak emission wavelength is between about 200 nanometers to about 300 nanometers, about 250 nanometers to about 350 nanometers, about 300 nanometers to about 400 nanometers, about 350 nanometers to about 450 nanometers, about 400 nanometers to about 500 nanometers, about 450 nanometers to about 550 nanometers, about 500 nanometers to about 600 nanometers, about 550 nanometers to about 650 nanometers, about 600 nanometers to about 700 nanometers, about 650 nanometers to about 800 nanometers, about 800 nanometers to about 900 nanometers, about 900 nanometers to about 1000 nanometers, about 1000 nanometers to about 1500 nanometers, about 1000 nanometers to about 2000 nanometers, about 1000 nanometers to about 2000 nanometers, about 1000 nanometers to about 3000 nanometers, about 1000 nanometers to about 4000 nanometers, about 1000 nanometers to about 5000 nanometers, about 1000 nanometers to about 6000 nanometers, about 1000 nanometers to about 7000 nanometers, about 1000 nanometers to about 8000 nanometers, about 1000 nanometers to about 9000 nanometers, to about 750 nanometers, about 700 nanometers to about 800 nanometers, about 750 nanometers to about 850 nanometers, about 800 nanometers to about 900 nanometers, about 850 nanometers to about 950 nanometers, about 900 nanometers to about 1000 nanometers, about 950 nanometers to about 1050 nanometers, about 1000 nanometers to about 1100 nanometers, about 1150 nanometers to about 1250 nanometers, or about 1200 nanometers to about 1300 nanometers.

[0049] As another example, some embodiments of the present disclosure provide nanoparticle transducers that exhibit sufficient stability for long-term in vivo analyte concentration monitoring, e.g., the nanoparticle transducers are capable of stably detecting analyte concentrations over long periods of time without substantial degradation. In various embodiments, the stability of the nanoparticle transducers is advantageous in ensuring that the transducers can be used in vivo for long periods of time without the need for replacement. In some embodiments, a population of nanoparticle transducers is considered to be "stable" if at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.5%, or at least 99.95% of the nanoparticle transducers in the population retain their ability to modulate fluorescence in response to fluctuations in analyte concentration over a specified period of time. In some embodiments, a nanoparticle transducer is considered to be "stable" if the emission intensity of the nanoparticle transducers retains their ability to measure fluctuations in analyte concentration over a specified period of time. In some embodiments, a nanoparticle transducer is considered to be "stable" if the intensity ratio of the two emission peaks retains the ability to measure fluctuations in analyte concentration over a specified period of time, even though the absolute emission intensity may decrease significantly. In some embodiments, a nanoparticle transducer is considered to be stable if the time constant (e.g., the time it takes for the fluorescence signal intensity to decay to 1 / e) 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 some embodiments, a nanoparticle transducer maintains sufficient signal intensity throughout a specified period of time to ensure that analyte detection can be performed.

[0050] In some embodiments of the present disclosure, the emission spectrum of the chromophore is selected or designed to exhibit narrowband emission characteristics at the peak emission wavelength to reduce or minimize overlap with other emission sources. For example, in certain embodiments, the chromophore has a peak emission bandwidth (e.g., the half-width (FWHM) of the emission peak) of about 5 nanometers, about 10 nanometers, about 15 nanometers, about 20 nanometers, about 25 nanometers, about 30 nanometers, about 35 nanometers, about 40 nanometers, about 45 nanometers, about 50 nanometers, about 60 nanometers, about 70 nanometers, about 80 nanometers, about 90 nanometers, or about 100 nanometers or less. Chromophore composition

[0051] Various types of chromophores are suitable for use with the methods and systems of the present disclosure, including but not limited to dyes, dyes, proteins, polymers, beads, particles, or combinations thereof. In some embodiments, the nanoparticle transducer comprises one or more chromophores (e.g., fluorophores). The chromophores described herein can be used to generate nanoparticle transducers according to various mechanisms. In some embodiments, the nanoparticle comprises a semiconducting polymer that fluoresces at one or more wavelengths in response to irradiation with light. The amount of fluorescence of the semiconducting polymer can depend on the concentration of reactant or product molecules. In some embodiments, the nanoparticle comprises a semiconducting polymer and a dye that fluoresces at one or more wavelengths. The amount of fluorescence of the dye depends on the concentration of reactant or product molecules. The dye can be physically doped or chemically attached to the semiconducting polymer to form the nanoparticle. The chromophore polymer can have energy transfer to the dye to enhance or amplify the fluorescence intensity of the dye.

[0052] In some embodiments, the nanoparticle transducer comprises at least one chromophore semiconducting polymer particle (also known as "polymer dot" or "Pdot"), which comprises one or more polymers (e.g., semiconducting polymer, non-semiconducting polymer, or combinations thereof) compressed into a stable submicron-sized particle. Semiconducting polymer particles are advantageous in certain embodiments for several reasons, compared to other types of chromophores: (1) semiconducting polymer particles are extremely bright, up to 30 times brighter than quantum dots, and are very photostable; (2) semiconducting polymer particles have fast photon emission rates, often with lifetimes of less than a nanosecond, making them well suited for high-speed optical detection; (3) semiconducting polymer particles have good biocompatibility and are not composed of cytotoxic heavy metals like quantum dots; (4) semiconducting polymer particles exhibit amplified energy transfer, so their fluorescence emission can be well modulated, for example, by photochromic molecules via energy transfer.

[0053] Chromophore polymer particles of various structures and compositions are applicable to the embodiments presented herein. The chromophore polymer particles provided herein are composed of a single polymer or alternatively comprise a blend of polymers. In certain embodiments, crushing, precipitation, and / or condensation of one or more polymers are performed to form a polymer matrix. In some embodiments, the properties of the chromophore polymer particles depend on the structure and / or properties of the constituent polymers. Thus, in certain embodiments, specific properties are obtained by changing the polymer backbone (main chain), side chains, terminal units, and substituents. In some embodiments, the optical properties of the chromophore polymer particles are adjusted by changing the structure of the polymer backbone (main chain).

[0054] In some embodiments, the chromophore polymer particles provided herein include one or more chromophores, also referred to herein as chromophore units. In certain embodiments, the chromophore absorbs light at a certain wavelength, for example, from the UV region to the near infrared region, and may or may not be emissive. In some embodiments, the chromophore units include, but are not limited to, structural units with delocalized pi-electrons, units of small organic dye molecules, and / or units of metal complexes. In various embodiments, the chromophore is part of the polymer matrix or is incorporated into the polymer matrix, for example, by blending, crosslinking, etc. In some embodiments, the chromophore polymer is a semiconducting polymer.

[0055] In certain embodiments, the chromophore polymer particles of the present disclosure include one or more chromophore polymers. In some embodiments, the chromophore polymer includes at least a portion that absorbs light at a certain wavelength, for example, ranging from the UV spectrum to the near infrared spectrum. The chromophore polymer according to the present disclosure may or may not be luminescent. In some embodiments, the chromophore polymer includes one or more chromophore units. Examples of chromophore polymers include, but are not limited to, polymers that include structural units with delocalized pi electrons (e.g., semiconducting polymers), polymers that include units of small organic dye molecules, polymers that include units of metal complexes, and polymers that include units of any combination thereof. In some embodiments, the chromophore units are incorporated into the polymer backbone. In some embodiments, the chromophore units are covalently attached to the side chains or terminal units of the polymer. In certain embodiments, the chromophore polymers are made using standard synthetic methods that are generally known in the art.

[0056] Various types of chromophoric polymer particles are suitable for use as a platform for the optical marking approach of the present disclosure. Chromophoric polymer particles can take various configurations, including but not limited to monolithic polymer particles with uniform and homogeneous composition, or polymer particles with distinct core and cap structures. Chromophoric polymer particles provided herein can be formed by any method known in the art, including but not limited to methods that rely on precipitation, methods that rely on the formation of emulsions (e.g., miniemulsions or microemulsions), and methods that rely on condensation. Examples of chromophore polymer particles suitable for use in the technology described herein can be found, for example, in PCT Application Nos. PCT / US2010 / 056079, PCT / US2012 / 071767, PCT / US2011 / 056768, PCT / US2013 / 024300, and PCT / US2013 / 063917, and U.S. Patent Publication No. 2013 / 0266957, each of which is incorporated herein by reference.

[0057] In some embodiments, the chromophore polymer particles are nanoparticles. In some embodiments, the size of the nanoparticles provided herein is defined in terms of a "critical dimension" that refers to the smallest dimension of the nanoparticle. Some nanoparticles are approximately spherical in shape, so that the critical dimension is the diameter of the spherical particle. In some embodiments, certain nanoparticles, such as nanospheres and nanocubes, are completely nanoscopic in size. In some embodiments, not all dimensions of the nanoparticles are nanoscale. For example, a nanocylinder may have a nanoscale diameter but a microscale length. A wide variety of nanoparticle shapes are applicable to the embodiments described herein, including, but not limited to, spheres, cylinders, ellipsoids, polyhedrons, prisms, rods, wires, or combinations thereof. As will be appreciated by those skilled in the art, in certain embodiments, the shape of the nanoparticle contributes to the optical properties (e.g., a nanorod may have different optical properties than a nanosphere).

[0058] In some embodiments, the typical size of the chromophore polymer particles is less than 100 nanometers. In certain embodiments, colloidal polymer nanoparticles are composed of a lyophobic polymer interior. Optionally, polyelectrolytes can be formed into nanoparticles. In certain embodiments, the chromophore polymer particles include at least one chromophore polymer formed into a stable particle. The particle size can vary, for example, from 5 nanometers to 500 nanometers. In some embodiments, the critical dimension (e.g., diameter) of the particle 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, less than 40 nanometers. In some embodiments, the critical dimension of the particle is less than 30 nanometers, less than 20 nanometers, or less than 10 nanometers.

[0059] In some embodiments, the chromophoric polymer particles described herein comprise a polymer matrix formed from one or more chromophoric polymers.Any suitable number and combination of chromophoric polymer types can be incorporated into the chromophoric polymer particles described herein, for example, one or more chromophoric polymers, two or more chromophoric polymers, three or more chromophoric polymers, four or more chromophoric polymers, five or more chromophoric polymers, six or more chromophoric polymers, seven or more chromophoric polymers, eight or more chromophoric polymers, nine or more chromophoric polymers, ten or more chromophoric polymers, fifty or more chromophoric polymers, or one hundred or more chromophoric polymers. The mass concentration of the chromophore polymer compared to the total chromophore polymer particle mass may vary from 1% to 99%, 10% to 99%, 20% to 99%, 30% to 99%, 40% to 99%, or 50% to 99%.

[0060] Chromophoric polymers of various types and compositions are applicable for use in accordance with the embodiments of the present disclosure. The chromophoric polymer may be a homopolymer or a heteropolymer. In various embodiments, the chromophoric polymer is a semiconducting polymer, a non-semiconducting polymer, or a combination thereof. For example, several semiconducting polymers are suitable for use in chromophoric polymer particles according to the present disclosure. Examples of semiconducting polymers include polyfluorene-based polymers, including but not limited to poly(9,9-dihexylfluorenyl-2,7-diyl) (PDHF)-based and poly(9,9-dioctylfluorenyl-2,7-diyl) (PFO)-based; poly[{9,9-dioctyl-2,7-divinylene-fluorenylene}-alt-co-{2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene ... poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1,3}-thiadiazole)] (PFBT) system, poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(4,7-di-2-thienyl-2,1,3-benzothiadiazole)] (PFTBT) system, and poly[(9,9-dioctylfluorenyl-2,7-diyl)- co-(4,7-di-2-thienyl-2,1,3-benzothiadiazole)] (PF-0.1TBT) based semiconducting polymers; phenylene vinylene polymers, including but not limited to poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene vinylene] (MEH-PPV) based semiconducting polymers and poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-(1-cyanovinylene-1,4-phenylene)] (CN-PPV) based semiconducting polymers; phenylene ethynylene based polymers, including but not limited to poly(2,5-di(3',7'-dimethyloctyl)phenylene-1,4-ethynylene (PPE) based semiconducting polymers; BODIPY based semiconducting polymers; squaraine based semiconducting polymers; or combinations thereof.

[0061] A wide variety of chromophore polymer structures are suitable for use in accordance with various aspects of the present disclosure. In some aspects, the chromophore polymer is a linear polymer. In other aspects, the chromophore polymer is a branched polymer. In certain aspects, the chromophore polymer is a dendrimer. In certain aspects, the chromophore polymer is a brush polymer. In certain aspects, the chromophore polymer is a star polymer.

[0062] In some embodiments, the chromophore polymer particles described herein contain a polystyrene-based comb polymer. Non-limiting examples of polystyrene-based comb polymers include polystyrene-grafted acrylic acid, polystyrene-grafted ethylene oxide, polystyrene-grafted butyl alcohol, and the like. In some embodiments, the chromophore polymer particles described herein contain a poly(methyl methacrylate)-based comb polymer. Non-limiting examples of poly(methyl methacrylate)-based comb polymers include poly(methyl methacrylate)-grafted acrylic acid, poly(methyl methacrylate)-grafted ethylene oxide, and the like. In some embodiments, the chromophore polymer particles described herein contain a comb polymer comprising a carboxyl group, an amine group, a thiol group, an ester group, a succinimidyl ester group, an azide group, an alkyne group, a cyclooctyne group, or a phosphine group.

[0063] In some embodiments, the chromophore polymer particles described herein contain polymers having terminal monomer units functionalized with, for example, carboxyl, amine, thiol, ester, succinimidyl 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, polydienes, polyphenylenes, polyethylene, poly(ethylene glycol), polylactides, polystyrenes, polysiloxanes, poly(vinylpyridine), poly(vinylpyrrolidone), polyurethanes, block copolymers thereof, random copolymers thereof, or alternating copolymers thereof, and the like.

[0064] In some embodiments, the chromophore polymer particles described herein are copolymers having one or more monomer units functionalized, such as poly((meth)acrylic acid)-based copolymers, 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(methacrylic acid-b-neopentyl methacrylate), poly(methyl methacrylate-b-acrylic acid), poly(methyl methacrylate-b-methacrylic acid), poly(methyl methacrylate-bN,N-dimethylacrylamide), poly(methyl methacrylate-b-sodium acrylate), poly(methyl methacrylate-b-methacrylic acid), poly(methyl methacrylate-bN,N-dimethylacrylamide), poly(methyl methacrylate-b-sodium acrylate), poly(methyl methacrylate-b-methacrylic acid), sodium), poly(neopentyl methacrylate-b-methacrylic acid), poly(t-butyl methacrylate-b-ethylene oxide), poly(2-acrylamido-2-methylpropanesulfonic acid-b-acrylic acid); polydiene copolymers such as poly(butadiene(1,2-addition)-b-ethylene oxide), poly(butadiene(1,2-addition)-b-methylacrylic acid, poly(butadiene(1,4-addition)-b-acrylic acid), poly(butadiene(1,4-addition)-b-ethylene oxide, poly(butadiene(1,4-addition)-b-sodium acrylate), poly(butadiene(1,4-addition)-bN-methyl 4-vinylpyridinium iodide), poly(isoprene-b-ethylene oxide), poly(isoprene-b-ethylene oxide), and poly(isoprene-bN-methyl 2-vinylpyridinium iodide);Poly(ethylene oxide)-based copolymers, such as poly(ethylene oxide-b-acrylic acid), poly(ethylene oxide-b-acrylamide), poly(ethylene oxide-b-butylene oxide), 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-b N,N-dimethylaminoethyl methacrylate), poly(ethylene oxide-b-propylene oxide), poly(ethylene oxide-b-t-butyl acrylate), poly(ethylene oxide-b-t-butyl methacrylate), poly(ethylene oxide-b-tetrahydrofurfuryl methacrylate), poly(ethylene oxide-b-2-ethyl oxazoline), poly(ethylene oxide-b-2-hydroxyethyl methacrylate), poly(ethylene oxide-b-2-methyl oxazoline); polyisobutylene-based copolymers, such as poly(isobutylene-b-acrylic acid), poly(isobutylene-b-ethylene oxide), poly(isobutylene-b-methacrylic acid);Polystyrene-based copolymers such as poly(styrene-b-acrylamide), poly(styrene-b-acrylic acid), poly(styrene-b-cesium acrylate), poly(styrene-b-ethylene oxide), poly(styrene-b-ethylene oxide) acid cleavable at block junctions, poly(styrene-b-methacrylic acid), poly(4-styrenesulfonic acid-b-ethylene oxide), poly(styrenesulfonic acid-b-methylbutylene), poly(styrene-bN,N-dimethylacrylamide), poly(styrene-bN-isopropylacrylamide), poly(styrene-bN-methyl 2-vinylpyridinium iodide), poly(styrene-bN-methyl-4-vinylpyridinium iodide), poly(styrene-b-propylacrylic acid), poly(styrene-b-sodium acrylate), poly(styrene-b-sodium methacrylate), poly(p-chloromethylstyrene-b-acrylamide), poly(p-chloromethylstyrene-b- ... poly(styrene-b-acrylamide), poly(styrene-co-p-chloromethylstyrene-b-acrylamide), poly(styrene-co-p-chloromethylstyrene-b-acrylic acid), poly(styrene-b-methylbutylene-co-isoprene sulfonate); polysiloxane-based copolymers, such as poly(dimethylsiloxane-b-acrylic acid), poly(dimethylsiloxane-b-ethylene oxide), poly(dimethylsiloxane-b-methacrylic acid); poly(ferrocenyldimethylsilane)-based copolymers, such as poly(ferrocenyldimethylsilane-b- ethylene oxide); poly(2-vinylnaphthalene)-based copolymers such as poly(2-vinylnaphthalene-b-acrylic acid), poly(vinylpyridine and N-methylvinylpyridinium iodide)-based copolymers such as poly(2-vinylpyridine-b-ethylene oxide), poly(2-vinylpyridine-b-methylacrylic acid), poly(N-methyl 2-vinylpyridinium iodide-b-ethylene oxide), poly(N-methyl 4-vinylpyridinium iodide-b-methyl methacrylate), poly(4-vinylpyridine-b-ethylene oxide) PEO end functionalized OH;as well as amphiphilic polymers including, but not limited to, poly(vinylpyrrolidone)-based copolymers, such as poly(vinylpyrrolidone-bD / L-lactide);

[0065] In some embodiments of the present disclosure, the chromophore polymer particles provided herein comprise CN-PPV, a polymer also known as poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-(1-cyanovinylene-1,4-phenylene)], which is a bright and compact orange-emitting semiconducting polymer particle. In certain embodiments, CN-PPV has excellent fluorescent properties, such as large absorption cross-section, high quantum yield, and fast emission rate. In some embodiments, the chromophore polymer particles comprise a polymer that is essentially composed of CN-PPV. In some embodiments, the particles comprise CN-PPV and at least one other material. For example, CN-PPV can be mixed with copolymers or other materials that provide additional functionality.

[0066] In some embodiments, the chromophore polymer particles of the present disclosure include semiconducting copolymers with at least two different chromophore units. For example, a conjugate copolymer can contain both fluorene and benzothiazole chromophore units present in a given ratio. Typical chromophore units used to synthesize semiconducting copolymers include, but are not limited to, fluorene units, phenylenevinylene units, phenylene units, phenyleneethynylene units, benzothiazole units, thiophene units, carbazolefluorene units, boron-dipyrromethene units, and derivatives thereof. Different chromophore units may be separate, as in block copolymers, or may be intermingled. In some embodiments, chromophore copolymers are represented by describing the identity of the major chromophore species. For example, PFBT is a chromophore polymer that contains fluorene and benzothiazole units in a certain ratio. In some cases, a dash is used to indicate the identity of the minor chromophore species next to the percentage of the minor chromophore species. For example, PF-0.1 BT is a chromophore copolymer containing 90% polyfluorene (PF) and 10% benzothiazole (BT).

[0067] In certain embodiments, the chromophore polymer particles comprise a blend of semiconducting polymers. The blend may comprise any combination of homopolymers, copolymers, and oligomers. The polymer blends used to form the chromophore polymer particles can be selected to tailor the properties of the resulting polymer particles, for example, to achieve a desired excitation or emission spectrum of the polymer particles.

[0068] In various embodiments of the present disclosure, the semiconducting chromophore polymer particles exhibit improved detection sensitivity, in part, because they exhibit higher quantum yields than other fluorescent reporters. In some embodiments, the quantum yields of the chromophore polymer particles used are 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 semiconducting chromophore polymer particles exhibit improved detection sensitivity, in part, because they exhibit larger absorption cross-sections. In various embodiments, the semiconducting chromophore polymer particles exhibit improved detection sensitivity, in part, because they exhibit faster emission rates than other fluorescent reporters. In certain embodiments, the emission rates of the chromophore polymer particles used are between about 100 picoseconds and about 50 nanoseconds.

[0069] In some aspects, the chromophoric polymer particles herein comprise polymers with units of small organic dye molecules, metal complexes, photochromic dyes, and any combination thereof, for example, optically inactive polymers such as polystyrene covalently linked or grafted with small organic dyes, metal complexes, photochromic dyes, or any combination thereof. In some aspects, the chromophoric polymer particles comprise semiconducting polymers covalently linked with small organic dye molecules, metal complexes, photochromic dyes, or any combination thereof as luminescent units. Such luminescent units can tune the emission color, increase the quantum yield, and improve the photostability of the chromophoric polymer particles. In some aspects, the small organic dyes or metal complexes have sensing functions, thus adding additional functionality to the chromophoric polymer particles, such as protein sensing capabilities.

[0070] In some embodiments, the nanoparticle transducer comprises one or more chromophores (e.g., fluorophores). The chromophores emit a fluorescence that is dependent on the fluid components. In some embodiments, the fluid components are reactive elements of a reaction catalyzed by the enzyme of the nanoparticle transducer, the reaction involving an analyte. In some cases, the fluid components are products of the reaction; in some cases, the fluid components are reactants of the reaction. In some embodiments, the reaction rate varies as a function of the analyte concentration, which changes the concentration of the fluid components and varies the fluorescence of the transducer accordingly.

[0071] In some embodiments, the chromophore comprises a dye. In some embodiments, the dye is sensitive to one or more fluid components. In some embodiments, the dye is sensitive to oxygen. Examples of oxygen-sensitive dyes that can be used in the nanoparticle transducers disclosed herein include Pt(II)- and Pd(II)-porphyrins, phosphorescent Ru(II) complexes, and Ir(III) complexes. Examples of oxygen-sensitive dyes include Pt(II) octaethylporphine (PtOEP), Pt(II) meso-tetra(pentafluorophenyl)porphine (PtTFPP), Pt(II) octaethylporphine ketone (PtOEPK), Pd(II) octaethylporphine (PdOEP), and Pd(II) meso-tetra(pentafluorophenyl)porphine (PdTFPP), Pd(II)-meso-tetra-(4-carboxyphenyl)porphyrin (PdTPCPP), Pd(II)-meso-tetra-(4-carboxyphenyl)tetrabenzoporphyrindene. Dorimer (PdTCPTBP), Pt(II)-coproporphyrin (PtCP), Pt(II)-meso-tetrabenzoporphyrin butyl octaester (PtTBP), Pt(II)-coproporphyrin-ketone (PtCPK), cyclometallated Ir(III) l-chloro-bridged dimeric coumarin complexes (Ir(III)(Cx)2(acac)), and [Ru(bpy)2(2-(4-carboxyphenyl)imidazo-[4,5-f][1,10]phenanthroline)H2)]2+ ([Ru(bpy)2(picH2)]2+).

[0072] In some embodiments, the chromophore comprises a dye sensitive to ions, pH, and temperature. Examples of dyes for constructing nanoparticle transducers include sodium-sensitive dyes, potassium-sensitive dyes, calcium-sensitive dyes, magnesium-sensitive dyes, iron-sensitive dyes, zinc-sensitive dyes, copper-sensitive dyes, manganese-sensitive dyes, pH-sensitive dyes, and temperature-sensitive dyes. Nanoparticles comprising chromophores sensitive to ions, pH, and temperature include, for example, those described in PCT / US2010 / 056079.

[0073] In some embodiments, the chromophore comprises a semiconducting chromophore polymer that is sensitive to one or more fluid components. The semiconducting polymer can be designed and synthesized to have a fluorescence that is sensitive to one or more fluid components. In some embodiments, the semiconducting chromophore polymer is sensitive to oxygen. Examples of strategies for synthesizing oxygen-sensitive semiconducting chromophore polymers include incorporating oxygen-sensitive units into the semiconducting polymer backbone or attaching oxygen-sensitive units to the side chains of the semiconducting polymer. Examples of semiconducting chromophore polymers to which oxygen-sensitive units can be attached include poly(9,9-dihexylfluorene) (PDHF) systems, poly(9,9-dioctylfluorene) (PFO) systems, poly{[9,9-di-(3-(3-methyloxetan-3-yl)methoxy)hexylfluorenyl-2,7-diyl-co-[9,9-dioctylfluorenyl-2,7-diyl]} (do-PFO) systems, poly[{9,9-dioctyl-2,7-divinylene-fluorenylene}-alt-co-{2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene}] (PFPV) systems, poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1,3} phenylene vinylene polymers, including, but not limited to, poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(4,7-di-2-thienyl-2,1,3-benzothiadiazole)] (PFBT) systems, poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene vinylene] (MEH-PPV) systems, poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-(1-cyanovinylene-1,4-phenylene)] (CN-PPV) systems, poly(2,5-di(3',7'-dimethyloctyl)phenylene-1,4-ethynylene (PPE) systems, BODIPY systems, and squaraine-based semiconducting polymers.

[0074] In some embodiments, the chromophore emits a fluorescence that depends on the concentration of hydrogen peroxide (H2O2). Hydrogen peroxide can be a product reactant. In some embodiments, the nanoparticles include a chromophore polymer that emits a fluorescence that depends on the concentration of hydrogen peroxide. In some embodiments, the nanoparticles include a chromophore polymer and a dye that emits fluorescence at one or more wavelengths. The amount of fluorescence of the dye can depend on the concentration of hydrogen peroxide. The dye can be physically doped or chemically attached to the chromophore polymer to form the nanoparticle, for example. The chromophore polymer can have an energy transfer 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 in the nanoparticle transducers disclosed herein include coumarin derivatives, fluorescein derivatives, rhodamine derivatives, cyanine derivatives, and boron-dipyrromethene (BODIPY) derivatives.

[0075] In some embodiments, the chromophore comprises a dye and a semiconducting chromophore polymer, and the dye and the semiconducting polymer interact to produce enhanced fluorescence. In some embodiments, the semiconducting polymer is insensitive to fluid components; the fluorescence from such a polymer can provide a stable internal standard, and thus can serve as a control for variable fluorescence signals at other wavelengths. The semiconducting chromophore polymer can have energy transfer to the dye to amplify and enhance the fluorescence of the dye. Examples of semiconducting chromophore polymers that can be used in the nanoparticle transducers disclosed herein include poly(9,9-dihexylfluorene) (PDHF) systems, poly(9,9-dioctylfluorene) (PFO) systems, and poly{[9,9-di-(3-(3-methyloxetan-3-yl)methoxy)hexylfluorenyl-2,7-diyl-co-[9,9-dioctylfluorenyl-2,7-diyl]} (do-PFO) systems, poly[{9,9-dioctyl-2,7-divinylene-fluorenylene}-alt-co-{2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene}] (PFPV) systems, poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1,3}-thiadiazole)] (PFBT) systems, poly[ phenylene vinylene polymers, including but not limited to (9,9-dioctylfluorenyl-2,7-diyl)-co-(4,7-di-2-thienyl-2,1,3-benzothiadiazole)] (PFTBT) systems, poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene vinylene] (MEH-PPV) systems, poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-(1-cyanovinylene-1,4-phenylene)] (CN-PPV) systems, poly(2,5-di(3',7'-dimethyloctyl)phenylene-1,4-ethynylene (PPE) systems, BODIPY systems, as well as squaraine-based semiconducting polymers. In some embodiments, the dye is sensitive to one or more fluid components. In some embodiments, the dye is sensitive to oxygen.Examples of oxygen-sensitive dyes that can be used in the nanoparticle transducers disclosed herein include Pt(II)-porphyrins and Pd(II)-porphyrins, phosphorescent Ru(II) complexes, and Ir(III) complexes. Other examples of oxygen-sensitive dyes include Pt(II) octaethylporphine (PtOEP), Pt(II) meso-tetra(pentafluorophenyl)porphine (PtTFPP), Pt(II) octaethylporphine ketone (PtOEPK), Pd(II) octaethylporphine (PdOEP), and Pd(II) meso-tetra(pentafluorophenyl)porphine (PdTFPP), Pd(II)-meso-tetra-(4-carboxyphenyl)porphyrin (PdTPCPP), Pd(II)-meso-tetra-(4-carboxyphenyl)tetrabenzoporphyrin decafluorophosphate (PTPCPP), and Pd(II)-meso-tetra-(4-carboxyphenyl)tetrabenzoporphyrin decafluorophosphate (PTPCPP). Examples of suitable dyes include, but are not limited to, Pt(II)-trimethylsilyltriphenylphosphine (PdTCPTBP), Pt(II)-coproporphyrin (PtCP), Pt(II)-meso-tetrabenzoporphyrin butyl octaester (PtTBP), Pt(II)-coproporphyrin-ketone (PtCPK), cyclometallated Ir(III) l-chloro-bridged dimeric coumarin complexes (Ir(III)(Cx)2(acac)), and [Ru(bpy)2(2-(4-carboxyphenyl)imidazo-[4,5-f][1,10]phenanthroline)H2)]2+ ([Ru(bpy)2(picH2)]2+). The dyes may be physically blended or chemically attached to the semiconducting chromophore polymer to form the nanoparticle transducer.

[0076] In some embodiments, the chromophore comprises multiple dyes. A first dye is sensitive to one or more fluid components, and the sensitive dye and the second dye can interact to generate enhanced fluorescence. In some embodiments, at least one dye is insensitive to fluid components, thus resulting in stable fluorescence as an internal standard. The multiple dyes can emit fluorescence at different wavelengths, allowing independent measurement of the fluorescence of each dye. The 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.

[0077] In some embodiments, the chromophoric polymer particles comprise semiconducting polymers physically mixed or chemically crosslinked with other chromophoric polymers, such as inert polymers covalently linked or grafted with small organic dyes, metal complexes, photochromic dyes, or any combination thereof, to provide additional functionality, such as protein sensing.

[0078] In some embodiments, the chromophore polymer particles comprise semiconducting polymers that are physically mixed or chemically crosslinked with other components, such as fluorescent dyes, inorganic luminescent materials, magnetic materials, metallic materials, etc., to tune the emission color, improve quantum yield and / or photostability, and / or provide additional functionality, such as, for example, magnetic functionality, plasmon resonance functionality, etc.

[0079] The optical properties, such as the absorption wavelength, for a given chromophore polymer particle can be tuned by modifying its composition and / or structure. Semiconducting polymers have been developed with absorption wavelengths ranging from the UV to the infrared, including the entire visible spectrum. In some embodiments, chromophore polymer particles are used that have a peak absorption wavelength between about 200 nanometers and about 300 nanometers, between about 250 nanometers and about 350 nanometers, between about 300 nanometers and about 400 nanometers, between about 350 nanometers and about 450 nanometers, between about 400 nanometers and about 500 nanometers, between about 450 nanometers and about 550 nanometers, between about 500 nanometers and about 600 nanometers, between about 550 nanometers and about 650 nanometers, between about 600 nanometers and about 700 nanometers, between about 650 nanometers and about 750 nanometers, between about 700 nanometers and about 800 nanometers, between about 750 nanometers and about 850 nanometers, between about 800 nanometers and about 900 nanometers, between about 850 nanometers and about 950 nanometers, or between about 900 nanometers and about 1000 nanometers.

[0080] Semiconductor polymers have been developed with emission wavelengths ranging from UV to infrared, including the entire visible spectrum. In some embodiments, the emission wavelengths are between about 200 nanometers and about 300 nanometers, between about 250 nanometers and about 350 nanometers, between about 300 nanometers and about 400 nanometers, between about 350 nanometers and about 450 nanometers, between about 400 nanometers and about 500 nanometers, between about 450 nanometers and about 550 nanometers, between about 500 nanometers and about 600 nanometers, between about 550 nanometers and about 650 nanometers, between about 600 nanometers and about 700 nanometers, between about 650 nanometers and about 750 nanometers, between about 700 nanometers and about 750 nanometers, between about 750 nanometers and about 800 nanometers, between about 800 nanometers and about 850 nanometers, between about 800 nanometers and about 900 nanometers, between about 900 nanometers and about 950 nanometers, between about 900 nanometers and about 1000 nanometers, between about 10 ... In some embodiments, chromophore polymer particles having a peak emission wavelength between about 00 nanometers and about 800 nanometers, between about 750 nanometers and about 850 nanometers, between about 800 nanometers and about 900 nanometers, between about 850 nanometers and about 950 nanometers, between about 900 nanometers and about 1000 nanometers, between about 950 nanometers and about 1050 nanometers, between about 1000 nanometers and about 1100 nanometers, between about 1150 nanometers and about 1250 nanometers, or between about 1200 nanometers and about 1300 nanometers are used.

[0081] In some embodiments, the present disclosure provides chromophoric polymer particles with narrow band emission. Narrow band emission is advantageous for certain applications, including but not limited to, the resolution of multiple fluorescent signals. The emission wavelength of the polymer particles can vary from ultraviolet to near infrared regions. In some embodiments, the FWHM of the emission band is less than about 100 nanometers, about 70 nanometers, about 65 nanometers, about 60 nanometers, about 55 nanometers, about 50 nanometers, about 45 nanometers, about 40 nanometers, about 35 nanometers, about 30 nanometers, about 25 nanometers, about 20 nanometers, or about 10 nanometers. In some embodiments, the FWHM of the polymer particles described herein can range between about 5 nanometers and about 100 nanometers, about 10 nanometers to about 70 nanometers, about 20 nanometers to about 60 nanometers, or about 30 nanometers to about 50 nanometers.

[0082] In some embodiments, the types of chromophore polymer particles of the present disclosure include polymers having narrow band emission units (e.g., narrow band monomers and / or narrow band units). For example, the present disclosure may include homopolymers or heteropolymers including narrow band monomers such as BODIPY and / or BODIPY derivative monomers, squaraines and / or squaraine derivative monomers, metal complexes and / or metal complex derivative monomers, porphyrins and / or porphyrin derivative monomers, metalloporphyrins and / or metalloporphyrin derivative monomers, phthalocyanines and / or phthalocyanine derivative monomers, lanthanide complexes and / or lanthanide complex derivative monomers, perylenes and / or perylene derivative monomers, cyanines and / or cyanine derivative monomers, rhodamines and / or rhodamine derivative monomers, coumarins and / or coumarin derivative monomers, and / or xanthenes and / or xanthene derivative monomers. In certain embodiments, the narrow band units are, for example, narrow band monomers or fluorescent nanoparticles embedded or attached to the polymer particles. The fluorescent nanoparticles may be, for example, quantum dots. Optionally, the narrow band unit comprises a polymer or a fluorescent dye molecule that provides a narrow emission of the polymer particles of the present disclosure.

[0083] In some embodiments of the present disclosure, the devices, compositions, systems, and methods provided herein utilize one or more chromophores (e.g., dyes or semiconducting chromophore polymers) that can generate fluorescence at one or more wavelengths in response to incident radiation, such as, for example, UV light, visible light, far-red light, near-infrared light, or other light. In some cases, the amount of fluorescence at a given wavelength from the chromophore varies as a function of the local concentration of a fluid component (signal chromophore); in other embodiments, the amount of fluorescence from the chromophore does not vary in response to said local concentration (control chromophore). In certain embodiments, the nanoparticles provided herein can incorporate both signal and control chromophores that emit fluorescence at signal and control wavelengths, respectively. Although various embodiments herein are described in terms of nanoparticles having one or two different emission wavelengths, it should be understood that the approach presented herein is also applicable to nanoparticles that emit more than two wavelengths. For example, nanoparticles can be provided that emit at two signal wavelengths and one or two control wavelengths, which can be used for multiple analyte measurement signals. A number of different nanoparticles may be provided, each with different signal / control wavelength pairs that respond to a distinctly different analyte (or optionally the same analyte, eg, in the case of redundant signaling).

[0084] In certain embodiments, the chromophore that generates the fluorescence at the signal wavelength exhibits different optical characteristics (e.g., emission spectrum, absorption spectrum, peak emission wavelength(s), peak excitation wavelength(s), emission intensity, emission lifetime, emission rate) when in different concentrations of a fluid component. For example, the chromophore may exhibit an increase (or decrease) in fluorescence in response to an increase in the concentration of a fluid component, such as a molecule. In some embodiments, the change in fluorescence may be ratiometric as a function of the concentration of the fluid component. The molecule may be, for example, oxygen, which may be a reactant or product of a reaction involving the analyte being measured and catalyzed by an enzyme. The enzyme may be associated with the nanoparticle that contains the chromophore such that the reaction catalyzed by the enzyme changes the local concentration of the molecule, thereby causing the fluorescence of the chromophore to change in response to changes in the concentration of the analyte. In some embodiments, such a change may be ratiometric. By generating fluorescence at a control wavelength, for example, the fluorescence ratio of control to signal can serve as a signal for analyte concentration, eliminating or reducing certain sources of noise and uncertainty in fluorescence intensity measurements. Enzyme composition

[0085] In certain embodiments disclosed herein, the detection of small molecules is based on the integration of nanoparticle oxygen transducers with oxygen-consuming enzymes that catalyze small molecule oxidation reactions. Optionally, the nanoparticle transducers can be directly mixed with the enzymes for measurement. Optionally, covalent conjugation is performed to link the nanoparticles to the enzymes, resulting in compact probes that can be used for intracellular sensing. Once the enzyme corona is formed on the nanoparticle surface, the nanoparticle-enzyme bioconjugates behave as nanoreactors that deplete their internal oxygen stores in the presence of small molecule analytes to which the enzymes are sensitive. Thus, as oxygen is depleted, the small molecule concentration is monitored by the optical signal of the oxygen transducer. The performance of this sensing scheme depends on factors including (1) whether the presence of the analyte can induce a distinct change in the oxygen distribution profile, and (2) whether the oxygen transducer can convert the change in oxygen into an optical signal. In addition, in vivo detection is closely related to issues such as local microvascular perfusion, oxygen availability in tissues, and enzyme activity. In the following sections, glucose is presented as an example that can illustrate the effectiveness of the nanoparticle transducers described herein in sensing analyte concentrations, with both theoretical analysis and experimental evidence for both in vitro and in vivo applications. Based on the examples described herein, nanoparticle transducers can be fabricated to generate fluorescent signals for the detection of a wide variety of analytes, including small molecules, large molecules, and other fluid constituents, by selecting appropriately reactive enzymes and corresponding chromophores that are sensitive to the reaction components of the reactions catalyzed thereby.

[0086] In many aspects, the nanoparticle transducers provided herein include an enzyme that catalyzes a reaction involving an analyte to be measured that produces a product and consumes a reactant, collectively referred to as a reaction element. In many aspects, the reaction element includes a fluid component, and the reaction changes the concentration of the fluid component. 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 its concentration. In some aspects, the fluid component is oxygen, and oxygen is a reactant. In some embodiments, the oxygen-consuming enzyme and the analyte each comprise one or more of the following pairs: glucose oxidase and glucose; ascorbic acid oxidase and ascorbic acid; glutamate oxidase and glutamate; dopamine beta-hydroxylase and dopamine; cholesterol oxidase and cholesterol; and alcohol oxidase and alcohol, lactate oxidase and lactate, and xanthine oxidase and xanthine, monoamine oxidase and monoamine, NADPH oxidase and NADPH, L-gulonolactone oxidase and L-gulonolactone, lysyl oxidase and lysine, laccases and their various substrates, such as phenol, and cytochrome P450 oxidase and its various substrates, including drugs. Optionally, the oxygen-consuming enzyme is an amine oxidase and the analyte is an amino acid. Optionally, the oxygen-consuming enzyme is a cytochrome P450 and the analyte is a drug that reacts with oxygen when catalyzed by cytochrome P450. Optionally, the oxygen consuming enzyme is NADPH oxidase (nicotinamide adenine dinucleotide phosphate oxidase) and the analyte is NADPH. Optionally, the oxygen consuming enzyme is xanthine oxidase and the analyte is xanthine. Optionally, the oxygen consuming enzyme is gulonolactone oxidase and the analyte is gulonolactone.

[0087] In some embodiments, the fluid component is other than oxygen; for example, the fluid component may be an ion, and the enzyme may catalyze a reaction that changes the ion concentration, and the chromophore may generate a fluorescence that is modulated by the ion concentration; the fluid component may be an acid or a base, and the enzyme may catalyze a reaction that changes the pH, and the chromophore may generate a fluorescence that is modulated by the pH; or the fluid component may be thermal energy, and the enzyme may catalyze a reaction that changes the temperature, and the chromophore may generate a fluorescence that is modulated by the temperature. In some embodiments, the fluid component may be hydrogen peroxide, and the enzyme may catalyze a reaction that changes the hydrogen peroxide concentration, and the chromophore may generate a fluorescence that is modulated by the hydrogen peroxide concentration. For example, hydrogen peroxide may be a product of the reaction.

[0088] In some embodiments, multiple enzymes are attached to the nanoparticle transducer to catalyze a respective one of the multiple reactions. The multiple reactions result in a chain of reactions in which one or more products of one reaction are reactants for another reaction. For example, multiple enzymes may result in an enzyme cascade, with each enzyme performing one step 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 modulation of the emission intensity of the chromophore. Device Components

[0089] In some aspects, the systems described herein include a computer with one or more processors and a memory device having executable instructions stored thereon. In some aspects, the computer is used to perform the methods described herein. In various aspects, the computer can be used to implement any of the systems or methods illustrated and described above. In some aspects, the computer includes a processor that communicates with several peripheral subsystems via a bus subsystem. These peripheral subsystems may include a storage subsystem, including a memory subsystem and a file storage subsystem, a user interface input device, a user interface output device, and a network interface subsystem.

[0090] In some aspects, the bus subsystem provides a mechanism by which the various components and subsystems of a computer can communicate with each other as intended. The bus subsystem may include a single bus or multiple buses.

[0091] In some aspects, the network interface subsystem provides an interface to other computers and networks. The network interface subsystem can be an interface for receiving data from a computer and transmitting data to other systems. For example, the network interface subsystem can connect a computer to the Internet and facilitate communication using the Internet.

[0092] In some embodiments, a computer includes user interface input devices, such as a keyboard, pointing devices, such as a mouse, trackball, touchpad, or graphics tablet, scanners, barcode scanners, touch screens integrated into a display, voice input devices, such as voice recognition systems, microphones, and other types of input devices. In general, use of the term "input device" is intended to include all possible types of devices and mechanisms for inputting information into a computer.

[0093] In some aspects, a computer includes a user interface output device, such as a display subsystem, a printer, a fax machine, or a non-visual display, such as an audio output device. The display subsystem may be a flat panel device, such as a liquid crystal display (LCD), or a projection device. In general, use of the term "output device" is intended to include all possible types of devices and mechanisms for outputting information from a computer.

[0094] In some aspects, the computer includes a storage subsystem that provides a computer-readable storage medium for storing basic programming and data structures. In some aspects, the storage subsystem stores software (programs, code modules, instructions) that, when executed by a processor, provide the functionality of the methods and systems described herein. These software modules or instructions can be executed by one or more processors. The storage subsystem can also provide a repository for storing data used in accordance with the present disclosure. The storage subsystem may include a memory subsystem and a file / disk storage subsystem.

[0095] In some embodiments, the computer includes a memory subsystem that may include several memories, including a main random access memory (RAM) for storage of instructions and data during program execution, and a read-only memory (ROM) in which fixed instructions are stored. The file storage subsystem provides non-transient persistent (non-volatile) storage of program and data files, and may include hard disk drives, USB sticks, solid state drives, optical drives, removable media cartridges, and other similar storage media.

[0096] The computers can be of various types, including smartphones, tablet computers, personal computers, handheld computers, workstations, network computers, mainframes, kiosks, servers, or any other data processing system. Because the nature of computers and networks is constantly changing, the descriptions of computers contained herein are intended only as specific examples intended to illustrate aspects of computers. Many other configurations are possible, having more or fewer components than the systems described herein. Further aspects of the invention

[0097] In various aspects, a nanoparticle transducer for analyte concentration measurement is provided. The nanoparticle transducer includes a nanoparticle including a chromophore, to which an enzyme is bound. The enzyme catalyzes a reaction including a plurality of reaction components. The reaction components include one or more reactants including an analyte and one or more products. The chromophore emits an amount of fluorescence determined by the concentration of a reaction component of the plurality of reaction components. The emission of fluorescence can be induced by irradiation with suitable light, such as ultraviolet light, visible light, far-red light, near-infrared light, or other light.

[0098] In some embodiments, one of the reaction elements is oxygen, and the amount of fluorescence emitted from the chromophore is determined by the concentration of oxygen. Optionally, the enzyme comprises glucose oxidase. The one or more reactants may comprise glucose and oxygen, and the one or more products comprise hydrogen peroxide and D-glucono-1,5-lactone.

[0099] In some aspects, the nanoparticle transducer further comprises catalase.

[0100] In some embodiments, the amount of fluorescence emitted from the chromophore is determined by the concentration of the reactants; in some embodiments, the amount of fluorescence emitted from the chromophore is determined by the concentration of the product.

[0101] In some embodiments, the nanoparticle comprises a Pdot. The enzyme is covalently attached to the nanoparticle.

[0102] In some embodiments, the chromophore comprises a semiconducting polymer. In some embodiments, the chromophore comprises a dye. The dye may be contained in the nanoparticle. The chromophore may comprise a semiconducting polymer and a dye, and the dye and the semiconducting polymer interact to generate enhanced fluorescence. In some cases, the chromophore comprises a blend of two or more semiconducting polymers.

[0103] In some embodiments, the fluorescence emitted from the chromophore comprises a signal fluorescence wavelength and a control fluorescence wavelength. The fluorescence emitted from the chromophore may comprise a fluorescence ratio 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. The signal fluorescence ratio may be determined by the concentration of the reaction component among the plurality of reaction components. In many cases, the fluorescence ratio varies ratiometrically with the concentration of the analyte. In some cases, the control wavelength fluorescence remains substantially constant while the signal wavelength fluorescence increases substantially linearly with the analyte concentration.

[0104] In various embodiments, the analyte is glucose and the analyte concentration range is within a range between 0 and about 20 mM of glucose. Optionally, the analyte concentration range is within a range between about 3 mM glucose and about 15 mM glucose.

[0105] In some embodiments, the nanoparticles contain at least 20 percent by weight of the chromophore. For example, the nanoparticles can contain at least 50 percent by weight of the chromophore, or at least 90 percent by weight of the chromophore. In some cases, the nanoparticles include about 100 percent by weight of the chromophore.

[0106] In some embodiments, the nanoparticles include a second chromophore for fluorescent detection of a second analyte and a second enzyme bound to the nanoparticles. The second enzyme catalyzes a second reaction including a second plurality of reaction elements. The second plurality of reaction elements includes a second reactant or reactants including the second analyte and a second product or products. The amount of fluorescence emitted from the second chromophore is determined by the concentration of a second reaction element of the second plurality of reaction elements. The fluorescence of the second chromophore includes a wavelength different from the fluorescence of the other chromophore.

[0107] In some embodiments, the nanoparticle transducer includes a second nanoparticle including a second chromophore and a second enzyme bound to the nanoparticle and configured to catalyze a second reaction including a second plurality of reaction elements. The second plurality of reaction elements includes a second reactant or reactants including a second analyte and a second product or products. The amount of fluorescence emitted from the second chromophore is determined by the concentration of a second reaction element of the second plurality of reaction elements.

[0108] Optionally, the enzyme is selected from the group consisting of ascorbic acid oxidase, glutamate oxidase, dopamine beta-hydroxylase, cholesterol oxidase, alcohol oxidase, amine oxidase, and cytochrome P450. Optionally, the analyte is selected from the group consisting of ascorbic acid, glutamate, dopamine, cholesterol, and alcohol. Optionally, the analyte can be an amino acid, a drug, a protein, a nucleic acid molecule, or a transmitter molecule. Optionally, the analyte is a carbohydrate, a lipid, or a metabolite. Optionally, the analyte is a sugar.

[0109] In some aspects, the analyte concentration is a blood concentration, hi some aspects, the analyte concentration is a concentration in lymph, saliva, tears, interstitial fluid, cerebrospinal fluid, or urine.

[0110] In some embodiments, the nanoparticle transducer includes a plurality of enzymes that catalyze a respective plurality of reactions, each of which includes a respective plurality of reaction components. The analyte is a reactant of one of the reactions, and the fluorescence emitted from the chromophore is determined by the concentration of the reaction components of at least one of the reactions. In some cases, one or more of the products of one enzymatic reaction are reactants of another enzymatic reaction, resulting in a chain of enzymatic reactions. Thus, the analyte concentration can affect the fluorescence of the chromophore by participating in a chain of enzymatic reactions.

[0111] In some cases, the critical dimension of the nanoparticle transducer is less than about 1000 nm, less than 700 nm, less than about 500 nm, or less than about 100 nm. In some cases, the critical dimension of the nanoparticle transducer is within the range of about 15 nm to about 45 nm.

[0112] In various aspects, an apparatus is provided for measuring a concentration of an analyte in a biological fluid. The apparatus comprises a plurality of fluorescent nanoparticle transducers prepared according to the aspects disclosed herein. The apparatus further comprises an optical sensor coupled to a processor and a memory, and an illumination source configured to irradiate the plurality of fluorescent nanoparticles with light to induce fluorescence therefrom. The memory includes instructions that, when executed, cause the processor to use the optical sensor to measure the fluorescence emitted by the plurality of fluorescent nanoparticle transducers.

[0113] In some aspects, the biological fluid is blood, lymph, saliva, tears, interstitial fluid, cerebrospinal fluid, or urine.

[0114] In some aspects, the plurality of fluorescent nanoparticle transducers are adapted to be positioned under the skin of a patient, and the optical sensor is adapted to detect fluorescence transmitted through the skin by the plurality of fluorescent nanoparticle transducers when facing the skin. Optionally, the optical sensor is configured to detect an amount of signal fluorescence at a signal fluorescence wavelength and an amount of control fluorescence at a control fluorescence wavelength, and the memory includes instructions for causing the processor to determine a measured fluorescence ratio based on said measured amounts of signal fluorescence and control fluorescence. Optionally, the memory includes instructions for causing the processor to determine a concentration of an analyte based on the measured fluorescence ratio.

[0115] In various aspects, a contact lens for detecting glucose concentration is provided. The contact lens comprises a permeable transparent membrane wearable on the eye, the lens containing a plurality of nanoparticle transducers according to the aspects disclosed herein. The nanoparticle transducers are configured to generate fluorescence in response to illumination by a scanner, the amount of fluorescence providing a signal detectable by the scanner for determining the concentration of glucose in a fluid at the surface of the eye. The membrane of the lens may be shaped to correct human vision when worn.

[0116] In various aspects, a device is provided for measuring glucose concentration from sweat of a subject. The device comprises a plurality of fluorescent nanoparticle transducers according to aspects disclosed herein, positioned by the device to contact the skin of the subject when worn. The device further comprises an illumination source configured to irradiate the plurality of fluorescent nanoparticles with light to induce fluorescence therefrom, and an optical sensor disposed within the device and oriented to detect fluorescence from the plurality of fluorescent nanoparticle transducers. A processor is coupled to the optical sensor. The processor is configured to determine a concentration of glucose in the sweat based on the fluorescence detected by the optical sensor. The processor may be further configured to determine a concentration of glucose in blood based on the concentration of glucose in the sweat.

[0117] In various aspects, a method of determining an analyte concentration in a fluid is provided. The fluid is contacted with a nanoparticle transducer according to aspects disclosed herein. The fluorescence of the nanoparticle transducer is measured, and the concentration of the analyte in the fluid is determined based on the measured fluorescence. Optionally, the method includes inducing fluorescence by irradiating the nanoparticle transducer with light.

[0118] In some aspects, the determining step includes comparing the measured fluorescence to a calibration curve of the nanoparticle transducer to determine an analyte concentration in the fluid. In some aspects, the measuring step includes measuring a plurality of fluorescence wavelengths, and the determining step is based on a ratio of the measured wavelengths. Optionally, the fluid is blood, sweat, or tears.

[0119] In some embodiments, a method is provided for measuring the concentration of an analyte in a fluid. The analyte undergoes a reaction in the fluid, which reaction changes the concentration of a fluid component. The fluid is contacted with Pdots that include a chromophore, and the chromophore emits an amount of fluorescence determined by the concentration of the fluid component. The chromophore emits fluorescence, and the concentration of the analyte in the fluid is determined based on the fluorescence.

[0120] In some aspects, the fluorescence emitted from the chromophore comprises 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, the fluorescence ratio being determined by the concentration of the fluid component. Optionally, determining the concentration comprises measuring the fluorescence at the signal fluorescence wavelength and the control fluorescence wavelength, determining a measured fluorescence ratio based on said measurements, and determining the concentration of the analyte based on the measured fluorescence ratio.

[0121] In some embodiments, the analyte is ascorbic acid and the fluid constituent is oxygen. Optionally, the determined concentration is between 1 mM and 20 mM of ascorbic acid. In some embodiments, the fluid is blood, sweat, or tears.

[0122] The specific dimensions of any of the disclosed apparatus, devices, and their components can be easily varied depending on the intended use, as will be apparent to those skilled in the art in light of the disclosure herein.Furthermore, it is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes therein may occur to those skilled in the art and are within the spirit and scope of this application and the appended claims.Many different combinations of the embodiments described herein are possible, and such combinations are considered part of this disclosure.

[0123] As used herein, A and / or B includes one or more of A or B, and combinations thereof, such as A and B. As used herein, when an embodiment is described in this disclosure as comprising one or more elements, embodiments consisting of said elements are also disclosed.

[0124] Any feature discussed in relation to any embodiment or embodiments herein can be readily adapted for use in other embodiments and embodiments herein. The use of different terms or reference numbers for similar features in different embodiments does not necessarily imply differences other than those explicitly stated. Thus, the present disclosure is intended to be described only by reference to the appended claims, and is not limited to the embodiments disclosed herein.

[0125] Unless otherwise specified, the methods and processes described herein may be performed in any order. For example, a method that describes steps (a), (b), and (c) may perform step (a) first, followed by step (b), and then step (c). Or, the method may be performed in a different order, such as first step (b), followed by step (c), and then step (a). Moreover, the steps may be performed simultaneously or separately, unless otherwise specified with specificity.

[0126] The details shown in this specification are examples and are presented only for the purpose of illustrative discussion of preferred embodiments of the present disclosure, and are presented to provide what is believed to be the most useful and easily understood description of the principles and conceptual aspects of various aspects of the present invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, and how several forms of the invention may be embodied in practice will become apparent to those skilled in the art upon reading this description in conjunction with the figures and / or examples.

[0127] Although preferred embodiments of the present disclosure have been shown and described herein, it is to be understood that the present disclosure is not limited to the particular embodiments of the disclosure described, since modifications of the particular embodiments may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology used is for the purpose of describing particular embodiments of the present disclosure and is not intended to be limiting. Rather, the scope of the present disclosure is defined by the appended claims.

[0128] Where a range of values ​​is provided, each intervening value between the upper and lower limits of that range, and any other stated or intervening value within that stated range, to the tenth of the unit of the lower limit unless otherwise clearly indicated by context, is encompassed within the disclosure provided herein. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges and are also encompassed within the invention, unless there is any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included within the disclosure provided herein.

[0129] All features discussed in relation to one embodiment or embodiments herein can be readily adapted for use in other embodiments and embodiments herein. The use of different terms or reference numbers for similar features in different embodiments does not necessarily imply differences other than those explicitly stated. Thus, the present disclosure is intended to be described solely by reference to the appended claims, and is not limited to the embodiments disclosed herein. EXAMPLES

[0130] The following examples are included to further describe some aspects of the disclosure and should not be used to limit the scope of the invention. Example 1 Working principle of nanoparticle transducers for analyte concentration measurement

[0131] In one example, the spatial and temporal changes in oxygen concentration in a typical sample configuration are simulated by a model that considers the glucose enzymatic reaction of glucose oxidase and oxygen diffusion. Figures 1A-1H illustrate the simulated O2 depletion kinetics and distribution profile for an exemplary nanoparticle transducer for glucose detection using an O2-modulated signal from a glucose oxidase-catalyzed reaction. A typical cuvette (1 cm x 1 cm x 3 cm) containing glucose oxidase (GOx) is used to model the oxygen consumption induced by glucose. Briefly, in a closed system (no oxygen diffusion), the oxygen consumption kinetics in the presence of GOx and glucose can be expressed as follows: a([O2]-[O2]0)+b(ln[O2]-ln[O2]0=-(t-t0)), where [O2]0 corresponds to [O2] in the air-saturated solution at time t0, just before the reaction begins. The parameters a and b can be calculated from the GOx concentration, the enzyme reaction rate constant for a given glucose concentration. This equation is valid when glucose is in excess compared to oxygen, which is generally true since physiological glucose concentrations (typically in the range of about mM) are significantly higher than the oxygen concentration in air-saturated solutions (about 250 μM) and in tissues (<100 μM). Based on the above equation, the kinetic changes in oxygen concentration in closed cuvettes containing GOx (5 nM) and varying concentrations of glucose are shown in Figure 1A. As can be seen, the initial oxygen concentration (about 250 μM, air-saturated solution) decreased as the enzyme reaction occurred, and then the differences induced by different glucose concentrations were distinguished. Due to limited oxygen availability in the solutions (closed systems with no oxygen supply and diffusion), oxygen was eventually depleted to similar equilibrium levels between solutions with different glucose contents.

[0132] Oxygen diffusion is relevant for the open cuvette configuration and for the quantification of glucose in subcutaneous tissue. To simplify the simulation, the Z-axis of the cuvette was discretized into a series of thin layers, and oxygen was modeled as diffusing only from the top opening. The temporal oxygen concentration in each layer was simulated by the combined effect of oxygen consumption and diffusion governed by Fick's law. Eventually an equilibrium between oxygen consumption and diffusion was established, and a flat concentration curve was obtained after a substantial reaction time, as shown in Figure 1B. Figure 1C illustrates a 3-D plot of the temporal and spatial oxygen distribution in a system containing 5 nM GOx and 20 mM glucose along the Z-axis of the open cuvette configuration, as well as the temporal evolution in each layer depending on the enzyme reaction time. At the 500 s time point, the glucose concentrations were well differentiated by oxygen mapping, especially in the bottom part of the cuvette. Figure 1D illustrates the oxygen distribution profile in an open cuvette with different glucose concentrations at the 500 s time point. The results demonstrate that glucose concentration in biological fluids can be effectively and efficiently measured in the open cuvette configuration of a fluorometer with the aid of an oxygen transducer.

[0133] Further simulations of oxygen mapping for a spherical sample in subcutaneous tissue, where the tissue oxygen concentration (<100 μM) is significantly lower than that in air-saturated solutions (~250 μM), are provided. To meet the requirements of in vivo real-time glucose monitoring, a 50 nM concentration of GOx was provided to achieve a rapid response time. As expected, the temporal oxygen changes in the center of this sample resulted in a shorter response time compared to those with lower GOx concentrations. Figure 1E illustrates these O2 depletion kinetics in a closed tissue oxygen environment. Oxygen was diffused from the circular edge to the interior to mimic a thin layer of a 3D spherical object in a real implant. Figure 1F illustrates the O2 depletion kinetics in tissue with oxygen diffusion. It is shown that the temporal evolution in the presence of oxygen diffusion showed a glucose enzymatic reaction resulting in a clearly different oxygen distribution curve in a relatively short time. Figure 1G illustrates the 2-D mapping of the circular configuration with oxygen diffusion at 20 seconds. As further shown by 2D mapping, the oxygen profiles are clearly differentiated at different glucose levels, indicating the feasibility of subcutaneous glucose measurement with oxygen transducers. It is noteworthy that some degree of oxygen diffusion resulted in significantly higher sensitivity in glucose quantification compared to the case without oxygen diffusion. This difference is shown in Figure 1H, which illustrates the sensitivity of oxygen depletion for glucose quantification with and without oxygen diffusion at the 20 second time point. Several strategies can be used to modulate oxygen or glucose diffusion in practical experiments, such as embedding the transducer in porous gels or other matrices with different encapsulation layers. Example 2 Creation of nanoparticle transducers for analyte concentration measurement

[0134] In this example, nanoparticle transducers were produced and characterized in an illustrative system for glucose detection. Aqueous dispersion of semiconducting polymer dots was performed using a reprecipitation method. In a typical preparation, the semiconducting polymer PDHF, the functional polymer PSMA, and the phosphorescent dye PtOEP were dissolved in anhydrous tetrahydrofuran (THF) by stirring overnight under inert atmosphere to make a stock solution of 1 mg / mL, respectively. The three solutions were diluted and mixed in THF to produce a solution mixture with a PDHF concentration of 100 μg / mL, a PtOEP concentration of 10 μg / mL, and a PSMA concentration of 10 μg / mL. A 2 mL aliquot of the solution mixture was immediately added to 10 mL of Milli-Q water in a bath sonicator while the mixture was sonicated, followed by an additional 100 s of sonication. The THF was removed by nitrogen stripping, and the solution was concentrated to 5 mL on a hot plate at 90° C. before being filtered through a 0.2 micron filter. During nanoparticle formation, the maleic anhydride units of the PSMA molecules hydrolyzed in an aqueous environment, generating carboxyl groups on the Pdots. The phosphorescent dye molecules were encapsulated inside the Pdots due to their hydrophobic nature, and the Pdot dispersions were clear and stable for several months without signs of aggregation.

[0135] The increase or decrease in oxygen concentration induced by glucose can be converted into an optical signal by using an oxygen-responsive transducer. Figure 2A-D illustrates the preparation and characterization of a nanoparticle transducer containing a Pdot-GOx assembly. This Pdot transducer contains a fluorescent semiconducting polymer [poly(9,9-dihexylfluorenyl-2,7-diyl), PDHF] doped with a chromophore containing an oxygen-sensitive phosphorescent dye (platinum(II) octaethylporphine, PtOEP). In this design, the conjugated polymer PDHF acts as a light harvester to transfer energy to the PtOEP dye, resulting in bright phosphorescence that is highly sensitive to oxygen concentration.

[0136] Figure 2A is a schematic representation of the formation of Pdot-GOx bioconjugates for in vivo glucose monitoring. As shown in Figure 2A, oxygen-sensitive Pdots were functionalized with surface carboxyl groups. The Pdots were coated with GOx using an EDC-catalyzed reaction between the carboxyl groups of the Pdots and the amine groups in the enzyme. Bioconjugation was performed by utilizing an EDC-catalyzed reaction between the carboxyl groups on the Pdot surface and the amine groups of the GOx enzyme. In this bioconjugation reaction, 80 μL of concentrated HEPES buffer (1 M, pH 6.5) was added to 4 mL of functionalized Pdot solution (50 μg / mL in MilliQ water), resulting in a Pdot solution in 20 mM HEPES buffer at pH 6.5. Then, 100 μL of glucose oxidase (20 mM EDC (10 μM in pH=6.5 HEPES) was added to the solution and mixed thoroughly by vortexing. 80 μL of freshly prepared EDC solution (5 mg / mL in MilliQ water) was added to the solution and the above mixture was placed on a rotary shaker at room temperature for 4 h. Finally, the resulting Pdot-bioconjugates were separated from free biomolecules by gel filtration using Sephacryl HR-300 gel medium. Varying the ratio of GOx to Pdots can generate Pdot-GOx sensors with different dynamic ranges.

[0137] Dynamic light scattering measurements showed that the hydrodynamic diameter of Pdots after bioconjugation increased from 24 nm to 32 nm, as illustrated in Figure 2B, while the surface zeta potential of Pdot-GOx changed from -31 mV to -20 mV, as illustrated in Figure 2C. Both particle size and surface potential measurements confirmed the successful conjugation and the presence of GOx on the particle surface. Transmission electron microscopy (TEM) showed that the Pdot-GOx nanoparticles were spherical and monodisperse. Figure 2D illustrates representative TEM images of carboxyl Pdots (left) and Pdot-GOx (right).

[0138] The Pdot-GOx bioconjugate also exhibited excellent colloidal stability in phosphate-buffered saline (PBS) solution for over 30 days. Figure 3A illustrates the colloidal stability of the Pdot-GOx transducer over 30 days. For the purpose of in vivo glucose monitoring, the Pdot-GOx conjugate had exceptional brightness and high sensitivity that allowed for easy detection of signals even from small amounts of implanted sensor material. The Pdot transducer provides sufficient luminescence signal for long-term transdermal detection while also meeting the biocompatibility requirements of implanted sensors.

[0139] Fluorescence spectroscopy demonstrated the high sensitivity, excellent selectivity, and tunable dynamic range of the Pdot-GOx assembly for glucose detection in biological fluids. The Pdot transducer exhibited a dominant absorption at 380 nm from PDHF and a major phosphorescence peak at 648 nm from PtOEP. Figure 3B shows the UV-visible (vis) absorption and photoluminescence spectra of the Pdot-GOx sensor. The red emission was well separated from the UV excitation, which is an excellent advantage for monitoring the emission signal with the naked eye. Figure 3C illustrates the spectral overlap between the fluorescence emission of PDHF and the absorption of the phosphorescent dye PtOEP. This overlap results in energy transfer between the chromophores, allowing the UV light absorbed by the PDHF donor to be pumped to the PtOEP acceptor. Figure 3D illustrates the emission spectra of undoped PDHF Pdots and PtOEP-doped Pdots with an excitation wavelength of 380 nm. The blue fluorescence of the PDHF polymer was significantly quenched due to efficient Förster resonance energy transfer occurring from the PDHF donor to the PtOEP acceptor. Example 3 Characterization of nanoparticle transducers

[0140] In this example, the spectroscopic and physical properties of the Pdot-GOx nanoparticle transducers were measured. Pdot particle size and morphology were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Dynamic light scattering was performed at 25 °C using a Malvern Nano ZS instrument with a 1 cm disposable polystyrene cuvette. Zeta potential measurements were performed on the same Malvern Nano ZS instrument. Samples for TEM measurements were prepared by drop casting of the Pdots dispersion onto a copper grid. The samples were dried at room temperature and then TEM images were obtained using a Hitachi H-600 microscope operating at 120 kV. UV-visible absorption spectra were recorded using a Schimadzu UV-2550 scanning spectrophotometer with a 1 cm glass cuvette. Fluorescence spectra were obtained using a Hitachi F-4500 fluorescence spectrophotometer. Fluorescence spectra of Pdot-GOx at different glucose concentrations were measured 10 min after adding glucose to the cuvette with an excitation wavelength of 380 nm. Intensity ratio of red emission to blue emission (I 648 / I 428 ) was calculated and the sensitivity curve was plotted.

[0141] Figure 4A shows the emission spectra of the Pdot-GOx transducer at different glucose concentrations. The curves vary from 0 mM to 20 mM glucose in increments of 2; at the peak at 648 nm, the curves are ordered in intensity from lowest to highest concentration, with 20 mM being the most intense. The Pdot-GOx bioconjugates exhibited bright red phosphorescence that was sensitive to glucose concentration, while the weak blue fluorescence remained constant. The constant blue fluorescence and sensitive red phosphorescence were useful for ratiometric sensing. This is useful for applications such as quantitative determination of glucose levels in cells and tissues. Figure 4B shows the ratiometric calibration plot (I 648 / I 428) as shown in Figure 4B. As shown in Figure 4B, the ratio of the emission at 648 nm to the emission at 428 nm showed a linear relationship with glucose concentration within the physiologically relevant range of about 4 mM to about 18 mM blood glucose. By defining the sensitivity as the slope of the linear portion of the curve, the Pdot-GOx sensor showed a 20% intensity change per mM, thus placing it among the most sensitive fluorescent glucose sensors. In addition, the dynamic range of the Pdot-GOx assembly can be tuned by changing the molar ratio of GOx to Pdot in the bioconjugation reaction.

[0142] Figure 4C illustrates the response curve of Pdot-GOx to glucose in aqueous suspension. The Pdot-GOx platform in aqueous environment exhibited a rapid glucose response within a few minutes. The sensor response was measured by a common fluorometer after adding glucose to the Pdot-GOx solution in a cuvette. The sensor response time was measured using a 1 cm glass cuvette at 25 °C in HEPES buffer (pH = 6.5) with a fluorescence spectrophotometer (Hitachi F-4500, Japan) equipped with a xenon lamp. All response curves were acquired using an excitation wavelength of 380 nm. The fluorescence intensity of Pdot-GOx (10 μg / mL) at 648 nm as a function of time was recorded by adding 30 μL of glucose solution to 3 mL of Pdot-GOx suspension. Keeping in mind that it takes time for glucose to diffuse into the excitation volume, the emission intensity at 648 nm increased to a constant value and reached a plateau within 10 min, indicating the rapid response of the sensor within a few minutes. To evaluate the selectivity of the Pdot-GOx sensor, the fluorescence spectra of Pdot-GOx were measured using 380 nm excitation 10 min after the addition of various carbohydrate species (10 mM). The response time was mainly determined by glucose diffusion and enzyme reaction.

[0143] The Pdot-GOx sensor shows excellent selectivity against potentially interfering substrates such as different carbohydrate derivatives due to the specific catalytic reaction of the GOx enzyme. Figure 4D illustrates the selectivity of the Pdot-GOx transducer for glucose over potentially interfering carbohydrates. The high selectivity provides a superior advantage in in vivo glucose monitoring, for example, compared to glucose sensing dyes based on boronic acid recognition. Furthermore, the operational stability was evaluated by reversible measurements, in which glucose was added to the Pdot-GOx solution for measurement and removed by a desalting column after each cycle. The sensor response remained unchanged for more than 10 repeated measurements. The rapid and reversible response allowed for continuous glucose measurement and easy incorporation into glucose sensors. After 2 months of storage at 25 °C and 4 °C, the sensor retained more than 95% of its initial response. These results demonstrated the excellent operational and storage stability of the Pdot-GOx bioconjugate. Example 4 Nanoparticle transducers sensitive to low analyte concentrations

[0144] In this example, a nanoparticle transducer with selected sensitivity is described, in particular, a nanoparticle transducer with analyte sensitivity in the low analytical range is provided. Figures 5A and 5B illustrate the use of nanoparticles densely coated with enzyme for sensing at low concentrations. Figure 5A illustrates the emission spectra of Pdots densely coated with GOx at various glucose concentrations, and Figure 5B shows the ratiometric calibration plot (I 648 / I 428 ) As shown in Figure 5A and Figure 5B, Pdots densely coated with GOx exhibit high sensitivity (25% per mM) in a relatively low analytical range (1-4 mM), which may be useful for glucose monitoring in hypoglycemia. Example 5 Biocompatibility of nanoparticle transducers

[0145] In this example, experiments are described that support the biocompatibility of the nanoparticle transducers described herein with cell lines. Biocompatibility is an important factor that determines whether nanoparticle transducers, such as Pdot-GOx nanoparticles, can be used as implantable sensors for in vivo glucose monitoring. A cell viability assay in HeLa cells was used to evaluate the cytotoxicity of Pdot-GOx nanoparticles. HeLa cell lines were used for cytotoxicity studies and intracellular glucose imaging. Dulbecco's Modified Eagle Medium (DMEM) with phenol red (Life Technologies Gibco, USA) supplemented with 10% fetal bovine serum (FBS), 50 U / mL penicillin, and 50 μg / mL streptomycin was used for cell culture. Cells were maintained in T75 cell culture flasks (NEST, Wuxi China) in an incubator (Thermo scientific, USA) in an atmosphere of air / CO2 (95:5) at 37°C. Cells were pre-cultured until confluence was reached before the experiment.

[0146] For cytotoxicity studies, cells were seeded in 96-well plates (7000 cells in 100 μL per well) for 24 h, and then Pdot, GOx, CAT (catalase), and Pdot-GOx (+CAT) (different final concentrations) were added into the cell culture medium, respectively. After incubating cells with various materials for 24 h, MTT (20 μL, 5 mg / mL, BioSharp, Hefei China) was added for 3 h. The medium was removed, and DMSO (150 μL) (Sigma-Aldrich, Shanghai China) was added to each well and gently shaken for 10 min at room temperature to dissolve any precipitates formed. The absorbance at 490 nm was measured by using a microplate reader (BioTek Cytation3, USA). Cell viability was expressed by the ratio of absorbance of cells incubated with Pdot-GOx solution to that of cells incubated with culture medium only.

[0147] Figures 6A-6D illustrate the cell viability of HeLa cells treated with various materials including Pdot-GOx transducers. Figures 6A, 6B, and 6C show the 24-hour cell viability of cells treated with various concentrations of Pdot-GOx transducers, GOx, and catalase, respectively. Figure 6A also illustrates the 24-hour cell viability of MCF-7 and GES-1 cells subjected to the same protocol as HeLa cells, showing that the viability is cell line independent. As shown by Figure 6B, GOx alone induced cell death due to the generation of hydrogen peroxide. However, as shown in Figure 6D, in the presence of catalase, Pdot-GOx nanoparticles (<10 μg / mL) are biocompatible with cells after 24 hours of incubation. In this concentration range, the Pdot-GOx sensor was able to enter the cells via endocytosis. HeLa cells were incubated with Pdot-GOx nanoparticles in sugar-free culture medium for 12 h.

[0148] For intracellular glucose sensing, 1.5 × 10 cells were plated on a 22-mm glass-bottom culture dish coated with poly-L-lysine (NEST, Wuxi China). 4 Hela cells were seeded and grown overnight (37°C, 5% CO2) in Dulbecco's modified Eagle's medium (DMEM). These cells were then cultured for 12 h in sugar-free DMEM containing Pdot-GOx (10 μg / mL) and catalase (CAT, 250 kDa, Sigma-Aldrich, Shanghai China) (300 nM). For 4 h of incubation, the cell culture was further supplemented with glucose (25 mM). The cells were then washed three times with warm PBS buffer before being viewed under a fluorescent microscope.

[0149] Fluorescence images were acquired with an inverted fluorescence microscope (Olympus IX71, Japan) equipped with a LUCPLFLN 20X objective with 0.45 NA. A mercury lamp generated excitation light that was filtered by a bandpass filter (Semrock FF01-377 / 50-25, Rochester, NY USA). Fluorescence signals were filtered by a bandpass filter (Semrock FF01-655 / 40-25, Rochester, NY USA) and imaged with an Andor iXon3 frame-transfer EMCCD (Andor, UK).

[0150] Figures 7A-7C illustrate intracellular glucose sensing in HeLa cells. Figure 7A shows HeLa cells as a control group without Pdot-GOx incubation, and Figure 7B shows cells incubated in sugar-free medium with Pdot-GOx nanoparticles for 24 h. As shown in Figure 7, fluorescence imaging showed obvious internalization of Pdot-GOx nanoparticles by cells. The medium of cultured cells was then supplemented with glucose. Figure 7C shows cells incubated with Pdot-GOx for 24 h and supplemented with glucose for 4 h. Compared with cells that were not fed with glucose, the intracellular luminescence was significantly enhanced, indicating the successful detection of intracellular glucose by the Pdot-GOx sensor. Example 6 Biocompatibility in an in vivo mouse model

[0151] In this example, we describe experiments using mouse subjects that support the biocompatibility of the nanoparticle transducers described herein for use in in vivo continuous analyte monitoring. All animal experiments used BALB / c male mice (Vital River Laboratories (VRL), Beijing China) weighing approximately 25 g and aged 8 weeks. Experimental group sizes included 3 animals per treatment, allowing for adequate reproducibility of results while reducing the number of animals. All imaged animals were included in the analysis. Animals were fasted for 8 hours prior to imaging for in vivo glucose monitoring. Each mouse was anesthetized with an intraperitoneal injection of 100 μL of chloral hydrate (10 wt%). Then, mice were subcutaneously injected with 200 μL of Pdot-GOx (50 μg / mL) on the back for continuous in vivo glucose monitoring.

[0152] Due to their exceptional brightness, Pdot transducers in the microgram range were detectable transcutaneously; three implantation sites with different concentrations were clearly distinguished using the small animal imaging system. Fluorescent animal imaging was acquired using a custom-built small animal imaging system equipped with an Andor iKon-M frame-transfer CCD (Andor iKon-M 934, UK) and a xenon light source (Asahi Spectra MAX-303, Japan). Fluorescent imaging was performed 25 min after anesthesia administration by using an excitation of 543 nm and an emission of 655 nm with an exposure time of 5 seconds. Immediately after imaging, blood samples were collected from the tail by quickly removing up to 1 cm of the mouse tail using a scalpel, and blood glucose concentrations were measured by using a standard glucose meter (Accu-Chek, Roche Diagnostics). Afterwards, the mice were intraperitoneally treated with 200 μL of sterile glucose solution (1 M) to increase blood glucose concentrations. After 15 min, 100 μL of sterile insulin saline solution (0.5 U / mL, Wanbang Biopharmaceuticals, Xuzhou China) was injected intraperitoneally to lower the blood glucose concentration. During this process, we captured fluorescent images and measured the blood glucose concentration every 5 min until the blood glucose concentration returned to the normal range. For the control group of animals, the mice were injected with the same dose of sterile saline instead of the simultaneous administration of glucose and insulin. Fluorescence imaging was performed following the same procedure. After the glucose monitoring experiment, the animals were euthanized by injecting an overdose of anesthesia.

[0153] Figure 8A and Figure 8B illustrate the fluorescence imaging of a mouse subcutaneously injected with Pdot-GOx transducer. Figure 8A illustrates the fluorescence imaging of three sites injected with Pdot-GOx transducer at different concentrations, and Figure 8B shows the fluorescence intensity of three different sites injected with Pdot-GOx. In fact, 0.25 μg (5 μL of 50 μg / mL) of Pdot-GOx was visible to the naked eye through the skin layer and hair under UV lamp excitation. Figure 9 shows images of a mouse subcutaneously injected with nanoparticle transducer under room light (top) and UV (bottom), where the fluorescence of the nanoparticle transducer is clearly visible. The ability to use a small amount of implant material helps reduce inflammation at the implant site and meet the biocompatibility requirements of implantable sensors.

[0154] The in vivo response of Pdot-GOx assemblies to increasing and decreasing blood glucose in live mice was demonstrated by whole-animal biophotonic imaging. Blood glucose levels in mice were increased to ~20 mM, which is within the hyperglycemic range, by intraperitoneal injection of glucose. Blood glucose levels were then decreased to ~10 mM, which is within the normoglycemic range, using insulin injections. For comparison, blood samples from cut tails were used and blood glucose concentrations were measured every 5 min using a commercial glucose meter. As a starting point, fluorescent images of Pdot-GOx sensors implanted in live mice were captured 25 min after anesthesia administration. The mice were then treated intraperitoneally with 200 μL of sterile glucose solution (1 M) to increase blood glucose levels. After 15 min, 100 μL of sterile insulin saline (0.5 U / mL) was administered intraperitoneally to decrease blood glucose levels.

[0155] During the whole process, fluorescence images were captured every 5 min to monitor the change in blood glucose concentration. Figure 10A and Figure 10B show the fluorescence images of mice with and without glucose / insulin treatment, respectively. As shown by the imaging results, the fluorescence signal increased immediately after glucose administration and decreased to the original level after insulin injection. In contrast, the fluorescence from the mice without glucose / insulin remains unchanged. Figure 10C shows the average luminescence intensity and standard deviation of the Pdot-GOx sensor as a function of blood glucose concentration for three mice administered glucose and insulin. As clearly shown in the figure, the luminescence intensity (circles) was closely correlated with the blood glucose level (squares) and always followed the concentration change due to blood glucose increase and decrease. For the mice that were not given glucose injection, both the luminescence intensity and glucose concentration remained relatively constant, as shown in Figure 10D. These observations clearly confirmed the excellent in vivo response of the Pdot-GOx sensor, which provided sufficient signal and sensitivity for transdermal monitoring of changes in blood glucose concentration. Example 7 Pharmacokinetics and stability of nanoparticle transducers

[0156] The pharmacokinetics and in vivo distribution of Pdot-GOx nanoparticles after subcutaneous injection were evaluated. Figure 11 illustrates the long-term glucose monitoring and in vivo distribution. Mice were sacrificed 30 days after sensor implantation. For biodistribution studies, mice were euthanized by an overdose of anesthesia after glucose monitoring experiments. Organs and tissues (heart, liver, spleen, lung, kidney, thigh muscle, and skin tissue near the implantation site) were removed for fluorescence imaging analysis. Finally, the excised organs and tissues were placed in formalin for histology and fluorescence analysis.

[0157] The excised organs and tissues were fixed overnight in 10% buffered formalin. The tissues were dehydrated through a series of graded ethanol baths, cleared using xylene, and then infiltrated with wax. The infiltrated tissues were then embedded in wax blocks. The tissues were then cut into 5 μm sections using a microtome and stained with hematoxylin and eosin (H&E). The histological sections were observed under an optical microscope. To assess the sensor distribution within the major organs and tissues after subcutaneous administration, fluorescent imaging was also used for analysis. Fluorescent images of tissue sections without H&E staining were captured using a fluorescent microscope equipped with a UV excitation filter (375 nm) and a red emission filter (655 nm). The subcutaneous tissues and various organs were excised for in vivo optical imaging.

[0158] Figure 11A shows fluorescence images of organs and skin tissues excised from mice subcutaneously injected with Pdot-GOx transducers (bottom) or sterile phosphate-buffered saline (top). As shown by the images, strong fluorescence was only observed from the subcutaneous tissue, indicating that Pdot-GOx remained at the implantation site for as long as one month. At this time point, no significant differences were observed in various organs, including liver, spleen, lungs, kidneys, heart, and muscle, compared to control animals, as shown in Figure 11B.

[0159] Further microscopic examination of tissue sections showed no detectable luminescence from these organs, consistent with the biophotonic imaging results. Figure 12 shows the histochemical analysis of tissue sections from mice 30 days after nanoparticle transducer injection. In vivo distribution studies showed that Pdot-GOx nanoparticles were mainly trapped within the subcutaneous implantation site and did not enter the peripheral blood. Water molecules in the injected solution may be rapidly absorbed by peripheral tissues, resulting in stable nanoparticle assemblies with little diffusion. This observation is consistent with the conclusion that Pdot aggregates on substrates have low solubility even in organic solvents.

[0160] Pdot-GOx assembly at the implantation site demonstrated sensitive glucose detection over an extended period of time, which was a major goal in the development of a continuous glucose monitor. In vivo optical imaging was performed at selected time intervals, i.e., 7, 15, and 30 days after injection of the Pdot-GOx sensor. Figures 13A-C show the fluorescence imaging of a live mouse at 7 days (Figure 13A), 15 days (Figure 13B), and 30 days (Figure 13C) after injection of the nanoparticle transducer.

[0161] The implanted Pdot-GOx assemblies exhibited excellent response without any noticeable signs of degradation over 7 and 15 days, as shown in Figure 11C and Figure 11D, respectively, where the fluorescence (circles) is closely aligned with glucose (squares). As shown in Figure 11E, after 30 days, the luminescence intensity of the sensor still correlated closely with blood glucose levels, always aligned with the concentration changes, with only a slight decrease in sensitivity from the relatively small intensity changes to glucose fluctuations in the up and down cycles. This slight degradation is likely due to the decrease in catalytic activity of the enzyme on the Pdot surface. Figure 11F illustrates the staining with hematoxylin and eosin of organ sections cut from mice injected with Pdot-GOx (right) and the PBS-injected control group (left). Compared to the control group, no noticeable organ damage or inflammation was observed. Histological analysis revealed no toxic effects in major organs and tissues, indicating biocompatibility with the Pdot material. Example 8 Nanoparticle transducers for glucose concentration monitoring using portable devices

[0162] As illustrated in this example, nanoparticle transducers enable quantitative measurement of in vivo glucose concentration without sophisticated imaging instrumentation. A portable fiber-optic based microspectrometer was used to detect the glucose response of subcutaneously implanted Pdot-GOx assemblies. Figure 14A shows a photograph of a mouse subcutaneously injected with Pdot-GOx under UV light (385 nm). The emission spectrum of the implanted Pdot-GOx sensor was measured by collecting transdermal fluorescence signals under the same protocol used for in vivo imaging. Figure 14B illustrates the kinetic changes in the fluorescence emission spectrum of a Pdot-GOx transducer implanted in a live mouse excited at 385 nm, together with an inset showing the change in the intensity ratio (650 nm to 480 nm) after glucose and insulin injection of the Pdot-GOx transducer implanted in a live mouse. As shown in Figure 14B, the emission intensity at 650 nm clearly reflected the increase and decrease of glucose levels due to the action of glucose and insulin. The highest peak is at 10 min, and each 10 min interval thereafter shows a gradual decline as glucose levels are lowered by insulin (except for the measurement at 0 min). The spectra and glucose levels at 0 and 40 min are nearly equal, and the lowest intensity curve corresponds to the measurement at 60 min. The blue emission at 480 nm remains constant and can be used as an internal reference for ratiometric measurements. Figure 14C shows the change in the intensity ratio (480 nm vs. 650 nm) of a Pdot-GOx sensor implanted in a live mouse after glucose and insulin injection. These results also closely follow the rise and fall of blood glucose measured by a glucometer using cut tail blood samples. Rapid and quantitative measurements using a compact, portable microspectrometer are particularly desirable for system integration with smartphones or the development of wearable devices. Example 9 Further chromophore and enzyme examples for nanoparticle transducers

[0163] This example illustrates some additional nanoparticle transducers using alternative chromophore configurations. The examples described are not exhaustive, but merely illustrative of the wide range of structures that can be formed using the techniques described herein. Although GOx is presented as an exemplary enzyme for the detection of glucose, alternative enzymes may be provided for monitoring other analytes; for example, the glucose-sensitive chromophore described herein may be substituted for alternative analytes by replacing the GOx enzyme with an enzyme that consumes oxygen by oxidizing the alternative analyte, resulting in a change in the fluorescence of the associated chromophore. Thus, any oxygen-sensitive chromophore can be used to provide a transducer fluorescent signal when combined with an oxygen-consuming enzyme for the detection of any analyte, provided that the enzyme catalyzes an oxygen-consuming reaction with the analyte.

[0164] 15A-15D show the fluorescence emission from a nanoparticle transducer with chromophores including do-PFO, 10% PdOEP, and 10% PSMA in a Pdot-GOx transducer for the detection of glucose. FIG. 15A shows the emission spectrum for multiple glucose concentrations. FIG. 15B shows a calibration plot of the transducer for detecting glucose for a concentration range of 0 mM to about 20 mM, showing a ratiometric response curve across the range. FIG. 15C shows in vivo mouse response data showing emission curves that substantially overlap the measured glucose concentrations. FIG. 15D shows images with time data for glucose-fed mice (top image) and a control group that was not fed glucose (bottom image). FIG. 15C shows in vivo mouse response data showing emission curves that substantially overlap the measured glucose concentrations. FIG. 15D shows images with time data for glucose-fed mice (top image) and a control group that was not fed glucose (bottom image).

[0165] Figures 16A and 16B show the fluorescence emission from a nanoparticle transducer with chromophores including PSMA, 1% PdOEP, and 0.1% coumarin 1 in a Pdot-GOx transducer for the detection of glucose. Figure 16A shows the emission spectrum for multiple glucose concentrations. Figure 16B shows a calibration plot of the transducer for detecting glucose for a concentration range of 0 to about 20 mM, showing the ratiometric response curve.

[0166] Figures 17A and 17B show the fluorescence emission from a nanoparticle transducer with chromophores including PSMA, 1% PtOEPK, and 0.1% Nile Red in a Pdot-GOx transducer for the detection of glucose. Figure 17A shows the emission spectrum for multiple glucose concentrations. Figure 17B shows a calibration plot of the transducer for detecting glucose for a concentration range of 0 to about 20 mM, showing the ratiometric response curve. Example 10 Nanoparticle sensors for the detection of reactive analytes

[0167] In a further aspect of the present disclosure, a nanoparticle sensor is provided that allows the detection of reactive analytes without the need for catalytic enzymes.The nanoparticle sensor can be used to detect ascorbic acid, for example, by providing nanoparticles such as Pdots that contain oxygen-sensitive chromophores.Ascorbic acid is a reducing agent, it spontaneously reacts and consumes oxygen.Therefore, the sensor can be used to detect the presence of ascorbic acid, for example, when ascorbic acid is present in pharmaceutical amounts.

[0168] 18A and 18B show fluorescence emission from an exemplary nanoparticle sensor having a chromophore containing PDHF, 10% PtOEP, and 10% PSMA in a Pdot nanoparticle sensor for the detection of ascorbic acid. FIG. 18A shows the emission spectrum for multiple ascorbic acid concentrations. FIG. 18B shows a calibration plot of the sensor detecting ascorbic acid for a concentration range of about 2 mM to about 20 mM, showing a ratiometric response curve over the entire range.

[0169] Figures 19A and 19B illustrate continuous in vivo monitoring of ascorbic acid in live mice using nanoparticle sensors. Figure 19A illustrates the fluorescence intensity of nanoparticle sensors injected into live mice administered different concentrations of ascorbic acid. Figure 19B shows in vivo fluorescence imaging of varying ascorbic acid concentrations in live mice injected with Pdot sensors.

[0170] Figures 20A and 20B illustrate the monitoring of ascorbic acid blood levels by a miniaturized optical detection system. Figure 20A shows the kinetic changes in the fluorescence emission spectrum of Pdots injected into a live mouse with respect to the blood concentration of ascorbic acid, excited at 385 nm. Figure 20B shows the response of fluorescence intensity to the blood concentration of ascorbic acid as a function of time after intravenous administration of ascorbic acid. Thus, in vivo detection of ascorbic acid using a nanoparticle sensor containing an oxygen-sensitive chromophore was demonstrated. Example 11 A wearable device for subject glucose monitoring using nanoparticle transducers

[0171] The long-term stability, high reliability, and rapid reversible response of the nanoparticle transducers described herein allow the use of Pdot-GOx transducers for in vivo glucose monitoring. In this example, as demonstrated in the in vivo studies described, Pdot-GOx transducers are injected subdermally to allow continuous transdermal monitoring with a probe that uses transdermal UV illumination and detects emitted fluorescence to continuously determine blood glucose levels. A wearable device is provided that includes an optical sensor that is sensitive to the fluorescent emission of the Pdot-GOx transducers and an illumination source that provides light of a wavelength that provides energy for the fluorescence of the nanoparticles. The optical sensor is adapted to detect the fluorescence transmitted through the skin of the subject to allow continuous transdermal monitoring of blood glucose. The optical sensor is coupled to a processor and memory located within the device. The memory includes instructions that, when executed, cause the processor to use the optical sensor to measure the fluorescence emitted by the multiple Pdot-GOx transducers in response to the presence of glucose and illumination from a light source. The device further includes a battery for providing power. Example 12 Nanoparticle transducers as glucose sensors for artificial pancreas

[0172] In this embodiment, the Pdot-GOx transducer is used as a glucose sensor in the artificial pancreas to provide a feedback loop that triggers insulin dispensing if the measured glucose is high or halts insulin dispensing if the measured glucose is low. The device includes an alarm function that is triggered in response to glucose rising above or falling below a predetermined threshold. The artificial pancreas is an implantable device that includes a glucose-sensitive nanoparticle transducer, an illumination source that provides UV light, and an optical detector that is adapted to detect fluorescence at the nanoparticle emission wavelength. The device further includes a processor for determining blood glucose levels from the measured fluorescence using a calibration curve similar to that described herein, and for controlling insulin dispensing from the storage chamber to the patient via an insulin pump. The storage chamber may be replenished from a source external to the patient as needed. The detector, processor, and pump provide a feedback loop to maintain blood glucose levels within a predetermined range, and an alarm may be triggered if the glucose level falls outside the predetermined range. The predetermined range is user adjustable to allow for tightening or loosening of glycemic control. The device also comprises a memory for logging glucose levels as a function of time for calibration purposes as well as for providing a glucose level history to a user.The device further comprises a transmitter to enable optional wireless communication with a mobile device and / or over a computer network. Example 13 Contact lenses containing nanoparticle transducers - Patents.com

[0173] In this example, a contact lens is provided that includes a nanoparticle transducer for detecting an analyte in the tear fluid of a subject wearing the lens on the subject's eye. When worn by the subject, tear fluid from the eye passes through the lens and the nanoparticle transducer contacts the tear fluid. The lens includes a nanoparticle transducer including glucose oxidase and a chromophore configured as described above for detecting glucose. The transducer is embedded in a substantially transparent membrane of the contact lens shaped to provide vision correction for the wearer. The subject is provided with a scanner that can be placed on the eye and includes a detector for providing illumination to stimulate fluorescence of the nanoparticle transducer and detecting the induced fluorescence. The nanoparticle transducer emits light of varying intensity in response to the concentration of the analyte, thereby providing a measurement of the concentration of the analyte in the subject's tear fluid. The concentration of the analyte in the tear fluid correlates with the concentration of the analyte in the blood; thus, this measurement provides a measurement of the concentration of the analyte in the blood. The scanner can also perform retinal scans for detection of conditions such as diabetic retinopathy. Example 14 Device for detecting blood glucose levels from sweat measurements

[0174] In this example, a device is provided for measuring blood glucose levels based on detecting glucose in a subject's sweat. The device is provided, comprising a wearable band, a plurality of nanoparticle transducers, an illumination source, and an optical sensor. The nanoparticle transducer comprises glucose oxidase and a chromophore configured as described above for detecting glucose. The nanoparticle transducer is disposed on a surface of the device such that when worn by a subject, the nanoparticle transducer contacts the subject's skin. As sweat is typically present on the skin, the nanoparticle transducer can thus contact the subject's sweat. The subject's sweat contains glucose that varies proportionally to glucose in the blood; thus, measuring the glucose concentration in the sweat allows the determination of the blood glucose level.

[0175] An illumination source of the device is positioned to provide illumination to a nanoparticle transducer in contact with the skin of the subject to induce fluorescence. The nanoparticle transducer produces fluorescence that varies based on the concentration of glucose in a thin film of sweat on the contacted skin. This fluorescence is detected by an optical sensor coupled to a processor. The processor determines the glucose concentration in the sweat based on the fluorescence detected by the optical sensor. The processor then determines the blood glucose concentration based on the glucose concentration in the sweat. Example 15 Nanoparticle transducer based on detection of product reaction element (H2O2)

[0176] In this example, nanoparticle transducers for detecting product reaction elements were produced and characterized in an illustrative system for detection of fluid constituents. Aqueous dispersions of nanoparticles were formed using a reprecipitation method. In one preparation, the functional polymer PSMA and hydrogen peroxide-sensitive dye, respectively, were dissolved in anhydrous tetrahydrofuran (THF) by stirring overnight under an inert atmosphere to make a stock solution of 1 mg / mL. These solutions were diluted and mixed in THF to produce a solution mixture, which was further added immediately to Milli-Q water in a bath sonicator while the mixture was sonicated. THF was removed by nitrogen stripping, and the solution was concentrated to 5 mL on a hot plate at 90°C before being filtered through a 0.2 micron filter. During nanoparticle formation, the maleic anhydride units of the PSMA molecules hydrolyzed in an aqueous environment, generating carboxyl groups on the Pdots. H2O2-sensitive dye molecules were either encapsulated inside the nanoparticles or associated with the polymer. Hydrogen peroxide (H2O2) is a by-product of cellular respiration and can be generated during many enzyme-catalyzed reactions. For example, H2O2 concentration can be influenced by the glucose oxidation reaction catalyzed by glucose oxidase. The change in hydrogen peroxide concentration induced by glucose can be converted into an optical signal by using a H2O2-responsive Pdot transducer. Figure 21 shows the spectral response of a H2O2-based nanoparticle transducer containing a nanoparticle-GOx assembly. Upon the addition of glucose, a clear fluorescence increase was observed due to the presence of H2O2 generated by the glucose oxidation reaction catalyzed by GOx. Thus, the nanoparticle-GOx assembly functions as a nanoparticle transducer for detecting an analyte (glucose in this example) based on the fluorescence mediated by the product of the reaction of the analyte catalyzed by the enzyme bound to the assembly.

[0177] Other nanoparticles or Pdot transducers can be prepared as described above, including ion or metal ion-based Pdot transducers, pH-based Pdot transducers, and heat-based Pdot transducers. Metal ions play an important role in the biological functions of many enzymes. Ions can function as electron donors or acceptors, Lewis acids, or structural regulators. Ions directly participate in catalytic processes. For example, carboxypeptidase A, hepatic alcohol dehydrogenase, aspartate transcarbamoylase, and alkaline phosphatase show different roles of metal ions in metalloenzymes, while zinc ions play an essential role in nucleotide polymerases to maintain normal growth and development. Thus, ion-sensitive Pdots can be used to construct nanoparticle transducers for the detection of analytes, which generate fluorescence that varies with analyte concentration by incorporating enzymes that catalyze reactions that change ion concentration. Similarly, proton donors and acceptors, such as acids and bases, can donate and accept protons in enzyme catalysis. For example, the first step of the serine protease catalytic mechanism involves an active site histidine accepting a proton from a serine residue. Thus, by incorporating an enzyme that catalyzes a pH-altering reaction, pH-sensitive Pdots can be used to construct nanoparticle transducers for the detection of analytes, generating fluorescence that varies with analyte concentration.

[0178] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous changes, modifications, and substitutions will occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of these claims and their equivalents are covered by the scope of the invention.

Claims

1. A nanoparticle transducer for measuring an analyte concentration, comprising: A nanoparticle comprising Pdots, the Pdots comprising one or more chromophores comprising a fused semiconducting chromophore polymer; an enzyme bound to the nanoparticle and configured to catalyze a reaction comprising a plurality of reaction elements, the enzyme being covalently bound to the Pdot, the enzyme being an oxygen-consuming enzyme; Including, the reaction elements include one or more reactants including the analyte and one or more products, and the amount of fluorescence emitted from the chromophore is determined by the concentration of a reaction element among the plurality of reaction elements; the fluorescence emitted from the one or more chromophores comprises a signal fluorescence wavelength and a control fluorescence wavelength; the fluorescence emitted from the one or more chromophores comprises a fluorescence ratio equal to a ratio of an amount of fluorescence emitted at the signal wavelength to an amount of fluorescence emitted at the control fluorescence wavelength, the fluorescence ratio being determined by a concentration of the reaction element among the plurality of reaction elements; the fluorescence ratio varies ratiometrically with the concentration of the analyte; a reaction element among the plurality of reaction elements includes oxygen, and the amount of fluorescence emitted from the chromophore is determined by a concentration of the oxygen; a chromophore of the one or more chromophores is chemically attached to another chromophore of the one or more chromophores, and a critical dimension of the nanoparticle transducer is less than about 1000 nm; The chromophore comprises a semiconducting polymer and a dye, the dye being an oxygen sensitive dye, the dye being contained in the Pdot, and the dye and the semiconducting polymer interact to produce enhanced fluorescence. Nanoparticle transducers.

2. The nanoparticle transducer of claim 1, wherein the analyte includes glucose.

3. A nanoparticle transducer as described in claim 1 or 2, wherein the enzyme includes glucose oxidase.

4. A nanoparticle transducer described in any of claims 1 to 3, wherein the one or more reactants include glucose and oxygen, and the one or more products include hydrogen peroxide and D-glucono-1,5-lactone.

5. A nanoparticle transducer described in any one of claims 1 to 4, further comprising catalase.

6. A nanoparticle transducer described in any one of claims 1 to 5, wherein the amount of fluorescence emitted from the chromophore is determined by the concentration of the reactant.

7. A nanoparticle transducer described in any one of claims 1 to 6, wherein the amount of fluorescence emitted from the chromophore is determined by the concentration of the product.

8. A nanoparticle transducer described in any one of claims 1 to 7, wherein the fluorescence emitted from the chromophore varies ratiometrically with the concentration of the analyte within a certain analyte concentration range.

9. A nanoparticle transducer as described in claim 8, wherein the analyte is glucose and the analyte concentration range is within a range between 0 and approximately 20 mM of glucose.

10. The nanoparticle transducer of claim 9, wherein the analyte concentration range is within the range of about 3 mM glucose to about 15 mM glucose.

11. A nanoparticle transducer described in any one of claims 1 to 10, wherein the nanoparticles contain at least 20 weight percent chromophores.

12. A nanoparticle transducer described in any one of claims 1 to 11, wherein the nanoparticles contain at least 50 weight percent chromophores.

13. A nanoparticle transducer described in any one of claims 1 to 12, wherein the nanoparticles contain at least 90 weight percent chromophores.

14. A nanoparticle transducer described in any one of claims 1 to 13, wherein the nanoparticles contain approximately 100 weight percent chromophore.

15. The method of claim 1, wherein the nanoparticle comprises a second chromophore for fluorescent detection of a second analyte; a second enzyme is bound to the nanoparticle and configured to catalyze a second reaction involving a second plurality of reaction elements; the second plurality of reaction elements includes a second reactant or reactants including the second analyte and a second product or products, and an amount of fluorescence emitted from the second chromophore is determined by a concentration of a second reaction element of the second plurality of reaction elements; the fluorescence of the second chromophore comprises a wavelength that is different from the fluorescence of other chromophores; A nanoparticle transducer according to any one of claims 1 to 14.

16. A method for producing a nanoparticle comprising: a second enzyme bound to the nanoparticle and configured to catalyze a second reaction comprising a second plurality of reaction elements; Further comprising: the second plurality of reaction elements includes a second reactant or reactants including a second analyte and a second product or products, and an amount of fluorescence emitted from the second chromophore is determined by a concentration of a second reaction element of the second plurality of reaction elements. A nanoparticle transducer according to any one of claims 1 to 15.

17. A nanoparticle transducer described in any one of claims 1 to 16, wherein the enzyme is selected from the group consisting of ascorbic acid oxidase, glutamate oxidase, dopamine beta-hydroxylase, cholesterol oxidase, alcohol oxidase, amine oxidase, and cytochrome P450.

18. A nanoparticle transducer described in any one of claims 1 to 17, wherein the analyte is selected from the group consisting of ascorbic acid, glutamate, dopamine, cholesterol, and alcohol.

19. A nanoparticle transducer as described in any one of claims 1 to 18, wherein the analyte is an amino acid.

20. A nanoparticle transducer described in any one of claims 1 to 18, wherein the analyte is a drug.

21. A nanoparticle transducer described in any one of claims 1 to 18, wherein the analyte is a protein, a nucleic acid molecule, or a transmitter molecule.

22. A nanoparticle transducer described in any one of claims 1 to 18, wherein the analyte is a carbohydrate, lipid, or metabolite.

23. A nanoparticle transducer as described in any one of claims 1 to 18, wherein the analyte is a sugar.

24. A nanoparticle transducer described in any one of claims 1 to 23, wherein the analyte concentration is a blood concentration.

25. A nanoparticle transducer as described in any of claims 1 to 24, comprising a plurality of enzymes which catalyze a plurality of respective reactions each comprising a respective plurality of reaction components, the analyte being a reactant of one of the plurality of reactions, and the fluorescence emitted from the chromophore being determined by the concentration of at least one of the reaction components of the reactions.

26. A nanoparticle transducer described in any one of claims 1 to 25, wherein the critical dimension of the nanoparticle transducer is less than about 100 nm.

27. ​​A nanoparticle transducer described in any one of claims 1 to 26, wherein the critical dimension of the nanoparticle transducer is in the range of about 15 to about 45 nm.

28. An apparatus for measuring a concentration of an analyte in a biological fluid, comprising a plurality of fluorescent nanoparticle transducers according to any one of claims 1 to 27, an optical sensor coupled to a processor and a memory; an illumination source configured to irradiate the plurality of fluorescent nanoparticles with light thereby inducing fluorescence therefrom; Further equipped with the memory includes instructions that, when executed, cause the processor to use the optical sensor to measure fluorescence emitted by the plurality of fluorescent nanoparticle transducers. Device.

29. The device described in claim 28, wherein the plurality of fluorescent nanoparticle transducers are adapted to be positioned under the patient's skin, and the optical sensor is adapted to detect fluorescence transmitted through the skin by the plurality of fluorescent nanoparticle transducers when facing the skin.

30. The apparatus of claim 28 or 29, wherein the optical sensor is configured to detect an amount of signal fluorescence at the signal fluorescence wavelength and an amount of control fluorescence at the control fluorescence wavelength, and the memory includes instructions for causing the processor to determine a measured fluorescence ratio based on the measured amounts of signal fluorescence and control fluorescence.

31. The apparatus of claim 30, wherein the memory includes instructions that cause the processor to determine a concentration of the analyte based on the measured fluorescence ratio.

32. The device described in any of claims 28 to 31, wherein the biological fluid is blood, lymph, saliva, tears, interstitial fluid, cerebrospinal fluid, or urine.

33. A contact lens for detecting glucose concentration, comprising a permeable transparent membrane wearable on the eye and containing a plurality of nanoparticle transducers as described in any one of claims 1 to 27, said nanoparticle transducers configured to generate fluorescence in response to illumination by a scanner, the amount of fluorescence providing a signal detectable by said scanner for determining the concentration of glucose in a fluid at the surface of the eye.

34. A contact lens as described in claim 33, wherein the membrane of the lens is shaped to correct human vision when worn.

35. A device for measuring glucose concentration from sweat of a subject, comprising: A plurality of fluorescent nanoparticle transducers according to any one of claims 1 to 27, positioned by the device to contact the skin of the subject when worn; an illumination source configured to irradiate the plurality of fluorescent nanoparticles with light thereby inducing fluorescence therefrom; an optical sensor disposed within the device and oriented to detect fluorescence from the plurality of fluorescent nanoparticle transducers; a processor coupled to the optical sensor and configured to determine a concentration of glucose in the sweat based on the fluorescence detected by the optical sensor; A device comprising:

36. The device of claim 35, wherein the processor is further configured to determine a concentration of glucose in the blood based on the concentration of glucose in the sweat.

37. A method for determining an analyte concentration in a fluid, comprising: contacting the fluid with a nanoparticle transducer according to any one of claims 1 to 27; measuring the fluorescence of the nanoparticle transducer; determining a concentration of the analyte in the fluid based on the measured fluorescence; A method comprising:

38. The method of claim 37, wherein the determining step includes a step of comparing the measured fluorescence to a calibration curve of the nanoparticle transducer to determine the analyte concentration in the fluid.

39. The method of claim 37 or 38, wherein the fluid is blood, sweat, or tears.

40. A method according to any one of claims 37 to 39, wherein the measuring step includes a step of measuring a plurality of fluorescent wavelengths, and the determining step is based on a ratio of the measured wavelengths.

41. A method for measuring a concentration of an analyte in a fluid, comprising: contacting the fluid with Pdots, the Pdots comprising an enzyme configured to catalyze a reaction comprising one or more chromophores comprising a fused semiconducting chromophore polymer and a plurality of reaction elements, the enzyme being covalently attached to the Pdots, the enzyme being an oxygen consuming enzyme, the reaction elements comprising one or more reactants and one or more products comprising the analyte, an amount of fluorescence emitted from the one or more chromophores being determined by a concentration of a reaction element among the plurality of reaction elements, the fluorescence emitted from the one or more chromophores comprising a signal fluorescence wavelength and a control fluorescence wavelength, the fluorescence emitted from the one or more chromophores being equal to a ratio of an amount of fluorescence emitted at the signal wavelength to an amount of fluorescence emitted at the control fluorescence wavelength. a fluorescence ratio, the fluorescence ratio being determined by a concentration of the reaction element of the plurality of reaction elements, the fluorescence ratio varying ratiometrically with a concentration of the analyte, a reaction element of the plurality of reaction elements comprising oxygen, the amount of fluorescence emitted from the chromophore being determined by the concentration of oxygen, a chromophore of the one or more chromophores being chemically attached to another chromophore of the one or more chromophores, the Pdot being included in a Pdot transducer, the critical dimension of the Pdot transducer being less than about 1000 nm, the chromophore comprising a semiconducting polymer and a dye, the dye being an oxygen sensitive dye, the dye being contained in the Pdot, and the dye and the semiconducting polymer interacting to produce enhanced fluorescence; measuring the fluorescence emitted by the one or more chromophores; determining a concentration of the analyte in the fluid based on the fluorescence; Including, The method, wherein the analyte undergoes a reaction in the fluid, the reaction changing a concentration of the fluid constituent.

42. The method of claim 41, wherein the determination of the concentration comprises measuring fluorescence at the signal fluorescence wavelength and a control fluorescence wavelength, determining a measured fluorescence ratio based on the measurements, and determining the concentration of the analyte based on the measured fluorescence ratio.

43. The method of any of claims 41 to 42, wherein the analyte is ascorbic acid and the fluid component is oxygen.

44. The method of claim 43, wherein the determined concentration is between 1 mM and 20 mM of ascorbic acid.

45. The method of any of claims 41 to 44, wherein the fluid is blood, sweat, or tears.

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