WO2008044138A1 - Optical nanosensor for detection of reactive oxygen species - Google Patents

Abstract

The invention relates generally to optical nanosensors of reactive oxygen species (ROS) and more particularly to nanosensors suitable for real-time monitoring of living cells. The. invention further relates to methods of use of such sensors, in particular, to methods of identifying compounds that stimulate or inhibit ROS production in particular cells.

Description

Optical nanosensor for detection of reactive oxygen species

The field of the invention relates generally to optical nanosensors of reactive oxygen species (ROS) and more particularly to nanosensors suitable for real-time monitoring of living cells. The invention further relates to methods of use of such sensors, in particular, to methods of identifying compounds that stimulate or inhibit ROS production in particular cells.

Background

Reactive oxygen species (ROS) are associated with a variety of pathological conditions, including cancer, diabetes, aging, tissue damage in ischemia and reperfusion, as well as other cardiovascular, pulmonary, inflammatory, neurodegenerative, renal, endothelial, and autoimmune disorders. (1-8). There is mounting interest in specialized anti-oxidant compounds as therapeutic interventions. However, details about how ROS contribute to disease etiology remain largely unknown. ROS have important signalling and gene regulatory roles in normal, healthy tissues. Accordingly, development of safe and effective anti-oxidant therapeutics depends on continuing research into ROS roles in normal and pathological physiology.

The term "reactive oxygen species" refers to oxygen radicals, or species that have an unpaired electron, as well as to hydrogen peroxide (H₂O₂), which can generate oxygen radicals through interactions with reactive transition metals. The reduction of atmospheric oxygen to H₂O during normal aerobic metabolism produces oxygen radicals as "by- products." An estimated 4-5% of respiratory oxygen consumption is ultimately released as intracellular "metabolic" ROS. (9). The best known "metabolic" ROS is the superoxide radical 02^", which is produced by reduction of atmospheric, molecular oxygen. Superoxide and other ROS are highly reactive and able to generate additional ROS in a potentially catastrophic "chain reaction." This "chain reaction" contributes to DNA damage, protein degradation and lipid peroxidation, which are implicated in etiology of atherosclerosis and other disease conditions. O₂ ^" is rapidly dismutated to the more stable H₂O₂ by ubiquitous superoxide dismutase enzymes. However, hydrogen peroxide, which can readily diffuse through cellular and intracellular membranes, is, itself, an important oxidant. H₂O₂ generates super-reactive hydroxyl radical (OH^') in interactions with water and transition me- tals, particularly iron and copper. Normal cells possess sophisticated anti-oxidant defence systems involving a large family of free radical quenching enzymes, endogenous anti-oxidants such as glutathione, and also dietary vitamins, such as vitamins C, E and beta-carotene (10-21). When these anti-oxidant defences are unable to fully quench ROS production, cells are said to be in "oxidative stress." However, "oxidative stress" is not universally pathological. Low levels of localized "oxidative stress" play a critical regulatory role in many instances. ROS are critical modulators of intracellular physiological processes. The best studied regulatory ROS is nitric oxide (NO-). However superoxide and hydrogen peroxide also play important regulatory and signalling roles. Unlike NO^', no specific target receptors for O₂" and H₂O₂ have yet been identified.

Although mechanisms of action remain unclear, O₂ ^'" and H₂O₂ modulate intracellular calcium homeostasis and play a general regulatory role in many tissues (22). Specialized NAD(P)H oxidases and other enzymes generate ROS in response to agonists or other stimuli. In vitro studies demonstrate that both O₂ ^'" and H₂O₂ inhibit protein phosphatases and modulate signalling pathways. For example, in vascular smooth muscle cells, inhibition of NADPH oxidase expression reduces levels of agonist-stimulated ROS production. This is associated with activation of a variety of protein kinases, including extra-cellular signal- regulated kinase, p38 mitogen-activated kinase, and cell survival kinase. (23-25). Reduction of agonist-stimulated ROS production was shown to reduce angiotensin-ll induced hypertrophy, serum-induced cell growth, and expression of platelet-derived growth factor. ROS also regulate expression of several classes of genes in vascular smooth muscle, including vasoactive molecules, chemotaxis factors, and cell surface adhesion molecules. (26).

O₂ ^'" and H₂O₂ play a particularly important role in regulation of inflammation processes: Targeting and accumulation of monocytes and macrophages is subject to ROS regulation (27-29) Activated neutrophils generate both O₂" and H₂O₂, as do polymorphonuclear and mononuclear cells (30).

Highly sensitive techniques for monitoring intracellular ROS generation are required to investigate the role of ROS in normal and pathological physiology. Because O₂ ^" is rapidly dismutated, H₂O₂ is generally used as an indirect measure, or "biomarker," of O₂" production. Previously, intracellular H₂O₂ measurements have relied primarily on direct use of specific, cell-permeable fluorophores as optical reporters. (31-38). These techniques have severe limitations. Direct loading of fluorescent dyes can be toxic to cells. Cell permeable dyes can reaccumulate in extracellular media, and may react with extracellular oxidants. Proteins and enzymes of the cellular milieu often interfere with free dye measurements, rendering the measurements effectively qualitative rather than precisely quantitative. Further, "free dye" measurements cannot provide any details about subcellular localization of ROS production.

Techniques for intracellular ROS measurement can be greatly improved through use of nanosensor devices which utilize an enzyme or other biological molecule as sensor, embedded within a protective nanoparticle. One such device is reported by Kim et. al, Analytical Chemistry (2005) 77:6828, who encapsulated horseradish peroxidase (HRP) within polyethylene glycol hydrogel spheres. In general, H₂O₂ detection by peroxidase- based assays relies on oxidation of a reporter dye that serves as reductant substrate for the peroxidase reaction. In the presence of H₂O₂, a suitable peroxidase enzyme, such as HRP, oxidizes hydrogen donors according to the following scheme:

(1 ) SP + H₂O₂ -> SP- H₂O₂

(2) SP- H₂O₂ + AH2 -> SP + H20 + A

where SP is suitable peroxidase, AH2 is reduced and A oxidized hydrogen donor (reporter dye).

An estimate of H₂O₂ can then be derived from decrease in fluorescence of initially fluorescent reporter dyes or from increase in fluorescent reaction product arising from oxidation of initially non-fluorescent reporter dyes. Spectroscopic signals other than fluorescence can also be used to monitor oxidation of some reporter dyes, although fluorescence is generally preferable. The nanosensor of Kim et al. catalysed oxidation of the reporter dye 10-acetyl-3,7,-dihydoxyphenoxazine, which was added to the cell culture surroundings. Fluorescence spectroscopy thereby provided a qualitative estimate of intracellular H₂O₂ concentrations in activated macrophages that had incorporated the nanosensors by direct phagocytosis. A significant disadvantage of this prior art ROS nanosensor is its comparatively large size, having a particle size distribution with average particle size 302.5 nm. Such large sized particles are more difficult to use with methods of targeting nanosensors to particular cells, other than macrophage phagocytosis. Also, in general, incorporation of such larger particles produces a larger disruption of intracellular milieu, which is important in most cells. ROS nanosensors of the prior art also do not protect cells from toxic effects of reporter dyes and do not provide much improvement over free dye in precision of quantitative measurements. Some embodiments of the present invention provide an optical ROS nanosensor suitable for intracellular use that provides the distinct advantage of smaller average particle size, achieved using acrylamide for biologically localised embedding. Some embodiments of the ROS nanosensor of the present invention further provide advantages of protecting cells from potential toxicity of reporter dyes as well as protecting reporter dyes from interference with cellular components, achieved by Incorporating both sensor enzyme and reporter dye within a nanoparticle. Optionally, incorporating within the nanoparticle one or more reference dyes that are unaffected by the peroxidase reaction provides an internal standard, or basis for normalization, that permits accurate, quantitative, ratiometric ROS measurements in vivo based on in vitro calibration. Ratiometric measurements are independent of nanosensor concentration and, also, independent of the ratio of active peroxidase, reporter and reference dye incorporated in the nanoparticle. Accordingly, some embodiments of the present invention provide a nanosensor that incorporates, within a nanoparticle, a suitable peroxidase enzyme, one or more reporter dyes and, optionally, one or more reference dyes.

Some embodiments of the invention further provide methods of use of the ROS nanosensor including methods of identifying compounds that stimulate or inhibit ROS production in particular cells.

Summary of the Invention

One aspect of some embodiments of the invention provides an optical nanosensor suitable for intracellular detection of ROS in vivo. A suitable peroxidase enzyme such as horseradish peroxidase is encapsulated, along with one or more reporter dyes, in a nanoparticle. The encapsulated peroxidase catalyses reduction of hydrogen peroxide or superoxide to water using a reporter dye as hydrogen donor. Differences in fluorescence or other spectroscopic signal between the reduced and oxidized species of a reporter dye provide a quantitative measurement of ROS. Optionally, a reference dye that is unaffected by the peroxidase reaction is also incorporated in the particle. This provides an internal standard, or basis for normalization, that permits accurate quantitative measurements in vivo based on in vitro calibration of relative signal between reporter and reference dyes.

In some embodiments, the nanosensors of the present invention can be produced with particle size distributions having average particle sizes of less than 50 nm in diameter. These nanoparticles can be incorporated within or adhered to living cells and provide measurements of intracellular or extracellular hydrogen peroxide at concentrations as low as 10 nM, with resolution of subcellular localization.

In other aspects, some embodiments of the invention provide methods of use of the optical nanosensor. As an intracellular or extracellular means for detection of reactive oxygen species, the nanosensor provides a sensitive tool with which to investigate ROS roles in normal and pathological physiology.

In one embodiment, a method is provided for identifying compounds that affect ROS production by a cell. As will be readily understood by those skilled in the art, therapeutic manipulation may involve either stimulation or inhibition of intracellular or extracellular ROS generation by a variety of specific cells. The nanosensor can be incorporated within or adhered to a cell which is then contacted with one or more test compounds to be screened. Optionally, such cells may be subject to one or more treatments that stimulate or suppress ROS production, in order to stimulate a response that an ideal test compound should enhance, counteract or otherwise modulate. The nanosensor can then be used to monitor the effects of test compounds in such cells. Compounds can then be selected that exert a desired effect on ROS production.

Brief description of the figures

Figure 1 is a schematic illustration of some embodiments of the nanosensors.

Figure 2 shows a fluorescence trace of catalysis of the peroxidase reaction by nanoparticle- embedded HRP using free (diffusible) (a) and dextran-linked (sterically hindered) (b) fluorescein as reductant substrate. The excitation wavelength was 470 nm and emission was obtained at 525 nm. As shown, nanoparticle-embedded HRP retains peroxidase activity and is effectively protected from contact with molecules larger than nanoparticle pore sizes.

Figure 3 shows a 340 nm absorbance trace of catalysis of the peroxidase-oxidase reaction by embedded HRP using NADH as reductant substrate. As shown, nanoparticle-embedded HRP retains peroxidase-oxidase activity. Figure 4 shows peroxidase activity of nanoparticle-embedded HRP at various time points during dialysis of nanoparticles. As shown, embedded HRP experiences almost no leaching from the nanoparticle.

Figure 5 shows a fluorescence trace of catalysis of the peroxidase reaction by a nanoparticle embedding both HRP and fluorescein, used as reductant substrate and reporter dye. Fluorescein is excited at 470 nm and emission is obtained at 525 nm. As shown, peroxidase activity can be readily monitored using the nanosensor comprising both HRP and reporter dye.

Figure 6 shows phase contrast and fluorescence microscope images of cultured liver cells in which similar nanosensors have been incorporated by gene gun insertion.

Figure 7 shows phase contrast and fluorescence microscope images of yeast cells in which similar nanosensors have been incorporated by electroporation.

Figure 8 shows phase contrast and fluorescence microscope images of yeast cells in which glucose nanosensors have been incorporated by electroporation.

Figure 9 shows a standard curve of analyte concentration vs the ratio of 600 nm (reporter dye) to 521 nm (reference dye) fluorescence obtained with a similar nanosensor comprising sensor enzyme, reporter dye and a reference dye.

Figure 10 shows a calibration curve for the nanosensors with fluorescein incorporated into polyacrylamide particles containing horseradish peroxidase to which various amounts of H₂O₂ were added.

Figure 11 shows a quantitative reaction of Texas Red with H₂O₂.

Figure 12 shows insensitivity of Alexa Fluor 488 to presence of reactive oxygen species. Detailed description of the preferred embodiments

As used herein, the following terms have the following meanings:

"peroxidase enzyme" is a molecule, or molecular assembly, that catalyses reduction of hydrogen peroxide to water. A "peroxidase enzyme" may be a synthetic, artificial enzyme, which may be adapted for immobilization on solid surfaces, or an engineered mutant, chimeric or other derivative polypeptide comprising some portion of amino acid sequence homologous to a naturally occurring polypeptide peroxidase.

"reporter dye" is a molecule that acts as reductant substrate for a "peroxidase enzyme" wherein differences in fluorescence or other spectroscopic signal between the reduced and oxidized species of the dye provide a measure of hydrogen peroxide concentration.

"reference dye" is a molecule that provides a constant spectroscopic signal against which the oxidation-sensitive signal of one or more reporter dyes may be normalized.

"average particle size" refers to the mean hydrodynamic diameter value of a distribution of particle hydrodynamic diameters determined by dynamic light scattering techniques.

"ROS" refers to reactive oxygen species, which include hydrogen peroxide, oxygen radicals and molecules that contain oxygen radicals.

"nanoparticle" refers to particles having an average diameter of less than 1 μm and to particles having longest dimension of less than 1 μm.

"candidate compounds" refers to any molecule, such as a protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide or the like, which has potential of directly or indirectly affecting ROS production in or around a cell.

"anti-oxidant" refers to both compounds that act directly to oxidize or reduce ROS and also to compounds that inhibit generation of ROS

"inhibit ROS production" refers to both attenuation of ROS generation and also to attenuation of ROS concentration levels by direct action to oxidize or reduce ROS "incorporated in a nanoparticle" refers to embedded within a nanoparticle matrix that permits diffusion of some molecules through the matrix. "Embedded" is neither synonymous to nor mutually exclusive of attachment to a surface of a nanoparticle.

Preparation of nanosensors

The invention may be practised using any material suitable for biologically localised embedding, where material to be embedded includes a peroxidase enzyme along with one or more reporter dyes and, optionally, one or more reference dyes. Embedding material is preferably an organic polymer or an inorganic sol-gel that is biocompatible with living cells. A suitable peroxidase enzyme can be embedded in a nanoparticle along with one or more reporter dyes in such manner that the enzyme and reporter dyes do not leak out of the particle, while ROS can readily diffuse through the particle. Suitable embedding materials may include polyethylene glycol hydrogels, poly-vinyl chloride, carboxylated poly-vinyl chloride, poly-vinyl chloride-co- vinyl acetate-co-vinyl alcohol, or, most preferably, polyacrylamide. Alternatively, a suitable peroxidase and one or more reporter dyes may be incorporated within liposomes.

A suitable peroxidase along with one or more reporter dyes and, optionally, one or more reference dyes may be embedded using any method known in the art, including but not limited to the methods described in the following references, each of which is incorporated by reference herein in entirety: US published application 2006/008924; PCT published application WO 99/02561; US patents 6,143,588; 6,471 ,968; 6, 485, 703; Clark, H. et al., "Optical nanosensors for chemical analysis inside single living cells," Analytical Chemistry (2005), 77:6828; Xu H. et al., "Fluorescent nano-PEBBLE sensors designed for intracellular glucose imaging," Analyst (2002), 127:1471; Sumner, J. and Kopelman, R., "Alexa fluor 488 as an iron sensing molecule and its application in PEBBLE nanosensors," Analyst (2005), 130: 528; Vamvakaki, V., et al., "Fluorescence detection of enzymatic activity within a liposome base nano-biosensor," Biosensors and Bioelectronics (2005), 21 :384; Clark, H. et al., Subcellular optochemical nanobiosensors: probes encapsulated by biologically localised embedding (PEBBLEs) Sensors and Actuators - B Chem (1998) 51:12; Clark, H. et al. Optochemical Nanosensors and Subcellular Applications in Living Cells, Mikrochimica acta (1999) 131:121 ; Aylott, J. Optical nanosensors-an enabling technology for intracellular measurements. Analyst (2003) 128(4):309-12; Clark, H. et al. Optical Nanosensors for Chemical Analysis inside Single Living Cells. 2. Sensors for pH and Calcium and the Intracellular Application of PEBBLE Sens Analytical Chemistry (1999) 71 : 4837 Any suitable peroxidase can be used that catalyses reduction of hydrogen peroxide to water using a reporter dye as hydrogen donor wherein differences in fluorescence or other spectroscopic signal between the reduced and oxidized species of the dye provide a quantitative measurement of hydrogen peroxide within a range of concentrations from 10. nM - 10 mM. In preferred embodiments, horseradish peroxidase (HRP) can be used or similar enzymes such as peroxidases from beet root (Beta vulgaris), turnip, vegetables of the Brassicaceae family, Arthromyces ramosus, soybeans Glycine max, sweet potatoes, tobacco, peanuts and other vegetable or animal sources. Suitable peroxidases may include, for example, eosinophil peroxidase, extensin peroxidase, guaiacol peroxidase, heme peroxidase, lactoperoxidase, myeloperoxidase, oxyperoxidase, protoheme peroxidase, pyrocatechol peroxidase, scopoletin peroxidase, thiocyanate peroxidase, or verdoperoxi- dase. A suitable peroxidase may be a synthetic, artificial enzyme, which may be adapted for immobilization on solid surfaces, or an engineered mutant, derivative or chimeric polypeptide comprising some portion of sequence homologous to a naturally occurring peroxidase. Peroxidase enzymes can be obtained from commercial sources, or obtained by any method known in the art, including, for example, by recombinant expression of cloned mutant or unmodified peroxidases or by any of the methods described by the following references, each of which is incorporated herein in entirety: Burke, J et al., "Expression of recombinant horseradish peroxidase C in Escherichia coli," Biochem. Soc. Trans., (1989), 17(6):1077; Ozaki, S., et al., "Molecular engineering of horseradish peroxidase: theioether sulfoxidation and styrene epoxidation by Phe-41 leucine and threonine mutants," J. Am. Chem. Soc. (1995), 117:7056; Singh, N. et al., "Purification of turnip peroxidase and its kinetic properties," Prep. Biochem. Biotechnol. (2002), 32(1 ):39; Kvaratskehelia, M., et al., "Purification and characterization of a novel class III peroxidase isoenzyme from tea leaves," Plant Physiol. (1997), 114(4):1237; Zilletti, L., et al., "Peroxidase catalysed formation of prostaglandins from arachidonic acid," Biochem. Pharmacol (1989) 38(15):2429.

A suitable peroxidase is preferably used in a substantially purified form with high specific activity. Specific activity can be expressed in terms of a common substrate of a suitable peroxidase as μmol substrate reacted at saturation/min/μmol peroxidase at pH 6.0 at 2O⁰C. For example, for HRP, in terms of the substrate purpurogallin, high specific activity is at least 150, or more preferably at least 375, or still more preferably at least 750 μmol substrate reacted/min/μmol peroxidase. For peroxidases such as HRP that contain hemin, purity can be expressed in RZ (Reinheitszahl), the absorbance ratio at concentrations of 25-50 μM in deionized water, which is a measure of hemin content. A substantially purified form has, for example, RZ > 2.0, or more preferably, RZ > 3.0. A suitable peroxidase may be purified from a tissue, recombinant or other source by any method known in the art.

Any suitable reporter dye can be used that acts as hydrogen donor in reduction of hydrogen peroxide to water catalysed by a suitable peroxidase and wherein differences in fluorescence or other spectroscopic signal between the reduced and oxidized species of the dye provide a measurement of ROS. For example, suitable reporter dyes include but are not limited to 10-AcetyI-3,7-dihydroxyphenoxazine; 2, 2-azino-di (3-ethylbenzthiazoline-6- sulfonate), diacetyldichlorofluorescein, p-hydroxyphenylacetate, 3-methoxy-4-hydroxy- phenylacetic acid, and N-acetyl-3,7-dihydroxyphenoxazine.

In some embodiments, a reference dye embedded in the nanoparticle provides a constant spectroscopic signal against which the redox sensitive signal of one or more reporter dyes may be normalized. Any suitable reference dye may be used, including, for example, 2',T- Difluorofluorescein, Texas Red, and sulphorhodamine 101.

In preferred embodiments, a suitable peroxidase enzyme, one or more reporter dyes and, optionally, one or more reference dyes are embedded within a polyacrylamide nanoparticle using a microemulsion polymerization method adapted from Daubresse, C, et al., J. Colloid and Interface Science (1994), 168:222, which microemulsion polymerization method is further described in detail by Clark, H. et al.

Optical Nanosensors for Chemical Analysis inside Single Living Cells. 2. Sensors for pH and Calcium and the Intracellular Application of PEBBLE Sens Analytical Chemistry (1999) 71: 4837. For some reference dyes it may be necessary to attach the dyes covalently to the matrix.

This microemulsion polymerization process is conducted by (i) preparing an aqueous phase of the microemulsion by adding an aqueous solution comprising materials to be embedded in nanoparticles to an aqueous solution comprising both acrylamide monomer and an appropriate cross-linking agent, such as N,N'-methylene-bisacrylamide, (ii) preparing an oil phase containing a hydrocarbon liquid and an appropriate surfactant or surfactant mixture to form an inverse microemulsion consisting of small aqueous monomer droplets dispersed in the continuous oil phase and (iii) subjecting the acrylamide monomer microemulsion to polymerization. Optionally, one or more additives may be included in the aqueous phase to extend storage stability or impart other properties to the nanoparticles. Such additives may include, for example, metal chelating agents, glycerol, urea, antimicrobial agents, sodium dodecyl sulfate or other agents.

The surfactant-coated, nanometer sized reverse micelles formed in the microemulsion act as nanoreactors for polymerization of acrylamide monomers and, also provide a steric barrier that inhibits polymerization between micelles. Acrylamide monomer, cross-linking agent, and materials to be embedded, such as HRP, one or more reporter dyes and, optionally, one or more reference dyes, are incorporated fully within the reverse micelles. The polymerisation reaction and formation of nanoparticles occurs in the aqueous core of the micelles. The final size of polymerized nanoparticles is approximately the size of this aqueous core.

The size of the reverse micelles and, thus, the final size of the polyacrylamide nanoparticles is primarily determined by the volume ratio of surfactant to aqueous phase. In general, a higher volume ratio of surfactant to aqueous phase results in formation of smaller micelles and, thus, in smaller polymerized nanoparticles. The use of two types of surfactants, for example, AOT (sodium bis-2-ethylhexylsulphosuccinate),and Brij30 (polyoxyethylene 4 lauryl ether), also helps keep initial monomer micelle sizes very small.

Typical surfactants useful in the practice of this invention may be anionic, cationic or nonionic. Preferred surfactants include sodium dioctyl sulfosuccinate, polyoxyethylene-4- lauryl ether, sorbitan monooleate, polyoxyethylene, sorbitan monooleate, sodium dioctyl- sulfosuccinate, sodium bis-2-ethylhexylsulphosuccinate ,oleamidopropyldimethyl amine, sodium isostearyl-2-lactate and other surfactants.

Any suitable organic solvent may be used to form the organic phase, preferably hexane.

In embodiments that utilize polyacrylamide as embedding material, pore size of the polyacrylamide matrix is primarily determined by acrylamide concentration and, to a lesser extent, N,N'-methylenebisacrylamide concentration in the microemulsion aqueous phase. Pore size can be minimized by keeping acrylamide concentration close to limits of aqueous solubility of acrylamide monomers. Smaller pore sizes prevent embedded peroxidases and dyes from leaching out of the nanoparticle matrix. Monomer concentration in the microemulsion aqueous phase can affect final average particle size. If monomer concentration is decreased, average particle size can also be decreased. The concentration of dyes and proteins in the microemulsion aqueous phase can affect monomer solubility and, thus, average particle size of the final polymerized particles. The presence of large amounts of dyes and/or proteins impedes polymerization. Furthermore, the concentration of components for incorporation can alter solubility of acrylamide monomers and thereby affect average particle size of the polymerized particles. The amounts of protein and reporter dye used can affect ROS detection limits. In one preferred embodiment, 2.00 mg reporter dye and 30 μM HRP are used in 2 ml of aqueous phase containing 3.45 M acrylamide and 0.94 M N.N'-methylenebisacrylamide in 10 mM, pH 7.2 phosphate buffer, which can provide nanosensors having average particle size of less than 50 nm diameter and limits of ROS detection from 50 nM and up.

The particle size distribution and average particle size of the polymerized nanoparticles can be determined by dynamic light scattering, for example, as described by any of the following references, each of which is incorporated by reference herein in entirety: ISO Reference, Particle size analysis - Photon correlation spectroscopy, ISO 13321 (1996); B. J. Berne and R. Pecora, Dynamic Light Scattering With Application to Chemistry, Biology, and Physics (General Publishing Company, Toronto, 1976).; C. S. Johnson, Jr. and D. A. Gabriel, Laser Light Scattering, (Dover, New York, 1994).

In preferred embodiments, average particle size of the nanoparticles is less than 250 nm diameter, preferably less than 225 nm diameter, still more preferably less than 200 nm diameter, even more preferably less than 175 nm diameter, still more preferably less than 150 nm diameter, even more preferably less than 125 nm diameter, still more preferably less than 100 nm diameter, even more preferably less than 75 nm diameter, still more preferably, less than 55 nm diameter, or most preferably, less than 50 nm diameter.

In other embodiments, targeting ligands useful in targeting the nanoparticle and its contents to particular cells may be directly incorporated in the polymer matrix. For example, acrylamide monomers or mini-polymers can be conjugated to polypeptide targeting sequences by any method known in the art, including, but not limited to, the methods disclosed by the following references, each of which is incorporated by reference herein in entirety: Bruggemeier, S., et al., "Use of protein-acrylamide copolymer hydrogels for measuring protein concentration and activity," Analytical Biochemistry (2004) 329(2): 180; Lu, Z., et al., "Antigen responsive hydrogels based on polymerizable antibody Fab fragment," Macromolecular Bioscience (2003), 3 (6): 296; Jackson, D., et al., "Free radical induced polymerization of synthetic peptides into polymeric immunogens," Vaccine (1997) 15(15): 1697; Monji, N., et al., "Activated, N-substituted acrylamide polymers for antibody coupling - application to a novel membrane-based assay," Journal of Biomaterials Science - Polymer Edition (1994) 5(5):407. Acrylamide monomers or mini-polymers derivatised with targeting sequences and/or other targeting agents can be added to the aqueous phase of the microemulsion prior to polymerization. Alternatively, targeting sequences and/or other targeting agents may be added to the nanoparticles after polymerization. For example, the nanoparticles may be coated with glycosylated and/ or unglycosylated polypeptide sequences as described by the following references, each of which is incorporated by reference herein in entirety: Arenkov, P. et al. Protein microchips: use for immunoassay and enzymatic reactions. Analytical Biochemistry 2000 Feb 15;278(2): 123-31; Weston, P. and Avrameas, S. Proteins coupled to polyacrylamide beads using glutaraldehyde. Biochemical and biophysical research communications 1971 Dec 17;45(6): 1574-80.

In one embodiment, the invention provides an optical nanosensor for detection of ROS comprising a peroxidase enzyme and one or more reporter dyes incorporated in a nanoparticle. In a preferred embodiment, the nanosensor may further comprise one or more reference dyes.

In one embodiment, the invention provides an optical nanosensor for detection of ROS comprising a peroxidase enzyme and one or more reporter dyes incorporated in a nanoparticle wherein the nanoparticle as produced has a size distribution having average particle size of less than 250 nm diameter. In a preferred embodiment, the nanosensor may further comprise one or more reference dyes. In other embodiments, the nanoparticle as produced has a size distribution having average particle size of any one of the following: less than 250 nm diameter, less than 225 nm diameter, less than 200 nm diameter, less than 175 nm diameter, less than 150 nm diameter, eferably less than 125 nm diameter, less than 100 nm diameter, less than 75 nm diameter, less than 55 nm diameter, or less than 50 nm diameter.

In one embodiment, the invention provides an optical nanosensor for detection of ROS comprising a peroxidase enzyme and one or more reporter dyes incorporated in a polyacrylamide nanoparticle. In a preferred embodiment, the nanosensor may further comprise one or more reference dyes. Incorporation of nanosensors within cells

Nanosensors of the present invention may be incorporated into cells by any method known in the art including but not limited to balistic insertion (gene gun), microinjection, electroporation, and targeted liposomal delivery, for example, as described in the following references, each of which is incorporated herein by reference in entirety: Roizenblat, R., et al., "Nanobiolistic delivery of indicators to the living mouse retina," J. Neuorsci. Methods (2006) 153(1):154; Longmuir, K., et al., "Effective targeting of liposomes to liver and hepatocytes in vivo by incorporation of a Plasmodium amino acid sequence," Pharmaceutical Research (2006) 23(4):759, Mehier-Humbert, S., and Guy, R., "Physical methods for gene transfer: Improving the kinetics of gene delivery into cells," Advanced Drug Delivery Reviews (2005) 57(5):733; McAllister, D., et al., "Microfabricated needles for transdermal delivery of macromolecules and nanoparticles: Fabrication methods and transport studies," PNAS (2003) 100 (24): 13755

Nanosensors of the present invention can be used in biopsy samples, organ slices, isolated perfused organs, organotypic cultures, organs in situ, whole animals in vivo and in other circumstances in which ROS measurements are desired in only one or some cell types present in a mixed cell system. For example, the nanosensors can be targeted for intracellular incorporation or for extracellular adherence to particular normal or cancerous cells by incorporation of targeting sequences in the nanoparticles, as described by the following references, each of which is incorporated by reference herein in entirety: US published application 2005/0042298, US published application 2006/0018826; US published application 2006/0034925.

In one embodiment, the invention provides a cell comprising an embodiment of the nanosensor as described herein.

Adherence of nanosensors to particular cells

The nanosensors can be targeted for extracellular adherence to particular cells by incorporation of targeting sequences in the nanoparticles, as described above. Detection of ROS using the nanosensor

Nanosensors incorporated within or adhered to cells are appropriately stimulated so as to elicit a spectroscopic signal that differs between oxidized and reduced species of a reporter dye. This spectroscopic signal from the nanosensor is then detected by means for detecting the spectroscopic signal. Methods of stimulating and detecting the spectroscopic signal from the nanosensor will vary depending on the choice of one or more reporter dyes and/or, optionally, one or more reference dyes incorporated in the nanoparticle. For any given reporter dye and/or reference dye, methods of stimulating and detecting the spectroscopic signal from the nanosensor are well known in the art.

Embodiments that incorporate one or more reference dyes can be calibrated in vitro for quantitiative, ratiometric measurements in vivo. Such calibrations and measurements in vivo can be performed, for example, as will be readily apparent to one skilled in the art, by modification of methods described by Sumner, J. and Kopelman, R., "Alexa fluor 488 as an iron sensing molecule and its application in PEBBLE nanosensors," Analyst (2005), 130: 528.

As will be readily apparent to one skilled in the art, as an intracellular or extracellular means for detection of reactive oxygen species, the nanosensor of the present invention provides a sensitive tool with which to investigate ROS roles in normal and pathological physiology.

As will be readily apparent to one skilled in the art, the nanosensor of the present invention can also be used to detect ROS in non-cellular samples, such as biological or other liquids, cellular extracts, culture media or other samples.

In one embodiment, the invention provides a method of measuring ROS comprising the steps of: i) providing a cell or other sample ii) contacting an embodiment of the nanosensor as described herein with said cell or other sample iii) providing detecting means for said nanosensor iv) detecting a response of said nanosensor in the presence of ROS by said detecting means, wherein the response is proportional to ROS concentration. Identification of compounds that stimulate or inhibit ROS production using the nanosensor

In one aspect, the present invention provides methods of identifying candidate compounds that are effective in modulating ROS production in or around a cell.

In one non-limiting example, a nanosensor of the present invention may be used to identify candidate compounds that inhibit ROS production in cells that express excitatory amino acid receptors. Generation of superoxide contributes to NMDA receptor mediated neurotoxicity by reacting with nitrous oxide to form peroxynitrite. This contributes to a common pathway of injury that is relevant to a variety of acute and chronic neurological disorders, including focal ischemia, Huntington's disease, Alzheimer's disease, amyotropic lateral sclerosis (ALS), AIDS dementia and other neurodegenerative diseases. Lewen, A., P. Matz, et al. (2000). "Free radical pathways in CNS injury." Journal of Neurotrauma 17(10): 871-890., Calabrese, V., R. Lodi, et al. (2005). Oxidative stress, mitochondrial dysfunction and cellular stress response in Friedreich's ataxia." Journal of the Neurological Sciences 233(1-2): 145-162. and references therein. Accordingly, inhibitors of ROS production in such cells are useful in development of anti-oxidant therapeutics.

Nanosensors of the present invention can be incorporated within suitable excitable cells, such as retinal ganglion cells, cortical cells, or primary myocytes. In some embodiments, cultured excitable cells containing a nanosensor of the present invention can be grown in 96-well plates and maintained by methods well known in the art. The cells can then be subjected to one or more treatments known to stimulate ROS production. For example, cells may be cultured in conditions that promote oxidative stress, such as ischemia-inducing conditions where cells are incubated in an air-tight hypoxia chamber in glucose-free medium. Test candidate compounds can be added before, during or after such stimulation. ROS production can then be compared between test compounds and no-effect controls using the nanosensor. Test compounds can then be identified that are effective in substan- tially reducing ROS production relative to controls in such cells.

In another non-limiting example, a nanosensor of the present invention may be used to identify candidate compounds that inhibit intracellular and/or extracellular ROS production in vascular smooth muscle cells (VSMC). The ROS "catastrophic" chain reaction plays an important role in low density lipoprotein (LDL) oxidation, which critically affects progression of atherosclerosis. ROS are also now widely believed to mediate initiation of atherosclerotic lesions by causing damage to vascular endothelium. (39) In aortic smooth muscle cells, severe oxidative stress has been associated with lipid peroxidation, depletion of NAD⁺ and ATP, DNA strand breakage, increased intracellular free Ca²⁺, and miscellaneous protein damage. (40-45). VSMC can also be injured by interactions with monocytes and macrophages. Targeting and accumulation of macrophages is subject to ROS regulation. (27-29). VSMC generate extracellular superoxide and H₂O₂ in oxidizing LDL for macrophage "targeting." Extracellular ROS can accumulate to levels that overwhelm local anti-oxidant defenses. In the presence of transition metal ions and thiol compounds, super- potent OH^'". and thiyl radicals can be generated, resulting in widespread damage to VSMC tissues. (38) Accordingly, inhibitors of intracellular and/or extracellular ROS production in such cells are useful in development of anti-oxidant therapeutics.

Many other examples will be readily apparent to one skilled in the art of disease conditions in which inhibitors or promoters of ROS production in various specific cell types will be useful in development of anti-oxidant or pro-oxidant therapeutics.

In one embodiment, the invention provides a method of identifying a compound that affects

ROS production comprising the steps of: i) incorporating an embodiment of the nansosensor within a cell or adhering an embodiment of the nansosensor onto a cell ii) optionally subjecting said cell to one or more treatments that stimulate or suppress ROS production iii) contacting said cell with one or more test compounds to be screened iv) using said incorporated nanosensor or said adhered nanosensor to determine whether ROS production within or around said cell is increased or decreased in the presence of said test compound, wherein an increase or decrease of ROS production is an indication that the test compound affects ROS production. EXAMPLES

1. Encapsulation of HRP in polyacrylamide nanoparticles

Reagents. Acrylamide, >99%, electrophoresis grade; N,N^'-methylenebis(acrylamide), >99%; N-hexane, >97%, HPLC-grade; AOT (sodium bis^-ethylhexylsulphosuccinate),

>96%, were obtained from Sigma-Aldrich (Steinheim, Germany). Brij30 (polyoxyethylene 4 lauryl ether) and trypsin from bovine pancreas, were obtained from Sigma-Aldrich (Saint

Louis, MO, USA). Horseradish peroxidase, (HRP) was obtained from Roche (Mannheim,

Germany), proteinase K from Novagen Inc, (Darmstadtt, Germany), H₂O₂, 30% solution, from J.T.Baker (Deventer, the Netherlands), and fluorescein linked to a 10.000 MW dextran from Molecular Probes (OR, USA).

Preparation of polymer particle. Horseradish peroxidase embedded in polyacrylamide particles was prepared by microemulsion polymerisation. The monomer solution contained 2.7 g acrylamide, 0.8 g N,N^'-methylenebis(acrylamide), and 9 ml of 10 mM sodium phosphate buffer, pH 7.2. The microemulsions were prepared by drop-wise addition of 1.9 ml monomer solution following addition of 150 μl 10 mM sodium phosphate buffer, pH 7.2 containing 380 μM HRP to a solution of 43 mL hexane, 3.08 g AOT, and 1.59 g Brij30 in a round bottom flask. The solution was stirred under argon throughout the preparation and deoxygenated by 3 freeze - vacuum - thaw cycles using liquid nitrogen as freezing medium. To initiate the polymerization, 50 μl of a 10% (w/w) sodium bisulfite solution was added. The solution was kept under argon and stirred at room temperature for two hours to ensure complete polymerization. Hexane was removed by rotary evaporation and the remaining solution was resuspended in 96% ethanol and transferred to an Amicon ultra- filtration cell model 8200 (Millipore Corp., Bedford, MA). The solution was washed with 600 ml 96% ethanol in order to separate surfactants, unreacted monomers, and excess dyes and proteins from the sensors using a 100 kDa filter under 2 bar of pressure. The polymer particles containing HRP were then resuspended in ethanol and passed through a suction filtration system (Millipore Corp., Bedford, MA) with a 0.025 μm nitrocellulose filter membrane and rinsed with 100 ml ethanol. The particles were collected when dry. The hydrodynamic particle diameter was determined to 49 nm by dynamic light scattering (DLS) at a fixed scattering angle of 90° using a BI-200SM from Brookhaven Instruments (NYC, USA): this incorporates a 632.8 nm HeNe laser. Sample temperature was kept constant with a thermostatting water bath operating at 25°C.

A schematic representation of the nanoparticles is shown in Figure 1. 2. Peroxidase activity of encapsulated HRP

The HRP-containing nanoparticles of example 1 were suspended in buffer (either 100 mM potassium phosphate buffer, pH 7.0, or 50 mM sodium acetate buffer, pH 5.0). The solution was then subjected to sonication for 25 minutes in order to obtain a homogenous solution. To test if sonication had any effect on enzyme activity, peroxidase activity was measured in a solution containing 0.5 μM HRP before and after 25 minutes of sonication. No change in activity due to sonication could be demonstrated.

Peroxidase activity was measured using the guaiacol/H₂O₂ assay as described by Bergmeyer, H., in Methods of enzymatic analysis, 2^nd English edition, Verlag Chemie, Academic Press, NY, 1974. The embedded HRP had at least 75% of the peroxidase activity of comparable amounts of free HRP at room temperature.

3. Efficacy of HRP encapsulation. Catalysis of the peroxidase reaction using non-encapsulated fluorescein as hydrogen donor was examined using the nanoparticles of example 1. Both free fluorescein and dextran- linked fluorescein were tested. Free HRP readily catalyses the peroxidase reaction using free or dextran-linked fluorescein. While free fluorescein can readily diffuse through the nanoparticle polyacrylamide matrix, dextran-linked fluorescein is sterically hindered from approach to HRP. To a solution containing 10 mg/ml nanoparticles, 1 μg/ml of either free or dextran-linked fluorescein was added along with 1O uM hydrogen peroxide. A time scan of the decaying fluorescence intensity of free fluorescein, excited at 470 nm is shown in Figure 2 (a) while fluorescence trace of the reaction using dextran-linked fluorescein is shown in Figure 2 (b). The vertical bars illustrate addition of fluorescein and H₂O₂ at times 140 and 400 s respectively in Figure 2 (a) and at times 400 and 650 s respectively in Figure 2 (b). As shown, free fluorescein is efficiently oxidized by embedded HRP in presence of hydrogen peroxide, while dextran-linked fluorescein is unaffected. This indicates that HRP is effectively embedded in the nanoparticles, protected from cellular components larger than nanoparticle matrix pores.

4. Peroxidase-Oxidase activity of encapsulated HRP.

The embedded HRP was also tested for peroxidase-oxidase (PO) activity, or catalysis of the reaction:

2YH₂ + O₂ --> 2Y + 2H₂O. (reaction 1) The PO reaction is much more complex than the classical peroxidase reaction (reaction 1 ) and may involve more than 20 individual reaction steps .The PO activity of HRP was measured as the oxidation rate of NADH as described by Scheeline, A. et al. The Peroxidase-Oxidase Oscillator and Its Constituent Chemistries. Chemical reviews 1997 May 8;97(3):739-756.

Peroxidase-containing polyacrylamide particles were suspended in 50 mM sodium acetate buffer, pH 5.0, at room temperature corresponding to a peroxidase concentration of 0.5 μM. 1 mL of the suspension was placed in a quartz cuvette (10 mm light path) and NADH was added to a final concentration of 200 μM. NADH concentration was measured by the absorbance at 350 nm. After approximately 500 s, 500 μM 4-hydroxybenzoic acid, an activator of the peroxidase-oxidase reaction. , was added to the solution.

Figure 3 shows the oxidation of NADH before and after addition of 0.5 mM A- hydroxybenzoic acid to a reaction mixture containing HRP-containing particles, NADH and oxygen. Rates of oxidation of NADH in the absence of a catalyst and in the presence of either free HRP or HRP-containing nanometer particles are shown in Table 1.

Table I. NADH oxidation rate by free horseradish peroxidase (HRP) and horseradish peroxidase-containing nanoparticles (HRP particles) in the presence and absence of A- hydroxybenzoic acid (HBA).

Catalyst Before HBA +- SD After HBA

None 0.77 +- 0.08 μM/min 0.77 +- 0.08 μM/min

Free HRP 4.89 +- 0.29 μM/min 217.0 +- 32.2 μM/min

Nanoparticles 2.19 +- 0.71 μM/min 90.0 +- 17.7 μM/min

As shown, embedded HRP retains more than 40% of the peroxidase-oxidase activity of free HRP.

5. Leaching of HRP from the nanoparticles.

Leaching of HRP, which has a molecular weight of 40 kDa, from the particles was measured by transferring 5 ml 100 mM sodium phosphate buffer, pH 7.0, with 10 mg HRP- containing nanoparticles into a dialysis tube with MWCO 50.000 Da (Spectra/Por dialysis membranes, California, USA). The tube was then placed in 50 ml 100 mM sodium phosphate buffer, pH 7.0, for several days at O⁰C. At intervals aliquots of 50 μl were removed from the solution inside the dialysis tube and 200 μl from the external solution and the peroxidase activity was measured as described for example 2.

Figure 4 shows peroxidase activity at various time points after the start of dialysis of the HRP-containing nanoparticles. The activity in the solution inside the dialysis tube is essentially the same after a week of dialysis, whereas the activity in the external solution increases slightly. The activity in the external solution is 500 times less than that of the solution in the dialysis tube suggesting that less than 1 % of the HRP in the tube is free in solution.

As shown, HRP is efficiently embedded in the nanoparticles, with negligible leaching.

6. Encapsulation of both reporter dye and HRP in the nanoparticles. Nanosensors, or nanoparticles containing both HRP and a reporter dye, fluorescein, were prepared as described for example 1, except that to the aqueous acrylamide monomer solution was added 150 μl buffer 10 mM sodium phosphate buffer, pH 7.2 containing 35 mM fluorescein in addition to 380 μM HRP. Nanoparticles of comparable size to those described in example 2 were obtained.

7. Efficacy of HRP and reporter dye encapsulation.

Catalysis of the peroxidase reaction using encapsulated fluorescein as hydrogen donor was examined using the nanoparticles of example 6. To a solution containing 10 mg/ml nanoparticles, 10 μM hydrogen peroxide was added. A time scan of the fluorescence trace is shown in Figure 5. the reporter dye is excited at 470 nm and emission is obtained at 525 nm. The vertical bar illustrates addition of H₂O₂ at time 1300 s. As shown, the nanosensor has a stable fluorescence baseline and efficiently catalyses the peroxidase reaction using embedded fluorescein as hydrogen donor.

8. Encapsulation of both reporter dye and reference dye in similar nanoparticles. Nanosensors, or nanoparticles containing both a reporter dye, the pH indicator , 2',7'- bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), and also a reference dye, e.g. Texas Red dextran, were prepared as described for example 1, except that to the aqueous acrylamide monomer solution was added 150 μl buffer 10 mM sodium phosphate buffer, pH 7.2 containing 35 mM BCECF and 1.6 mM Texas Red dextran instead of 380 μM HRP. Nanoparticles of comparable size to those described in example 2 were obtained. 9. Intracellular detection using similar nanosensors in cultured liver cells.

The nanosensors of example 8 were inserted into cultured liver cells using a gene gun. Figure 6 shows phase contrast and fluorescence microscopy images of liver cells containing the nanosensors. Figure 6 (a) shows ordinary phase contrast microscopic image of the cells. 6 (b) shows BCECF fluorescence microscopic image of the liver cells, excitation at 580±20 nm and emission at 525±15 nm. Illuminated spots correspond to cells that have incorporated the nanosensors, in which reporter dye fluorescence provides a measure of pH. 6 (c) shows fluorescence microscopic image of the stimulated liver cells, excitation at 560±20 nm and emission at 645±30 nm. Illuminated spots correspond to cells that have incorporated the nanosensor, in which Texas Red dextran fluorescence provides a ratiometric reference against which reporter dye fluorescence can be normalized. As shown, cells that exhibit BCECF fluorescence also exhibit Texas Red dextran fluorescence.

Cells that exhibit Texas Red dextran fluorescence also exhibit fluorescein fluorescence. In this case, the ratio of BCECF to Texas Red dextran fluorescence provides a quantitative measure of intracellular pH. Similar nanosensors can be prepared that incorporate HRP, an

ROS reporter dye and Texas Red dextran. Where Texas Red dextran fluorescence signal is unaffected by the peroxidase reaction, the ratio of ROS reporter dye fluorescence to Texas

Red dextran fluorescence can provide a quantitative measure of intracellular ROS that is independent of nanosensor concentration and, also, independent of the ratio of active peroxidase, reporter and reference dye incorporated in the nanoparticle. 6 (d) shows an overlay of 6 (a) and 6 (c) that identifies cells that have incorporated the nanosensors of example 8. As shown, using a gene gun for intracellular delivery, about 10% of exposed cells incorporate the nanosensors of example 8.

10. Encapsulation of reporter dye, reference dye and sensor enzyme in similar nanoparticles.

Nanosensors, or nanoparticles containing the enzyme glucose oxidase, a reporter dye, an oxygen-sensitive Ru-complex, and also a reference dye, Texas Red dextran, were prepared as described for example 1, except that to the aqueous acrylamide monomer solution was added 150 μl buffer 10 mM sodium phosphate buffer, pH 7.2 containing 3.7 mM RU- sensitive complex, and 1.6 mM Texas Red and 10 mg glucose oxidase (from Aspergillus niger, SIGMA-Aldrich, Steinheim, Germany) instead of 380 μM HRP. Nanoparticles of comparable size to those described in example 2 were obtained. 11. Intracellular incorporation of similar nanosensors into yeast cells by electroporation. The nanosensors of example 10 were inserted into cultured yeast cells by electroporation. Figure 8 shows phase contrast and fluorescence microscopy images of yeast cells containing the nanosensors. Figure 8 (a) shows ordinary phase contrast microscopic image of the cells. 8 (b) shows fluorescence microscopic image of the yeast cells. The oxygen- sensitive Ru-complex is excited at 450±20 nm and emission at 610±20 nm. Illuminated spots correspond to cells that have incorporated the nanosensor, in which reporter dye fluorescence provides a measure of intracellular glucose. Comparison of 8 (a) and 8 (b) shows that, using electroporation for intracellular delivery, about 50% of exposed cells incorporate the nanosensors of example 10.

12. Calibration of similar nanosensor for quantitative, ratiometric, intracellular measurements.

The nanosensors of example 11 were calibrated for quantitative glucose determination by measuring both 600 (Ru-complex) and 521 (Texas Red dextran) nm fluorescence at glucose concentrations between 0 and 5 mM. The ratio F600/F521 provides a quantitative estimate of glucose concentration, as shown in Figure 9 (a). Figure 9 (b) shows fluorescence spectra of the nanosensors between 500 and 680 nm at various glucose concentrations.

Similar nanosensors can be prepared that incorporate HRP, an ROS reporter dye and Texas Red. Where Texas Red fluorescence signal is unaffected by the peroxidase reaction, the ratio of ROS reporter dye fluorescence to Texas Red fluorescence can provide a quantitative measure of intracellular ROS similar to the exemplified quantitative measure of intracellular glucose.

13. Insertion of nanosensors of the present invention into cells.

Nanosensors of the present invention can be inserted into living cells by use of a cell penetrating peptide (CPP). Following preparation of the ROS-sensors CPP are attached to the sensor surface and the ROS-sensors are inserted into cells by incubation of cells with sensors according to the method of (48). 14. Calibration of nanosensors of the present invention.

Nanosensors of example 6 were prepared with fluorescein incorporated into polyacrylamide particles containing horseradish peroxidase. A calibration curve for the nanosensors is shown in Figure 10. The solution contained 5 mg/ml nanoparticles in 100 mM phosphate buffer, pH 7.0, to which we have added various amounts of H₂O₂. The standard deviation is shown and is based on three individual repetitions. The plot shows change in fluorescence due to fluorescein as a function of the H₂O₂ concentration. Fluorescein was excited at 470 nm and emission was measured at 525 nm. 50 μl fluorescein, 25 mg/ml was added to 2 ml of the HRP containing monomer solution.

15. Ratiometric quantification.

Quantification of ROS in living cells using nanosensors of the present invention can be based upon ratiometric measurements. Two dyes are incorporated into the polymer particle. While the first dye changes its fluorescence according to the ROS concentration, the second dye is a reference dye which has constant emission.

Horseradish peroxidase can react with a variety of different fluorescent dyes when reactive oxygen species are present. Accordingly, one skilled in the art can readily choose a suitable dye for incorporation in a nanosensor of the present invention even if a cell possesses some natural fluorescence at certain wavelengths. For example, as shown in

Figure 11 , Texas Red dextran (Mw 10.000 g/mol) is oxidized by H₂O₂ in the presence of

HRP. 20 μg/ml Texas Red dextran and 5 μM HRP are dissolved in 100 mM phosphate buffer, pH 7.0. H₂O₂ is added at various concentrations. The dye is excited at 570 nm and emission is measured at 590 nm.

Ideally, the reference dye does not react with horseradish peroxidase when reactive oxygen species or other metabolic components are present. One skilled in the art can readily choose a suitable dye for incorporation in a nanosensor of the present invention. For example, as shown in Figure 12, Alexa Fluor 488 Dextran (Mw 10.000 g/mol) does not react with HRP when ROS are present and is a suitable reference dye. 20 μg/ml of the fluorescent dye Alexa Fluor 488 dextran (Mw 10.000 g/mol) is dissolved in 100 mM phosphate buffer, pH 7.0. 5 μM horseradish peroxidase is added at time T=160 sec. At time T=290 sec. 0.05 μM H₂O₂ is added. HRP absorbs light at dye excitation wavelength, which results in the decrease in fluorescence signal when HRP is added. Excitation is fixed at 495 nm and emission at 519 nm using 2.5 nm slit width.

Each of the references cited herein is incorporated by reference herein in entirety.

As will be readily understood by one skilled in the art, the aspects, descriptions, features, advantages, embodiments and examples of the present invention disclosed herein are representative only and not considered to limit the invention as defined by the claims.

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Claims

What is claimed:

1. An optical nanosensor for detection of ROS comprising a peroxidase enzyme and one or more reporter dyes incorporated in a nanoparticle.

2. The nanosensor of claim 1 further comprising one or more reference dyes.

3. An optical nanosensor for detection of ROS comprising a peroxidase enzyme and one or more reporter dyes incorporated in a nanoparticle wherein the nanoparticle as produced has a size distribution having average particle size of less than 250 nm diameter.

4. The nanosensor of claim 3 further comprising one or more reference dyes.

5. An optical nanosensor for detection of ROS comprising a peroxidase enzyme and one or more reporter dyes incorporated in a polyacrylamide nanoparticle.

6. The nanosensor of claim 3 further comprising one or more reference dyes.

7. A method of measuring ROS comprising the steps of: i) providing a cell or sample ii) contacting a nanosensor of claim 1 or 2 with said cell or sample iii) providing detecting means for said nanosensor iv) detecting a response of said nanosensor in the presence of ROS by said detecting means, wherein the response is proportional to ROS concentration.

8. A method of measuring ROS comprising the steps of: i) providing a cell or sample ii) contacting a nanosensor of claim 3 or 4 with said cell or sample iii) providing detecting means for said nanosensor iv) detecting a response of said nanosensor in the presence of ROS by said detecting means, wherein the response is proportional to ROS concentration.

9. A method of measuring ROS comprising the steps of: i) providing a cell or sample ii) contacting a nanosensor of claim 5 or 6 with said cell or sample iii) providing detecting means for said nanosensor iv) detecting a response of said nanosensor in the presence of ROS by said detecting means, wherein the response is proportional to ROS concentration.

10. A method of identifying a compound that affects ROS production comprising the steps of: i) Incorporating a nansosensor of claim 1 or 2 within a cell or adhering a nansosensor of claim 1 or 2 onto a cell ii) optionally subjecting said cell to one or more treatments that stimulate or suppress ROS production iii) contacting said cell with one or more test compounds to be screened iv) using said incorporated nanosensor or said adhered nanosensor to determine whether

ROS production within or around said cell is increased or decreased in the presence of said test compound, wherein an increase or decrease of ROS production is an indication that the test compound affects ROS production.

11. A method of identifying a compound that affects ROS production comprising the steps of: i) Incorporating a nansosensor of claim 3 or 4 within a cell or adhering a nansosensor of claim 3 or 4 onto a cell ii) optionally subjecting said cell to one or more treatments that stimulate or suppress ROS production iii) contacting said cell with one or more test compounds to be screened iv) using said incorporated nanosensor or said adhered nanosensor to determine whether

ROS production within or around said cell is increased or decreased in the presence of said test compound, wherein an increase or decrease of ROS production is an indication that the test compound affects ROS production.

12. A method of identifying a compound that affects ROS production comprising the steps of: i) Incorporating a nansosensor of claim 5 or 6 within a cell or adhering a nansosensor of claim 5 or 6 onto a cell ii) optionally subjecting said cell to one or more treatments that stimulate or suppress ROS production iii) contacting said cell with one or more test compounds to be screened iv) using said incorporated nanosensor or said adhered nanosensor to determine whether

ROS production within or around said cell is increased or decreased in the presence of said test compound, wherein an increase or decrease of ROS production is an indication that the test compound affects ROS production.

13. A cell comprising the nanosensor of any one of claims 1-6.