Abstract:
The invention provides ion-selective sensors comprising quantum dots capable of selectively measuring ions, e.g., Na + , K + , Cl − , etc., in various environments, including in the cytosol of a living cell. Quantum dots are attractive probes for microscopy due to their photophysical advantages over fluorescent dyes, including prolonged photostability, brightness and quantum efficiency. In certain embodiments, a sensor comprises one or more quantum dots, a pH-sensitive dye, and optionally an ion-selective component such as an ionophore. These elements may, for example, be disposed in a polymer matrix. In certain embodiments, the sensors may detect ionic analytes by selective ion extraction by the polymer, thereby inducing a pH change within the sensor which in turn changes the absorbance of the pH-sensitive dye. The change of absorbance may in turn attenuate the intensity of detectable emissions, e.g., fluorescence, from the quantum dot by directly absorbing its fluorescence emission.

Description:
CROSS-REFERENCE TO RELATED CASES 
     This application claims the benefit of U.S. Provisional Patent Application No. 60/834,973, entitled “Ion-Selective Quantum Dots,” filed on Jul. 31, 2006, the entire disclosure of which is hereby incorporated by reference as if set forth herein in its entirety. 
    
    
     BACKGROUND OF THE INVENTION 
     Ion channels lie at the heart of the dynamics of excitable cells and control the action potential responsible for the beat of a cardiac cell or the firing of a synapse. Traditionally, calcium has been the most studied ion in the intracellular space, and as a result a good deal is understood about calcium ion dynamics, regulation and impact on the cell. While it is generally believed that sodium is essential to the function of excitable cells, its role is not as well understood. Sodium channels occur at sites of action potential generation in neurons and are responsible for the upstroke of the action potential in cardiac cells. Sodium channelopathies are known to be responsible for major classes of disease, including epilepsy and hypertension, as well as leading to potentially fatal arrhythmias in Long QT syndrome. In addition, calcium dynamics are often dependent upon sodium, through the sodium/calcium exchanger. The exchanger is responsible for the rapid export of calcium from the cell and is driven by the transmembrane sodium gradient. While channel labeling studies have elucidated the location of sodium channels, little is known about the function, dynamics and impact of sodium channels. 
     There is a wide selection of methods for measuring ion flux in cells including, for example, patch clamping, fluorescent indicator dyes, polymer-based ion-selective nanosensors and dye-loaded liposomes. There are, however, disadvantages to the available methods of ion monitoring. For example, whole-cell patch clamp monitoring which involves contacting cells with a pulled glass capillary to electrically monitor ion channels, can be performed on single cells but not reliably in a high-throughput fashion. Fluorescent ion-indicator dyes while effective for monitoring certain ions, lack selectivity for other physiological ions and are easily photobleached. Ion-selective nanosensors (e.g., probes encapsulated by biologically localized embedding (PEBBLEs)) which comprise a fluorescent indicator dye display fast response time to ions as they diffuse into the polymer matrix, however, the components of the nanosensors, such as the fluorescent dye, are also prone to photobleaching. Dye-loaded liposomes are highly biocompatible due to their lipid construction but are limited in their range of detectable analytes particularly to gases. 
     New methods are needed for monitoring ion fluctuations in cells that are selective for a particular ionic analyte such as sodium, are biocompatible and have prolonged sensor lifespans. Further, sensors that are small enough to enter the cell but emit a strong enough signal to be measured extracellularly would be highly desired. 
     SUMMARY OF THE INVENTION 
     The invention provides ion-selective sensors comprising quantum dots capable of selectively measuring ions, e.g., Na + , K + , Cl − , etc., in the cytosol of a single living cell. Quantum dots are attractive probes for microscopy due to their photophysical advantages over fluorescent dyes, including prolonged photostability, brightness and quantum efficiency. In certain embodiments, a sensor comprises one or more quantum dots, a pH-sensitive dye, and optionally an ion-selective component such as an ionophore. These elements may, for example, be disposed in a polymer matrix. In certain embodiments, the sensors may detect ionic analytes by selective ion extraction by the polymer, thereby inducing a pH change within the sensor which in turn changes the absorbance of the pH-sensitive dye. The change of absorbance may in turn attenuate the intensity of detectable emissions, e.g., fluorescence, from the quantum dot by directly absorbing its fluorescence emission. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG. 1  is a schematic diagram of a cell assay system according to an illustrative embodiment of the invention. 
         FIG. 2  is a representation of two exemplary modes of operation of the quantum dot incorporated sensor for the detection of cationic analytes. In Mode A, the sensor fluoresces in the presence of the ionic analyte, In Mode B, the sensor fluoresces in the absence of ionic analyte. 
         FIG. 3  is a representation of two exemplary modes of operation of the quantum dot incorporated sensor for the detection of anionic analytes. In Mode A, the sensor fluoresces in the presence of the ionic analyte, In Mode B, the sensor fluoresces in the absence of ionic analyte. 
         FIG. 4  is a is a representation of a sensor coated with a surface modifier such as PEG. 
         FIG. 5  shows cross sections of various optical sensor arrangements suitable for use in various implementations of a cell assay system: a) optical ion sensor film adhered to a support containing a layer of cells; b) optical ion sensor particles located within cells; c) optical ion sensor film and particles contacting cells from exterior and interior respectively; d) sensor with removable optical sensor film and electrode. 
         FIG. 6  presents an embodiment of a microfluidic device in accord with the present invention: a) microfluidic device with wells that lead to branched channels where cells flow and detect analytes within such channels; b) depiction of imaging fluorescence microfluidic device of  6   a  wherein black bars represent fluorescence in channels of the device visualized as an optical bar code. 
         FIG. 7  shows the selectivity of the nanosensor of the invention for ion detection. 
         FIG. 8  depicts the experimental response to sodium. a) Spectral response of immobilized sensors to increasing concentrations of sodium. b) Calibration curve of ratiometric sensors. 
         FIG. 9  depicts the spectral overlap of a quantum dot that fluoresces at 655 nm and the absorbance of a chromoionophore at varying sodium concentrations. 
         FIG. 10  depicts biocompatibility of nanosensors in HEK cells. HEK cells were incubated with either control (water), nanosensors without quantum dots (nans), quantum dot nanosensors, 100 nm gold nanoparticles, or 20 nm latex beads (a negative control). 
         FIG. 11  is a confocal image of nanosensors without quantum dots loaded into an HEK 293 cell. 
         FIG. 12  shows a LIVE/DEAD assay wherein nanosensors with quantum dots were loaded into HEK 293 cells overnight and then stained. The green indicates healthy cells, while the red stains the nuclei of dead cells. No difference in the ratio of live to dead cells was noted between nanosensor loaded cells and control (no nanosensors). 
         FIG. 13  depicts a. fluorescence image of an isolated neonatal rat ventricular myocyte loaded with sodium-selective nanosensors. b. the fluorescence collected from a nanosensor in a cardiac cell during stimulation 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     In brief overview, embodiments of the present invention provide systems, methods, and devices for measuring ionic analytes. In exemplary embodiments, sensors are placed inside or outside a cell. Changes in emissions from the sensor indicate the ion concentrations and fluxes from the cell. In certain aspects, the sensors comprise a polymer, a fluorescent semiconductor nanocrystal (also known as a quantum dot™ particle) that fluoresces at a first wavelength, and a chromoionophore that absorbs photons of the first wavelength in one state and does not absorb photons of the first wavelength in a second state. In monitoring ionic analytes, the chromoionophore changes state in response to proton concentration (i.e., the protonated chromoionophore is one state while the deprotonated chromoionophore is a second state). To monitor a specific analyte, an ionophore that selectively associates with specific ions or groups of ions is included in the sensor. Once the ionophore associates with a cationic analyte (e.g., Na +  associates with a Na + -selective ionophore), for example, protons are displaced from the sensor to equilibrate charge, altering the state of the chromoionophore. The fluorescence emitted from the sensor indicates the state of the chromoionophore which correlates to the presence and/or concentration of the ionic analyte. Sensors that use fluorescent dyes instead of quantum dots are disclosed in U.S. patent application Ser. No. 11/522,169, filed Sep. 15, 2006, the disclosure of which is incorporated herein by reference. 
     In certain embodiments, the sensor includes an ionophore, a chromoionophore, a quantum dot, and optionally one or more additives. The components are typically embedded in a polymer. In certain embodiments, the polymer comprises poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacralate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides, polyorthoesters, poly(ester amides), polyamides, poly(ester ethers), polycarbonates, silicones, polyalkylenes such as polyethylene, polypropylene, and polytetrafluoroethylene, polyalkylene glycols such as poly(ethylene glycol) (PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such as poly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinyl ethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halides such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes, polystyrene (PS), polyurethanes, derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose, polymers of acrylic acids, such as poly(methyl(meth)acrylate) (PMMA), poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate), poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) (jointly referred to herein as “polyacrylic acids”), and copolymers and mixtures thereof, polydioxanone and its copolymers, polyhydroxyalkanoates, poly(propylene fumarate), polyoxymethylene, poloxamers, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), trimethylene carbonate, polyvinylpyrrolidone, and the polymers described in Shieh et al., 1994, J. Biomed. Mater. Res., 28, 1465-1475, and in U.S. Pat. No. 4,757,128, Hubbell et al., U.S. Pat. Nos. 5,654,381; 5,627,233; 5,628,863; 5,567,440; and 5,567,435. Other suitable polymers include polyorthoesters (e.g. as disclosed in Heller et al., 2000, Eur. J. Pharm. Biopharn., 50:121-128), polyphosphazenes (e.g. as disclosed in Vandorpe et al., 1997, Biomaterials, 18:1147-1152), and polyphosphoesters (e.g. as disclosed in Encyclopedia of Controlled Drug Delivery, pp. 45-60, Ed. E. Mathiowitz, John Wiley &amp; Sons, Inc. New York, 1999), as well as blends and/or block copolymers of two or more such polymers. The carboxyl termini of lactide- and glycolide-containing polymers may optionally be capped, e.g., by esterification, and the hydroxyl termini may optionally be capped, e.g., by etherification or esterification. In certain embodiments, the polymer comprises or consists essentially of polyvinyl chloride (PVC), polymethyl methacrylate (PMMA) and decyl methacrylate or copolymers or any combination thereof. 
     In certain embodiments, the polymer comprises a biocompatible polymer, e.g., selected from poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(ethylene glycol) (PEG), poly(vinyl acetate) (PVA), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), polyalkyl cyanoacrylate, polyethylenimine, dioleyltrimethyammoniumpropane/dioleyl-sn-glycerolphosphoethanolamine, polysebacic anhydrides, polyurethane, nylons, or copolymers thereof. In polymers including lactic acid monomers, the lactic acid may be D-, L-, or any mixture of D- and L-isomers. The terms “biocompatible polymer” and “biocompatibility” when used in relation to polymers are art-recognized. For example, biocompatible polymers include polymers that are neither themselves toxic to the host (e.g., a cell, an animal, or a human), nor degrade (if the polymer degrades) at a rate that produces monomeric or oligomeric subunits or other byproducts at toxic concentrations in the host. Consequently, in certain embodiments, toxicology of a biodegradable polymer intended for intracellular or in vivo use, such as implantation or injection into a patient, may be determined after one or more toxicity analyses. It is not necessary that any subject composition have a purity of 100% to be deemed biocompatible. Hence, a subject composition may comprise 99%, 98%, 97%, 96%, 95%, 90% 85%, 80%, 75% or even less of biocompatible polymers, e.g., including polymers and other materials and excipients described herein, and still be biocompatible. 
     The polymer phase may comprise a plasticizer, such as dioctyl sebacate (DOS), o-nitrophenyl-octylether, dimethyl phthalate, dioctylphenyl-phosphonate, dibutyl phthalate, hexamethylphosphoramide, dibutyl adipate, dioctyl phthalate, diundecyl phthalate, dioctyl adipate, dioctyl sebacate, or other suitable plasticizers. In certain embodiments, the plasticizer is poly(glycerol sebacate), PGS. 
     In certain embodiments, e.g., particularly where the polymer is biocompatible, a biocompatible plasticizer is used. The term “biocompatible plasticizer” is art-recognized, and includes materials which are soluble or dispersible in the relevant polymer, which increase the flexibility of the polymer matrix, and which, in the amounts employed, are biocompatible. Suitable plasticizers are well known in the art and include those disclosed in U.S. Pat. Nos. 2,784,127 and 4,444,933. Specific plasticizers include, by way of example, acetyl tri-n-butyl citrate (c. 20 weight percent or less), acetyltrihexyl citrate (c. 20 weight percent or less), butyl benzyl phthalate, dibutylphthalate, dioctylphthalate, n-butyryl tri-n-hexyl citrate, diethylene glycol dibenzoate (c. 20 weight percent or less) and the like. 
     The ionophore is a compound, typically an electrically neutral compound, that associates (e.g., forms a complex, chelate, or other non-covalent association) with a target ion, and is selective for the target ion relative to other ions. The ionophore is selected to be lipid-soluble and does not emit light in the visible spectrum in either of its complexed and non-complexed states. In certain aspects, the ionophore of the mixture included herein is chosen to selectively bind an ionic analyte, for example, K + , Na + , Ca 2+ , H + , Ba 2+ , Li + , Cl − , NH 4   + , or NO 3   − . Potassium ion ionophores include, for example, valinomycin, crown ethers, e.g., dimethyldibenzo-30-crown-10, dicyclohexyl-18-crown, dimethyldicyclohexyl-18-crown-6, tetraphenyl borate, tetrakis(chlorophenyl)borate. Sodium ion ionophores include, for example, methyl monensin, N,N′,N″-triheptyl-N,N′,N″-trimethyl-4,4′,4″-propylidintris-(3-oxabutyramide), N,N,N′,N′-tetracyclohexyl-1,2-phenylenedioxydiacetamide, 4-octadecanoyloxymethyl-N,N,N′,N′-tetracyclohexyl-1,2-phenylenedioxydiacetamide, bis[(12-crown-4)methyl]dodecylmethylmalonate. Exemplary calcium ion ionophores include, for example, bis(didecylphosphate), bis(4-octylphenylphosphate), bis(4-(1,1,3,3-tetramethylbutyl)phenylphosphate tetracosamethylcyclododecasiloxane, N,N′-di(11-ethoxycarbonyl)undecyl)-N,N′,4,5-tetramethyl-3,6-dioxaoctane diamide. Barium ion ionophores include, for example, calcium di(2-ethylhexyl)phosphate+decan-1-ol, barium complex of nonylphenoxypoly(ethyleneoxy)ethanol in ortho-nitrodiphenyl ether. Chloride ion ionophores include, for example, {μ-[4,5-dimethyl-3,6-bis(octyloxy)-1,2-phenylene]}bis(trifluoroacetato-O)dimercuri (ETH 9009), {μ-[4,5-dimethyl-3,6-bis(dodecyloxy)-1,2-phenylene]}bis(mercury chloride) (ETH 9033), 5,10,15,20-tetraphenyl-21H,23H-porphin manganese (III) chloride (MnTPPCl), tributyltin chloride (TBTCl) and trioctyltin chloride (TOTCl). Bicarbonate ion ionophores of the invention include, for example, quaternary ammonium ion exchanger p-octodecyloxy-meta-chlorophenyl-hydrazone-mesoxalonitrile. Ammonium ion ionophores include, for example, nonactin and monactin. Nitrate ion ionophores include, for example, tridodecylhexadecylammonium nitrate+n-octyl-ortho-nitrophenyl, 1:10 phenanthroline nickel (II) nitrate+para-nitrocymene. Lithium ion ionophores include, for example, N,N′-diheptyl-N,N′,5,5-tetramethyl-3,7-dioxononanediamide), 12-crown-4,6,6-dibenzyl-14-crown-4. 
     A chromoionophore is an ionophore that changes its optical properties in the visible spectrum depending on the state of complexation. Chromoionophores for use in sensors are typically proton-sensitive dyes that change absorbance (and fluorescence in many cases) depending on the degree of protonation, although chromoionophores that change absorbance in response to other ions can also be used. The chromoionophores are preferably highly lipophilic to inhibit leaching from the sensor matrix. Suitable chromoionophores include Chromoionophore I (i.e., 9-(Diethylamino)-5-(octadecanoylimino)-5H-benzo[a]phenoxazine), Chromoionophore II (i.e., 9-Dimethylamino-5-[4-(16-butyl-2,14-dioxo-3,15-dioxaeicosyl)phenylimino]benzo[a]phenoxazine) and Chromoionophore III (i.e., 9-(Diethylamino)-5-[(2-octyldecyl)imino]benzo[a]phenoxazine). Chromoionophore II exhibits light absorbance peaks at 520 nm and 660 nm and a fluorescent emission peak at 660 nm. Chromoionophore III has light absorbance peaks at 500 nm and 650 nm and fluorescent emission peaks at 570 nm and 670 nm. 
     Quantum dots are fluorescent semiconductor nanocrystals having a characteristic spectral emission, which is tunable to a desired energy by selection of the particle size, size distribution and composition of the semiconductor nanocrystal. The emission spectra of a population of quantum dots have linewidths as narrow as 25-30 nm, depending on the size distribution heterogeneity of the sample population, and lineshapes that are symmetric, gaussian or nearly gaussian with an absence of a tailing region. Advantageously, the range of excitation wavelengths of the quantum dots is broad. Consequently, this allows the simultaneous excitation of varying populations of quantum dots in a system having distinct emission spectra with a single light source, e.g., in the ultraviolet or blue region of the spectrum. 
     In certain embodiments, quantum dots of the sensor described herein are, for example, inorganic crystallites between 1 nm and about 1000 nm in diameter, preferably between about 2 nm and about 50 nm, more preferably about 5 nm to 20 nm, such as about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nm. Such quantum dots include a “core” of one or more first semiconductor materials, and which may be surrounded by a “shell” of a second semiconductor material. A semiconductor nanocrystal core surrounded by a semiconductor shell is referred to as a “core/shell” semiconductor nanocrystal. The surrounded “shell” will most preferably have a bandgap greater than the bandgap of the core material and can be chosen so to have an atomic spacing close to that of the “core” substrate. The core and/or the shell material can be a semiconductor material including, but not limited to, those of the group II-VI (ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe, MgTe and the like) and III-V (GaN, GaP, GaAs, GaSb, InN, InP, InAs, InSb, AlAs, AlP, AlSb, AlS, and the like) and IV (Ge, Si, Pb and the like) materials, and an alloy thereof, or a mixture thereof. 
     In certain aspects, a sensor comprises exactly one quantum dot. In certain embodiments, a sensor comprises more than one quantum dot, for example, 2, 3, 4, or 5 quantum dots. In certain embodiments wherein the sensor comprises more than one quantum dot, the sensor comprises two or more types of quantum dots, each type having a distinct emission wavelength, e.g., independently selected from, for example, 490, 520, 545, 560, 580, 620, 655 nm. The availability of two distinct wavelength emissions (e.g., one or more quantum dots of wavelength 545 nm and one or more quantum dots with emission wavelength of 655 nm) may allow improvements in recording of changes in ion concentration by using the ratio of the two distinct signals. Fluctuations in fluorescence that are common to both signals should theoretically cancel in a ratio. The detectable fluorescence emission of the quantum dot particles may fluctuate depending on variables including number of quantum dots, quantum dot location within the cell, photobleaching, and possible changes in excitation light intensity, all effects that can occur slowly and are not related to ion presence or concentration. Therefore, effects including number of quantum dots, quantum dot location within the cell, photobleaching, and possible changes in excitation light intensity, may be attenuated. 
     In certain embodiments, the fluorescence signal of the quantum dot may trigger a detectable event within the cell. For example, fluorescence may in turn excite a secondary dye or quantum dot in the particle that easily generates reactive oxygen species (ROS). The ROS would then attack the cell, effectively stimulating necrosis (cell death), which may then be detected either visually or using markers sensitive to cell death. Alternatively, instead of including a secondary component within the particle, another particle may be added to the cell or cell culture. This additional particle may, for example, comprise a photo-degradable polymer membrane. When the primary sensor fluoresces, the emitted light will rupture the secondary particle, releasing its contents. The contents may, for example, be a drug that is therapeutic or apoptotic, e.g., triggering another detectable event. 
     The sensor may comprise an additive, e.g., to embed charge sites within the polymer phase and/or to help enforce charge neutrality within the sensor  112 . For sensors targeting cations, the additive can be any inert and preferably lipophilic component that has a negative charge associated with it. For sensors targeting anions, the additive is positively charged and preferably lipophilic. The additive allows the polymer phase to carry a corresponding amount of oppositely charged particles while maintaining overall charge neutrality of the sensor. The concentration ratio of additive to chromoionophore is preferably 1:1, thereby allowing the chromoionphore to become completely protonated or deprotonated. One suitable additive for sensors targeting negative ions is potassium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (KTFPB). The lipophilic, anionic component TFPB molecules are retained by the polymer phase, and the potassium ions are either complexed by the ionophore or expelled into the sample solution through diffusion. In one particular implementation, the sensor film is composed of a suspension produced from about 60 mg of DOS, 30 mg of PVC, and up to about 5 mg of additive, ionophore, and chromoionophore. 
     In a sample solution, the sensor continuously extracts or expels, for example, analyte cations depending on ion activity in the sample solution. The ion activity of a sample solution can be monitored by observing the fluorescence of a sensor of the invention in the sample solution. As depicted in  FIG. 2 , the sensor may fluoresce in the presence of a cationic analyte  21 , and not in the absence of said analyte, Mode A. In such embodiments, the chromoionophore  24 , of the sensor absorbs photons  25 , of a quantum dot  26 , when the cationic analyte  21  is not bound to the ionophore  22 . In such embodiments, the wavelength of photons  25  emitted from the quantum dot  26  when excited with a light source such as UV or visible light fall within the absorbance range, e.g., maximum absorbance range, of the chromoionophore  24  bound to a proton  23 , such that the fluorescence of the quantum dot is attenuated or completely undetectable from outside of the polymer matrix  20  (Mode A, sensor on the left). As the target ion  21  increases in concentration in solution, the ions  21  are drawn into the polymer matrix  20  to bind with the ion-selective ionophore  22 . To maintain charge neutrality within the polymer matrix  20 , protons  23  dissociate from the chromoionophore  24  in the sensor and diffuse out of the polymer matrix  20  into the sample solution, altering the absorbance properties of the chromoionophore  24 . The deprotonated chromoionophore  24  has a shifted absorbance region such that the photons  25  emitted by the quantum dot  26  are no longer absorbed by the chromoionophore  24  (Mode A, sensor on the right). The sensor then emits a detectable signal indicating the presence of the analyte. 
     In an alternate embodiment for detecting cationic analytes,  FIG. 2 , Mode B, the quantum dot  27  of the sensor emits photons  25  that are not absorbed by the chromoionophore  24  in the absence of the cationic analyte  21 . In certain such embodiments, the chromoionophore  24  absorbs photons  25  of the quantum dot  27  when the cationic analyte  21  is bound to the ionophore  22 . In such embodiments, the wavelength of emitted photons  25  from the quantum dot  27  when excited with a light source such as UV or visible light, do not fall within the absorbance range, e.g., the maximum absorbance range, of the chromoionophore  24  when bound to a proton  23 , such that the fluorescence of the quantum dot  27  is emitted from the polymer matrix  20  (Mode B, sensor on the left). As the target ion  21  increases in concentration in solution, the ions  21  are drawn into the polymer matrix  20  to bind with the ion-selective ionophore  22 . To maintain charge neutrality within the polymer matrix  20  of the sensor, protons  23  dissociate from the chromoionophore  24  of the sensor and diffuse out of the polymer matrix  20  into the sample solution, altering the absorbance properties of the chromoionophore  24 . The deprotonated chromoionophore  24  has a shifted absorbance region such that the photons  25  emitted by the quantum dot  27  are absorbed by the chromoionophore  24  (Mode B, sensor on the right). The sensor signal is attenuated or extinguished indicating the presence of the analyte. 
     In an embodiment for detecting anionic analytes, depicted in  FIG. 3 , Mode A, the ionophore  22  of the sensor selectively binds an anionic analyte  28  or a group of anionic analytes. In certain such embodiments, the sensor comprises a chromoionophore  24  which absorbs photons  25  emitted from the quantum dot  26  upon excitation, e.g., by light such as UV or visible, when the ionic analyte  28  is not bound to the ionophore  22  of the sensor. In such a state, the wavelengths of the photons  25  emitted by the quantum dot  26  are within the absorbance range, e.g., the maximum absorbance range, of the chromoionophore  24  in a deprotonated state and the fluorescence detected outside of the polymer matrix  20  is attenuated or undetectable from outside the sensor ( FIG. 3 , Mode A, sensor on the left). As the target ion  28  increases in concentration in the sample solution, the anionic analyte  28  is drawn into the polymer matrix  20 , binding with the ion-selective ionophore  22 . To maintain charge neutrality within the polymer matrix  20 , protons  23  diffuse from the sample solution into the polymer matrix  20 , protonating the chromoionophores  24  such that the absorbance properties are altered. The protonated chromoionophore  24  has a shifted absorbance region such that the photons  25  of the quantum dot  26  are not absorbed by the chromoionophore  24  ( FIG. 3 , Mode A, sensor on the right). The sensor emits a detectable fluorescence signal indicating the presence of the analyte  28 . 
     In an alternate embodiment for detecting anionic analytes, depicted in  FIG. 3 , Mode B, the ionophore of the sensor selectively binds an anionic analyte  28  or a group of anionic analytes. In certain such embodiments, the sensor comprises a chromoionophore  24  which does not absorb photons  25  emitted from the quantum dot  26 , upon excitation, e.g., by light such as UV or visible, when the ionic analyte  28  is not bound to the ionophore  22  of the sensor. In such a state, the wavelengths of the photons  25  emitted by the quantum dot  26  are outside of the absorbance range, e.g., the maximum absorbance range, of the chromoionophore  24  in a deprotonated state and the fluorescence detected outside of the polymer matrix  20  is attenuated or absent ( FIG. 3 , Mode B, sensor on the left). As the target ion  28  increases in concentration in the sample solution, the anionic analyte  28  is drawn into the polymer matrix  20 , binding with the ion-selective ionophore  22 . To maintain charge neutrality in the polymer matrix  20 , protons  23  diffuse from the sample solution into the polymer matrix  20 , protonating the chromoionophores  24  such that the absorbance properties are altered. The protonated chromoionophore  24  has a shifted absorbance region such that the photons  25  of the quantum dot  26  are not absorbed by the chromoionophore  24  ( FIG. 3 , Mode B, sensor on the right). The sensor signal is attenuated or extinguished indicating the presence of the analyte  28 . 
     The following is a non-limiting, illustrative list of target ion ( 21  or  28 )/ionophore  22  pairings suitable for use in the sensors: potassium/Potassium Ionophore III (i.e., BME-44, 2-Dodecyl-2-methyl-1,3-propanediyl bis[N-[5′-nitro(benzo-15-crown-5)-4′-yl]carbamate]), sodium/Sodium Ionophore IV (i.e., 2,3:11,12-Didecalino-16-crown-5 2,6,13,16,19 Pentaoxapentacyclo [18.4.4.4 7,12 .0 1,20 .0 7,12 ]dotriacontane), sodium/Sodium Ionophore V (i.e., 4-Octadecanoyloxymethyl-N,N,N′,N′-tetracyclohexyl-1,2-phenylenedioxydiacetamide), sodium/Sodium Ionophore VI (i.e., Bis[(12-crown-4)methyl]dodecylmethylmalonate Dodecylmethylmalonic acid bis[(12-crown-4)methyl ester]), sodium/Sodium Ionophore X (4-tert-Butylcalix[4]arene-tetraacetic acid tetraethylester), calcium/Calcium Ionophore III (i.e., Calimycin), and calcium/Calcium ionophore IV (i.e., N,N-Dicyclohexyl-N′,N′-dioctadecyl-diglycolic diamide). For target anions, illustrative target ion/ionophore pairings include chloride/Chloride Ionophore III (i.e., 3,6-Didodecyloxy-4,5-dimethyl-o-phenylene-bis(mercury chloride) and nitrite/Nitrite Ionophore I (i.e., Cyanoaqua-cobyrinic acid heptakis(2-phenylethyl ester)). 
     In certain embodiments, the sensor further comprises a surface modifier (SM). In certain embodiments, the SM comprises a molecule that promotes the delivery or localization of the sensor within a cell. SMs of the invention include molecules with a hydrophilic portion  40  and a hydrophobic portion  42 ,  FIG. 4 . In certain embodiments, the hydrophobic portion  42  of the SM anchors the SM to the hydrophobic polymer matrix  41 . In certain embodiments, the SM is disposed on the surface of the sensor, e.g., covers a portion of the surface or covers the entire surface. Exemplary hydrophobic portions  42  of the SM include but are not limited to, lipids and hydrophobic polymers. In certain embodiments, the hydrophilic portion  40  of the SM is disposed on the surface of the sensor. An exemplary hydrophilic portion  40  includes, but is not limited to, polyethylene glycol (PEG). In certain embodiments, the hydrophilic portion (PEG) is bound to the hydrophobic portion (lipid) through a linker (e.g., phosphate, ceramide). 
     In certain embodiments, the sensor further comprises a targeting moiety. In certain embodiments, the targeting moiety is bound to the polymer matrix. In certain embodiments, the targeting moiety is bound to the SM on the surface of the polymer matrix. The targeting moiety, which assists the sensor in localizing to a particular target area, entering a target cell(s), and/or locating proximal to an ion channel, may be selected on the basis of the particular condition or site to be monitored. The targeting moiety may further comprise any of a number of different chemical entities. In one embodiment, the targeting moiety is a small molecule. Molecules which may be suitable for use as targeting moieties in the present invention include haptens, epitopes, and dsDNA fragments and analogs and derivatives thereof Such moieties bind specifically to antibodies, fragments or analogs thereof, including mimetics (for haptens and epitopes), and zinc finger proteins (for dsDNA fragments). Nutrients believed to trigger receptor-mediated endocytosis and therefore useful targeting moieties include biotin, folate, riboflavin, carnitine, inositol, lipoic acid, niacin, pantothenic acid, thiamin, pyridoxal, ascorbic acid, and the lipid soluble vitamins A, D, E and K. Another exemplary type of small molecule targeting moiety includes steroidal lipids, such as cholesterol, and steroidal hormones, such as estradiol, testosterone, etc. 
     In another embodiment, the targeting moiety may comprise a protein. Particular types of proteins may be selected based on known characteristics of the target site or target cells. For example, the probe can be an antibody either monoclonal or polyclonal, where a corresponding antigen is displayed at the target site. In situations wherein a certain receptor is expressed by the target cells, the targeting moiety may comprise a protein or peptidomimetic ligand capable of binding to that receptor. Proteins ligands of known cell surface receptors include low density lipoproteins, transferrin, insulin, fibrinolytic enzymes, anti-HER2, platelet binding proteins such as annexins, and biological response modifiers (including interleukin, interferon, erythropoietin and colony-stimulating factor). A number of monoclonal antibodies that bind to a specific type of cell have been developed, including monoclonal antibodies specific for tumor-associated antigens in humans. Among the many such monoclonal antibodies that may be used are anti-TAC, or other interleukin-2 receptor antibodies; 9.2.27 and NR-ML-05 to the 250 kilodalton human melanoma-associated proteoglycan; and NR-LU-10 to a pancarcinoma glycoprotein. An antibody employed in the present invention may be an intact (whole) molecule, a fragment thereof, or a functional equivalent thereof. Examples of antibody fragments are F(ab′) 2 , Fab′, Fab, and Fv fragments, which may be produced by conventional methods or by genetic or protein engineering. 
     Other preferred targeting moieties include sugars (e.g., glucose, fucose, galactose, mannose) that are recognized by target-specific receptors. For example, instant claimed constructs can be glycosylated with mannose residues (e.g., attached as C-glycosides to a free nitrogen) to yield targeted constructs having higher affinity binding to tumors expressing mannose receptors (e.g., glioblastomas and gangliocytomas), and bacteria, which are also known to express mannose receptors (Bertozzi, C R and M D Bednarski Carbohydrate Research 223:243 (1992); J. Am. Chem. Soc. 114:2242, 5543 (1992)), as well as potentially other infectious agents. Certain cells, such as malignant cells and blood cells (e.g., A, AB, B, etc.) display particular carbohydrates, for which a corresponding lectin may serve as a targeting moiety. 
     The nanosensors of the invention can be incorporated into cells to detect ionic analytes and determine the presence and/or concentration, e.g., with a fluorescence signal.  FIG. 1  is a schematic diagram of a cell assay system  100  according to an illustrative embodiment of the invention. The cell assay system  100  includes a support  102 , a biological sample holder  104 , an excitation light source  106 , a light sensor  108 , and a computing device  111 . 
     The support  102  supports a sensor  112 , e.g., a film having a suspension of sensor matrices as described above, for positioning in the biological sample holder  104 . In various implementations, the sensor  112  is adhered to the support  102  by deposition in a solution of sensor matrices dissolved or dispersed in a solvent, such as in a polar organic solvent like tetrahydrofuran (THF). In such implementations, the support  102  is preferably formed from a material resistant to the solvent. Materials resistant to THF include, without limitation,  304  stainless steel;  316  stainless steel; acetal polymer (marketed as DELRIN by E.I. du Pont de Nemours and Company); bronze; carbon graphite; carbon steel; ceramic Al 2 O 3 , a perfluoroelastomer compound, such as CHEMRAZ marketed by Greene, Tweed; epoxy; HOSTELRY Calloy (marketed by Haynes International, Inc.); KALES elastomer (marketed by DuPont Performance Elastomers); polychlorotrifluoroethylene; NYLON (marketed by E.I. du Pont de Nemours and Company); polyetherether ketone (PEEK); polyphenylene sulfide; and PTFE. 
     The film of the sensor can be produced in various ways. In one implementation, as described above, a predetermined amount of the sensor mixture (e.g., the combined polymer phase, ionophore, quantum dots, additive, and chromoionophore) is dissolved in a solvent, such as THF. The solution is then deposited, sprayed, or spun onto a surface. The solvent evaporates, leaving the sensor film on the surface. 
     In another implementation, the film is formed from a deposition of sensor microspheres. To produce the microspheres, a sensor emulsion is formed by injecting a sensor suspension dissolved in THF (e.g., 16 mL THF/100 mg PVC) into a pH buffered solution. The sensor suspension includes approximately 60 mg of DOS, 30 mg of PVC, and up to approximately 5 mg of chromoionophore, additive, and ionophore. The emulsion is then submerged in a sonicating water bath. Typically, 50 μL of the sensor suspension/THF solution is injected into 1,000-1,500 μL of buffered solution. The resulting emulsion contains a mixture of spherical sensor particles ranging in size from 200 nm to 20 pm in diameter. In certain embodiments, the nanosensors range in size from about 5 nm to about 300 nm in diameter, such as about 20 nm to about 200 nm in diameter, e.g., about 100 nm. In certain embodiments, the nanosensors that comprise only one quantum dot range in size from about 5 nm to about 50 nm in diameter, such as about 5 nm to about 25 nm in diameter, e.g., 20 nm. In certain embodiments wherein the particles are non-spherical, the diameter is measured at the widest dimension of the nanosensor. Particles of larger dimension are, of course, readily prepared. 
     Sensor materials as discussed herein can be sized and shaped in any suitable configuration that can be achieved using the polymer. For example, in certain embodiments, the nanosensors are non-spherical, such as a disk or a cube, or even sculpted or molded into a utilitarian or aesthetic shape. A sensor emulsion can be spun, sprayed, or evaporated onto any surface to create a porous sensor membrane. In certain embodiments, the sensor film can be of a size suitable for the application, such as the coating of a glass slide, the bottoms of wells of a 96-well plate, or even a beverage dispenser, such as a pitcher, tank, or bottle. Films formed from microspheres tend to expose a greater surface area of sensor to a given sample, yielding improved performance characteristics. 
     In certain aspects, a film of the sensor particles is deposited on the surface of a support. In certain embodiments, the support is an instrument that can be placed in a solution such as a glass rod, a stirring bar, a straw, or glass beads. In certain embodiments, the support is a container in which the ionic solution to be evaluated can be contained. In certain embodiments, the surface of the support is partially coated with the sensor particles while in other embodiments, the support surface is entirely coated with the sensor particles. In certain embodiments, the sensors are incorporated within the support and the support is sculpted into a desired shape such as a stir bar, a film, or a bead. 
     In certain embodiments, the sensors are used to detect ions in water or other aqueous solutions. In certain embodiments, the support deposited with the sensor particles is used to detect the presence of ions in an aqueous solution. In certain exemplary embodiments, the sensors are used to detect ions in water, e.g., tap water or ground water, to determine the levels of toxic ions in solution or to determine the hardness of the aqueous solution. In certain exemplary embodiments, the sensors are added to manufacturing solutions to measure ions during production of, e.g., the mass production of soda, ion-restoring beverages or other ionic drinks. In certain embodiments, the sensors are used in the laboratory to monitor the ion content of a reaction mixture or stock solution. 
     The biological sample holder  104  holds a biological sample for analysis by the cell assay system  100 . The biological sample can include cells adhered to the walls of the biological sample holder  104 , for example, in a monolayer, or cells suspended in a liquid buffer. The biological sample holder  104  is preferably transparent, or at least includes a transparent region through which the sensor  112  can be excited and through which the results of such excitation can be monitored. 
     The sensor  112  is illuminated with a light source  106  to excite the quantum dots. The light source can be in the UV or visible portion of the electromagnetic spectrum, or the light source may generate a wide spectrum light. In one implementation, the light source  106  is coupled to the support  102 . 
     The emissions of the sensor  112  is detected, e.g., the fluorescence of the sensor is detected by a light sensor  108 . The light sensor  108  may include a charge-coupled device, a fluorometer, a photomultiplier tube, or other suitable device for measuring fluorescence. In one implementation, a spectrophotofluorometer is as both the light source  106  and the light sensor  108 . The light sensor  108  may also be coupled to the support  102 . 
     The support  102  may include an agent introduction port  118 . The agent introduction port  118  can include a pipette or an electro-mechanical dispenser device, such as a solenoid or electrostatically driven plunger or syringe. 
     The computing device  111  controls the various components of the cell assay system  100 . The computing device  111  may be a single computing device or multiple computing devices providing the various functionalities used to control the cell assay system. These functionalities are provided by an excitation control module  126 , an agent introduction module  130 , and an analysis module  134 . The excitation control module  126  controls the light source  108  to emit a wider or narrower wavelength range of excitation light. The agent introduction module  130  controls the introduction of an agent into the biological sample holder  104  via an agent introduction means  118 . The analysis module  134  analyzes the output of the light sensor  108 , e.g., before and after an agent is introduced into the biological sample holder  104  to determine the effect of the agent on the cells in the biological sample holder  104 . The analysis module  134  may also control the other modules in the computing device, i.e., the excitation control module  126  and the agent introduction module  130 , to coordinate an assay protocol. The computing device  111  and/or devices may also include various user interface components, such as a keyboard, mouse, trackball, printer, and display. 
     A module may be implemented as a hardware circuit comprising custom VLSI circuits or gate arrays, off-the-shelf semiconductors such as logic chips, transistors, or other discrete components. A module may also be implemented in programmable hardware devices such as field programmable gate arrays, programmable array logic, programmable logic devices or the like. 
     Modules may also be implemented in software for execution by various types of processors. An identified module of executable code may, for instance, comprise one or more physical or logical blocks of computer instructions which may, for instance, be organized as an object, procedure, or function. Nevertheless, the executables of an identified module need not be physically located together, but may comprise disparate instructions stored in different locations which, when joined logically together, comprise the module and achieve the stated purpose for the module. A module of executable code may be a single instruction, or many instructions, and may even be distributed over several different code segments, among different programs, and across several memory devices. 
     The various modules are in communication with the various devices they control or from which they obtain data. They may be connected over a local area network, wirelessly, over a bus, or over typical cables known in the art of computer interfaces for connecting computing devices with peripherals. 
       FIGS. 5A-5D  depict sensor arrangements suitable for use in various implementations of the cell assay system  100  of  FIG. 1 .  FIG. 5A  shows a first sensor arrangement that includes a support  202  and a biological sample holder  204 . The biological sample holder  204  includes a monolayer of cells  206  adhered to the biological sample holder  204 . Alternatively, the biological sample holder  204  holds cells suspended in a buffer. The support  202  and biological sample holder  204  correspond to the support  102  and biological sample holder  104  of  FIG. 1 . A sensor film  210  is coupled to the distal end of the support  202 . 
       FIG. 5B  illustrates an alternative sensor arrangement  250 , which includes a biological sample holder  254  having therein a monolayer of cells  256  adhered to the surfaces of the biological sample holder  254 , or cells suspended in a buffer. Instead of including a sensor film adhered to a support, the sensor arrangement  250  relies upon nanosensor particles  258  introduced into the cells  256  adhered to the biological sample holder  254 . 
     The nanosensor particles  258  may be introduced into the cells  256  in any suitable manner. In one method, the particles  258  are introduced into a buffer liquid deposited in the biological sample holder  254 . A voltage source then generates a voltage sufficiently strong to electroporate the cells  256 , thereby allowing the nanosensor particles  258  to enter directly into the cells. In another approach, the surfaces of the nanosensor particles  258  are first coated with a substance such as a hydrophilic coating, for example polyethyleneglycol (PEG), which assist the particles  258  in crossing through cell membranes. The nanosensor particles  258  contact the cells  256  which bring the particles  258  into their interior in vesicles via endocytosis, pinocytosis, phagocytosis, or similar biological processes. In certain embodiments, a substance applied to the nanosensor particles  258  breaks down the vesicle membrane, releasing the nanosensor particles  258  into the cell cytoplasm. In still other approaches, the particles  258  may be introduced into cells  256  using a glass needle or through ballistic bombardment. 
     To determine compartmentalization of nanosensors within the cells TEM and fluorescence staining can be used. TEM can be used to determine location of the nanosensor in a cell, may provide a good understanding of nanosensor transport in the cell and serve as a validation of the co-localization staining. The second method, co-localization staining, can be used to determine endosomal release. 
     Dyes suitable for performing co-localization studies include: FM1-43, FM4-64, Fluorescein Transferrin, and Lysotracker Red. FM1-43 is a lipophilic dye that readily stains cell membranes. Previous studies have shown the effectiveness of FM1-43 to stain endosomes. Its fluorescence emission is typically greatly increased upon incorporation into a hydrophobic environment. FM1-43 will typically stain the plasma membrane of a cell and remain associated with the lipid bilayer as it forms an endosome. Dye that is not taken into the cell and remains on the plasma membrane can be easily removed by gentle washing. FM4-64 is an analog of FM1-43 and behaves in a very similar fashion. It is more hydrophobic then FM1-43 and therefore may be more suitable for endocytosis studies. FM4-64 has been well characterized as an endosomal stain. The long wavelength emission of FM4-64 may be advantageous when using sensors of different spectral properties similar to the other fluorescent stains being utilized. 
     In some embodiments, the sensor is attached to the exterior of a cell rather than introduced into the interior. If, for example, the activity of an ion channel is to be studied, the sensor may be attached to the cell surface or placed in close proximity to the cell surface in a location where ion concentrations are in flux, such as adjacent to an ion channel. The sensor may be positioned adjacent to the ion channel of a cell, for example, by covalently linking one or more antibodies that selectively bind the ion channel of interest to a sensor particle as described above. The antibody-linked sensor particles may be added to a cell suspension to bind to the ion channel. This approach can be used to link ion-specific sensors to any feature on the exterior of the cell membrane to which antibodies selectively bind. Alternatively, the sensors may be attached to the cell membrane by other suitable coupling chemistries, e.g., biotin-(strept)avidin complexing or polysaccharide binding. See the thesis “High Throughput Optical Sensor Arrays for Drug Screening” by Daniel I. Harjes (2006), available from the Massachusetts Institute of Technology and incorporated herein by reference. 
       FIG. 5C  illustrates a second alternative sensor arrangement  270  that includes a support  272  and a biological sample holder  274 . A sensor film  276  is coupled to the distal end of the support  272 . A cell monolayer  278  adheres to the surfaces of the biological sample holder  274 . Alternatively, cells are suspended in a buffer. In addition, nanosensor particles  280  are introduced into the cells. In certain embodiments, the quantum dots used in the sensor film  276  differ from the quantum dots used in the nanosensor particles  280 . In particular, the different quantum dots desirably have distinguishable fluorescence characteristics such that an analysis module analyzing the output of a light sensor monitoring the sensor arrangement  270  can differentiate between the output of the sensor film  272  and the nanosensor particles  280 . As a result, the analysis module can differentiate between intracellular target ion concentration and extracellular target ion concentration. In an exemplary embodiment, the sensor film comprises quantum dots of a selected fluorescence wavelength, e.g., 560 nm, and the nanosensor particles comprise quantum dots of a selected fluorescence wavelength, e.g., 655 nm. In addition, the sensor film  272  may include ionophores different from those included in the sensor particles  280 , e.g., nanosensor particles comprising sodium ionophores and sensor films comprising potassium ionophores. Thus, the sensor arrangement  270  can monitor the concentrations of two different target ions. 
       FIG. 5D  illustrates a third alternative sensor arrangement  290  that includes an electrode support  292  and a biological sample holder  294 . The biological sample holder  294 , in addition to a cell monolayer  296  or cells suspended in a buffer, includes a removable sensor film  298 . The removable sensor film  298 , for example, can be a glass cover slip or other transparent surface coated with a sensor film. 
     In still another embodiment, the sensor film is coated onto the inner surface of the biological sample holder. And in another approach, to accommodate multiwell plates, such as the 96-well plate format often used in assays, one embodiment of the present invention utilizes round glass coverslips coated with the sensor film along with the cells to be monitored. In certain embodiments, larger multiwell plates such as 384- and 1536-well plates are applied with a layer of sensor film disposed on a surface of some or all of the wells. In these embodiments, each well contains a single sensor type to track a specific species of interest; the various sensor types may differ in the ionophore employed and utilize quantum dots with fluorescence wavelengths that are the same or similar. The compound of interest is then added directly to the well. The multiwell plate is then placed in a fluorometer and the fluorescence intensity is monitored with time. 
     In a typical implementation, a plurality of biological sample holders holding biological samples is provided. Biological samples introduced into the holders may include cells suspended in a buffer solution, but alternatively cells may be adhered to the walls of the biological sample holders. Next, sensors are introduced into the biological sample holders as shown in  FIGS. 5A and 5C , and/or are introduced into the cells themselves. Alternatively, the sensors can coat the walls of the biological sample holders. As described above, nanosensor particles can be introduced either by electroporating the cells via electrodes positioned in the biological sample holders or by the chemistry applied to the nanosensor particles breaching vesicle membranes within the cells. Similarly, the sensors can be introduced into the cells using pico-injection, bead loading, a gene gun, or through liposomal delivery techniques known in the art. 
     An agent, such as a therapeutic, toxin, biological macromolecule (such as a nucleic acid, an antibody, a protein or portion thereof, e.g., a peptide), small molecule (of 2000 amu or less, 1000 amu or less or 500 amu or less), protein, virus, bacteria, chemical compound, mixture of chemical compounds, or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues, or other biologically active agent may be introduced into one or more of the biological sample holders. In one particular implementation using an array of biological sample holders, no agent is introduced into a first row of biological sample holders to preserve a control. A first agent is introduced into a second row of biological sample holders. Additional agents are added to additional rows of the array of biological sample holders. The fluorescence of the sensors introduced into the biological sample holders may be monitored. The monitoring preferably begins prior to introduction of the agents and continues thereafter. Changes in ion concentration resulting from the introduced agents are then determined. By comparing the changes in ion concentration after adding an agent, one can determine the effect of the agent on the cells being tested. 
     The sensors of the invention can be used to monitor the effects of pharmaceutical agents on biological systems such as the cardiovascular system or the circulatory system. Action potentials generated by cardiac or neural cells in culture are defined by a flux of sodium and potassium into and out of the cell. In certain embodiments, the sensors of the invention measure this ion flux in cardiac cells accurately and spatially in a high throughput manner. 
     In certain aspects, the sensors are used in the drug discovery process. In certain such embodiments, the sensors are used to measure the efficacy of a therapy. For example, ion-selective sensors may be employed to monitor the effect of ion channel-modulating drugs. In alternative embodiments, sensors are used to screen for cytotoxic substances by, for example, determining ionic flux in cardiac cells in response to a cytotoxic agent and using these values as a comparison for testing novel therapeutic agents. 
     In certain aspects, the sensors of the invention are implanted into small animals to monitor biological responses to new therapeutic agents. In certain embodiments, the implantable sensors are used to study the mechanism of disease in small animals. In certain such embodiments, the animals, such as rats or mice, are, for example, infected with a disease and the biological functions are monitored by detecting the signal of the implanted optical sensors. In such embodiments, the animal is placed within a monitoring element, e.g., a fluorescent monitoring cell similar to a monitoring element used to take X-rays of small animals, wherein the quantum dots of the sensors are excited, e.g., with UV light, and fluorescence emitted from the sensors within the animal may be detected. 
     In various embodiments the invention may be constructed to directly detect the presence of particular ions. As illustrated in the tables below, it is known in the art that certain diseases affect particular ion channels in a cell. Accordingly, assays for those ions utilizing the present invention may furnish a diagnostic tool to determine the presence of particular diseases. Accordingly, the scope of the present invention should be understood to also include the application of the heretofore-described subject matter to measure the ions set forth in the following tables, as well as their application to diagnose the presence of the associated diseases also appearing in the following tables. 
     
       
         
               
               
               
               
               
             
               
             
               
               
               
               
               
             
               
             
               
               
               
               
               
             
               
             
               
               
               
               
               
             
               
             
               
               
               
               
               
             
               
             
               
               
               
               
               
             
               
             
               
               
               
               
               
             
               
             
               
               
               
               
               
             
           
               
                   
               
             
             
               
                   
                   
                 Channel-forming 
                   
                   
               
               
                 Channel 
                 Gene 
                 unit/ligand 
                 OMIM 
                 Disease 
               
               
                   
               
             
          
           
               
                 Cation channels: 
               
             
          
           
               
                 CHRNA1/ACHRA 
                 CHRNA1 
                 α, ACh 
                 100690 
                 Myasthenia congenita 
               
               
                 CHRNA4 
                 CHRNA4 
                 α, ACh 
                 118504 
                 Autosomal dominant nocturnal frontal lobe epilepsy 
               
               
                 CHRNB2 
                 CHRNB2 
                 β, ACh 
                 118507 
                 Autosomal dominant nocturnal frontal lobe epilepsy 
               
               
                 Polycystin-2 
                 PKD2 
                 α 
                 173910 
                 Autosomal dominant polycystic kidney disease (ADPKD) 
               
               
                 CNGA3 
                 CNGA3 
                 α, cGMP 
                 60053 
                 Achromatopsia 2 (color blindness) 
               
               
                 CNGB1 
                 CNGB1 
                 β, cGMP 
                 600724 
                 Autosomal recessive retinitis pigmentosa 
               
               
                 CNGB3 
                 CNGB3 
                 β, cGMP 
                 605080 
                 Achromatopsia 3 
               
             
          
           
               
                 Sodium channels: 
               
             
          
           
               
                 Na.1.1 
                 SCN1A 
                 α 
                 182389 
                 Generalized epilepsy with febrile seizures (GEFS+) 
               
               
                 Na.1.2 
                 SCN2A 
                 α 
                 182390 
                 Generalized epilepsy with febrile and afebrile seizures) 
               
               
                 Na.1.4 
                 SCN4A 
                 α 
                 603967 
                 Paramyotonia congenital, potassium aggressive myotonia, 
               
               
                   
                   
                   
                   
                 hyperkalemic periodic paralysis 
               
               
                 Na.1.5 
                 SCN5a 
                 α 
                 600163 
                 Long-QT syndrome, progressive familial heart block type 1, 
               
               
                   
                   
                   
                   
                 Brugada syndrome (idiopathic ventricular arrhythmia) 
               
               
                 SCNIB 
                 SCN1B 
                 β 
                 600235 
                 Generalized epilepsy with febrile seizures (GEFS+) 
               
               
                 ENACα 
                 SCNNIA 
                 α 
                 600228 
                 Pseudohypoaldosteronism type 1 (PHA1) 
               
               
                 ENaCβ 
                 SCNN1B 
                 β 
                 600760 
                 PHA1, Liddle syndrome (dominant hypertension 
               
               
                 ENaCγ 
                 SCNN1G 
                 γ 
                 600761 
                 PHA1, Liddle syndrome 
               
             
          
           
               
                 Potassium channels: 
               
             
          
           
               
                 K, 1.1. 
                 KCNA1 
                 α 
                 176260 
                 Episodic ataxia with myokymia 
               
               
                 KCNQI/K, LQT1 
                 KCNQ1 
                 α 
                 192500 
                 Autosomal dominant long-QT syndrome (Romano-Ward) 
               
               
                   
                   
                   
                   
                 Autosomal recessive long-QT syndrome with deafness (Jervell- 
               
               
                   
                   
                   
                   
                 Lange-Nielsen) 
               
               
                 KCNQ2 
                 KCNQ2 
                 α 
                 602235 
                 BFNC (epilepsy), also with myokymia 
               
               
                 KCNQ3 
                 KCNQ3 
                 α 
                 602232 
                 BFNC (epilepsy) 
               
               
                 KCNO4 
                 KCNQ4 
                 α 
                 603537 
                 DFNA2 (dominant hearing loss) 
               
               
                 HERG/KCNH2 
                 KCNH2 
                 α 
                 152427 
                 Long-QT syndrome 
               
               
                 Kir1.1/ROMK 
                 KCNJ1 
                 α 
                 600359 
                 Bartter syndrome (renal salt loss, hypokalemic alkalosis) 
               
               
                 Kir2.1/IRK/KCNJ2 
                 KCNJ2 
                 α 
                 600681 
                 Long-QT syndrome with dysmorphic features (Andersen 
               
               
                   
                   
                   
                   
                 syndrome) 
               
               
                 Kir6.2/KATATP ATP   
                 KCNJ11 
                 α 
                 600937 
                 Persistent hyperinsulinemic hypoglycemia of infancy (PHHI) 
               
               
                 SURI 
                 SURI 
                 β 
                 600509 
                 PHHI 
               
               
                 KCNE1/Mink/ISK 
                 KCNE1 
                 β 
                 176261 
                 Autosomal dominant long-QT syndrome (Romano-Ward) 
               
               
                   
                   
                   
                   
                 Autosomal recessive long-QT syndrome with deafness (Jervell- 
               
               
                   
                   
                   
                   
                 Lange-Nielson) 
               
               
                 KCNE2/MiRP1 
                 KCNE2 
                 β 
                 603796 
                 Long-QT syndrome 
               
               
                 KCNE3/MiRP2 
                 KCNE3 
                 β 
                 604433 
                 Periodic paralysis 
               
             
          
           
               
                 Calcium channels: 
               
             
          
           
               
                 Ca, 1.1 
                 CACNA1S 
                 α 
                 114208 
                 Hypokalemic periodic paralysis, malignant hyperthermia 
               
               
                 Ca, 1.4 
                 CACNA1F 
                 α 
                 300110 
                 X-linked congenital stationary night blindness 
               
               
                 Ca, 2.1 
                 CACNA1A 
                 α 
                 601011 
                 Familial hemiplegic migraine, episodic staxia, spinocerebella 
               
               
                   
                   
                   
                   
                 ataxia type 6 
               
               
                 RyRI 
                 RYR1 
                 α 
                 180901 
                 Malignant hyperthermia, central core disease 
               
               
                 RyR2 
                 RYR2 
                 α 
                 180902 
                 Catecholaminergic polymorphic ventricular tachycardia, 
               
               
                   
                   
                   
                   
                 arrhythmogenic right ventricular dysplasia type 2 
               
             
          
           
               
                 Chloride channels: 
               
             
          
           
               
                 CFTR 
                 ABCC7 
                 α 
                 602421 
                 Cystic fibrosis, congenital bilateral asplasia of vas deference 
               
               
                 CIC-1 
                 CLCN1 
                 α 
                 118425 
                 Autosomal recessive (Becker) or dominant (Thomsen myotonia 
               
               
                 CIC-5 
                 CLCN5 
                 α 
                 300008 
                 Dent&#39;s disease (X-linked proteinuria and kidney stones) 
               
               
                 CIC-7 
                 CLCN7 
                 α 
                 602727 
                 Osteopetrosis (recessive or dominant) 
               
               
                 CIC-Kb 
                 CLCNKB 
                 α 
                 602023 
                 Bartter syndrome type III 
               
               
                 Barttin 
                 BSND 
                 β 
                 606412 
                 Bartter syndrome type IV (associated with sensorineural 
               
               
                   
                   
                   
                   
                 deafness) 
               
               
                 GLRA1 
                 GLRA1 
                 α, glycine 
                 138491 
                 Hyperekplexin (startle desease) 
               
               
                 GABAα1 
                 GABRA1 
                 α GABA 
                 137160 
                 Juvenile myoclonus epilepsy 
               
               
                 GABAγ2 
                 GABRG2 
                 γ, GABA 
                 137164 
                 Epilepsy 
               
             
          
           
               
                 Gap junction channels: 
               
             
          
           
               
                 Cx26 
                 GJB2 
                   
                 121011 
                 DFNB3 (autosomal dominant hearing loss) 
               
               
                   
                   
                   
                   
                 DFNB1 (autosomal recessive hearing loss) 
               
               
                 Cx30 
                 GJB4 
                   
                 605425 
                 DFNA3 
               
               
                 Cx31 
                 GJB3 
                   
                 603324 
                 DFNA2 
               
               
                 Cx32 
                 GJB1 
                   
                 304040 
                 CMTX (X-linked Charcot-Mari-Tooth neuropathy) 
               
               
                 AChR α7 
                   
                   
                   
                 Inflammation 
               
               
                 ClC7 
                   
                   
                   
                 Osteoporosis 
               
               
                 Ether-a-go-go (eag, 
                   
                   
                   
                 Cancer 
               
               
                 erg, elk) 
                   
                   
                   
                   
               
               
                 Gardos channel 
                   
                   
                   
                 Sickle cell anemia 
               
               
                 P2X7 
                   
                   
                   
                 Immune disorders 
               
               
                 TRPC6 
                   
                   
                   
                 Asthma, COPD 
               
               
                 TRPM1 
                   
                   
                   
                 Melanoma 
               
               
                 TRPM2 
                   
                   
                   
                 Asthma 
               
               
                 TRPM4 
                   
                   
                   
                 Immune disorders 
               
               
                 TRPM7 
                   
                   
                   
                 Stroke 
               
               
                 TRPM8 
                   
                   
                   
                 Prostate cancer 
               
               
                 TRPV1 
                   
                   
                   
                 Urinary incontinence, pain 
               
               
                   
               
             
          
           
               
                 The third column classifies channel proteins into α, β, and γ subunits, where α subunits are 
               
               
                 always directly involved in pore formation, Several β subunits are only accessory (i.e., do not 
               
               
                 form pores), as is the case, for example, with SCN1B and barttin. Others (e.g. of ENaC and 
               
               
                 GABA receptors) participate in pore formation. For ligand-gated channels, the ligand is given. 
               
               
                 Note that GABA and glycine act from the extracellular side, whereas cGMP is an intracellular 
               
               
                 messenger. 
               
             
          
           
               
                 Gene 
                 Accession ID 
                 Gene Locus 
                   
                 Tissue Expression 
               
               
                   
               
               
                   
                   
                   
                 Sodium Channel Type/Disease 
                   
               
               
                 SCN1A 
                 GDB: 118870 
                 2q24 
                 SCN1, vg type 1, α-subunit (280 KDa) 
                 Brain 
               
               
                   
                 S71446 
                   
                   
                   
               
               
                 SCN1B 
                 GDB: 127281 
                 19q13.1 
                 Hs.89634, vg type 1 β 1  subunit 
                 Brain, heart, skeletal 
               
               
                   
                 U12188-12104 
                   
                 (38 KDa) 
                 muscle 
               
               
                   
                 L16242, L10338 
                   
                   
                   
               
               
                 SCN2A1 
                 GDB: 120367 
                 2q23 
                 SCN2A, HBSCI, vg type II, α 1 - 
                 Brain, peripheral nerve 
               
               
                   
                   
                   
                 subunit (280(KDa) 
                   
               
               
                 SCN2A2 
                 CDB: 133727 
                 2q23-24.1 
                 HBSCH, vg type II, α 2 -subunit vg 
                 Brain 
               
               
                   
                   
                   
                 type II, β 2 -subunit (33 KDa) 
                   
               
               
                 SCN2B 
                 GDB: 118871 
                   
                   
                   
               
               
                   
                 AF019498 
                   
                   
                   
               
               
                 SCN3A 
                 GDB: 132151 
                 2q24-31 
                 vg type III, α-subunit (280 kDa) 
                 Brain 
               
               
                   
                 S69887 
                   
                   
                   
               
               
                 SCN4A 
                 GDB: 125181 
                 17q23.1-25.3 
                 SkM1, vg type IV α-subunit (260 kDa), 
                 Skeletal muscle 
               
               
                   
                 L04216-L04236 
                   
                 hyperkalemic periodic 
                   
               
               
                   
                   
                   
                 paralysis, paramyotonia congentia, 
                   
               
               
                   
                   
                   
                 potassturn-aggravated myotonia 
                   
               
               
                 SCN4B 
                 GDB: 125182 
                 3q21 
                 vg type IV, β-subunit, 
                 Heart, fetal skeletal 
               
               
                 SCN5A 
                 GDB: 132152 
                   
                 SkM2, hH1, vg type V, α-subunit, 
                 muscle 
               
               
                   
                   
                   
                 long Q-T syndrome 3 
                   
               
               
                 SCN6A 
                 GDB: 132153 
                 2q21-23 
                 Hs99945, vg type VI, α-subunit 
                 Heart, uterus, fetal and 
               
               
                   
                   
                   
                   
                 denervated skeletal muscle 
               
               
                   
                   
                   
                 Calcium Channel Type/Disease 
                   
               
               
                 SCN7A 
                 GDB: 228137 
                 12q13 
                 vg type VII, α-subunit 
                 Brain, spinal cord 
               
               
                 SCN8A 
                 GDB: 631695 
                   
                 vg type VIII, α-subunit, motor 
                   
               
               
                   
                   
                   
                 end-plate disease + ataxia in mice 
                   
               
               
                 SCN9A 
                 GDB: 3750013 
                   
                 vg type IX, α-subunit 
                 Thyroid and adrenal 
               
               
                   
                   
                   
                 neuroendocrine type 
                 gland 
               
               
                 SCN10A 
                 GDB: 750014 
                 1pter-p36.3 
                 hPN3, vg type X 
                 Sensory neurons, dorsal 
               
               
                   
                   
                   
                   
                 root ganglia 
               
               
                 SCNN1A 
                 GDB: 366596 
                 12pt3 
                 SCNN1, nvg type 1 α-subunit of 
                 Kidney, lung colon 
               
               
                   
                 Z92978 
                   
                 ENaC 
                   
               
               
                 SCNN1B 
                 GDB: 434471 
                 16p12.2-p12.1 
                 nvg 1 β-subunit, Liddle&#39;s syndrome 
                 Kidney, lung colon 
               
               
                   
                   
                   
                 pseudohypoaldosterontsm I 
                   
               
               
                 SCNN1D 
                 GDB: 6053678 
                 1p36.3-p36.2 
                 DnaCh, nvg 1 δ-subunit 
                 Kidney, lung, colon 
               
               
                 SCNN1G 
                 GDB: 568769 
                 16p122-p12.1 
                 nvg 1 γ-subunit, Liddle&#39;s syndrome 
                 Kidney, lung, colon 
               
               
                   
                 X87160 
                   
                 pseudohypoaldosterontsm I 
                   
               
               
                 CACNA1A 
                 GDB: 126432 
                 19p13 
                 P/Q type α 1A- subunit, eqisodic ataxia 
                 Brain (cortex, bulbus, 
               
               
                 CACNL1A4 
                 Z80114-Z80155, 
                 19p13.1 
                 2, familial hemiplegic migraine, 
                 olfacorius, 
               
               
                   
                 X99697, U79666 
                   
                 spinocerebellar ataxia 6; tottering, 
                 hippocarnpus, 
               
               
                   
                   
                   
                 leaner, and rolling mice 
                 cerebellum, brain stem), 
               
               
                   
                   
                   
                   
                 motoneurons, kidney 
               
               
                 CACNA1B 
                 GDB: 580689 
                 9q34 
                 CACNN, N-type α 1A- subunit 
                 Central, peripheral 
               
               
                 CACNL1A5 
                 M94172, M94173 
                   
                   
                 nervous system 
               
               
                 CACNA1C 
                 GDB: 126094 
                 12p13 
                 CCHL1A1, L-type α 1A- subunit 
                 Heart, fibroblasts, lung, 
               
               
                 CACNL1A1 
                 L29636, L29634, 
                 12p13.3 
                   
                 smooth muscle (2 splice 
               
               
                   
                 L29629 
                   
                   
                 variants) 
               
               
                 CACNA1D 
                 GDB: 128872 
                 3p14.3 
                 CCHL1A2, L-type α 1D -subunit 
                 Brain, pancreas, 
               
               
                 CACNL1A2 
                   
                 3p21.3.2? 
                   
                 neuroendocrine 
               
               
                 CACNA1E 
                 GDB: 434408 
                 1q25-31 
                 R-type α 1C- subunit 
                 Brain, skeletal muscle 
               
               
                 CACNL1A6 
                   
                   
                   
                 (end plate) 
               
               
                 CACNA1F 
                 GDB: 6053864 
                 Xp11.23-11.22 
                 α 1F -Subunit 
                 Retina 
               
               
                 GACNIAG 
                 AF27964 
                 17q22 
                 T-type α 1G -subunit 
                 Brain 
               
               
                 CACNA1S 
                 GDB: 126431 
                 1q31-32 
                 L-type α 1B -subunit (5% 212, 95% 
                 Skeletal muscle (brain, 
               
               
                 CACNL1A8 
                 Z22672, L33798 
                   
                 190 kDa), malignant hyperthermia 
                 kidney) 
               
               
                   
                 U30666-U30707 
                   
                 5, hypokalernic periodic paralysis 
                   
               
               
                 CACNA2 
                 GDB: 132010 
                 7q21-22 
                 CACNA2, CACNA2D1, α 8     8   -subunit 
                 α 2A ; skeletal muscle, 
               
               
                 CACNL2A 
                 Z28613, Z28609 
                   
                 (175 kDa), MHS3 
                 heart, brain, ileum; α 2B ; 
               
               
                   
                 Z28605, Z28602 
                   
                   
                 brain; α 2CVD ; aorta 
               
               
                   
                 Z28699, M76559 
                   
                   
                   
               
               
                 CACNB1 
                 GDB: 132012 
                 17q21-22 
                 β 1 -Subunit (524 aa, 54 kDa) 
                 β 1 A/M; skeletal muscle 
               
               
                 CACNLB1 
                 GDB: 1073281 
                   
                   
                 β 1 B/C; brain, heart, 
               
               
                   
                 U86952-U86961 
                   
                   
                 spleen 
               
               
                   
                 M76560, L06111 
                   
                   
                   
               
               
                   
                 GDB: 193328 
                   
                   
                   
               
               
                 CACNB2 
                 GDB: 132014 
                 10p12 
                 MYSB, β 2 -subunit 
                 β 2 A/B/E; brain, heart, 
               
               
                 CACNLB2 
                 Q08289 
                   
                   
                 lung, aorta 
               
               
                 CACNB3 
                 GDB: 341023 
                 12q13 
                 β 2 -subunit (482 aa) 
                 Brain, heart, lung, spleen, 
               
               
                 CACNLB3 
                 L27584 
                   
                   
                 skeletal and smooth 
               
               
                   
                   
                   
                   
                 muscle, aorta, trachea, 
               
               
                   
                   
                   
                   
                 ovary, colon 
               
               
                 CACNB4 
                 GDB: 6028693 
                 2q22-23 
                 β 2 -subunit, lethargic mice 
                 Brain, kidney 
               
               
                 CACNG 
                 GDB: 132015 
                 17q24 
                 γ-Subunit (222 aa, 30 kDa) 
                 Skeletal muscle, lung 
               
               
                 CACNLG 
                 L07738 
                   
                   
                   
               
               
                 CACNG2 
                   
                   
                 γ2-Subunit, stargazin, absence 
                 Brain 
               
               
                   
                   
                   
                 epilepsy stargazer, waggler mice 
                   
               
               
                 RYR1 
                 GDB: 120359 
                 19q13.1 
                 Ryanodine receptor 1, Ca release 
                 Skeletal muscle, testis, 
               
               
                   
                   
                   
                 channel, 3 splice variants, 
                 brain, submaxillary and 
               
               
                   
                   
                   
                 malignant hyperthermia 1, central 
                 adrenal glands, spleen 
               
               
                   
                   
                   
                 core disease 
                   
               
               
                 RYR2 
                 GDB: 125278 
                 1pter-qter 
                 RYR2, calcium release channel 
                 Heart, smooth muscle 
               
               
                   
                   
                 1q42.1-43 
                   
                   
               
               
                 RYR3 
                 GDB: 138451 
                 15q14 
                 RYR3, calcium release channel 
                 Brain, neonatal skeletal 
               
               
                   
                   
                 15q14-15 
                   
                 muscle, adult diaphragm 
               
               
                   
                   
                   
                 Potassium Channel Type/Disease 
                   
               
               
                 KCNA1 
                 GDB: 127903 
                 12p13 
                 RBK1, HUK1, MBK1, AEMK, 
                 Brain, nerve, heart, 
               
               
                   
                 LO2750 
                   
                 Kv1.1, Shaker homolog 1, Shaker, 
                 skeletal muscle, retina, 
               
               
                   
                   
                   
                 episodic ataxia 1 (with myokymia) 
                 pancreatic islet 
               
               
                 KCNA1B 
                   
                 3q26.1 
                 Kvβ1.1, Kvβ1.3 (splice product), 
                   
               
               
                   
                   
                   
                 β-subunit 
                   
               
               
                 KCNA2 
                 GDB: 128062 
                 12pter-qter 
                 HK4, Kv1.2, Shaker homolog 2 
                 Brain, nerve, heart, 
               
               
                   
                 X17622 
                   
                   
                 pancreatic islet 
               
               
                 KCNA2B 
                   
                 1p36.3 
                 Kvβ1.2, β-subunit 
                   
               
               
                 KCNA8 
                 GDB: 128079 
                 1p13.3 
                 Hs.1750, MK3, HLK3, HPCN3, 
                 Skeletal muscle, 
               
               
                   
                 L23499 
                   
                 Kv1.3, Shaker homolog 3 
                 lymphocytes (brain, 
               
               
                   
                   
                   
                   
                 lung, thymus, spleen) 
               
               
                 KCNA4 
                 GDB: 126730 
                 11p14 
                 Hs.89647, Hs.1854, HK1, HPCN2, 
                 Brain, nerve, heart, fetal 
               
               
                   
                 M60450 
                   
                 Kv1.4, Shaker homolog 4 
                 skeletal muscle, 
               
               
                   
                 M55514 
                   
                   
                 pancreatic islet 
               
               
                 KCNA4L 
                 GDB: 386059 
                 11q14 
                 Shaker homolog type 4-like 
                   
               
               
                 KCNA5 
                 GDB: 127904 
                 12p13.3-13.2 
                 Hs.89509, HK2, HPCNI, Kv1.5 
                 Brain, heart, kidney, lung, 
               
               
                   
                 M83254 
                 12p13 
                 Shaker homolog 5 
                 skeletal muscle, 
               
               
                   
                 M60451 
                 12p13.33-12.31 
                   
                 pancreatic islet 
               
               
                 KCNA6 
                 GDB: 128080 
                 12p13 
                 HBK2, Kv1.6, Shaker homolog 6 
                 Brain, pancreatic islet 
               
               
                   
                 X17622 
                   
                   
                   
               
               
                 KCNA7 
                 GDB: 127905 
                 19q13.3 
                 HAK6, Kv1.7 Shaker homolog 7 
                   
               
               
                 KCNA8 
                   
                   
                 see KCNQ1 
                   
               
               
                 KCNA9 
                   
                   
                 see KCNQ1 
                   
               
               
                 KCNA10 
                 GDB: 5885822 
                   
                 Shaker homolog type 10, cGMP 
                   
               
               
                   
                   
                   
                 activated 
                   
               
               
                 KCNB1 
                 GDB: 128081 
                 20q13.2 
                 Kv2.1, Shab homolog 1 
                 Brain, heart, kidney, 
               
               
                   
                   
                   
                   
                 retina, skeletal muscle 
               
               
                 KCNB2 
                   
                   
                 Kv2.2, Shab homolog 2 
                 Brain, heart, retina 
               
               
                 KCNC1 
                 GDB: 128082 
                 11p15.1 
                 Kv3.1, Shaw homolog 1 
                 Brain, skeletal muscle, 
               
               
                   
                 S56770 
                   
                   
                 spleen, lymphocytes 
               
               
                   
                 M96747 
                   
                   
                   
               
               
                 KCNC2 
                 GDB: 127906 
                 19q13.3-13.4 
                 Kv3.2, Shaw homolog 2 
                 Brain 
               
               
                 KCNC3 
                 GDB: 127907 
                 19q13.3 
                 Kv3.3, Shaw homolog 3 
                 Brain, liver 
               
               
                 KCNC4 
                 GDB: 127908 
                 1p21 
                 Kv3.4, HKSHIIIC, Shaw homolog 4 
                 Brain, skeletal muscle 
               
               
                 KCND1 
                 GDB: 128083 
                   
                 Kv4.1, Shal hormolog 1 
                 Brain 
               
               
                 KCND2 
                 GDB: 134771 
                   
                 RK5, Kv4.2, Shal homolog 2 
                 Brain, heart, aorta 
               
               
                 KCND3 
                 GDB: 134772 
                   
                 Kv4.3, KSHIVB, Shal homolog 3 
                   
               
               
                 KCNE1 
                 GDB: 127909 
                 21q22.1-22.2 
                 MinK, ISK, vg Isk homolog 1 (129 
                 Kidney, submandibular 
               
               
                   
                   
                   
                 aa), long Q-T syndrome 5 
                 gland, uterus, heart, 
               
               
                   
                   
                   
                   
                 cochlea, retina 
               
               
                 KCNMA1 
                 GDB: 386031 
                 10pter-qter 
                 SLO, Hs.62679, α-subunit member 
                 Fetal skeletal muscle 
               
               
                   
                 U09383-4 
                 7q32.1 
                 1, α-subunit of maxiK or BK 
                   
               
               
                   
                 U02632 
                   
                 channel 
                   
               
               
                 KCNMB1 
                 GDB: 6099615 
                 5q34 
                 hSLO-β, β-subunit member 1 (191 
                 Smooth, fetal skeletal 
               
               
                   
                 U42600 
                   
                 aa), β-subunit of max1K or BK 
                 muscle, brain 
               
               
                   
                   
                   
                 channel 
                 (hippocampus, corpus 
               
               
                   
                   
                   
                   
                 callosum) 
               
               
                 KCNN1 
                 U69883 
                   
                 SK(Ca)1, small-conductance Ca- 
                 Brain, heart 
               
               
                   
                   
                   
                 activated K channel, apamin- 
                   
               
               
                   
                   
                   
                 insensitive 
                   
               
               
                 KCNN2 
                   
                   
                 SK(Ca)2, apamin sensitive 
                 Brain, adrenal gland 
               
               
                 KCNN3 
                 Y08263 
                 1q? 
                 SK(Ca)3, small-conductance Ca- 
                 Brain, heart, (human 
               
               
                   
                 AA285078 
                   
                 activated K channel, intermediate 
                 embryonic) skeletal 
               
               
                   
                   
                   
                 apamin sensitivity 
                 muscle, liver 
               
               
                 KCNN4 
                 AF022150 
                 19q13.2 
                 IK1, intermediate-conductance 
                 T lymphocytes, colon, 
               
               
                   
                 AF022797 
                   
                 Ca-activated K channel, KCa4, 
                 smooth muscles, 
               
               
                   
                 AF033021 
                   
                 SK4, Gantos channel 
                 prostata, red blood cells, 
               
               
                   
                 AF000972 
                   
                   
                 neurons 
               
               
                 KCNQ1 
                 GDB: 741244 
                 11p15.5 
                 KCNA9, (KV)LQT1, KQT-like 
                 Heart, cochlea, kidney, 
               
               
                   
                 U40990 
                   
                 subfamily member 1, long Q-T 
                 lung, placenta, colon 
               
               
                   
                   
                   
                 syndrome 1 
                   
               
               
                 KCNQ2 
                 GDB: 9787229, 
                 20q13.3 
                 KQT-like subfamily member 2 (872 
                 Brain 
               
               
                   
                 Y15065, 
                   
                 aa) 
                   
               
               
                   
                 AF033348 
                   
                   
                   
               
               
                 KCNQ3 
                 GDB: 9787230 
                 8q24.22-24.3 
                 KQT-like subfamily member 3 (825 
                 Brain 
               
               
                   
                 AF033347 
                   
                 aa) 
                   
               
               
                 HERG 
                 GDB: 407638 
                 7q35-36 
                 HERG, similar to ether-a-go go 
                 Brain, heart 
               
               
                   
                   
                   
                 (eag), Ikr, long Q-T syndrome 2 
                   
               
               
                 KCNJ1 
                 GDB: 204206 
                 11q24 
                 ROMK1, Kirl.1, Hs.463, 
                 Kidney, pancreatic islets 
               
               
                   
                 U65406, U12541 
                   
                 Bartter/hyperprostaglandin 
                   
               
               
                   
                   
                   
                 E syndrome 
                   
               
               
                 KCNJ2 
                 GDB: 278964 
                 17pter-qter 
                 IRK1, Kir2.1, Hs.1547 
                 Muscle, neural tissue, 
               
               
                   
                 U12507 
                   
                   
                 heart 
               
               
                 KCNJ3 
                 GDB: 278325 
                 2q24.1 
                 GIRK1, Kir3.1 
                 Heart, cerebellum 
               
               
                   
                 U50964 
                   
                   
                   
               
               
                 KCNJ4 
                 GDB: 374080 
                 22q13.1 
                 HIR, HIRK1, HIRK2, Kir2.3 
                 Heart, skeletal muscle, 
               
               
                   
                 Z97056 
                   
                   
                 brain 
               
               
                 KCNJ5 
                 GDB: 547948 
                 11q24 
                 CIR, KATP1, GIRK4, Kir3.4 
                 Heart, pancreas 
               
               
                 KCNJ6 
                 GDB: 547949 
                 21q22.1 
                 KCNJ7, GIRK2, KATP2, BIR1, 
                 Cerebellum, pancreatic 
               
               
                   
                 U24660 
                   
                 Kir3.2, ataxia, weaver mice 
                 islet 
               
               
                 KCNJ8 
                 GDB: 633096 
                 12p11.23] 
                 Kir6.1, uKATP, ubiquitious K ATP   
                 Brain, heart, skeletal, 
               
               
                   
                   
                   
                 α-subunit 
                 smooth muscle, others 
               
               
                 KCNJ10 
                 GDB: 3750203 
                 1q22-23] 
                 Kir1.2, Kir4.1 
                 Glia 
               
               
                 KCNJ11 
                 GDB: 7009893 
                 [11p15.1] 
                 Kir6.2, BIR, K(ATP) α-subunit, 
                 Pancreatic islets 
               
               
                   
                   
                   
                 hyperinsulinemic hypoglycemia 
                   
               
               
                 KCNJ12 
                 GDB: 4583927 
                 [17p11.1] 
                 Kir2.2 
                   
               
               
                 KCNJ15 
                 GDB: 6275865 
                 [21q22.2] 
                 Kir4.2 
                   
               
               
                 KCNJN1 
                 GDB: 6108062 
                 [ ] 
                 Kir2.2v, subfamily inhibitor 1 
                   
               
               
                 SUR1 
                 GDB: 591970 
                 [11p15.1] 
                 SUR(1), sulfonylurea receptor, 
                 Pancreatic islets 
               
               
                   
                   
                   
                 K(ATP) β-subunit, 
                   
               
               
                   
                   
                   
                 hyperinsulinemic hypoglycemia 
                   
               
               
                 SUR2 
                   
                 12p12.1] 
                 SUR2, SUR2A, B, sulfonylurea 
                 2A: heart, 2B: brain, liver, 
               
               
                   
                   
                   
                 receptor 2 (1545-aa), β-subunit of 
                 skeletal, smooth muscle, 
               
               
                   
                   
                   
                 K(ATP) 
                 urinary bladder 
               
               
                 KCNK1 
                 GDB: 6045446 
                 1q42-43 
                 DPK, TWIK1 
                 Kidney 
               
               
                 KCNK2 
                   
                 1q41 
                 TREK1 
                 Brain 
               
               
                 KCNK3 
                 GDB: 9773281 
                 2p23 
                 TASK 
                 Kidney 
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
               
             
           
               
                   
               
               
                 Therapeutic Target 
                 Enzyme Family 
                 Assay 
               
               
                   
               
             
             
               
                 Alzheimer&#39;s 
                 CMGC 
                 ERK2 (P42mapk) 
               
               
                 Alzheimer&#39;s 
                 Phospholipase 
                 PLA2 
               
               
                 Alzheimer&#39;s 
                 Cyclooxygenases 
                 COX2 
               
               
                 Alzheimer&#39;s 
                 CaMK 
                 MARKI 
               
               
                 Alzheimer&#39;s 
                 CaMK 
                 MARK2 
               
               
                 Alzheimer&#39;s 
                 AGC 
                 PKCalpha 
               
               
                 Alzheimer&#39;s 
                 AGC 
                 PKCgamma 
               
               
                 Alzheimer&#39;s 
                 AGC 
                 PKCgamma 
               
               
                 Alzheimer&#39;s 
                 Cysteine proteases 
                 caspase-3 
               
               
                 Alzheimer&#39;s 
                 Cysteine proteases 
                 caspase-6 
               
               
                 Alzheimer&#39;s 
                 Aspartic proteases 
                 BACE-1 (beta-secretase) 
               
               
                 Alzheimer&#39;s 
                 Aspartic proteases 
                 cathepsin D 
               
               
                 Alzheimer&#39;s 
                 Aspartic proteases 
                 cathepsin E 
               
               
                 Alzheimer&#39;s 
                 Metalloproteases 
                 ACE 
               
               
                 Alzheimer&#39;s 
                 Metalloproteases 
                 ACE 
               
               
                 Alzheimer&#39;s 
                 Metalloproteases 
                 TACE 
               
               
                 Alzheimer&#39;s 
                 NO synthases 
                 constitutive NOS 
               
               
                   
                   
                 (cerebellar) 
               
               
                 Alzheimer&#39;s 
                 Monoamine 
                 acetylcholinesterase 
               
               
                   
                 &amp;neurotransmitter 
                   
               
               
                   
                 synthesis &amp; metabolism 
                   
               
               
                 Alzheimer&#39;s 
                 Monoamine 
                 COMT (catechol-O-methyl 
               
               
                   
                 &amp;neurotransmitter 
                 transferase) 
               
               
                   
                 synthesis &amp; metabolism 
                   
               
               
                 Alzheimer&#39;s 
                 Monoamine 
                 MAO-A 
               
               
                   
                 &amp;neurotransmitter 
                   
               
               
                   
                 synthesis &amp; metabolism 
                   
               
               
                 Alzheimer&#39;s 
                 Monoamine 
                 MAO-B 
               
               
                   
                 &amp;neurotransmitter 
                   
               
               
                   
                 synthesis &amp; metabolism 
                   
               
               
                 Alzheimer&#39;s 
                 Monoamine 
                 tyrosine hydroxylase 
               
               
                   
                 &amp;neurotransmitter 
                   
               
               
                   
                 synthesis &amp; metabolism 
                   
               
               
                 Alzheimer&#39;s 
                 Phospholipase C 
                 PLC 
               
               
                 Alzheimer&#39;s 
                 Miscellaneous enzymes 
                 xanthine oxidase/ 
               
               
                   
                   
                 superoxide 02-scavenging 
               
               
                 Dependence/Addiction 
                 AGC 
                 PKA 
               
               
                 Dependence/Addiction 
                 AGC 
                 PKCalpha 
               
               
                 Dependence/Addiction 
                 AGC 
                 PKCbeta 1 
               
               
                 Dependence/Addiction 
                 AGC 
                 PKCbeta 2 
               
               
                 Dependence/Addiction 
                 AGC 
                 PKCdelta 
               
               
                 Dependence/Addiction 
                 Monoamine 
                 GABA transaminase 
               
               
                   
                 &amp;neurotransmitter 
                   
               
               
                   
                 synthesis &amp; metabolism 
                   
               
               
                 Dependence/Addiction 
                 Cyclases 
                 adenylyl cyclase 
               
               
                   
                   
                 (stimulated) 
               
               
                 Dependence/Addiction 
                 Phospholipase C 
                 PLC 
               
               
                 Dependence/Addiction 
                 ATPase 
                 ATPase (Na + /K + ) 
               
               
                 Inflammation/Arthritis/Allergy 
                 RTK 
                 EGFR kinase 
               
               
                 Inflammation/Arthritis/Allergy 
                 RTK 
                 FLT-1 kinase (VEGFR1) 
               
               
                 Inflammation/Arthritis/Allergy 
                 RTK 
                 KDR kinase (VEGFR2) 
               
               
                 Inflammation/Arthritis/Allergy 
                 CTK 
                 Fyn kinase 
               
               
                 Inflammation/Arthritis/Allergy 
                 CTK 
                 HCK 
               
               
                 Inflammation/Arthritis/Allergy 
                 CTK 
                 Lek kinase 
               
               
                 Inflammation/Arthritis/Allergy 
                 CTK 
                 Lyn kinase 
               
               
                 Inflammation/Arthritis/Allergy 
                 CTK 
                 ZAP70 kinase 
               
               
                 Inflammation/Arthritis/Allergy 
                 CMGC 
                 ERK2 (P42mapk) 
               
               
                 Inflammation/Arthritis/Allergy 
                 CMGC 
                 JNK 1 
               
               
                 Inflammation/Arthritis/Allergy 
                 CMGC 
                 JNK 2 
               
               
                 Inflammation/Arthritis/Allergy 
                 CMGC 
                 P38alpha kinase 
               
               
                 Inflammation/Arthritis/Allergy 
                 Phospholipase 
                 PLA2 
               
               
                 Inflammation/Arthritis/Allergy 
                 Cyclooxygenases 
                 COX1 
               
               
                 Inflammation/Arthritis/Allergy 
                 Cyclooxygenases 
                 COX2 
               
               
                 Inflammation/Arthritis/Allergy 
                 TXA2 synthetase 
                 TXA2 synthetase 
               
               
                 Inflammation/Arthritis/ 
                 CaMK 
                 MAPKAPK2 
               
               
                 Allergy 
                   
                   
               
               
                 Inflammation/Arthritis/ 
                 AGC 
                 PKA 
               
               
                 Allergy 
                   
                   
               
               
                 Inflammation/Arthritis/ 
                 Lipoxygenases 
                 12-lipoxygenase 
               
               
                 Allergy 
                   
                   
               
               
                 Inflammation/Arthritis/ 
                 Lipoxygenases 
                 15-lipoxygenase 
               
               
                 Allergy 
                   
                   
               
               
                 Inflammation/Arthritis/ 
                 Serine proteases 
                 elastase 
               
               
                 Allergy 
                   
                   
               
               
                 Inflammation/Arthritis/ 
                 Serine proteases 
                 cathepsin G 
               
               
                 Allergy 
                   
                   
               
               
                 Inflammation/Arthritis/ 
                 Serine proteases 
                 kallikrein 
               
               
                 Allergy 
                   
                   
               
               
                 Inflammation/Arthritis/ 
                 Serine proteases 
                 tryptase 
               
               
                 Allergy 
                   
                   
               
               
                 Inflammation/Arthritis/ 
                 Cysteine proteases 
                 caspase-1 
               
               
                 Allergy 
                   
                   
               
               
                 Inflammation/Arthritis/ 
                 Cysteine proteases 
                 caspase-4 
               
               
                 Allergy 
                   
                   
               
               
                 Inflammation/Arthritis/ 
                 Cysteine proteases 
                 caspase-5 
               
               
                 Allergy 
                   
                   
               
               
                 Inflammation/Arthritis/ 
                 Cysteine proteases 
                 cathepsin B 
               
               
                 Allergy 
                   
                   
               
               
                 Inflammation/Arthritis/ 
                 Cysteine proteases 
                 cathepsin X 
               
               
                 Allergy 
                   
                   
               
               
                 Inflammation/Arthritis/ 
                 Aspartic proteases 
                 cathepsin E 
               
               
                 Allergy 
                   
                   
               
               
                 Inflammation/Arthritis/ 
                 Metalloproteases 
                 MMP-1 
               
               
                 Allergy 
                   
                   
               
               
                 Inflammation/Arthritis/ 
                 Metalloproteases 
                 MMP-2 
               
               
                 Allergy 
                   
                   
               
               
                 Inflammation/Arthritis/ 
                 Metalloproteases 
                 MMP-3 
               
               
                 Allergy 
                   
                   
               
               
                 Inflammation/Arthritis/ 
                 Metalloproteases 
                 MMP-7 
               
               
                 Allergy 
                   
                   
               
               
                 Inflammation/Arthritis/ 
                 Metalloproteases 
                 MMP-8 
               
               
                 Allergy 
                   
                   
               
               
                 Inflammation/Arthritis/ 
                 Metalloproteases 
                 MMP-9 
               
               
                 Allergy 
                   
                   
               
               
                 Inflammation/Arthritis/ 
                 Metalloproteases 
                 MMP-13 
               
               
                 Allergy 
                   
                   
               
               
                 Inflammation/Arthritis/ 
                 Metalloproteases 
                 MT1-MMP (MMP-14) 
               
               
                 Allergy 
                   
                   
               
               
                 Inflammation/Arthritis/ 
                 Metalloproteases 
                 TACE 
               
               
                 Allergy 
                   
                   
               
               
                 Inflammation/Arthritis/ 
                 Phosphatases 
                 phosphatase CD45 
               
               
                 Allergy 
                   
                   
               
               
                 Inflammation/Arthritis/ 
                 Phosphodiesterases 
                 PDE2 
               
               
                 Allergy 
                   
                   
               
               
                 Inflammation/Arthritis/ 
                 Phosphodiesterases 
                 PDE4 
               
               
                 Allergy 
                   
                   
               
               
                 Inflammation/Arthritis/ 
                 Phosphodiesterases 
                 acid sphingomyelinase 
               
               
                 Allergy 
                   
                   
               
               
                 Inflammation/Arthritis/ 
                 Monoamine &amp; 
                 HNMT (histamine N- 
               
               
                 Allergy 
                 neurotransmitter 
                 methyltransferase) 
               
               
                   
                 synthesis &amp; metabolism 
                   
               
               
                 Inflammation/Arthritis/ 
                 Miscellaneous enzymes 
                 myeloperoxidase 
               
               
                 Allergy 
                   
                   
               
               
                 Inflammation/Arthritis/ 
                 Miscellaneous enzymes 
                 xanthine oxidase/ 
               
               
                 Allergy 
                   
                 superoxide 02-scavenging 
               
               
                 Neuroprotection 
                 RTK 
                 TRKB 
               
               
                 Neuroprotection 
                 CMGC 
                 CDK5 
               
               
                 Neuroprotection 
                 CMGC 
                 DYRKla 
               
               
                 Neuroprotection 
                 CMGC 
                 ERK1 
               
               
                 Neuroprotection 
                 CMGC 
                 ERK2 (P42mapk) 
               
               
                 Neuroprotection 
                 MCGC 
                 JCK 3 
               
               
                 Inflammation/Arthritis/ 
                 Metalloproteases 
                 MMP-13 
               
               
                 Allergy 
                   
                   
               
               
                 Inflammation/Arthritis/ 
                 Metalloproteases 
                 MT1-MMP (MMP-14) 
               
               
                 Allergy 
                   
                   
               
               
                 Inflammation/Arthritis/ 
                 Metalloproteases 
                 TACE 
               
               
                 Allergy 
                   
                   
               
               
                 Inflammation/Arthritis/ 
                 Phosphatases 
                 phosphatase CD45 
               
               
                 Allergy 
                   
                   
               
               
                 Inflammation/Arthritis/ 
                 Phosphodiesterases 
                 PDE2 
               
               
                 Allergy 
                   
                   
               
               
                 Inflammation/Arthritis/ 
                 Phosphodiesterases 
                 PDE4 
               
               
                 Allergy 
                   
                   
               
               
                 Inflammation/Arthritis/ 
                 Phosphodiesterases 
                 acid sphingomyelinase 
               
               
                 Allergy 
                   
                   
               
               
                 Inflammation/Arthritis/ 
                 Monoamine &amp; 
                 HNMT (histamine N- 
               
               
                 Allergy 
                 neurotransmitter 
                 methyltransferase) 
               
               
                   
                 synthesis &amp; metabolism 
                   
               
               
                 Inflammation/Arthritis/ 
                 Miscellaneous enzymes 
                 myeloperoxidase 
               
               
                 Allergy 
                   
                   
               
               
                 Inflammation/Arthritis/ 
                 Miscellaneous enzymes 
                 xanthine oxidase/ 
               
               
                 Allergy 
                   
                 superoxide 02-scavenging 
               
               
                 Neuroprotection 
                 RTK 
                 TRKB 
               
               
                 Neuroprotection 
                 CMGC 
                 CDK5 
               
               
                 Neuroprotection 
                 CMGC 
                 DYRKla 
               
               
                 Neuroprotection 
                 CMGC 
                 ERK1 
               
               
                 Neuroprotection 
                 CMGC 
                 ERK2 (P42mapk) 
               
               
                 Neuroprotection 
                 MCGC 
                 JCK 3 
               
               
                 Neuroprotection 
                 Cyclooxygenases 
                 COXI 
               
               
                 Neuroprotection 
                 Cyclooxygenases 
                 COX2 
               
               
                 Neuroprotection 
                 CaMK 
                 CaMK2alpha 
               
               
                 Neuroprotection 
                 AGC 
                 PKA 
               
               
                 Neuroprotection 
                 Cysteine proteases 
                 caspase-3 
               
               
                 Neuroprotection 
                 Phosphodiesterases 
                 PDEI 
               
               
                 Neuroprotection 
                 Phosphodiesterases 
                 PDE6 
               
               
                 Neuroprotection 
                 NO synthases 
                 constitutive NOS 
               
               
                   
                   
                 (endothelial) 
               
               
                 Neuroprotection 
                 NO synthases 
                 constitutive NOS 
               
               
                   
                   
                 (cerebellar) 
               
               
                 Neuroprotection 
                 Monoamine &amp; 
                 acetylcholinesterase 
               
               
                   
                 neurotransmitter 
                   
               
               
                   
                 syntheses &amp; metabolism 
                   
               
               
                 Neuroprotection 
                 Monoamine &amp; 
                 COMT (catechol-O-methyl 
               
               
                   
                 neurotransmitter 
                 transferase) 
               
               
                   
                 syntheses &amp; metabolism 
                   
               
               
                 Neuroprotection 
                 Monoamine &amp; 
                 GABA transaminase 
               
               
                   
                 neurotransmitter 
                   
               
               
                   
                 syntheses &amp; metabolism 
                   
               
               
                 Neuroprotection 
                 Monoamine &amp; 
                 HNMT (histamine N- 
               
               
                   
                 neurotransmitter 
                 methyltransferase) 
               
               
                   
                 syntheses &amp; metabolism 
                   
               
               
                 Neuroprotection 
                 Monoamine &amp; 
                 MAO-A 
               
               
                   
                 neurotransmitter 
                   
               
               
                   
                 syntheses &amp; metabolism 
                   
               
               
                 Neuroprotection 
                 Monoamine &amp; 
                 MAO-A 
               
               
                   
                 neurotransmitter 
                   
               
               
                   
                 syntheses &amp; metabolism 
                   
               
               
                 Neuroprotection 
                 Monoamine &amp; 
                 PNMT 
               
               
                   
                 neurotransmitter 
                 (phenylethanoiamine-N- 
               
               
                   
                 syntheses &amp; metabolism 
                 methyl 
               
               
                   
                   
                 transferase) 
               
               
                 Neuroprotection 
                 Monoamine &amp; 
                 tyrosine hydroxylase 
               
               
                   
                 neurotransmitter 
                   
               
               
                   
                 syntheses &amp; metabolism 
                   
               
               
                 Neuroprotection 
                 Cyclases 
                 guanylyl cyclase (basal) 
               
               
                 Neuroprotection 
                 Cyclases 
                 guanylyl cyclase 
               
               
                   
                   
                 (stimulated) 
               
               
                 Neuroprotection 
                 ATPase 
                 ATPase (Na+/K+) 
               
               
                 Neuroprotection 
                 Miscellaneous enzymes 
                 xanthine 
               
               
                   
                   
                 oxidase/superoxide 02- 
               
               
                   
                   
                 scavenging 
               
               
                 Parkinson 
                 CMGC 
                 JNK 1 
               
               
                 Parkinson 
                 Phospholipase 
                 PLA2 
               
               
                 Parkinson 
                 Cyclooxygenases 
                 COX2 
               
               
                 Parkinson 
                 Cysteine proteases 
                 caspase-3 
               
               
                 Parkinson 
                 NO synthases 
                 constitutive NOS 
               
               
                   
                   
                 (cerebellar) 
               
               
                 Parkinson 
                 Monoamine &amp; 
                 acetylcholinesterase 
               
               
                   
                 neurotransmitter 
                   
               
               
                   
                 syntheses &amp; metabolism 
                   
               
               
                 Parkinson 
                 Monoamine &amp; 
                 COMT (catechol-O-methyl 
               
               
                   
                 neurotransmitter 
                 transferase 
               
               
                   
                 syntheses &amp; metabolism 
                   
               
               
                 Parkinson 
                 Monoamine &amp; 
                 MAO-A 
               
               
                   
                 neurotransmitter 
                   
               
               
                   
                 syntheses &amp; metabolism 
                   
               
               
                 Parkinson 
                 Monoamine &amp; 
                 MAO-B 
               
               
                   
                 neurotransmitter 
                   
               
               
                   
                 syntheses &amp; metabolism 
                   
               
               
                 Cancer 
                 RTK 
                 Axl kinase 
               
               
                 Cancer 
                 RTK 
                 c-kit kinase 
               
               
                 Cancer 
                 RTK 
                 c-kit kinase 
               
               
                 Cancer 
                 RTK 
                 EGFR kinase 
               
               
                 Cancer 
                 RTK 
                 EphAl kinase 
               
               
                 Cancer 
                 RTK 
                 EphA3 kinase 
               
               
                 Cancer 
                 RTK 
                 EphA4 kinase 
               
               
                 Cancer 
                 RTK 
                 EphB2 kinase 
               
               
                 Cancer 
                 RTK 
                 FGFR1 kinase 
               
               
                 Cancer 
                 RTK 
                 FGFR2 kinase 
               
               
                 Cancer 
                 RTK 
                 FGFR3 kinase 
               
               
                 Cancer 
                 RTK 
                 FGFR4 kinase 
               
               
                 Cancer 
                 RTK 
                 FLT-1 kinase (VEGFRl) 
               
               
                 Cancer 
                 RTK 
                 FLT-3 kinase 
               
               
                 Cancer 
                 RTK 
                 FLT-4 kinase (VEGFR3) 
               
               
                 Cancer 
                 RTK 
                 Fms/CSFR kinase 
               
               
                 Cancer 
                 RTK 
                 HER2/ErbB2 kinase 
               
               
                 Cancer 
                 RTK 
                 HER4/ErbB4 kinase 
               
               
                 Cancer 
                 RTK 
                 KDR kinase (VEGFR2) 
               
               
                 Cancer 
                 RTK 
                 PDGFRalpha kinase 
               
               
                 Cancer 
                 RTK 
                 PDGFRbeta kinase 
               
               
                 Cancer 
                 RTK 
                 Ret kinase 
               
               
                 Cancer 
                 RTK 
                 TIE2 kinase 
               
               
                 Cancer 
                 RTK 
                 TRKA 
               
               
                 Cancer 
                 CTK 
                 Abl kinase 
               
               
                 Cancer 
                 CTK 
                 BLK 
               
               
                 Cancer 
                 CTK 
                 BMX (Bk) kinase 
               
               
                 Cancer 
                 CTK 
                 BRK 
               
               
                 Cancer 
                 CTK 
                 BTK 
               
               
                 Cancer 
                 CTK 
                 CSK 
               
               
                 Cancer 
                 CTK 
                 FAK 
               
               
                 Cancer 
                 CTK 
                 Fes kinase 
               
               
                 Cancer 
                 CTK 
                 Fyn kinase 
               
               
                 Cancer 
                 CTK 
                 JAK2 
               
               
                 Cancer 
                 CTK 
                 JAK3 
               
               
                 Cancer 
                 CTK 
                 Lck kinase 
               
               
                 Cancer 
                 CTK 
                 PYK2 
               
               
                 Cancer 
                 CTK 
                 Src kinase 
               
               
                 Cancer 
                 CTK 
                 Syk 
               
               
                 Cancer 
                 CTK 
                 Yes kinase 
               
               
                 Cancer 
                 CMGC 
                 CDC2/CDK1 (cycB) 
               
               
                 Cancer 
                 CMGC 
                 CDK2 (cycE) 
               
               
                 Cancer 
                 CMGC 
                 CDK4 (cycDl) 
               
               
                 Cancer 
                 CMGC 
                 CDK5 
               
               
                 Cancer 
                 CMGC 
                 CK2 (casein kinase 2) 
               
               
                 Cancer 
                 CMGC 
                 DYRKla 
               
               
                 Cancer 
                 CMGC 
                 ERK1 
               
               
                 Cancer 
                 CMGC 
                 ERK2 (P42mapk) 
               
               
                 Cancer 
                 CMGC 
                 HIPK2 
               
               
                 Cancer 
                 CMGC 
                 IKKalpha 
               
               
                 Cancer 
                 CMGC 
                 IKKbeta 
               
               
                 Cancer 
                 CMGC 
                 JNK 1 
               
               
                 Cancer 
                 CMGC 
                 JNK 2 
               
               
                 Cancer 
                 CMGC 
                 NEK1 
               
               
                 Cancer 
                 CMGC 
                 NEK2 
               
               
                 Cancer 
                 CMGC 
                 NEK4 
               
               
                 Cancer 
                 CMGC 
                 p38alpha kinase 
               
               
                 Cancer 
                 CMGC 
                 p38beta 2 kinase 
               
               
                   
                   
                 (SAPK2b2) 
               
               
                 Cancer 
                 CMGC 
                 p38delta kinase 
               
               
                 Cancer 
                 CMGC 
                 p38ganuna kinase 
               
               
                 Cancer 
                 Cyclooxygenases 
                 COX2 
               
               
                 Cancer 
                 CaMK 
                 CaMKldelta 
               
               
                 Cancer 
                 CaMK 
                 CaMK 
               
               
                 Cancer 
                 CaMK 
                 CHK1 
               
               
                 Cancer 
                 CaMK 
                 CHK2 
               
               
                 Cancer 
                 CaMK 
                 DAPK1 
               
               
                 Cancer 
                 CaMK 
                 DAPK2 
               
               
                 Cancer 
                 CaMK 
                 MAPKAPK2 
               
               
                 Cancer 
                 CaMK 
                 MAPKAPK3 
               
               
                 Cancer 
                 CaMK 
                 MAPKAPK5 (PRAKO 
               
               
                 Cancer 
                 CaMK 
                 MAARK1 
               
               
                 Cancer 
                 CaMK 
                 MARK2 
               
               
                 Cancer 
                 CaMK 
                 MARK4 
               
               
                 Cancer 
                 CaMK 
                 Pim 1 kinase 
               
               
                 Cancer 
                 CaMK 
                 Pirn2 kinase 
               
               
                 Cancer 
                 AGC 
                 Aktl/PKBalpha 
               
               
                 Cancer 
                 AGC 
                 Akt2/PKBbeta 
               
               
                 Cancer 
                 AGC 
                 Akt3/PKBgamma 
               
               
                 Cancer 
                 AGC 
                 AurA/Aur2 kinase 
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
               
             
           
               
                   
               
               
                 Therapeutic Target 
                 Enzyme Family 
                 Assay 
               
               
                   
               
             
             
               
                 Cancer 
                 AGC 
                 AurB/Aurl kinase 
               
               
                 Cancer 
                 AGC 
                 AurC/Aur3 kinase 
               
               
                 Cancer 
                 AGC 
                 P70S6Ke 
               
               
                 Cancer 
                 AGC 
                 PDK1 
               
               
                 Cancer 
                 AGC 
                 PKA 
               
               
                 Cancer 
                 AGC 
                 PKCalpha 
               
               
                 Cancer 
                 AGC 
                 PKCbeta 1 
               
               
                 Cancer 
                 AGC 
                 PKCbeta 2 
               
               
                 Cancer 
                 AGC 
                 PKCdelta 
               
               
                 Cancer 
                 AGC 
                 PKCgamma 
               
               
                 Cancer 
                 AGC 
                 PKG2 
               
               
                 Cancer 
                 AGC 
                 ROCK1 
               
               
                 Cancer 
                 AGC 
                 ROCK2 
               
               
                 Cancer 
                 AGC 
                 RSK2 
               
               
                 Cancer 
                 AGC 
                 SGKI 
               
               
                 Cancer. 
                 Lipoxygenases 
                 12-lipoxygenase 
               
               
                 Cancer 
                 TKL 
                 RAF-1 kinase 
               
               
                 Cancer 
                 STE 
                 MEK1/MAP2KI 
               
               
                 Cancer 
                 STE 
                 MKK4/JNK1 
               
               
                 Cancer 
                 STE 
                 MKK6 
               
               
                 Cancer 
                 STE 
                 PAK1 
               
               
                 Cancer 
                 STE 
                 PAK2 
               
               
                 Cancer 
                 Serine proteases 
                 elastase 
               
               
                 Cancer 
                 Serine proteases 
                 cathepsin G 
               
               
                 Cancer 
                 Cysteine proteases 
                 caspase-2 
               
               
                 Cancer 
                 Cysteine proteases 
                 caspase-3 
               
               
                 Cancer 
                 Cysteine proteases 
                 caspase-8 
               
               
                 Cancer 
                 Cysteine proteases 
                 caspase-9 
               
               
                 Cancer 
                 Cysteine proteases 
                 cathepin B 
               
               
                 Cancer 
                 Cysteine proteases 
                 cathepsin H 
               
               
                 Cancer 
                 Cysteine proteases 
                 cathepsin L 
               
               
                 Cancer 
                 Cysteine proteases 
                 cathepsin X 
               
               
                 Cancer 
                 Aspartic proteases 
                 cathepsin D 
               
               
                 Cancer 
                 Aspartic proteases 
                 cathepsin E 
               
               
                 Cancer 
                 Metalloproteases 
                 MMP-1 
               
               
                 Cancer 
                 Metalloproteases 
                 MMP-2 
               
               
                 Cancer 
                 Metalloproteases 
                 MMP-3 
               
               
                 Cancer 
                 Metalloproteases 
                 MMP-7 
               
               
                 Cancer 
                 Metalloproteases 
                 MMP-8 
               
               
                 Cancer 
                 Metalloproteases 
                 MMP-9 
               
               
                 Cancer 
                 Metalloproteases 
                 MMP-12 
               
               
                 Cancer 
                 Metalloproteases 
                 MMP-13 
               
               
                 Cancer 
                 Metalloproteases 
                 MT1-MMP (MMP-14) 
               
               
                 Cancer 
                 Metalloproteases 
                 TACE 
               
               
                 Cancer′ 
                 Metalloproteases 
                 MMP-1 
               
               
                 Cancer 
                 Phosphatases 
                 Phosphatase 1B 
               
               
                 Cancer 
                 Phosphatases 
                 Phosphatase 2B 
               
               
                 Cancer 
                 Phosphodiesterases 
                 PDE2 
               
               
                 Cancer 
                 Phosphodiesterases 
                 PDE4 
               
               
                 Cancer 
                 Phosphodiesterases 
                 PDES 
               
               
                 Cancer 
                 Phosphodiesterases 
                 acid spingomyelinase 
               
               
                 Cancer 
                 NO synthases 
                 constitutive NOS 
               
               
                   
                   
                 (endothelial) 
               
               
                 Cancer 
                 NO synthases 
                 constitutive NOS 
               
               
                   
                   
                 (cerebellar) 
               
               
                 Cancer 
                 Cyclases 
                 adenylyl cyclase (basal) 
               
               
                 Cancer 
                 Cyclases 
                 adenylyl cyclase 
               
               
                   
                   
                 (stimulated) 
               
               
                 Cancer 
                 Phospholipase C 
                 PLC 
               
               
                 Cancer 
                 Miscellaneous enzymes 
                 myeloperoxidase 
               
               
                 Cancer 
                 Miscellaneous enzymes 
                 xanthine 
               
               
                   
                   
                 oxidase/superoxide 02- 
               
               
                   
                   
                 scavenging 
               
               
                 Diabetes 
                 RTK 
                 Ax1 kinase 
               
               
                 Diabetes 
                 RTK 
                 EGFR kinase 
               
               
                 Diabetes 
                 RTK 
                 IGFIR kinase 
               
               
                 Diabetes 
                 CMGC 
                 ERK2 (P42mapk) 
               
               
                 Diabetes 
                 CMGC 
                 Jnk1 
               
               
                 Diabetes 
                 Cyclooxygenases 
                 COX2 
               
               
                 Diabetes 
                 TXA2 synthetase 
                 TXA2 synthetase 
               
               
                 Diabetes 
                 CaMK 
                 AMPKalpha 
               
               
                 Diabetes 
                 AGC 
                 Aktl/PKBalpha 
               
               
                 Diabetes 
                 AGC 
                 Akt2/PKBbeta 
               
               
                 Diabetes 
                 AGC 
                 Akt3/PKBgamma 
               
               
                 Diabetes 
                 AGC 
                 PDK1 
               
               
                 Diabetes 
                 AGC 
                 PKA 
               
               
                 Diabetes 
                 AGC 
                 PKCalpha 
               
               
                 Diabetes 
                 AGC 
                 PKCbeta I 
               
               
                 Diabetes 
                 AGC 
                 PKCbeta 2 
               
               
                 Diabetes 
                 AGC 
                 PKCgamma 
               
               
                 Diabetes 
                 AGC 
                 SGK2 
               
               
                 Diabetes 
                 Metalloproteases 
                 ACE 
               
               
                 Diabetes 
                 Metalloproteases 
                 MMP-1 
               
               
                 Diabetes 
                 Metalloproteases 
                 MMP-2 
               
               
                 Diabetes 
                 Metalloproteases 
                 MMP-3 
               
               
                 Diabetes 
                 Metalloproteases 
                 MMP-7 
               
               
                 Diabetes 
                 Metalloproteases 
                 MMP-8 
               
               
                 Diabetes 
                 Metalloproteases 
                 MMP-9 
               
               
                 Diabetes 
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       FIGS. 6   a - b  present another embodiment  300  of a sensor in accord with the present invention. The sensor comprises a well  310  and micro-channels, indicated generally at  315 , branching successively therefrom. The cells are placed in the well  310 , sensors with different specificities line each of the channels  315 , and a solution is flowed from the well  310  through the channels  315 . When the fluorescence from the channels  315  is imaged, the pattern formed by changes in intensity may be visualized as an optical barcode, as depicted in  FIG. 6   b , indicating the presence or absence of the various sensed materials in the solution. 
     The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention in any way. 
     EXAMPLES 
     Ion-Selective Polymer Solution. The ion-selective polymer solution was made from the following components: 30 mg High Molecular Weight Polyvinyl Chloride, 60 mg Bis-2-Sebacate, 0.1 mg Sodium Ionophore X, 0.1 mg Sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate, and 0.1 mg Chromoionophore I. The combined reagents were stirred in 500 μL of tetrahydrofuran (THF) to afford a homogenous solution.
 
Ion-Selective Sensor Fabrication. Quantum dots (ITK organic 655, Invitrogen) were flocculated in a methanol/isopropanol mixture with the addition of toluene in a 1:1 (v:v) ratio of toluene:quantum dot solution. The supernatant was removed and the quantum dots were resuspended in THF containing 3.3 mM 1-decanethiol. To the quantum dot solution (0.2 nMoles) was added the ion-selective polymer solution (17.2 nMoles Chromoionophore I, 50 μl) and the mixture was stirred.
         Immobilized Polymer Matrix of Sensors. To form an immobilized polymer matrix of sensors, 1 μl of the polymer/quantum dot mixture was dropped onto a 5 mm glass coverslip to afford a thin homogenous matrix. Individual coverslips could then be placed in 96-well plates and experiments carried out.   Particle Sensors. To form particle sensors, dichloromethane was added to the polymer/quantum dot mixture in a 1:1 (v:v) ratio. This solution was then added directly to 5 mL of H 2 O containing 200 μg of dissolved 1,2-Diacyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethyleneglycol)-550] (PEG-lipid) by injecting through a pipette tip during sonication. Sonication was performed with a probe-tip sonicator (Branson) at 15% amplitude during and after addition for a total of 3 minutes, resulting in plasticized polymer nanosphere formation. The solution was then passed through a 0.2 μm syringe filter (Acrodisc, Gelman Laboratory) to remove large particles.
 
Particle Sizing and Zeta Potential. Particle size and zeta potential of the sensors were determined using a nanosizer (Nano Series ZS90, Malvern).
 
Calibration and Selectivity. Selectivity was determined using immobilized polymer matrix sensors. Parallel measurements were taken during addition of increasing ion concentration in HEPES (10 mM) buffered solution (pH=7.4) for response to sodium and potassium (n=4 each). Response was determined by expressing the data as α=(I−I min )/(I max −I min ). I is the intensity at the given ion concentration, I min  is the intensity at the minimum signal (0 Na + ), and I max  is the intensity at the maximum signal (1 M Na + ). Data were acquired in a Spectramax Gemini EM microplate fluorometer (Molecular Devices) exciting at 405 nm and emitting at 655 nm. The immobilized polymer matrix sensors were pretreated with HEPES buffered solution at pH=5.0, the pH of the sensors in solution. The baseline was then established in HEPES buffered solution at pH=7.4. Sodium and potassium solutions in the range of 1 mM to 1 M were added and the sensor was allowed to equilibrate before measurements were made. Response was determined by fitting a sigmoidal curve to the plot of a vs. ion concentration using Origin software,  FIG. 7 .
       

     In order to prevent dilution effects, the polymer matrix with no biocompatible coating was immobilized to a glass coverslip for calibration and selectivity measurements. The dynamic range of the sensor was found to be 1 mM to 1 M, shown spectrally in  FIG. 8   a . By adjusting the ratio of components, the concentration range was tuned to maximize the resolution at typical levels of intracellular sodium. In this case, the resolution was 80 μM at 17 mM, the center of the dynamic range. This means that a change in fluorescence intensity of 1% would correspond to a change in concentration of 80 μM, as measured on a fluorescence plate reader. 
     Ratiometric Measurements Immobilized polymer matrix sensors containing both 545 nm and 655 nm quantum dots was made in a similar fashion to the method described above. In this case, 1 nmole of each colored (i.e., emission wavelength) quantum dot was used, giving a total of 2 nmoles of quantum dots just as above. The sensors were calibrated with sodium ions while recording emission at 545 and 655 nm in the fluorometer, using an excitation wavelength of 405 nm. The ratio of 655/545 was taken, averaged over the data set and plotted using the graphing software program Origin. A sigmoidal curve was fit to the data and a half-maximal response was determined ( FIG. 8   b ). 
     A control ion-selective polymer matrix was made similarly to polymer matrix described above, however the control polymer matrix did not contain Chromoionophore I. Immobilized polymer matrix sensors were made as described above. The response was determined by measuring fluorescence over 30 minutes after addition of 200 mM sodium solution. (This was performed in parallel with standard immobilized polymer matrix sensors to analyze the response time). 
     Spectral Overlap. Absorbance characteristics of the chromoionophore were obtained by creating sensors without quantum dots. The sensors were placed in a 96-well plate and absorbance was measured at wavelengths ranging from 500 nm to 700 nm. This was performed in the presence of 0, 1, 10, 100, and 1000 mM sodium solution in pH 7.4 Hepes buffer. From these spectral results, quantum dots were selected that emit fluorescence at a wavelength that coincides with the chromoionophore absorbance wavelengths. The overlap was confirmed by creating a sensor without chromoionophore but with quantum dots at the ratio mentioned above. A fluorescent spectrum was then measured by exciting at 400 nm and collecting emission from 600 nm to 700 nm in steps of 5 nm. In the absence of the chromoionophore, the quantum dot sensors fluoresced at an intensity that was not affected by the presence or absence of sodium ions. 
     A quantum dot with fluorescence maximum at 655 nm and a chromoionophore in a sensor that absorbs fluorescence at low sodium ion concentrations is depicted in  FIG. 9 . The absorbance of the chromoionophore at 655 nm (gray lines,  FIG. 9 ) decreases as sodium concentration increases, resulting in an increase in fluorescent signal directly related to increasing sodium concentration. Note the preferred overlap of the quantum dot emission (red line,  FIG. 9 ).
         Particle sensors without quantum dots: A polymer cocktail was formulated using, for example, traditional amounts of the sensor components (PVC, plasticizer, ionophore, chromoionophore and ionic additive) in a 1:1 (v:v) THF/dichloromethane solution. A probe-tip sonicator was used to sonicate an aliquot of 100 μL of polymer solution in 5 mL of buffer containing 0.1% PEG modified lipid. The nanosensors were dried by passing the particle solution through a nitrogen feed airbrush.
 
Surface Chemistry: The surface chemistry of the sensor can be changed by varying the concentration of the functionalized PEG incorporated onto the sensor. As above, zeta potential can be used to analyze the effectiveness of the coating at any given functionalized PEG concentration. The concentration of the functionalized PEG may be changed to optimize the properties of the sensors to their intended use.
 
Incorporation of Surface modifier onto Nanosphere Surface: The PEG lipid molecule PEG2000 PE {1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-2000](Ammonium Salt)} (Avanti Polar Lipids, Alabaster Ala.) can be used to attach functional groups. The amine functional group is also available through Avanti Polar Lipids, DSPE-PEG(2000)Amine {1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-N-[Amino(Polyethylene Glycol)2000](Ammonium Salt)}, and requires no alterations.
       

     The functionalized PEG can be dissolved into HEPES Buffered Saline (HeBS) and added to a solution containing sensor. The mixture may be mixed, e.g., with a vortexer for 1-2 minutes, to ensure ample interaction time and destabilization of aggregates. The resulting sensor-PEG can be subjected to acidification (decrease of solution pH from 7.4-5.5) and mechanical stress (trituration). The zeta potential can be measured (zetasizer) before and/or after acidification and mechanical stress to determine the surface concentration of the SM. This may correlate to the ability of the functionalized PEG to coat the surface of the sensor and withstand chemical and mechanical changes associated with the endocytotic pathway. 
     Biocompatibility. Biocompatibility was determined by incubating the sensors with HEK 293 cells (ATCC). The cells were trypsonized from normal culture and pipetted into a clear 96-well plate at a concentration of 30,000 cells/well. The cells were grown overnight in 300 μL of media to allow attachment to the plate. An aqueous solution (20 μL) containing 10 11  particle sensors/mL was added to each well. For control experiments 20 μL of distilled water was used. Different particles were used to compare to the particle sensors. Particles used for comparison include: gold nanoparticles (colloidal gold 100 nm, SPI) and FluoSpheres (20 nm carboxylate modified microspheres, Invitrogen). Each type of nanosensor was tested on 8 wells of cells. The nanosensors were incubated with the cells overnight and the media was changed the following day. 
     At 24, 48, and 72 hours following washing, an MTT assay (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was performed (In vitro toxicology assay kit, Sigma). The cells were incubated with MTT for 2 hours. The MTT reduction product formazan was then dissolved and the absorbance of each well was read at 570 and 690 nm. The 690 nm absorbance served as background and was subtracted from the 570 nm value. The data was then averaged and compared to control to generate  FIG. 10 . 
     Loading sensors without quantum dots into cells: One method used for loading the cell involved incubating a solution of sensors in media with the cells overnight. The following day, the cells were washed 3× with PBS buffer. The cells were then imaged on the confocal microscope, with excitation 633 nm and emission 670-690 nm indicating that some of the polymer nanosensor had loaded into the cell, with plenty in the cytosol, but the image indicated a distribution in larger bundles clustered around the nucleus,  FIG. 11 .
 
Loading sensors into cells: A method for loading sensors into cells involves injecting the sensors into the cytosol of the HEK 293 cells via a 2 MOhm resistance patch pipette. The sensor solution may be diluted, e.g., one to one with a 2× intracellular solution. Once a GOhm seal has been achieved, the membrane ruptures and the sensors diffuse into the cytosol of the cell. No pressure need be applied to the patch pipette solution; rather the sensors enter the cytoplasm by simple diffusion. Time course experiments can be performed by acquiring sensor signal every minute over the course of 30 minutes to evaluate the rate of diffusion out of the patch pipette and homogeneity of distribution once inside the cytosol. Patching of cells is discussed in detail below.
 
Ability to Load into Cell: The sensors can be loaded into the test cell, such as HEK 293 or HL-1 cells. HEK 293 cells are maintained by standard cell culture and HL-1 cells are maintained similarly with alterations. Cells may be plated, e.g., onto 25 mm glass cover slips in 6 well plates at 25% confluence the day before experiments are to be carried out. When using HEK 293 cells the cover slips may be coated with poly-L-lysine, while cover slips for HL-1 cell experiments may be coated with gelatin and fibronectin.
 
     The sensors in a HeBS solution may be added to 2 mL of cell culture media (at volumes not exceeding a ratio of 1:10) to replace the media in each well. The sensors can be incubated with the cells at 37° C. 5% CO 2 , e.g., 10 min-24 hrs. Following incubation, the cover slips may be washed with HeBS containing 10 mM Glucose warmed to 37° C. and transferred to a microscope chamber. The chamber may be filled with HeBS with glucose and placed onto the microscope (microscope experiments may be performed on the LSCM). Data may be acquired using LSCM. Images may be acquired using a plan-apochromatic 63×1.4 NA oil immersion lens. Excitation/Emission settings may be selected according to the type of sensors being tested, e.g., for particle sensors-PEG Ex/Em of 633/670-690 nm. Loading may be evaluated by determining the quantity of sensors in each cell. A lack of nuclear loading may indicate either intracellular loading or plasma membrane incorporation. 
     Ability to Locate Into cytosol: The sensors may be analyzed for their ability to release from endosomes and enter the cytosol. Both HEK 293 and HL-1 cells may be prepared for microscopy according to the methods above. Additionally, the sensors may be loaded as described above, optionally with organelle-specific dyes. LSCM images may be acquired using the same microscope configuration described above. Cells may be loaded with sensors as described above and fixed for analysis of cytosolic location by TEM.
 
Imaging of sensors: After loading via patch pipette the sensors for both simultaneous patch experiments and independent imaging experiments may be performed. Images can be acquired with a standard CCD camera (CoolSnap HQ, Roper Scientific) or a high speed camera (Cooke, for channel experiments) attached to a Zeiss Axiovert 200 microscope. A standard FITC cube (Chroma) may be used when imaging CoroNa Green. Channel activity may be controlled with the patch setup and fluorescence detection may be timed to coordinate with channel opening.
 
Intracellular calibration: Calibration experiments can be performed in HEK 293 cells. The SENSORs can be calibrated in the cytosol after injection loading with a pipette. The sodium ionophore gramicidin (10 μM, Sigma) can be used to transport sodium across the membrane and Strophanthidin (100 μM, Sigma) can be used to deactivate any Na—K ATPase in the cells. Two solutions can be made that contain 140 mM Sodium (30 mM NaCl and 110 mM sodium gluconate) and zero Sodium (30 mM KCl and 110 mM potassium gluconate, to maintain ionic balance). Both solutions may also contain 10 mM HEPES, 10 mM glucose and, 1 mM EGTA (sigma) and a pH of 7.4. They can be mixed to achieve the desired concentration of sodium and perfused into the microscope chamber. Acquisitions of data may be made every 5 seconds and the sodium concentration may only be switched after a plateau in signal has been achieved for over two minutes. Selectivity can be determined by performing a calibration response to potassium. In this case, however, valinomycin (Sigma) may be used instead of gramicidin, all other conditions being the same.
 
     Response repeatability can be determined using the conditions in the sodium calibration. The extracellular concentration of sodium can be switched back and forth from zero sodium to 50 mM sodium every ten minutes over the course of an hour. 
     Intracellular Response to Ion of Interest: Experiments may be performed to determine responsiveness of the nanosensors in the cytosol. Cells prepared for microscope experiments and functionalized nanosensors may be loaded as described above. Nanosensors may be imaged on a LSCM as described above. A description of methods suitable to characterize sodium nanosensor response follows; when using other ion-specific nanosensor, different ionophores can be employed. 
     LSCM images may be from nanosensor-loaded cells in HeBS containing zero sodium. A sodium ionophore such as Valinomycin (10 μM) (Sigma) may be added to equilibrate the concentration of sodium between the extracellular solution and the cytosol. Sodium concentrations may be increased in a step-wise manner by addition of a high sodium (1 M) stock HeBS. Images may be acquired after the system is allowed to reach equilibrium (˜2-3 min) and intensity of the nanosensors can be measured. The sodium concentration may be raised, e.g., to 1 M, to establish a maximum concentration value of intensity. The data may then be correlated to the both the minimum intensity and maximum intensity and a calibration curve can be generated. 
     In a similar fashion, selectivity of the cytosolic nanosensor may be determined using interfering ions and their corresponding ionophores. The addition of interfering ions and the ionophore may be added while performing the calibration mentioned above. The acquired calibration curve may then be compared to that generated from sodium ion alone and a selectivity coefficient can be determined. 
     Nanosensor-loaded cells may also be subjected to whole-cell patch-clamp. The cells can be exposed to a drug to induce sodium flux across plasma membrane channels. Channel activity using patch-clamp can be recorded simultaneously with nanosensor fluorescence. This allows a direct comparison of this method to measure ion flux with the instant method discussed herein. 
     Whole Cell Patching: Recombinant HEK 293 cells expressing Na V 1.7 can be used to analyze sodium detection of intracellular nanosensors. Standard whole cell voltage clamp protocols may be employed to assess channel current density, voltage-dependent activation, inactivation, and the time course of recovery from inactivation. Ionic currents may be recorded with whole-cell voltage clamp methods, using an Axopatch-200B amplifier (Axon Instruments). Borosilicate glass electrodes with tip resistances 1-3 Mohm can be used. Command pulses may be generated by 12-bit digital-to-analog converter controlled by pCLAMP6 software (Axon Instruments). Experiments may be conducted at 36° C. To measure activity of the voltage-gated sodium channels, currents can be recorded following a step change of the holding potential from −120 mV to −20 mV test potential. The external solution may contain (mmol/L): 30 NaCl, 110 CsCl, 1.8 CaCl 2 , 2 CdCl 2 , 1 MgCl 2 , 10 HEPES, 10 glucose, 1 4-AP. Intracellular solution may contain (mmol/L): 10 NaCl, 130 CsCl, 5 EGTA, 10 HEPES, 10 glucose. Inactivation and activation kinetics can be analyzed. Nanosensors may be introduced via patch pipette in the whole cell configuration, and maximum amplitude of the sodium current can be measured simultaneously with measurement of sodium flux optical recording from the nanosensors. 
     The same experiments may be repeated with recombinant K V  1.3 HEK 293 cells to demonstrate specificity of the sodium-sensitive nanosensors. In this case the cells may be hyperpolarized to below the reversal potential for potassium to allow for potassium influx into the cell. To measure the potassium currents, the extracellular solution may contain (mmol/L) NaCl 136, KCl 5.4, MgCl 2  1, CaCl 2  1, NaH 2 PO 4  0.33, HEPES 5, and dextrose 10 (pH 7.35, NaOH). For delayed rectifier current recording, nifedipine (5 μmol/L), 4-aminopyridine (2 mmol/L), and atropine (200 nmol/L) can be added to suppress L-type calcium current (I Ca,L ), transient outward current (I to ), and 4-aminopyridine-dependent muscarinic K +  currents. Dofetilide (1 μmol/L) can be added for I Ks  recording. For I to  recording, nifedipine may be replaced by CdCl 2  (200 μmol/L). Na +  current (I Na ) contamination may be avoided by substitution of equimolar Tris-HCl for NaCl and use of tetrodotoxin. A suitable internal solution for K + -current recording may contain (mmol/L) K-aspartate 110, KCl 20, MgCl 2  1, MgATP 5, LiGTP 0.1, HEPES 10, Na-phosphocreatinine 5, and EGTA 5.0 (pH 7.3, KOH). 
     Validation of sensor response with molecular dyes: The same experiments carried out on sensors may be performed using a fluorescein detection microscope cube. The cell impermeant CoroNa Green salt can be loaded through the patch pipette to determine if patch pipette loading alters the location and response of the dye.
 
Characterization of sensor function: Blockade of sodium channel using lidocaine and STX can reduce sodium flux into the cell, resulting in decrease in signal amplitude using optical recordings from the sensors. Recombinant Na V 1.7 HEK 293 cells may be subject to whole cell patch clamp, and the sensors may be introduced via the patch clamp. Lidocaine or STX may be perfused into the patch chamber and peak amplitude of the sodium current can be determined both by patch clamp (using standard voltage-dependent activation protocol) and optical recording (sensors) before and after infusion of the agent.
 
Live/Dead Assays After Nanosensor Loading: A fluorescence live/dead assay, consisting of calcein to stain living cells and ethidium bromide to stain the nuclei of dead cells, was performed in order to determine the viability of cells loaded with nanosensors,  FIG. 12 . The staining procedure included incubation of nanosensor-loaded cells (overnight loading, as above) in 8 μM of calcein and 8 μM of ethidium bromide for 20 minutes at 37° C. 5% CO 2 , then rinsing 3×. The cells were then imaged on the confocal microscope. In  FIG. 12  the green indicates healthy cells, while the red stains the nuclei of dead cells. No difference in the ratio of live to dead cells was noted between nanosensor loaded cells and control (no nanosensors). Following an implementation of this method, it was noted that the number of living cells was not different from control cells (not shown) which contained no nanosensors.
 
Nanosensors in Cardiac Cells: Isolated neonatal rat ventricular myocytes were plated onto laminin coated 25 mm coverslips. Electroporation was performed in a custom chamber with custom parallel electrodes spaced at 1 cm. The cells were porated in a Ringer&#39;s solution containing 1:10 volume ratio of nanosensor solution. 800 V pulses were applied for 20 μseconds 8 times using an electroporator (Harvard Apparatus). The cells were then allowed to recover for 10 minutes before imaging. Images were taken on a confocal microscope (LSM 510 Meta, Zeiss) exciting at 488 nm and emitting at 650-690, 63× oil immersion objective. As can be seen in  FIG. 13   a , the nanosensors loaded into the cardiac cells efficiently using electroporation.
 
     As the cells “beat” during electrical stimulation in cardiac cells, the fluorescence from the sodium sensors was collected and was seen to oscillate at 1 Hz, the pacing frequency. In  FIG. 13   b , the fluorescence collected from a nanosensor during stimulation, is charted. The changes of fluorescence observed are attributed to oscillation of sodium, as the changes occur at the pacing frequency of the cells. The data is the average of three time segments of the same experiment (data from time 0-3 seconds was averaged with data 3-6 seconds and data 6-9 seconds), and base-line corrected to account for photobleaching. 
     Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.