A Real-Time Ratiometric Method for the Determination of Molecular

Anal. Chem. 2001, 73, 4124-4133

A Real-Time Ratiometric Method for the Determination of Molecular Oxygen Inside Living Cells Using Sol-Gel-Based Spherical Optical Nanosensors with Applications to Rat C6 Glioma Hao Xu,† Jonathan W. Aylott,†,§ Raoul Kopelman,*,† Terry J. Miller,‡ and Martin A. Philbert‡

Department of Chemistry and Department of Environmental Health Sciences, University of Michigan, Ann Arbor, Michigan 48109-1055

The first sol-gel-based, ratiometric, optical nanosensors, or sol-gel probes encapsulated by biologically localized embedding (PEBBLEs), are made and demonstrated here to enable reliable, real-time measurements of subcellular molecular oxygen. Sensors were made using a modified Sto1 ber method, with poly(ethylene glycol) as a steric stabilizer. The radii of these spherical PEBBLE sensors range from about 50 to 300 nm. These sensors incorporate an oxygen-sensitive fluorescent indicator, Ru(II)tris(4,7-diphenyl-1,10-phenanthroline) chloride ([Ru(dpp)3]2+), and an oxygen-insensitive fluorescent dye, Oregon Green 488-dextran, as a reference for the purpose of ratiometric intensity measurements. The PEBBLE sensors have excellent reversibility, dynamic range, and stability to leaching and photobleaching. The small size and inert matrix of these sensors allow them to be inserted into living cells with minimal physical and chemical perturbations to their biological functions. Applications of sol-gel PEBBLEs inserted in rat C6 glioma cells for real-time intracellular oxygen analysis are demonstrated. Compared to using free dyes for intracellular measurements, the PEBBLE matrix protects the fluorescent dyes from interference by proteins in cells, enabling reliable in vivo chemical analysis. Conversely, the matrix also significantly reduces the toxicity of the indicator and reference dyes to the cells, so that a wide variety of dyes can be used in optimal fashion. Oxygen is a key parameter in biological systems and is either produced by autotrophic photosynthesis in the presence of light or consumed by different metabolic processes. For example, oxygen plays a crucial role in the cellular aerobic energy metabolism since it is used as an electron acceptor at the end of the aerobic pathway of glucose oxidation. Thus, knowledge of oxygen gradients in complex biological samples is of paramount importance for the understanding and quantification of these processes.1,2 The numerous biological roles of molecular oxygen create the need for noninvasive, sensitive, and selective detection methods that are capable of real-time oxygen measurements. †

Department of Chemistry. Department of Environmental Health Sciences. § Present address: Department of Chemistry, University of Hull, Hull, HU6 7RX, U.K. ‡

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Optical oxygen sensors offer advantages over conventional electrochemical methods in that they are fast, do not consume oxygen, and are not easily poisoned.3-9 This is particularly true for optical nanosensors.5,6 Sensor operation is usually based on the quenching of fluorescence in the presence of oxygen. Thus, it is important that the matrix preclude other quenchers. In medical and biochemical research, when the domain of the sample is reduced to micrometer regimes, e.g. living cells or their subcompartments, the real-time measurement of chemical and physical parameters with high spatial resolution and negligible perturbation of the sample becomes extremely challenging. A traditional strength of chemical sensors (optical, electrochemical, etc.) is the minimalization of chemical interference between sensor and sample, achieved with the use of inert, “biofriendly” matrixes or interfaces. However, when it comes to penetrating individual live cells, the mere size of the sensor results in physical interference, accompanied by serious biological damage and resultant biochemical consequences. To overcome this problem, the past decade saw significant progress in miniaturization of electrodes and fiber-optic optodes.10,11 However, even reductions to tips with 100-nm radius or below are not really satisfactory.11 In contrast, individual molecular probes (free sensing dyes) are physically small enough but usually suffer from the chemical interference between probe and cellular components. Thus, no viable molecular probes for oxygen sensing have been demonstrated. A recent development in sensor design attempts to combine the advantages of sensor tips and molecular probes, i.e., simultaneously avoid both physical and chemical interference between sensor (probe) and sample (cell or organelle). These spherical nanosensors have been termed PEBBLEs (probes encapsulated by biologically (1) Klimant, I.; Ruchruh, F.; Liebsch, G.; Stangelmayer, A.; Wolfbeis, O. S. Mikrochim. Acta 1999, 131, 35-46. (2) Lahdesmaki, I.; Scampavia, L. D.; Beeson, C.; Ruzicka, J. Anal. Chem. 1999, 71, 5248-5252. (3) McDonagh, C.; MacCraith, B. D.; McEvoy, A. K. Anal. Chem. 1998, 70, 45-50. (4) Demas, J. N.; DeGraff, B. A. Anal. Chem. 1991, 63, 829A-837A. (5) Rosenzweig, Z.; Kopelman, R. Anal. Chem. 1995, 67, 2650-2654. (6) Rosenzweig, Z.; Kopelman, R. Sens. Actuators, B 1996, 36, 475-483. (7) Lee, S. K.; Okura, I. Anal. Chim. Acta 1997, 342, 181-188. (8) McNamara, P. K.; Li, X.; Stull, A. D.; Rosenzweig, Z. Anal. Chim. Acta 1998, 361, 73-78. (9) Bacon, J. R.; Demas, J. N. Anal. Chem. 1987, 59, 2780-2785. (10) Buhlman, P.; Pretsch, E.; Bakker, E. Chem. Rev. 1998, 98, 1593-1647. (11) Tan, W.; Kopelman, R.; Barker, S. L. R.; Miller, M. T. Anal. Chem. 1999, 71, 606A-612A. 10.1021/ac0102718 CCC: $20.00

© 2001 American Chemical Society Published on Web 08/03/2001

localized embedding) and, due to their chemically inert matrixes and their small physical sizes, provide an almost nonperturbing measurement inside intact biological systems. A prime advantage of PEBBLE nanosensors is that their matrix provides a protective coating for the indicator dyes, and thus their response is not affected by interferences, such as protein binding and/or membrane/organelle sequestration in the biological sample.12 Conversely, the nanosensor matrix also provides protection to the cellular contents, enabling dyes that would usually be toxic to cells to be entrapped in the nanosensor matrix and used for intracellular sensing. The small size and lack of deleterious response due to interferences suggest that spherical nanosensors are well suited for intracellular applications. As mentioned, no reliable optical method for the real-time and quantitative measurement of intracellular oxygen levels is currently available. This is largely due to the constraints of using sensors of much larger size, in comparison to PEBBLEs, or using single-intensity-based free dye solutions that may be toxic to or highly perturbed by cell contents, or both. The lack of such a method means that the numerous important biological roles of oxygen and the biochemical events related to the change of intracellular oxygen cannot be fully studied and understood. The advantages of PEBBLE sensors suggest that they would provide a reliable method for monitoring oxygen inside living cells. Very preliminary qualitative reports from our group, on acrylamide-based oxygen-sensing PEBBLEs, demonstrated their insertion into live cells.13,14 It is necessary to point out that, in the present paper, we deal with an ensemble or a suspension of PEBBLEs, not just one single PEBBLE. In intracellular measurements, to obtain a certain satisfactory signal level, the use of many small PEBBLE sensors is preferred over the use of one larger sensor. This is because the small size of PEBBLE sensors causes minimal physical perturbations to the cells. Since a small PEBBLE sensor only takes up ∼1 ppm of the volume of a cell, even the insertion of many of them still causes only negligible perturbation. Using a single larger sensor may cause more perturbation to cells than using a number of small ones. Also, using many small sensors, compared to using one larger sensor, enables better distributions for imaging and chemical analysis. Furthermore, it is more difficult to deliver only one single, larger PEBBLE into a cell. However, when necessary, intracellular measurements can be carried out using a single small PEBBLE, as has been demonstrated in a previous paper.14 A number of different optical sensors have been reported for the detection of both gaseous and aqueous oxygen.1,3,5-9,15 Most of these sensors were based on measurements of a single fluorescence intensity, which are known to be problematic in most practical applications due to signal changes resulting from factors such as light scattering by the sample and excitation source fluctuations, in addition to differing oxygen concentrations.16 Ratiometric sensors have been one answer to the problems posed (12) Graber, M. L.; DiLillo, D. C.; Friedman, B. L.; Pastoriza-Munoz, E. Anal. Biochem. 1986, 156, 202-212. (13) Clark, H. A.; Hoyer, M.; Parus, S.; Philbert, M. A.; Kopelman, R. Mikrochim. Acta 1999, 131, 121-128. (14) Clark, H. A.; Barker, S. L. R.; Brasuel, M.; Miller, M. T.; Monson, E.; Parus, S.; Shi, Z. Y.; Song, A.; Thorsrud, B.; Kopelman, R.; Ade, A.; Meixner, W.; Athey, B.; Hoyer, M.; Hill, D.; Lightle, R.; Philbert, M. A. Sens. Actuators, B 1998, 51, 12-16. (15) Hartmann, P.; Leiner, M. J. P.; Lippitsch, M. E. Anal. Chem. 1995, 67, 88-93.

by intensity measurements,16-19 since they compensate for the effect of these factors by taking the ratio of the indicator peak intensity over the reference peak intensity. Another solution has been the measurement of fluorescence lifetimes;1,20-22 however, ratiometric methods are experimentally simpler than lifetime measurements. A number of ratiometric nanosensors for extraand intracellular studies have been reported previously, using the emission ratios of self-referencing fluorescent dyes, e.g., CNF and SNAFL, or incorporating an indicator dye and a reference dye, for monitoring cellular pH, calcium, and NO levels,17-19,21-26 though there have been no reports of quantitative measurements of intracellular oxygen levels using ratiometric methods. Several ratiometric oxygen sensors have been described in the literature,27-29 but they cannot be applied to intracellular studies due to their comparatively large sizes and configurations. To minimize sensor sizes, for minimal perturbations, dye-encapsulating liposomes were recently developed by McNamara et. al. as fluorescence-based oxygen nanosensors.30 However, though effective in vitro, they cannot be used inside cells, where the liposome would usually open up and release the dye into the cell, with the ensuing interference problems. We report here on a novel ratiometric method for the detection of molecular oxygen inside living cells using the coimmobilization of two fluorescent dyes within a sensing matrix to yield ratiometric emission measurements. The sensors are ratiometric, due to a reference fluorescent dye, Oregon Green 488-dextran. The fluorescence intensity of Oregon Green is not affected by changing oxygen concentrations, making it ideal as a reference dye. The conjugation of the Oregon Green dye to dextran makes the complex highly water soluble, which helps it dissolve better in the sol-gel matrix. Also the high molecular weight of dextran helps prevent the Oregon Green dye molecules from leaching out of the sensing matrix. It should be noted though that Oregon Green becomes pH sensitive when the pH is below 6, so it is necessary that the pH of the sensing environment is above 6 or, alternatively, remains constant, or is monitored separately during the analysis. The fluorescent ruthenium complex Ru(II)-tris(4,7diphenyl-1,10-phenanthroline) chloride ([Ru(dpp)3]2+) is an ideal candidate for use as the indicator dye because it is photostable, (16) Lakowicz, J. R. In Topics in Fluorescence Spectroscopy; Lakowicz, J. R., Ed.; Plenum Press: New York, 1994; Vol. 4, p 3. (17) Tan, W.; Shi, Z. Y.; Smith, S.; Birnbaum, D.; Kopelman, R. Science 1992, 258, 778-781. (18) Tan, W.; Shi, Z. Y.; Kopelman, R. Anal. Chem. 1992, 64, 2985-2990. (19) Tan, W.; Shi, Z. Y.; Kopelman, R. Sens. Actuators, B 1995, 28, 157-163. (20) Lakowicz, J. R. In Topics in Fluorescence Spectroscopy; Lakowicz, J. R., Ed.; Plenum Press: New York, 1994; Vol. 4, pp 3-6, 432-445. (21) Chen-Esterlit, Z.; Aylott, J. W.; Kopelman, R. SPIE Proc.-Int. Soc. Opt. Eng. 1999, 3540, 19-27. (22) Barker, S. L. R.; Clark, H. A.; Swallen, S. F.; Kopelman, R.; Tsang, A. W.; Swanson, J. A. Anal. Chem. 1999, 71, 1767-1772. (23) Barker, S. L. R.; Kopelman, R. Anal. Chem. 1998, 70, 4902-4906. (24) Clark, H. A.; Kopelman, R.; Tjalkens, R.; Philbert, M. A. Anal. Chem. 1999, 71, 4837-4843. (25) Kopelman, R.; Philbert, M. A. Book of Abstracts, 218th ACS National Meeting, New Orleans, 1999; Abstr, ANYL-131. (26) Ji, J.; Rosenzweig, N.; Griffin, C.; Rosenzweig, Z. Anal. Chem. 2000, 72, 3496-3503. (27) Kane, J.; Martin, R.; Schilling, A. PCT Int. Appl., WO 94-US10139, 1995. (28) Dourado, S.; Kopelman, R. Proc. SPIE-Int. Soc. Opt. Eng. 1999, 3540, 224234. (29) Kostov, Y.; Van Houten, K. A.; Harms, P.; Pilato, R. S.; Rao, G. Appl. Spectrosc. 2000, 54, 864-868. (30) McNamara, K. P.; Rosenzweig, Z. Anal. Chem. 1998, 70, 4853-4859.

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has a long excited-state lifetime (5.3 µs) and a high luminescence quantum yield (∼30%), and is readily quenched by oxygen.31 Sol-gel glass was used as the matrix for the fabrication of PEBBLE nanosensors because of the superior properties it has over organic polymers. Sol-gel glass is a porous, high-purity, optically transparent and homogeneous material,32 thus making it an ideal choice as a sensor matrix for quantitative spectrophotometric measurements. Also, it is chemically inert, photostable, and thermally stable compared with polymer matrixes. The preparation of sol-gel glasses is technically simple, and tailoring the physicochemical properties (i.e., pore size or inner-surface hydrophobicity) of sensor materials can be achieved easily by varying the processing conditions and the amount or type of reactants used. This enables the pore sizes to be optimized such that the analyte is able to diffuse easily and interact with the sensing molecules while the latter are prevented from leaking out of the matrix. A range of sol-gel sensor configurations have been described in the literature, including monoliths, thin films, miniaturized probe tips, and powders.32 Immobilization of the sensing reagent in a supportive matrix is a critical step in the fabrication of optical sensors. It can be achieved by either chemical or physical entrapment of the fluorescent dye molecules in the pore structures of the sol-gel network. An important advantage of physical entrapment is the minimal alteration in the spectral and binding properties of the sensing molecules due to interactions with the supporting matrix. A potential disadvantage is the leaching of the dye molecules out of the matrix; but for short time or disposable use of the sensors, as described here for biological cells, this is not of much concern. In comparison with film-based optodes, altering the matrix of a nanosphere sensor presents a greater challenge. The PEBBLE sensor principle requires the indicator dye to be immobilized inside the sensor, rather than on the surface. Thus, no commercial nanobeads can be utilized. Previous PEBBLE sensors (with a polyacrylamide matrix) were prepared by using a microemulsion technique.24 However, this was found not to work for sol-gel matrixes. The present paper describes the first fabrication of solgel-based ratiometric PEBBLE sensors, using a tailor-made method in which the sensor size distribution and other properties are modified by the inclusion of PEG. These nanosensors contain two fluorescent dyes: an oxygen-responsive sensor dye and an oxygen-insensitive reference dye. Sensor characteristics, stability, immunity to interference, and applications to real-time oxygen analysis inside rat C6 glioma cells are detailed. EXPERIMENTAL SECTION Reagents. All reagents and solvents were purchased from Aldrich (Milwaukee, WI) unless otherwise noted. The reference dye, Oregon Green 488-dextran was purchased from Molecular Probes (Eugene, OR) and the indicator dye, Ru(II)-tris(4,7diphenyl-1,10-phenanthroline) chloride, was purchased from GFS Chemicals, Inc. (Columbus, OH). Ethanol (200 proof) was obtained from Pharmco Products Inc. (Brookfield, CT). Gases. O2 (99.99%, extra dry grade) and N2 (99.99%, extra dry grade) were obtained from Cryogenic Gases (Detroit, MI). Cell Culture. Rat C6 glioma cells were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 4500 mg/L D(31) Mills. A.; Thomas, M. Analyst. 1997, 122, 63-68. (32) Uhlmann, D. R.; Teowee, G.; Boulton, J. J. Sol-Gel Sci. Technol. 1997, 8, 1083-1091.

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glucose, 2 mM L-glutamine, and 20% fetal bovine serum. Cells were released from culture dishes by trypsin treatment 1 day prior to experiments and plated on uncoated 22-mm glass cover slips in 35-mm culture disks. PEBBLE Delivery. A Biolistic PDS-1000/He system (benchtop model) from BioRad (Hercules, CA) with grade 5 helium (Cryogenics, Detroit, MI) was used to inject cells with sol-gel PEBBLEs. Sample preparation for the particle delivery system required dispersion of the PEBBLEs in ethanol and the careful application of a thin layer of PEBBLEs onto the target membrane (5.5 cm above the cells). The optimum firing pressure was found to be 650 psi, with a vacuum of 15 Torr on the system. Culture medium (DMEM) was removed from the cells by pipet prior to PEBBLE delivery. Following biolistic delivery of PEBBLEs, the cells were rinsed with Dulbecco’s phosphate-buffered saline (DPBS) and incubated with DPBS during measurements. Preparation of Sol-Gel PEBBLEs. The typical reaction solution consists of poly(ethylene glycol) (PEG) MW 5000 monomethyl ether (3 g), ethanol (200 proof) (6 mL), Oregon Green 488-dextran MW 10 000 (0.1 mM), [Ru(dpp)3]2+ (0.4 mM), and 30 wt % ammonia water (3.9 mL) with ammonia serving as catalyst and water being one of the reactants. Upon mixing, the solution became transparent and tetraethyl orthosilicate (TEOS) (0.5 mL) was added dropwise to initiate the hydrolysis of TEOS. The solution was then stirred at room temperature for 1 h to allow the sol-gel reaction to reach completion. A liberal amount of ethanol was then added to the reaction solution, and the mixture was transferred to an Amicon ultrafiltration cell (Millipore Corp., Bedford, MA). A 100-kDa membrane was used to separate the reacted sol-gel particles (PEBBLEs) from the unreacted monomers, PEG, ammonia, and dye molecules, under a pressure of 10 psi. The PEBBLEs were further rinsed with 500 mL of ethanol to ensure that all unreacted chemicals had been removed from the PEBBLEs. The PEBBLE solution was then passed through a suction filtration system (Fisher, Pittsburgh, PA) with a 2-µm filter membrane to separate the larger size particles from the smaller ones. The filtrate (containing the smaller particles) was filtered again, this time with a 0.02-µm filter membrane, to collect the particles which were then dried to yield a final product consisting of sol-gel PEBBLEs in the size range of 100-600 nm in diameter. Optical Apparatus for Ratiometric Sensor Measurements. The excitation source was a 488-nm Ar ion laser (Ion Laser Technology, Salt Lake City, UT) operated at 40 mW. A highprecision single-mode optical fiber coupler (Newport) was used to direct the laser beam into a single-mode optical fiber (Polymicro Technologies, Phoenix, AZ), to excite the sensor reagent. An Hg lamp (Olympus, Melville, NY) was used as the light source in the dissolved oxygen calibration and the intracellular oxygen measurements. Sensor fluorescence spectra were collected through the optics of an Olympus inverted fluorescence microscope and sent to an Acton Research Corp. spectrograph. The detector was a liquid nitrogen-cooled charge-coupled device (CCD) from Princeton Instruments, interfaced with a DFI P166 computer. In some of the aqueous-phase experiments (i.e., Figure 2b), measurements were taken on a FluoroMax-2 spectrofluorometer (ISA Jobin Yvon-Spex, Edison, NJ), slits set to 2 nm for both the emission and excitation. Nomarski and fluorescence images of

cells loaded with PEBBLE sensors were obtained using a laser scanning confocal microscope system composed of an Olympus IX 70 fluorescence microscope and an Olympus Fluoview confocal unit. This system is equipped with an Ar-Kr and a He-Ne laser. Data were acquired with an Olympus fluoview software package and analyzed using Photoshop Adobe Systems (Mountainview, CA) software packages. SEM Imaging. Sol-gel PEBBLEs were dispersed in water and sonicated for several minutes to prevent aggregation of particles. Then a drop of the PEBBLE solution was placed on a piece of glass microslide (Arthur H. Thomas Co., Philadelphia, PA) attached to a metal grid coated with carbon film and dried gradually at room temperature. The sample was then sputter coated with gold and visualized using a Hitachi S-3200N scanning electron microscope. Sensor Calibration. Sensor calibrations were made in a sealed glass chamber with three stoppered ports. For the calibration of gaseous oxygen, several drops of a PEBBLE water solution were put onto the bottom of the chamber and the PEBBLEs adhered to the surface after the water evaporated. Laser light was introduced to illuminate these sensors by a single-mode optical fiber (Polymicro Tech., Phoenix, AZ), placed through the central stopper of the chamber. Gas was introduced into the chamber through the stopper on the left side, and the stopper on the right side was used for ventilation, by insertion of a hypodermic needle through it. A combination of cylinders of oxygen and nitrogen and a gas blender (Cole-Parmer Instrument Co., Vernon Hills, IL) was used to achieve precise gas mixtures that were passed at selected flow rates through the sealed glass chamber. The accuracy of mixing the gases using this gas blender was provided by the manufacturer to be (2% full scale. For the calibration of dissolved oxygen (DO), 2 mL of a 1 mg/mL PEBBLE solution was placed in the same glass chamber. The same experimental setup was used as in the gaseous-phase calibration, except that the excitation source used was an Hg lamp (Olympus, Melville, NY). The excitation light was introduced through the inverted microscope objective from underneath the chamber. Different DO concentrations were obtained by flowing, from the gas blender into the PEBBLE solution, a gas mixture of predetermined nitrogen and oxygen concentrations (The dissolved oxygen concentration was expressed in units of ppm. The oxygen concentration in an oxygen-saturated solution was calculated to be ∼42.5 ppm. This calculation was based on the solubility constant of oxygen in water at 21 °C (0.004 252 g of O2 in 100 g of water).33) RESULTS AND DISCUSSION Sol-Gel PEBBLE Formation. In our method of producing monodisperse, spherical, and porous nanoparticles using a solgel process, tetraethyl orthosilicate (TEOS, or Si(OEt)4) is used as the precursor and proceeds through the following two steps to form the final particles:

(1) hydrolysis reaction: tSisOEt + H2O f tSisOH + EtOH (I) In this reaction, as the TEOS precursor and water are immiscible, a cosolvent such as ethanol is often used as a medium (33) http://jcbmac.chem.brown.edu/myl/hen/oxygenHenry.html.

in which the hydrolysis reaction takes place; i.e., alkoxide groups are replaced with hydroxyl groups. The above equation is only the first step of hydrolysis, and the degree of hydrolysis is controlled by the water/precursor molar ratio, r. The higher the value of r, the more complete the hydrolysis is. In an excess amount of water, Si(OH)4 will be formed, favoring the formation of a three-dimensional network. The hydrolysis reaction is accelerated by the use of a catalyst, which can be either an acid or a base.

(2) condensation reactions: tSisOEt + HOsSit f tSisOsSit + EtOH or,

tSisOH + HOsSit f tSisOsSit + H2O

(II)

Condensation reactions involving the silanol groups produce siloxane bonds (Si-O-Si) and form the gel network. The conditions used in our method, i.e., pH above 8 and excess water (with an r value greater than 50), result in the formation of compact structures rather than extended polymeric networks, favoring the formation of silica particles. In the early stages of this work, we used a method developed by Sto¨ber34 to produce sol-gel nanoparticles. However, an average particle size of several micrometers was often obtained using this method, largely due to aggregation between the particles when collected as a dry solid. A modification was made to the method whereby the addition of PEG MW 5000 monomethyl ether, which acts as a steric stabilizer, reduced the particle diameter to the range of about 0.1-1.5 µm. As will be described later in the paper, the addition of PEG to the sensing matrix also greatly helped improve the sensor performance in oxygenated water. Further separation of the sol-gel particles using a suction filtration system resulted in a particle diameter distribution of 100-600 nm. A scanning electron micrograph of these particles is shown in Figure 1. The size distribution as measured by SEM is summarized in Table 1 showing that more than 50% of the particles have diameters smaller than 200 nm. According to previous literature data, most of the doped PEG side chains are believed to be anchored onto the surface of the silica particles and extend from the particle surface to contribute a steric stabilization function.35 Thus, the large degree of aggregation between primary nanosize particles, which was the main cause for the formation of micrometersize particles in our earlier experiments, was prevented by the repulsion force and solvation layer of the PEG surface moiety on silica particles.36 Parameters including the water/precursor molar ratio, the type and amount of catalyst, the pH, and the concentration and molecular weight of the PEG added were studied for their effects on the size and size distribution of particles. The optimum conditions were determined and used for sensor preparation. Calibration of Sol-Gel PEBBLEs. The oxygen quenching process is ideally described by the linear Stern-Volmer equation: (34) Sto ¨ber, W.; Fink, A.; Bohn, E. Colloid Interface Sci. 1968, 26, 62-69. (35) Coombes, A. G. A.; Tasker, S.; Lindblad, M.; Holmgren, J.; Hoste, K.; Toncheva, V.; Schacht, E.; Davies, M. C.; Illum, L.; Davis, S. S. Biomaterials 1997, 18, 1153-1161. (36) Kim, K. S.; Cho, S. H.; Shin, J. S. Polym. J. 1995, 27, 508-514.


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Figure 1. Scanning electron micrographs showing the size distribution of sol-gel-PEG particles produced by the modified Sto¨ber method. Scale bars: (a) 5 µm; (b) 1 µm.

Figure 2. (a) Gaseous-phase emission spectra of sol-gel oxygen PEBBLE sensors excited at 488 nm. Top line, purged with N2; middle line, in air; bottom line, purged with O2. The peak on the left is the fluorescence emission of the reference dye, and the peak on the right is the fluorescence emission of the indicator dye. (b) Aqueous-phase emission spectra of sol-gel oxygen PEBBLEs excited at 488 nm. Top line, PEBBLE solution purged with N2; middle line, PEBBLE solution purged with air; bottom line, PEBBLE solution purged with O2.

QG ) (IN2 - IO2)/IN2

Table 1. Size Distribution of Sol-Gel PEBBLEs in Figure 1a, with a Total of 230 Particles diameter (nm)

percentage

diameter (nm)

percentage

100-150 150-200 200-300

36.1 18.3 7.8

300-400 400-500 500-600

4.8 8.7 24.3

I0/I ) 1 + KSVp[O2] where I0 and I are the luminescence intensities in the absence and presence of oxygen at a partial pressure of p[O2], respectively, and KSV is the Stern-Volmer constant, which depends directly upon the diffusion constant of oxygen, the solubility of oxygen, and the quenching efficiency and lifetime of the excited-state of the fluorophore.4 The fluorescence emission spectra of the sol-gel PEBBLE sensors, in different gaseous oxygen concentrations, are shown in Figure 2a. The sensor response was determined from the ratio (R) of the fluorescence intensities of [Ru(dpp)3]2+ to Oregon Green 488-dextran. These plots illustrate a number of features that are important to the optimization of the sensor design. The overall gas-phase quenching response, QG, is given by 4128


where IN2 and IO2 denote intensities in 100% N2 and 100% O2, respectively. The measured value of QG (the higher, the better) for the sol-gel PEBBLEs is ∼92%. These data are in agreement with thin film sol-gel oxygen sensors3 and have the advantage of being a ratiometric measurement. The calibration curve for the ratiometric sol-gel PEBBLEs showing the response to gaseous oxygen is displayed in Figure 3a. Fluorescence emission intensity maximums of Ru(dpp)3 and Oregon Green 488-dextran were used to determine the ratios. However, the possible problem of the overlap of the two peaks was minimized by the use of deconvoluted spectra obtained from raw data, where peaks are well separated; thus, the effects of peak overlap on the sensor calibration are minimized. The quasi-linearity (r2 ) 0.995) of the Stern-Volmer plot implies a single fluorophore class accessible to molecular oxygen.37 Having some dye molecules not accessible to oxygen, because of the depth of penetration of oxygen into the matrix, is a main reason given for the nonlinearity of the SternVolmer plot of sol-gel thin-film sensors.3 This problem could be (37) Xu, W.; Schmidt, R.; Whaley, M.; Demas, J. N.; DeGraff, B. A.; Karikari, E. K.; Famer, B. A. Anal. Chem. 1995, 67, 3172-3180.

Figure 3. (a) Stern-Volmer plot of relative fluorescence intensity ratios for ratiometric sol-gel oxygen PEBBLEs in gaseous phase. (b) Stern-Volmer plot of relative fluorescence intensity ratios for ratiometric sol-gel oxygen PEBBLEs in aqueous phase. R0 is the ratio between the fluorescence intensities of Ru(dpp)3 and Oregon Green 488-dextran in the absence of oxygen, and R is the ratio at a given oxygen concentration.

alleviated in the sol-gel oxygen PEBBLEs since they have very small sizes; so it is comparatively easier for oxygen to have access to all the dye molecules in the PEBBLEs. In other words, the small size of the PEBBLEs allows the oxygen to interact uniformly with a greater proportion of fluorophore molecules, thus resulting in a larger linear range in the Stern-Volmer plot. The reversibility of the previously reported Ru-based sol-gel sensors in the gas phase has also been retained in these nanosensors, as evidenced in Figure 4a. Alternating measurements were taken by filling the reaction chamber with oxygen, air, and nitrogen. It should be noted that this figure cannot be used to deduce the sensor response time as the transition regions are mostly indicative of the time taken to achieve manually selected stable gas concentrations. Nonoptimized measurements indicate that response times for a 90% oxygen concentration change should be well below 1 s, which is a direct result of the small size of the PEBBLEs. The sensors showed at least 98% recovery each time after the sensing environments were changed among air-, oxygen-, and nitrogen-saturated conditions. Figure 2b shows the response of the TEOS-based sol-gel PEBBLEs to DO. The QDO for the sol-gel oxygen PEBBLEs is ∼80%. (Quenching response to dissolved oxygen, QDO, is defined in a way similar to QG, where IN2 and IO2 are replaced by I in fully deoxygenated water and I in fully oxygenated water, respectively.) This value represents a slight reduction in performance vis-a`-vis

Figure 4. (a) Reversibility of PEBBLE sensor response to gaseous oxygen. Alternating measurements were taken of sensors in a chamber purged with air, O2, and N2. (b) Reversibility of PEBBLE sensor response to dissolved oxygen. Alternating measurements were taken in air-, O2- and N2-saturated PEBBLE sensor solutions.

gaseous oxygen; however, it is a great improvement with regard to TEOS-based sol-gel films.38 These sol-gel films, made using TEOS as the precursor, had an excellent response to oxygen in the gas phase, i.e., QG ) 90%, but a poor quenching response to dissolved oxygen, QDO, of only ∼20%. MacCraith and co-workers have subsequently reported significant improvements to the QDO ratio by preparing organically modified sol-gel (Ormosil) films using methyltriethoxysilane (MTEOS) and ethyltriethoxysilane (ETEOS) as the precursors. The success of the Ormosil films in raising the QDO ratio to 70-80% was largely due to the increased hydrophobicity of the film, which reduced the water solubility in the film and caused the partitioning of oxygen out of solution and into the film.3 This highly increased the accessibility of oxygen molecules to the indicator dye molecules. It is thought that the high QDO response of the TEOS sol-gel PEBBLEs might be caused by the PEG content of the sensing matrix playing a role analogous to the Ormosil precursors and thus partitioning the oxygen preferentially into the sol-gel PEBBLEs. It is well known that oxygen has a higher solubility in organic liquids than in water,39 so it should dissolve much better in an organic phase compared to an aqueous phase. In summary, doping the sol-gel PEBBLEs with PEG adds organic components to the sensing matrix and thus encourages the partitioning of oxygen into the matrix and increases the accessibility of oxygen to the entrapped (38) McEvoy, A. K.; McDonagh, C.; MacCraith, B. D. Analyst 1996, 121, 785788. (39) The Merck Index, 12th ed.; Budavari, S., Ed.; Merck & Co., Inc.: Rahway, NJ, 1996; p 1195.


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indicator dye molecules. This is in addition to the role of PEG in preventing particle aggregation in the PEBBLE sensor fabrication. Figures 3b and 4b show the Stern-Volmer plot of fluorescence intensity ratios versus oxygen concentrations and the reversibility of the sol-gel PEBBLEs versus changing dissolved oxygen concentrations, respectively. These data show that although the performance of the sol-gel PEBBLEs is slightly reduced in the aqueous phase relative to the gas phase, the sensors still demonstrate good reversibility and reproducibility as evidenced by the error bars in the calibration curve. The dashed line (the linear range of the Stern-Volmer plot) in Figure 3b covers the extent of the biologically relevant regime of intracellular oxygen concentrations. As is well known, all primary animal cells are aerobic, and oxygen is constantly being consumed inside cells. So, usually the oxygen concentrations in the intracellular aqueous environment is lower than the oxygen concentration (∼8-9 ppm) in the air-saturated aqueous environment outside the cells, because of the salt content and metabolic activities inside living cells. Thus, the biologically relevant regime of intracellular oxygen levels ranges approximately from 0 to 9 ppm. We note that in this regime, the Stern-Volmer plot is quasi-linear (r2 ) 0.988). The sensors showed at least 95% recovery each time that the sensing environments were changed among air-, O2-, or N2saturated sensor solutions. The measured transition times in Figure 4b are on the order of 20-30 s, but these times are much longer than the intrinsic response time of the PEBBLEs, due to the significant contribution of the time used to saturate the solution with O2 or N2. It is difficult to measure the exact response time, because changing between oxygenated and deoxygenated PEBBLE solutions takes time itself and measured transition times (including the time of saturating the solution) are only an upper limit of the response time. We believe that the PEBBLE sensors should intrinsically have shorter response times than previously reported thin-film and fiber-optic sol-gel sensors (on the order of seconds or minutes) simply because of the small sizes of our sensors. According to the Einstein diffusion equation, where X2 ) 2Dτ, shorter diffusion length X (which is directly related to the size of the sensor) results in shorter time for oxygen molecules to diffuse through the sensing matrix (which is basically the response time). A lower limit can be estimated using D ≈ 2 × 10-9 m2/s (diffusion constant of oxygen in water) and X ≈ 3 × 10-7 m, giving τ ≈ 20 × 10-6 s, i.e. a response time in the microsecond range. An upper limit can be estimated considering that our sensor dimensions are 10-100 times smaller than thin film sensors and have a spherical shape. This should give our PEBBLE sensors a response time in the millisecond range. Photostability. The photostability of the sol-gel PEBBLEs was also studied. In this experiment, sol-gel PEBBLEs located on the bottom of a glass container were constantly illuminated by a 488-nm laser light through a single-mode optical fiber (∼100µm i.d.). The light intensity emitted from the tip of the fiber was ∼200 µW (corresponding to a power density of ∼ 2 W/cm2), which is significantly higher than the excitation intensity normally used for our experiments (50-100 µW). The duration of this experiment was over 100 min, and only a variation of at most 3% in the fluorescence intensity ratios was observed. The overall decrease in the absolute fluorescence intensity of [Ru(dpp)3]2+ was ∼11% and for Oregon Green it was ∼9%. The decrease in the intensity 4130


of both dyes is much lower in real applications since usually a much lower excitation intensity is used. Taking into account our short exposure time (usually 0.2 s) per measurement, this decay would occur after 30 000 independent measurements and an even larger number of measurements at the lower power density usually used in real experiments. It should also be noted that, for the intended applications of these sol-gel PEBBLEs, i.e., to facilitate intracellular measurements, the lifetime of the cells outside an incubator environment is of the order of 90 min. Also, usually only 10 or fewer measurements are taken per minute. Thus, the operational lifetime of the sensors is much longer than the lifetime of the sampling medium. In summary, these sol-gel PEBBLEs are highly photostable over their operational time scale. Sensor Stability with Respect to Dye Leaching. Leaking of dye molecules from the sol-gel matrix of PEBBLE sensors is a major concern and is very dependent on the dye. Factors such as the molecular size of the dye (small dyes can more readily diffuse through the pores and leak out of the matrix) and the solubility of the dye in the matrix and in water play a significant role. These factors must be taken into consideration during the fabrication of the sensors, since the pore size and microstructure (e.g., the hydrophobicity of the inner surface of the pores within the solgel matrix) of the matrix can easily be tailored by varying the type of precursor, the water-to-precursor ratio (r), or the pH of the reaction solution during the sol-gel preparation process. Generally, sol-gel supports provide excellent stability with respect to dye leaching.40 In particular, ruthenium complexes often have excellent stability inside the sol-gel matrix, and in agreement with previous reports,1,3,40-42 the indicator dye [Ru(dpp)3]2+ shows no signs of leaching. For the reference dye, the large size of the dextran molecular backbone to which the Oregon Green dye molecules are bound should greatly prevent leaching. A study into the stability of the sol-gel PEBBLEs was performed. A 2 mg/mL PEBBLE solution (in 5 mL of pH 7 buffer) was prepared and was monitored over a 3-day period. After each day, all the PEBBLEs in the solution were collected by a suction filtration system using a 20-nm pore size membrane, and a spectrum was taken of the filtrate using 488-nm laser excitation. In each spectrum, there was no signal observed which was 3 times higher than the background noise at the emission wavelengths of the two dyes entrapped within the PEBBLEs, indicating that there was no detectable leaching of the dye molecules out of the sensor matrix. A solution containing free [Ru(dpp)3]2+ and Oregon Green dye molecules (same concentration as in PEBBLEs) was diluted until the fluorescence spectrum of the solution became similar to the spectrum of the filtrate in the leaching experiment. According to the dilution factor, a rough estimate provides an upper limit of 1% for the amount of dye molecules leached out of the sensing matrix. This result shows the excellent stability of the sol-gel PEBBLE sensors with respect to dye leaching. In addition, we note again that the typical biological application is performed over a time scale of only a few hours. Interferences. We have found that the sol-gel matrix of PEBBLE sensors prevents macromolecules, such as proteins, from (40) Ingersoll, C. M.; Bright, F. V. CHEMTECH 1997, 27, 26-31. (41) Bossi, M. L.; Daraio, M. E.; Aramendia, P. F. J. Photochem. Photobiol. A: Chem. 1999, 120, 15-21. (42) Murtagh, M. T.; Shahriari, M. R.; Krihak, M. Chem. Mater. 1998, 10, 38623869.

Figure 5. (a) Effect of protein on sol-gel PEBBLEs and free dye solution. Adding a very little amount of 5% BSA to a solution of Ru(dpp)3 and Oregon Green 488-dextran dye molecules changed the fluorescence intensity ratio of the two dyes dramatically, while for PEBBLE sensors this ratio remained almost constant. (b) Effect of protein on free dye molecules in solution. It shows that the fluorescence intensity of [Ru(dpp)3]2+ changed dramatically upon interaction with BSA, while the intensity of Oregon Green remained largely unchanged. So, Ru(dpp)3 is responsible for the large change in fluorescence intensity ratios in (a).

diffusing through the matrix (as expected). The matrix thus protects the entrapped dyes from the intracellular environment, preventing interference with the fluorescent properties of the dyes.12 Without this shielding of a dye, its fluorescence would behave unpredictably inside a given cell, making calibration of even ratiometric dyes difficult or impossible.43 The effects of nonspecific protein binding were investigated by the addition of bovine serum albumin (BSA, 5% solution), and the results are shown in Figure 5a. Adding as little as 0.14% BSA to a solution containing [Ru(dpp)3]2+ and Oregon Green 488-dextran dye (at the same molar ratio in solution as inside the PEBBLEs) changed the fluorescence intensity ratio of the two dyes by a factor of more than 2.3 (i.e., an increase of over 130%). This change was mostly due to the change in the fluorescence intensity of [Ru(dpp)3]2+ after the addition of BSA, while the intensity of Oregon Green remained basically unchanged (Figure 5b). However, under the same conditions, the PEBBLE sensors containing these two dyes were not affected by the addition of BSA and a change in fluorescence intensity ratios of at most 4% was observed when even an increased concentration of BSA (0.23%) was added (Figure 5a). This difference in the effect of nonspecific protein binding (43) Clark, H. A.; Hoyer, M.; Philbert, M. A.; Kopelman, R. Anal. Chem. 1999, 71, 4831-4836.

between the dye molecules in solution and those entrapped in the PEBBLE matrix is of critical importance. This is one of the prime advantages of using PEBBLEs for intracellular analysis. One of the major, often unreported, drawbacks of intracellular fluorescence studies is that frequently the dyes used show no signs of interference when tested with single contaminating analytes, but when subjected to a concoction of unknown, biologically derived contaminants, the fluorescent response can be significantly different. However, with the protection of the matrix, this problem can be largely eliminated and the in vitro calibration of PEBBLEs still remains valid in the intracellular environment. At the same time, the matrix also significantly reduces the toxicity of the dye to the cells, so a large variety of dyes can be used. The results from the nonspecific protein-binding assay show the value of incorporating the dye inside the protective matrix. The susceptibility of the sol-gel PEBBLEs to heavy metal ions (Hg2+, Ag+) and to one of the notorious collisional quenchers (I-) was also examined. Hg(NO3)2, Ag(NO)3, and KI were added respectively to a PEBBLE solution and to a free dye solution of [Ru(dpp)3]2+ (same concentrations as in PEBBLEs) up to a concentration of ∼200 ppm. There was a 5-10% decrease in the fluorescence intensity of the [Ru(dpp)3]2+ free dye each time, while for the PEBBLEs, no measurable effect could be observed. Measurements Inside Living Cells. To demonstrate how sol-gel PEBBLEs can be used for intracellular analysis, it is first necessary to introduce the sensors into the cells. Our group has experience of introducing acrylamide PEBBLE sensors into cells,13,24 and drawing on this previous work, we chose to insert the sol-gel PEBBLEs using gene gun insertion. The gene gun uses a burst of helium to fire PEBBLEs from a target membrane into adherent cells in a culture dish. Figure 6a shows the image of C6 glioma cells containing solgel PEBBLEs under Nomarski illumination. Sol-gel PEBBLEs were inserted by gene gun injection under a firing pressure of 650 psi. It can be seen that the cells still maintained their morphology after the injection of PEBBLEs and showed no sign for cell death (no swelling or rounding up of the cells). The cytotoxicity of the PEBBLEs using gene gun injection was determined previously in our group, using a Trypan Blue viability assay, giving ∼98% cell viability13 (with the most destructive aspect of the technique being the necessity to carry out the insertion in partial vacuum). The fluorescence confocal images of these cells are demonstrated in Figure 6b-e. Green fluorescence of Oregon Green 488-dextran and red fluorescence of [Ru(dpp)3]2+ were excited by reflecting the 488- (Ar-Kr) and 543-nm (He-Ne) laser lines, respectively, onto the specimen using a double dichroic mirror (Due to the limitations of the specific filter configuration and the controlling software on the confocal microscope, we were not able to excite both the indicator and reference dye at the same wavelength (488 nm) and then collect the emission fluorescence at two different wavelengths simultaneously. The 543-nm HeNe laser was used for excitation of Ru(dpp)3 (in order to collect its red emission). However, in other experiments using the inverted fluorescence microscope, only the Ar+ laser was used to excite both dyes). The Oregon Green fluorescence from the PEBBLEs inside cells (Figure 6b) was detected by passage through a 510-nm long-pass and a 530-nm short-pass filter and the fluorescence of [Ru(dpp)3]2+ (Figure 6c) through a 605-nm Analytical Chemistry, Vol. 73, No. 17, September 1, 2001

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Figure 6. Confocal images of rat C6 glioma cells loaded with sol-gel PEBBLEs by gene gun injection. (a) Nomarski illumination. (b) Oregon Green fluorescence of PEBBLEs inside cell. (c) [Ru(dpp)3]2+ fluorescence of PEBBLEs inside cells. (d) Overlaid images of (a) and (b). (e) Overlaid images of (a) and (c).

(45-nm band-pass) barrier filter. A 40×, 1.4 NA oil immersion objective was used to image Oregon Green and [Ru(dpp)3]2+ fluorescence. The distribution of PEBBLEs shown in the overlaid images (Figure 6d and e) shows that the green and red fluorescence in Figure 6b and c emanated from the PEBBLEs inside cells. It should be noted that most of the PEBBLEs were collected in the cytoplasm, but some were observed in the nucleus. In gene gun delivery, it is not possible to direct the sensors to a particular region of the cell, since the PEBBLEs are shot in a random pattern. However, changing the firing pressure, or the distance between the target membrane (with PEBBLEs on it) and the cells, will result in different momenta with which the PEBBLEs 4132 Analytical Chemistry, Vol. 73, No. 17, September 1, 2001

are shot into the cells. By controlling this momentum, PEBBLEs will selectively stay either in the nucleus or in the cytosol of the cells. Or, when other methods are used, e.g., liposomal delivery,13,43 PEBBLEs can be selectively delivered into the cytoplasm of cells. Cells can then be selected with the PEBBLE(s) lodged in the desired location. (We note that, in preliminary experiments done in the authors’ laboratories, in vivo toxicity has been shown to be minimal.) Figure 7 shows the response of oxygen-sensitive sol-gel PEBBLEs, inserted inside rat C6 glioma cells, to changing intracellular oxygen concentrations. After gene gun injection, the cells were immersed in DPBS and a spectrum was taken of these

oxygen concentration in this case is 7.9 ( 2.1 ppm.) The comparatively large errors are due to the lower resolution of the spectrometer. We note that our intracellular oxygen value (when cells were in air-saturated DPBS) is comparable with the value of ∼7.1 ppm measured electrochemically inside the much larger islets of Langerhans.44 These results show that the PEBBLE sensors are responsive when loaded into cells and that they retain their spectral characteristics, enabling a ratiometric measurement to be made.

Figure 7. Fluorescence spectra of a typical ratiometric sensor measurement of molecular oxygen inside rat C6 glioma cells. Bottom line, cells (loaded with sol-gel PEBBLEs) in air-saturated DPBS; middle line, cells in N2-saturated DPBS, 25 s after replacing the airsaturated DPBS; top line, cells in N2-saturated DPBS, after 2 min. Table 2. Real-Time Measurements of Intracellular Oxygen Inside Rat C6 Glioma Cells conditions

average intracellular oxygen concn (ppm)

cells in air-saturated buffer cells in N2-saturated buffer (after 25 s) cells in N2-saturated buffer (after 120 s) air-saturated buffer solution

7.9 ( 2.1 6.5 ( 1.7 e1.5 8.8 ( 0.8

cells using 480 ( 10-nm excitation light. The air-saturated DPBS was then replaced by nitrogen-saturated DPBS to cause a decrease in the intracellular oxygen concentration, and the response of the oxygen PEBBLE sensors inside the cells was monitored during a time period of 2 min. As can be seen, the fluorescence intensity of [Ru(dpp)3]2+ went up gradually as the oxygen level inside the cells decreased. We note that the fluctuation in the fluorescence intensity of Oregon Green 488-dextran in Figure 7 is most likely due to optical alignment changes that occurred while the airsaturated buffer was changed to a N2-saturated buffer (from the bottom spectrum to the middle spectrum), not due to chemical instability. This problem was alleviated by the ratiometric measurements used in this experiment, and thus did not affect the precision of calibration. Average intracellular oxygen concentrations were determined on the basis of the Stern-Volmer calibration curve obtained using the fluorescence microscope-Acton spectrometer system and are summarized in Table 2. (The intracellular oxygen levels were obtained as follows: For example, when the PEBBLEs were calibrated in a solution, the R0 value was determined to be ∼2.86 by taking a spectrum of the nitrogenated PEBBLE solution. The ratio R between the intensities of Ru(dpp)3 and Oregon Green 488-dextran in the cells when exposed to air-saturated solution (the bottom spectrum in Figure 7) was ∼1.75. So, (R0/R - 1) is ∼0.64. Using this value and the dissolved oxygen calibration curve, Figure 3b, the intracellular (44) Jung, S. K.; Gorski, W.; Aspinwall, C. A.; Kauri, L. M.; Kennedy, R. T. Anal. Chem. 1999, 71, 3642-3649.

CONCLUSIONS This report details the preparation of oxygen-sensitive solgel PEBBLE nanosensors and their characterization. The solgel particles were synthesized using a modified Sto¨ber method incorporating PEG monomethyl ether as a steric stabilizer to reduce aggregation between the particles, resulting in 100-600nm-diameter particles containing co-immobilized [Ru(dpp)3]2+ and Oregon Green 488-dextran. The combination of the dyes enabled a ratiometric fluorescent determination of molecular oxygen in both gaseous and dissolved phases. The nanosensor devices were shown to have a reversible response to oxygen and to be photostable for a period in excess of 30 000 measurements. The nonresponse of the sol-gel oxygen PEBBLEs to a nonspecific binding protein demonstrates the advantage for cellular analysis of entrapping the fluorescent recognition species within, rather than on the outside of, a matrix. This advantage is 2-fold; it prevents the cellular contents from interfering with the measurement and it protects the cell from possible toxic effects of the recognition species. The result is a reliable, nonperturbative tool for measuring intracellular oxygen. Using gene gun injection, ratiometric sol-gel PEBBLEs were inserted into rat C6 glioma cells and responded to differing oxygen concentrations. These results demonstrate the utility of sol-gel-based PEBBLE sensors for real-time intracellular analysis. Direct microscopic measurements of individual cells would allow correlation of oxygen levels with various cellular activities. The methods described here indicate that such measurements are feasible and that extended applications of these sensors could provide valuable additional information about intracellular oxygen. Continued investigations to further improve cell-loading techniques and widen the number of analytes that can be determined using sol-gel PEBBLE sensors are in progress. Also, the same fabrication method is being applied to produce photodynamic nanoplatforms. ACKNOWLEDGMENT We thank the University of Michigan Electron Microbeam Analysis Laboratory (funded in part by NSF Grant EAR-9628196) for use of the SEM. We also gratefully acknowledge NIH Grants 2R01-GM50300-04A1 (R.K.) and R01-ES08846 (M.A.P.) for funding, as well as NCI Contract N01-CO-07013. Received for review March 6, 2001. Accepted June 2, 2001. AC0102718


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A Real-Time Ratiometric Method for the Determination of Molecular

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