Development and Critical Evaluation of Fluorescent Chloride

Jul 29, 2008 - Anja Graefe,*,† Sarmiza E. Stanca,‡ Sandor Nietzsche,§ Lenka Kubicova,| ... Friedrich-Schiller-University, Jena, Germany, and Inst...
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Anal. Chem. 2008, 80, 6526–6531

Development and Critical Evaluation of Fluorescent Chloride Nanosensors Anja Graefe,*,† Sarmiza E. Stanca,‡ Sandor Nietzsche,§ Lenka Kubicova,| Rainer Beckert,| Christoph Biskup,*,‡ and Gerhard J. Mohr† Institute of Physical Chemistry and Institute of Organic Chemistry and Macromolecular Chemistry, Friedrich-Schiller-University, Jena, Germany, and Institute of Physiology II and Center of Electron Microscopy, Universita¨tsklinikum, Jena, Germany In this study, we describe the preparation and evaluation of new fluorescent sensor nanoparticles for the ratiometric measurement of chloride concentrations. Both a chloridesensitive dye (lucigenin) and a reference dye (sulforhodamine derivative) were incorporated into polyacrylamide nanoparticles via inverse microemulsion polymerization and investigated for their response to chloride ions in buffered suspension as well as in living cells. The fluorescence intensity of lucigenin reversibly decreased in the presence of chloride ions due to a collisional quenching process, which can be described with the Stern-Volmer equation. The determined Stern-Volmer constant KSV for the quenching of lucigenin incorporated into particles was found to be 53 M-1 and is considerably smaller than the Stern-Volmer constant for quenching of free lucigenin (KSV ) 250 M-1) under the same conditions. To test the nanosensors in living cells, we incorporated them into Chinese hamster ovary cells and mouse fibroblasts by using the conventional lipofectamin technique and monitored the response to changing chloride concentrations in the cell. The ionic composition of the intracellular and extracellular space is highly regulated. This applies to cations as well as to anions. Among the anions, especially chloride plays a central role in many biological processes. Chloride concentration in the serum is high and varies between 98 to 106 mM.1 Intracellularly, chloride is largely replaced by polyvalent anions such as proteins and organic phosphate compounds. Chloride concentrations range from 2 mM in skeletal muscle, 20-40 mM in epithelial cells, to 90 mM in erythrocytes.2 Chloride movement across the plasma membrane is involved in the regulation of the cell volume and intracellular pH. Sodium and chloride transport across the epithelia of the intestine and kidney is tightly regulated and plays * To whom correspondence should be addressed. E-mail: [email protected]; [email protected]. † Institute of Physical Chemistry, Friedrich-Schiller-University. ‡ Institute of Physiology II, Universita¨tsklinikum Jena. § Center of Electron Microscopy, Universita¨tsklinikum Jena. | Institute of Organic and Macromolecular Chemistry, Friedrich-SchillerUniversity. (1) Fauci, A. S.; Braunwald, E.; Isselbacher, K. J.; Wilson, J. D.; Martin, J. B.; Kasper, D. L.; Hauser, S. L.; Longo, D. L. Harrison’s Principles of Internal Medicine, 14th ed.; McGraw-Hill: New York, 1998. (2) Hladky, S. B.; Rink, T. J. Body Fluid and Kidney Physiology; Hodder Arnold: London, 1986.

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a major role in maintaining the extracellular fluid volume of our body. Moreover, the composition of fluids secreted by glandular organs is modified by chloride reabsorption or secretion in the glandular ducts.3 Many of these transport processes can be affected by genetic diseases.4 Among them, cystic fibrosis is the most common genetic disease, which is caused by a mutation in the cystic fibrosis transmembrane regulator.5 It affects several epithelial organs, such as the exocrine pancreas, the lungs, the intestine, and the sweat glands. The most serious symptoms are generally observed in the lungs, where the fluid layer covering the airways becomes viscous and impedes mucociliary clearance. This causes obstruction of small airways and promotes infections.1 Other diseases caused by mutations in chloride channels are the autosomal dominant myotonia congenital (Thomsen’s disease) and the autosomal recessive generalized myotonia (Becker’s disease). Also some forms of nephrolithiasis and Bartter’s syndrome are caused by chloride channel mutations.4 To measure the chloride concentration in body fluids can be a first step in establishing the diagnosis. For example, a high chloride concentration in the sweat is an almost pathognomic sign for cystic fibrosis, since other diseases associated with elevated sweat electrolytes are rare. This was recognized a long time ago6,7 and is still in the genomic era an important step toward establishing the diagnosis.8 Apart from this, the measurement of chloride in the blood and urine is useful to assess renal function and acid-base homeostasis.9 To understand the underlying pathomechanisms, however, it would be desirable to determine the chloride concentration not only in body fluids but also in the cytosol and intracellular compartments. In biological samples, chloride can be determined by various techniques.10 Among them ion-selective electrodes are most commonly used.10–12 For intracellular chloride measurements, (3) Boron, W. F.; Boulpaep, E. L. Medical Physiology; Elsevier Saunders: Philadelphia, 2005. (4) Ashcroft, F. M. Ion Channels and Disease; Academic Press: San Diego, 2000. (5) Riordan, J. R. Science 1989, 245, 1066–1073. (6) Di Sant’Agnese, P. A.; Darling, R. C.; Perera, G. A.; Shea, E. Pediatritrics 1953, 12, 549–563. (7) Gibson, L. E.; Cooke, R. E. Pediatrics 1959, 23, 545–599. (8) Mishra, A.; Greaves, R. J. Clin. Biochem. Rev. 2005, 26, 135–153. (9) Koch, S. M.; Taylor, R. W. Crit. Care Med. 1992, 20, 227–240. (10) Geddes, C. D. Meas. Sci. Technol. 2001, 12, R53–R88. (11) Wegmann, D.; Weiss, H.; Ammann, D.; Morf, W. E.; Pretsch, E.; Suggahara, K.; Simon, W. Microchim. Acta 1984, 3, 1–16. (12) Barbour, H. M. Ann. Clin. Biochem. 1991, 28, 150–154. 10.1021/ac800115u CCC: $40.75  2008 American Chemical Society Published on Web 07/29/2008

however, most of these techniques cannot be employed. This gap can be filled by fluorescent techniques, which allow monitoring the concentration of ions in living cells on the stage of a microscope. Fluorescent ion indicators can be easily loaded to cells13 and allow measuring the ion concentration in many cells simultaneously.14 For chloride, quinolinium derivatives have been introduced as indicators, whose fluorescence is quenched by Clby a collisional mechanism.15,16 The relationship between the fluorescence intensity and the concentration of quenching Cl- anions can be described by the Stern-Volmer equation: F0 τ0 ) ) 1 + KSV[Cl-] F τ

(1)

where F0 and τ0 are the fluorescence intensity and lifetime of the fluorophore in absence and F and τ are the fluorescence intensity and lifetime in presence of the quenching Cl- ion, respectively. KSV is the Stern-Volmer quenching constant. Fluorescence intensities, however, depend not only on the concentration of the quenching Cl- ion but also on the concentration of the indicator itself. The indicator concentration inside a cell is not easy to control and may vary during the course of an experiment due to leaching and bleaching. This can considerably hamper any approach to measure intracellular Cl- concentration by simple fluorescence intensity measurements. One way to circumvent this problem is to measure the fluorescence lifetime of the indicator, which shows the same dependence on the concentration of the quencher (see eq 1) but is independent of the indicator concentration. The feasibility of this approach has been nicely shown by Kaneko et al., who measured in this way the chloride concentration in olfactory sensory neurons.17 However, lifetime measurements are technically demanding and require, when performed in the time-domain, pulsed lasers and fast photomultipliers, which are not available in a standard fluorescence microscope setup. The detection of chloride inside cells using (chromo)ionophores was described by Barker and Brasuel et al.18,19 A chlorideselective ionophore together with a pH indicator dye were both embedded inside hydrophobic nanoparticles. The selective extraction of chloride into the nanoparticles led to a coextraction of protons, causing changes in fluorescence. A limitation of these sensor particles is their size of ∼600 nm and their hydrophobicity, which is not compatible with the hydrophilic environment in biological samples and complicates endocytosis. An approach to circumvent the problems inherent to fluorescence intensity measurements is to incorporate the indicator dye and a reference dye, whose fluorescence is not quenched by Clin the matrix of nanosized polymer beads (Figure 1). The fluorescence signal of the indicator dye can then be related to Tsien, R. Y. Nature 1981, 290, 527–528. Tsien, R. Y. Annu. Rev. Neurosci. 1989, 12, 227–253. Verkman, A. S. Am. J. Physiol. 1990, 259, C376–C388. Biwersi, J.; Verkman, A. S. Biochemistry 1991, 30, 7879–7883. Kaneko, H.; Putzier, I.; Frings, S.; Kaupp, U. B.; Gensch, T. J. Neurosci. 2004, 24, 7931–7938. (18) Barker, S. L. R.; Thorsrud, B. A.; Kopelman, R. Anal. Chem. 1998, 70, 100–104. (19) Brasuel, M. G.; Miller, T. J.; Kopelman, R.; Philbert, M. A. Analyst 2003, 128, 1262–1267. (13) (14) (15) (16) (17)

Figure 1. Schematic drawing of a sensor nanoparticle and its interaction with ions and proteins. The white and the black dots represent the indicator dye and the reference dye, respectively. Ions can freely diffuse into the matrix of the nanobeads where they interact with the indicator dye, whereas bigger proteins cannot enter the matrix.

Scheme 1. (a) Structure of the Indicator Dye Lucigenin and (b) of the New Rhodamine-Based Reference Dye (R3)

the fluorescence signal of the reference dye and signal changes that are due to fluctuations or heterogeneities of the nanobead concentration can be eliminated in this way. Moreover, this approach has the advantage that the polymer matrix protects the dyes from proteins, which can interact with the dye and introduce fluorescence changes that bias the determination of the analyte concentration. Vice versa the polymer matrix also protects the cell from potentially harmful dyes. In this study, we used the acridinium-based dye lucigenin (Scheme 1) to measure the Cl- concentration. The fluorescence of lucigenin can be effectively quenched by halides, cyanate, thiocyanate, and amines.20 The Stern-Volmer constant of lucigenin is greater than that of other quinolinium- and acridinium-based dyes.21,22 We expected that lucigenin would confer an equally high chloride sensitivity to our nanosensors, while the polymer matrix would prevent the dye from interacting with cellular components such as proteins or DNA, thereby reducing the toxic effects of lucigenin.23 As reference dye we choose the rhodamine-based dye R3 (Scheme 1), which has a polymerizable function and can be covalently attached to the polymer matrix. At this stage of the study, we did not try to attach polymerizable groups to lucigenin, since previous studies had shown that the efficiency of quenching of bis-acridinium derivatives is diminished by larger substituents at the nitrogen atom.22 The attention of the following experiments is on the critical evaluation of the optical and structural properties of the nanosen(20) Legg, K. D.; Hercules, D. M. J. Phys. Chem. 1970, 74, 2114–2118. (21) Biwersi, J.; Tulk, B.; Verkman, A. S. Anal. Biochem. 1994, 219, 139–143. (22) Huber, C.; Fa¨hnrich, K.; Krause, C.; Werner, T. J. Photochem. Photobiol. A 1999, 128, 111–120. (23) Wu, H. L.; Li, W. Y.; He, X. W.; Miao, K.; Liang, H. Anal. Bioanal. Chem. 2002, 373, 163–168.

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Figure 2. Structural properties of the sensor nanoparticles. (a) Size distribution obtained from DLS of the sensor nanoparticles suspended in phosphate buffer (pH 7.2). (b) Transmission electron micrograph of nanoparticles negatively stained by loaded with uranyl acetate. (c) Micrograph of the sensor negatively stained by obtained from atomic force microscopy (AFM). (d) Height distribution of the AFM micrograph shown in (c).

Figure 3. Fluorescence properties of the sensor nanoparticles. Fluorescence spectra of sensor nanoparticles suspended in phosphate buffer pH 7.2 and their response to increasing concentrations of 0, 1.5, 3.5, 6, 8.4, 10.9, 13.3, and 18.2 mM chloride. Table 1. Experimental Quenching Constants (KSV) and Slope (IR3/Ilucigenin vs [Cl-]) of the Calibrations Plots for the Nanoparticles upon Reaction with Halides and Pseudohalides (Measured in Phosphate Buffer pH 7.2)

chloride bromide isothiocyanate iodide

KSV (M-1)

slope (M-1)

53 89 125 137

80 135 198 214

sors, their cross-reactivity and on the influence of proteins on the fluorescence of the dyes. 6528

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EXPERIMENTAL SECTION Materials. See Supporting Information. Preparation of the Sensor Nanoparticles. Polyacrylamide nanoparticles were prepared by inverse microemulsion polymerization as described by Clark et al.24 A solution of 3.08 g of Brij 30 and 1.59 g (3.6 mmol) of AOT in 43 mL of hexane was purged with nitrogen for 1 h. To this mixture, 2 mL of a solution consisting of 0.4 g (2.6 mmol) of N,N-methylene-bis-acrylamide and 1.85 g (26 mmol) of acrylamide in 4.5 mL of phosphate buffer (pH 7.2) was added slowly under extensive stirring and purging with nitrogen. To the resulting inverse microemulsion, 140 µL of a buffered 1 × 10-6 M solution of lucigenin and 120 µL of a buffered 7.4 × 10-7 M solution of R3 were added. Afterward, the polymerization was started by adding 24 µL of an aqueous APS solution (15%) and 8 µL of TEMED. To finish the polymerization process, the emulsion was stirred at room temperature for 2 h. Then hexane was removed by evaporation, and the cloudy residue suspended in 100 mL of ethanol. This suspension was filtered with a Millipore system using filter membranes with pore size of 20 nm. For removal of unreacted monomers, remaining surfactants, and dyes, the particles were washed with 300 mL of ethanol until the filtrate was colorless. After drying in vacuum, the solid nanoparticles were purified via dialysis of a particle suspension (20 mg/10 mL buffer) against 1 L of phosphate buffer solution (pH 7.2) for 24 h. (24) Clark, H. A.; Hoyer, M.; Philbert, M. A.; Kopelman, R. Anal. Chem. 1999, 71, 4831–4836.

Figure 4. Effect of pH on fluorescence of lucigenin. (a) Lucigenin in solution; (b) lucigenin incorporated in particles. Fluorescence was measured in phosphate buffer at the indicated pH with a potassium chloride concentration of 0 (b) and of 12 mM (O).

Figure 5. Effect of BSA on fluorescence of lucigenin. Lucigenin (9) and rhodamine R3 (0) in solution; lucigenin (b) and rhodamine R3 (O) incorporated into particles. The fluorescence of the solutions and suspensions was excited at a wavelength of 430 nm, to avoid autofluorescence of BSA. All spectra were recorded in a phosphate buffer of pH 7.2.

Figure 6. Confocal micrographs of CHO cells incubated with nanosensors. (a) Fluorescence of lucigenin. (b) Fluorescence of the reference dye R3. (c) Overlap of fluorescence of both dyes. (d) Intensity profile of both dyes along the line indicated in panel c.

Cell Culture and Transfection Procedure. Chinese hamster ovary (CHO) and mouse fibroblasts cells were grown on coverslips and transfected with the nanosensors by using the conventional lipofectamin technique. In brief, 3 mg of ratiometric nanobeads were mixed with 100 µL of EC buffer, vortexed for 1 min, and sonicated for 3 min. Then 12 µL of Effecten and 300 µL of Optimem were added, and the dispersion was again sonicated for 3 min. Cells were superfused with 300 µL of this mixture after

removal of the cell culture medium. After an incubation period of 3 h in a 5% CO2 atmosphere at 37 °C, the nanobead/liposome mixture was removed by extensive washing. For confocal imaging, the coverslips were transferred to a custom-built microscope chamber that was superfused with phosphate-buffered solutions (pH 7.4) containing 9 mM Na2HPO4, 2.1 mM NaH2PO4, 10 mM glucose, 1 mM MgSO4, and potassium chloride in the indicated concentrations. The chloride concentration was complemented with potassium gluconate to yield a constant osmolarity of 300 osmol/L. To ensure equilibration of [Cl-] with the extracellular solution, tributyltin (40 µM, Sigma, St.Louis, MO) and nigericin (10 µM) were added. RESULTS AND DISCUSSION Optical Properties of Indicator and Reference Dye in Solution. In aqueous solution lucigenin has an excitation maximum at 368 nm and an emission maximum at a wavelength of 505 nm. The sulforhodamine R3 has its maximum in excitation at 583 nm and emits at 602 nm (Figure S-1, Supporting Information). The fluorescence intensity of lucigenin in solution is reversibly quenched by chloride. As described above, the dynamic quenching process can be described by a collisional mechanism and follows Stern-Volmer kinetics. In a 67 mM phosphate buffer (pH 7.2),we determined a Stern-Volmer constant (KSV) of 250 M-1. Structural Properties of the Sensor Nanoparticles. The nanoparticles prepared via microemulsion polymerization show a bimodal size distribution in aqueous suspension, consisting of a major part with a hydrodynamic diameter of 24 nm and a minor fraction with a diameter of 200 nm (Figure 2a). The resulting broadness of the distribution is indicated by a polydispersity index of 0.32. The size distribution of the nanoparticles is supported by the results obtained by transmission electron microscopy (Figure 2b) and atomic force microscopy (Figure 2c,d). The latter methods, however, have the disadvantage that the particles are not imaged in their native, wet state. Occasionally, aggregates are formed upon drying, and the particles appear to be flattened with a larger diameter and a smaller height than in the wet state. Thus, the size distribution obtained by the imaging methods does not entirely reflect the size distribution obtained by dynamic light scattering (DLS), especially for the larger diameters. Optical Properties of the Nanoparticles. The excitation and emission spectra of lucigenin and the dye R3 are only slightly changed, when incorporated into the polyacrylamide nanoparticles. The Stern-Volmer constant for quenching of lucigenin by chloride inside the particles was found to be 53 M-1. This decrease of the Analytical Chemistry, Vol. 80, No. 17, September 1, 2008

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Figure 7. Intracellular chloride calibration. (a) Overlay of the transmission and lucigenin and rhodamine fluorescence images obtained from mouse fibroblasts incubated with the ratiometric nanosensors. (b) Time course of lucigenin (orange circles) and rhodamine (red circles) fluorescence in the cytosol of the cell marked in panel a during superfusion with phosphate buffers containing chloride in the indicated concentrations. Cells were permeabilized with tributyltin and nigericin.

quenching rate in the particles compared to the rate in solution can be explained by the immobilization of lucigenin in the polymer matrix. In the polymer, the access to the dye is hindered, which leads to a lower probability of encounters between dye and quencher and thus to smaller quenching constants.25 Another reason for lower chloride quenching rates might be that a Donnan potential builds up between the polymer and the surrounding media which impedes anions such as chloride from entering into the matrix. By plotting the ratio of fluorescence intensities against the chloride concentration the sensitivity of the particles can be determined. Figure 3 shows the fluorescence spectra of the sensor particles in phosphate buffer with chloride concentrations ranging from 0 to 18.2 mM. In case of chloride as quencher ion, the slope of the calibration curve is 79.7 ± 3.5 M-1 (intercept 1.67 ± 0.03, R ) 0.994, standard deviation 0.058, N ) 8). Lucigenin fluorescence can also be quenched by other halide ions and pseudohalides.16,20,22 The efficiency of the quenching process increases with a lower ionization potential of the quencher ion, and as a result, the sensitivity of the nanoparticles to bromide, iodide, and thiocyanate is higher than for chloride (Table 1). This, however, does not impede the determination of chloride in biological tissues, since the concentration of these ions is by far too low to have a significant impact on the quenching of lucigenine.10 Cross-Reactivity of the Nanosensors. The pH of living tissues can vary during the course of an experiment. This change in pH can also bias the fluorescence signal of lucigenin: In the absence of chloride, the fluorescence of lucigenin in solution decreases by ∼33%, if the pH of the buffer solution increases from 4.9 to 8.1. In contrast, the fluorescence does not change significantly if lucigenin is dissolved in a 12 mM potassium chloride solution, because the quenching constant for chloride in solution is higher than that for hydroxyl ions (Figure 4a). If lucigenin is incorporated into the sensor particles, fluorescence decreases by ∼58% when pH is varied from 4.9 to 8.1. In the presence of chloride, the fluorescence intensity decreases by the same amount (Figure 4b). In other words, the Stern-Volmer constant for hydroxyl ions is higher if lucigenin is incorporated into the polymer matrix. In the polymer matrix, the impact of hydroxyl (25) Huber, C.; Krause, C.; Werner, T.; Wolfbeis, O. S. Microchim. Acta 2003, 142, 245–253.

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ions on lucigenin fluorescence is only slightly reduced by the presence of chloride ions. This can be explained by the fact that hydroxyl ions can better enter the hydrophilic polymer matrix and get better access to lucigenin than chloride, due to the formation of hydrogen bonds within the network of water molecules inside the polymer bead. Thus, in the polymer matrix, quenching by hydroxyl ions is almost not diminished and dominates over the reduced quenching of chloride, whereas in solution quenching by chloride is by far more effective. Phosphate, which was used in this study as buffer, did not have any significant effect on lucigenin fluorescence in concentrations ranging from 0 to 22 mM. The same applies to sulfate, which was tested in concentrations ranging from 0 to 14 mM sulfate. Influence of BSA on Nanosensor Fluorescence. The spectral properties of fluorescence dyes can be changed by the presence of proteins, which can form aggregates with dyes.26 This interaction can seriously influence measurements with ion indicators in biological tissues where proteins are omnipresent. Figure 5 shows that free lucigenin and free rhodamine R3 fluorescence are considerably quenched by bovine serum albumin (BSA). Especially the fluorescence intensity of lucigenin decreases by ∼70% at a BSA concentration of 5%. However, when the same amount of BSA was added to a nanoparticle suspension, the fluorescence intensity of the protected dyes decreased only slightly. Absorption of the excitation beam by BSA and scattering of the emitted fluorescence by BSA contribute to this decrease. Thus, the dyes might be even better shielded by the polymer matrix than it appears from this plot. Application of the Nanosensors in Living Cells. To test the nanosensors in living cells, we incorporated them into CHO cells and mouse fibroblasts by using the conventional lipofectamin technique, which is usually used to deliver DNA to cells (see Supporting Information). By adding the liposome-nanosensor solution to the cell culture, a large number of cells could be efficiently loaded with the nanosensors. A confocal cross section of CHO cells incubated with the nanosensors shows that the nanosensors are distributed in the cytosol leaving the nucleus blank (Figure 6). The intensity profile along the line in panel c shows that both dyes are equally distributed in the cytosol. To assess the response of the nanoparticles in living cells, we superfused loaded cells subsequently with solutions containing (26) Aylott, J. W. Analyst 2003, 128, 309–312.

10 and 50 mM chloride. Intracellular [Cl-] was set equal to extracellular [Cl-] by using tributyltin and the ionophore nigericin. Tributyltin acts as a Cl-/OH- antiporter, which exchanges Clfor OH- ions.27 To prevent a shift of the intracellular pH, which would have been invariably caused by the Cl-/OH- exchange, the K+/H+ antiporter nigericin was added, which clamped the intracellular pH to the extracellular pH. Figure 7 shows the time course of the lucigenin and rhodamine R3 fluorescence during such an experiment. In this experiment, the cell shifts only slightly so that the reference signal is almost constant. As expected, lucigenin fluorescence decreases at higher Cl- concentrations and reaches the original value after an exchange of the superfusing solution to the initial Cl- concentration. However, for this type of experiment, special care must be taken to prevent phototoxic damage of the cells. Images had to be acquired at a high scan speed (2.6 µs/pixel, 1.6 s/image) and a low repetition rate (1 image/min). If light exposure was increased, cells were damaged and membrane blebs could be observed. CONCLUSIONS Ratiometric fluorescent nanosensors for chloride were prepared by inverse microemulsion polymerization and characterized in vitro and in vivo. In vitro, the particles show cross-reactivity to other halide ions. But, because of their low concentrations in biological samples, this effect can be neglected in biological measurements. The nanosensors are also sensitive to changes in pH. This might play a role in experiments where intracellular pH (27) Krapf, R.; Berry, C. A.; Verkman, A. S. Biophys. J. 1988, 53, 955–962.

changes. Here, the pH has to be monitored and taken into account for the determination of the chloride concentration. Sulfate and phosphate have only little effect on lucigenin fluorescence. Thus, phosphate is an ideal buffer for experiments with lucigenin. Lucigenin fluorescence can be also quenched by proteins. This can be effectively prevented by incorporating the dye into the polymer matrix of nanobeads. This approach has the additional benefit that a reference dye can be added to the polymer matrix. The fluorescence signal of this dye can then be used to correct for fluctuations in the concentration of the nanobeads. ACKNOWLEDGMENT We thank Jochen Schmidt, Institute of Physical Chemistry Friedrich-Schiller-University Jena, for performing the BET-surface measurements. This work was supported by the Heisenberg Fellowship MO 1062/1-2, the research grant MO 1062/2-1 of Deutsche Forschungsgemeinschaft and the European Union project “Sensor Nanoparticles for Ions and Biomolecules” (MTKDCT-2005-029554). This support is most gratefully acknowledged. A.G. and S.E.S. contributed equally to this paper. SUPPORTING INFORMATION AVAILABLE Supplementary figures, synthesis of the reference dye, and experimental details. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review January 16, 2008. Accepted June 17, 2008. AC800115U

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