Development of an Optical Nanosensor Incorporating a pH-Sensitive

Letter pubs.acs.org/ac

Development of an Optical Nanosensor Incorporating a pH-Sensitive Quencher Dye for Potassium Imaging Ali Sahari,† Timothy T. Ruckh,† Richard Hutchings,‡ and Heather A. Clark*,† †

Department of Pharmaceutical Sciences, Northeastern University, Boston, Massachusetts 02115, United States Berry and Associates, Inc., Dexter, Michigan 48130, United States

‡

S Supporting Information *

ABSTRACT: One of the key challenges in the design of a sensor for measuring extracellular changes in potassium concentration is selectivity against the competing cation, sodium. Here, we present an optode-based nanosensor selective to potassium ions, owing to the addition of a pH-sensitive quencher molecule paired with a static fluorophore. The nanosensor was fabricated using emulsification and characterized in solution by absorbance and fluorescence spectroscopy. The resulting nanosensor detected potassium with nearly 1 order of magnitude higher selectivity compared to our chromoionophore-based optode nanosensors. In addition to the improved selectivity, the nanosensor has the following properties required for measurements in a biological environment: (1) a physiologically relevant dynamic range, (2) response to potassium ions at a physiological ionic strength, and (3) response to serum potassium in the presence of fouling biological components. The potassium nanosensor described in this study is envisioned to have application in cellular imaging and drug screening.

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sensitivity, wavelength, and selectivity. Nano-optode sensors have been shown to image sodium and potassium changes in cellular environments and in vivo.6,23,24 Top-down nanoemulsion methods16,25 provide a fast and easy synthesis compared to the earlier fabrication methods that created nanoscale sensors by first polymerizing nanospheres and then introducing sensor materials postfabrication.26,27 The disadvantage of using top-down methods is the difficulty in maintaining the ratio of components during fabrication, which can result in reduced performance. Our previous work on producing sodium-selective nanosensors demonstrated reduced selectivity in the presence of potassium5 compared to other sodium optodes,21,28,29 but the poorer selectivity was still adequate for use in cellular measurements.23 Unfortunately, our initial attempts at achieving adequate selectivity for potassium measurements with chromoionophore-based sensors fell short (Figure S1) compared to the results achieved with alternative fabrication methods.26,27 In addition, as the dynamic range of the sensor for potassium measurements was tuned to the extracellular range, it is possible to exacerbate the selectivity problem further, potentially leading to inaccurate cellular measurements. The goal of this work is to refine the topdown nanoemulsion-based ion-selective optode formulation to

maging ions in a cellular environment became a reality with the invention of fluorescent calcium imaging indicators.1 The field expanded quickly as new ion indicators, including fluo-4 and fluo-5N, overcame the key challenges of short wavelength, weak emission, and nanomolar detection range.2,3 However, the array of fluorescent indicators for other ions, such as potassium and sodium, has been less expansive. There are commercially available fluorescent indicators for potassium, such as PBFI,4 but overall the choices are limited.5,6 The difficulty in synthesizing selective potassium indicators arises from the background levels of sodium,4,7,8 where the resting level of extracellular potassium is only 3.5−5.5 mM and the resting level of extracellular sodium is 135−145 mM. Thus, a fluorescent sensor that can detect extracellular changes in potassium concentration with selectivity against background cations is needed for cellular imaging and physiological monitoring. One solution to cellular imaging was the invention of nanoparticle sensors for ion detection.9−15 Nanoscale optodes, the equivalent of ion-selective electrodes, have shown potential to overcome the issues experienced by molecular indicators for measuring ions in a biological environment.16−22 One of the distinct advantages of these sensors is the modularity of the sensing components. Unlike molecular indicators, the recognition modality and the optical reporter are separate moieties that are confined within the boundaries of the plasticized polymer matrix. This modularity enables the easy exchange of components to tune the sensor response for dynamic range, © XXXX American Chemical Society

Received: August 11, 2015 Accepted: October 7, 2015

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DOI: 10.1021/acs.analchem.5b03080 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry

Figure 1. (A) pH-based mechanism of the potassium nanosensor and (B) molecular structure of blueberry-C6-ester-652 quencher dye.

intensity). The nanosensor solution was finally filtered with a 0.8 μm syringe filter to remove excess polymer. The hydrodynamic diameter of the fabricated nanoparticles was measured by dynamic light scattering (DLS) using a 90 Plus particle size analyzer (Brookhaven Instruments Corp., Holtsville, NY) by 1:10 dilution of the nanosensors in 10 mM HEPES buffer (pH 7.4). The effective diameter of the nanoparticles measured by the detector was 128 nm. The zeta potential of the nanoparticles was measured to be −47.25 ± 2.24 mV using a zeta potential analyzer (Brookhaven Instruments Corp.). Calibration solutions of 1 μM−1 M potassium chloride (KCl) were prepared in 10 mM HEPES buffer (pH 7.4) in order to characterize the response of the fabricated nanosensors to potassium. A plate reader was used to read the emitted fluorescent of the nanosensors (excitation, 650 nm; emission, 675 nm) in the KCl solutions as well as in 0.1 M HCl and 0.1 M NaOH solutions for fully protonated and deprotonated end point states, respectively. The calibration curves representing the nanosensor response in the presence of different concentrations of potassium were provided by normalizing the data with respect to the fully protonated/deprotonated states and plotting the fluorescent intensities against the log of the analyte concentration (Figure 2). The nanosensor

produce a selective nanosensor for the detection of potassium flux into the extracellular space. The general formulation of ion-selective optodes contains a matrix of plasticized polymer, a pH-responsive chromoionophore, an optically inactive ionophore, and a hydrophobic charge-carrying molecule to facilitate ion exchange within the core of the nanosensor.17,18,30 For example, in the potassiumdetecting nanosensor, potassium ionophore selectively carries potassium ions into the core leading to deprotonation of the chromoionophore in order to maintain charge neutrality in the core. This process results in a change in the fluorescent characteristics of the chromoionophore which can be quantified by a fluorometer. Quencher molecules have been previously reported use in making ion-selective sensors.15,31,32 In this work, the formulation of potassium-specific optodes was modified with the substitution of blueberry-C6-ester-652 quencher dye (BLU 00652), synthesized by Berry and Associates (Dexter, MI), for the commonly used chromoionophore III (CH III). In addition, a static fluorophore was paired with the quencher molecule to produce a fluorescent signal from the nanosensor construct.33 As the potassium ions are extracted, the absorbance of the quencher changes leading to a change in the FRET between the quencher and the fluorophore. The sensor mechanism is illustrated in Figure 1A. The molecular structure of the quencher molecule, characterized by 1H NMR, is shown in Figure 1B. The absorbance of the quencher dye at the wavelength of 665 nm was plotted against a range of concentrations of the quencher dye dissolved in CH3CN (pH 9.5) (Figure S2A) and the extinction coefficient was measured to be 25440 M−1 cm−1 in a 1 cm cell. The quencher dye was taken through a pH titration in 1:1 CH3CN/buffer, and the pKa was determined to be 7.85 at 665 nm (Figure S2B). We fabricated the potassium-sensitive nanosensors using the nanoemulsion method we previously established.34 First, an optode was formulated by mixing 30 mg of poly(vinyl chloride), 66 μL of bis(2-ethylhexyl) sebacate, 12.5 mg of potassium ionophore III, 4 mg of blueberry-C6-ester-652, 1 mg of sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (NaTFPB), and 0.5 mg of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD) in 500 μL of THF. Next, in order to fabricate the particle nanosensors, 1.25 mg of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-550] (ammonium salt) (DSPE-PEG) was dried in a scintillation vial and then resuspended in 4 mL of 10 mM HEPES buffer (pH 7.4) with a probe tip sonicator for 30 s at 20% intensity. A volume of 50 μL of the optode solution was diluted with 75 μL of dichloromethane, and the mixture was added to the PEG-lipid solution while under probe tip sonication (3 min, 20%

Figure 2. Characterization of the potassium nanosensors in two separate solutions of KCl and NaCl exhibits selectivity of −2.2 immediately after sensor fabrication. Data represented as mean values with error bars for the standard deviations.

responded dynamically to changes in potassium concentration by an 83% change between 1 μM and 100 mM KCl where the lower and upper levels of detection were 0.1 and 180.0 mM, respectively. Nanosensors were also sensitive to physiologically relevant changes; the fluorescence changed by nearly 28% between 1 and 8 mM potassium with the concentration of 3.8 ± 0.2 mM at half protonation of the quencher molecule. In order to characterize the sensor selectivity for potassium over the primary interfering ion, sodium, nanosensor response to B

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Analytical Chemistry sodium was measured in the presence of 10 mM−2.5 M sodium chloride (NaCl) and the calibration curve was generated (Figure 2). Nanosensors responded to sodium concentration of 575.8 ± 26.4 mM at half protonation. The lower and upper levels of detection of sodium were 95.0 mM and 3492.6 mM, respectively, indicating that the nanosensor was minimally responsive to sodium with normal physiologicalrelevant concentrations (i.e., 135−145 mM). The NicolskiiEisenman equation17 was used to calculate the selectivity coefficient for potassium over sodium. The nanosensor containing the blueberry-C6-ester-652 quencher dye measured potassium ion concentrations with the selectivity coefficient of −2.2 over the interfering sodium ions to overcome the relatively low selectivity of our regular chromoionophorebased potassium nanosensors (−1.4, see Figure S1). The absorbance spectra and peaks of the nanosensor response at different concentrations of potassium and sodium were also measured and plotted in Figures S3 and S4, respectively, which further corroborates the selective response of the nanosensors to potassium. To demonstrate the shelf life of the nanosensors, four batches of nanoparticles were formulated and mixed together. The fluorescent response to potassium was evaluated on days 0, 1, 4, and 7, as described previously, while stored at room temperature and protected from exposure to ambient light. The calibration curves demonstrating the stability of the nanosensor response are illustrated in Figure 3. The potassium concen-

Figure 4. Characterization of the effect of ionic strength on nanosensor response.

potentially fouling components. First, the off-the-clot serum solution was diluted at the ratio of 4:1 serum:water, adjusted to pH 7.4, and spiked with 0, 1, 2, 3, and 4 mM potassium and/or 5, 10, 20, and 30 mM sodium. Potassium and sodium concentrations as well as pH of the serum solutions were determined using an i-STAT portable clinical analyzer. The nanosensors were fabricated as described above and concentrated 20× by centrifugation. The nanosensor response was measured in both buffered calibration solutions and serum solutions. The result of Clarke Error Grid analysis was illustrated in Figure 5, which shows that the nanosensors are

Figure 5. Clarke Error Grid Analysis of the potassium nanosensor in diluted and spiked serum solutions. Potassium and sodium concentrations are (K+/ Na+): (1) 3.2/109, pH 7.36; (2) 4.2/108, pH 7.35; (3) 5.3/108, pH 7.43; (4) 6.2/108, pH 7.39; (5) 7.3/108, pH 7.38; (6) 3.2/113, pH 7.36; (7) 3.2/119, pH 7.37; (8) 3.1/130, pH 7.38; (9) 3.2/137, pH 7.37; (10) 5.1/120, pH 7.49; (11) 7.1/139, pH 7.50.

Figure 3. Calibration curves representing the time-stability of the nanosensor response.

tration at half protonation changed to 3.7 ± 0.2, 4.2 ± 0.1, and 5.3 ± 0.3 after 1, 4, and 7 days, respectively. Although the ANOVA statistical model determined that the effect of time was significant (p-value < 0.01), the sensor detects the desired physiological range. To evaluate the effect of ionic strength on the nanosensor response, KCl was complemented with lithium chloride (LiCl) to yield a constant ionic strength (i.e., [K+] + [Li+] = 145 mM). 35 Thus, calibration solutions of KCl with the concentrations described earlier were prepared, with background lithium ions. The resulting calibration curve is shown in Figure 4. The nanosensors were responsive to potassium ions in a constant ionic strength condition with a slight shift in the degree of protonation for the quencher molecule which resulted in the concentration of 1.9 ± 0.1 mM at half protonation, implying the effect of ionic strength was not significant in detection of potassium. Finally, we compared the analytical parameters achieved in buffered calibration solution to those in diluted and spiked human serum, which is composed of a complex matrix with

responsive to potassium in serum solutions, with the best accuracy in the physiological range. Of note, similar to the optodes based on chromoionophores, the sensor response is dependent on the sample pH. In the normal biological range, the nanosensor based on the blueberry-C6-ester-652 dye showed insignificant changes in response to pH changes (Figure S5). This behavior does not contradict the demonstrated pH-dependent mechanism of the nanosensor because the pKa of the nanosensor is outside the physiological pH. This is very similar to the pKa observed for CH III in an optode, where the reported pKa is >10.36,37 In summary, we fabricated an optical nanosensor for detection of potassium ions substituting the commonly used chromoionophores with a pH-sensitive quencher molecule. The resulting nanosensor exhibited an improved selectivity to potassium in the presence of the primary interfering ion, sodium. This will help overcome the challenge of having poor selectivity associated with the currently existing molecular C

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(11) Fernández-Argüelles, M. T.; Yakovlev, A.; Sperling, R. A.; Luccardini, C.; Gaillard, S.; Sanz Medel, A.; Mallet, J.-M.; Brochon, J.C.; Feltz, A.; Oheim, M. Nano Lett. 2007, 7, 2613−2617. (12) Clark, H. A.; Hoyer, M.; Philbert, M. A.; Kopelman, R. Anal. Chem. 1999, 71, 4831−4836. (13) Clark, H. A.; Kopelman, R.; Tjalkens, R.; Philbert, M. A. Anal. Chem. 1999, 71, 4837−4843. (14) Xie, X.; Bakker, E. ACS Appl. Mater. Interfaces 2014, 6, 2666− 2670. (15) Wolfbeis, O. S. Chem. Soc. Rev. 2015, 44, 4743. (16) Dubach, J. M.; Harjes, D. I.; Clark, H. A. Nano Lett. 2007, 7, 1827−1831. (17) Bakker, E.; Bühlmann, P.; Pretsch, E. Chem. Rev. 1997, 97, 3083−3132. (18) Bühlmann, P.; Pretsch, E.; Bakker, E. Chem. Rev. 1998, 98, 1593−1688. (19) Si, D.; Epstein, T.; Koo Lee, Y.-E.; Kopelman, R. Anal. Chem. 2012, 84, 978−986. (20) Xie, X.; Mistlberger, G. n.; Bakker, E. Anal. Chem. 2013, 85, 9932−9938. (21) Shortreed, M.; Bakker, E.; Kopelman, R. Anal. Chem. 1996, 68, 2656−2662. (22) Dubach, J. M.; Balaconis, M. K.; Clark, H. A. J. Visualized Exp. 2011, DOI: 10.3791/2896. (23) Dubach, J. M.; Das, S.; Rosenzweig, A.; Clark, H. A. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 16145−16150. (24) Dubach, J. M.; Lim, E.; Zhang, N.; Francis, K. P.; Clark, H. J. Integr. Biol. 2011, 3, 142−148. (25) Xie, X.; Bakker, E. Anal. Bioanal. Chem. 2015, 407, 3899−3910. (26) Ruedas-Rama, M. J.; Hall, E. A. Analyst 2006, 131, 1282−1291. (27) Brasuel, M.; Kopelman, R.; Miller, T. J.; Tjalkens, R.; Philbert, M. A. Anal. Chem. 2001, 73, 2221−2228. (28) Brasuel, M.; Kopelman, R.; Kasman, L.; Miller, T. J.; Philbert, M. In Sensors, 2002, Proceedings of IEEE; IEEE: New York, 2002; pp 288−292. (29) Buck, S. M.; Xu, H.; Brasuel, M.; Philbert, M. A.; Kopelman, R. Talanta 2004, 63, 41−59. (30) Bakker, E.; Simon, W. Anal. Chem. 1992, 64, 1805−1812. (31) Huber, C.; Krause, C.; Werner, T.; Wolfbeis, O. S. Microchim. Acta 2003, 142, 245−253. (32) Oehme, I.; Wolfbeis, O. S. Microchim. Acta 1997, 126, 177−192. (33) Krause, C.; Werner, T.; Huber, C.; Klimant, I.; Wolfbeis, O. S. Anal. Chem. 1998, 70, 3983−3985. (34) Cash, K. J.; Clark, H. A. Trends Mol. Med. 2010, 16, 584−593. (35) Dietrich, S.; Stanca, S. E.; Cranfield, C. G.; Hoffmann, B.; Benndorf, K.; Biskup, C. In BiOS; International Society for Optics and Photonics (SPIE): Bellingham WA, 2010; pp 756914−756919. (36) Bakker, E.; Lerchi, M.; Rosatzin, T.; Rusterholz, B.; Simon, W. Anal. Chim. Acta 1993, 278, 211−225. (37) Lindner, E.; Rosatzin, T.; Jeney, J.; Cosofret, V. V.; Simon, W.; Buck, R. P. J. Electroanal. Chem. 1993, 352, 309−312.

indicators for potassium detection at the extracellular levels of 3.5−5.5 mM in the presence of interfering sodium ions. Moreover, the potassium nanosensor developed in this study is extending the field of ion-selective optodes that are made using top-down nanoemulsions detecting potassium with nearly 1 order of magnitude higher selectivity than achieved through the use of a single chromoionophore. Also, the nanosensor is brighter owing to the incorporated static fluorophore and does not require external “activation” for measurement while in equilibrium with the environment, advantageous for dynamic cellular measurements. We also showed the effect of ionic strength on the nanosensor response is minimal and the nanosensor is responsive to potassium in human serum. In future studies, this nanosensor will be modified to enable ratiometric measurements which is a critical step toward cellular studies. Incorporating a reference dye, the ratiometric sensor will have an advantage for extracellular imaging in that the ratio of the two fluorescence intensities can provide a built-in correction factor for sensor concentration, photobleaching, and environmental changes.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b03080. Calibration curve of a sample regular chromoionophorebased nanosensor, extinction coefficient and pH titration of the quencher molecule, absorbance spectra of the nanosensor, and nanosensor response to change in pH (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +1 (617) 373 3091. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to acknowledge Christopher Skipwith for insightful discussions. This project was supported by the National Institutes of Health (Grant 5R01NS081641).

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REFERENCES

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DOI: 10.1021/acs.analchem.5b03080 Anal. Chem. XXXX, XXX, XXX−XXX