196 CHIMIA 2015, 69, No. 4 Laureates: Junior Prizes, SCS Fall Meeting 2014 doi:10.2533/chimia.2015.196 Chimia 69 (2015) 196–198 © Schweizerische Chemische Gesellschaft Potassium Sensitive Optical Nanosensors Containing Voltage Sensitive Dyes

Xiaojiang Xie§a, Agustín Gutiérreza, Valentin Trofimovb, Istvan Szilagyia, Thierry Soldatib, and Eric Bakker*a

§SCS-Metrohm Award for best oral presentation

Abstract: Ionophore-based ion-selective optical nanosensors have been explored for a number of years. Voltage sensitive dyes (VSDs) have been introduced into this type of sensors only very recently, forming a new class of analytical tools. Here, K+-sensitive nanospheres incorporating a lipophilic VSD were successfully fabricated and characterized. The nanosensors were readily delivered into the social amoeba Dictyostelium discoideum in a non-invasive manner, forming a promising new platform for intracellular ion quantification and imaging. Keywords: Nanosphere · Optical sensor · Potential · Valinomycin · Voltage sensitive dye

1. Introduction tion mode that can overcome the pH cross- times of several microseconds. However, response in a specific pH window.[11,12] the relative signal change in absorbance Established nanoscale ionophore- However, an exhaustive detection mode or fluorescence is much smaller, render- based optical ion-selective sensors contain will always cause a dramatic perturbation ing them not sufficiently sensitive to ion several major components that include of the sample and is, unfortunately, not a imaging applications. This work focuses a chromoionophore (a hydrophobic pH universal sensing scheme that could be ap- therefore on redistributing solvatochromic indicator), a lipophilic ion exchanger, an plied in biological imaging. dyes to design ion imaging reagents. ionophore (ion receptor), a matrix material The root cause of the pH cross-re- and a surfactant to form and stabilize the sponse is in the chromoionophore itself structure.[1–3] because it is a receptor for hydrogen ions, 2. Method and Materials Similar to so-called bulk optodes, a making H+ inevitably the reference ion. classical cation nanosensor functions on Eliminating the reference ion H+ should 2.1 Materials the basis of ion exchange between hydro- potentially help to overcome the pH depen- Pluronic® F-127 (F127), bis(2-ethyl- gen ions and the cationic analyte, and for dence of this type of sensor. Recently, we hexyl) sebacate (DOS), tetrahydrofuran an anion-responsive sensor, on the coex- have demonstrated that voltage-sensitive (THF), methanol, potassium tetrakis- traction of hydrogen ions and the anionic dyes (VSDs) can be applied to ionophore- [3,5-bis(trifluoromethyl)phenyl]borate analyte.[4–6] In this type of sensors, the based ion-selective nanospheres and result (KTFPB), 3,3'-dioctyldecyloxacarbocya- chromoionophore indicates the level of the in sensor responses that are indeed pH in- nine perchlorate (VSD) and reference dye hydrogen ions in the organic sensing ma- dependent.[13] Lumogen Red were obtained from Sigma- terial and thereby indirectly quantifies the VSDs have been useful optical indica- Aldrich. Cell culture media (HL5-C medi- analyte in the aqueous sample.[7–10] tors for membrane potential measurements um including glucose supplemented with While abundant research work has been in cells and organelles that are too small vitamins and micro-elements, and LoFlo dedicated to the fundamental understanding for electrodes.[14] VSDs are generally cat- medium) were obtained from ForMedium. and application of the chromoionophore- egorized by their response to the change in All solutions were prepared by dissolv- containing ion-selective sensors, their pH local electric field, and one distinguishes ing appropriate salts into deionized water cross-response always remained a key fast-response and slow-response probes.­[15] (Mili-Q). All salts used were analytical drawback. Recently, it was demonstrated Most slow-response VSDs exhibit polar- grade or better. that one can operate nanoscale sensor sus- ity-dependent optical characteristics. The pensions in a so-called exhaustive detec- absorption and/or emission spectra in polar 2.2 Nanosensor Preparation and nonpolar solvents can be very different, The K+-selective nanospheres were making these dyes solvatochromic. As the prepared by dissolving 0.3 mg of KTFPB, electric field changes, the slow-response 0.01 mg of VSD, 0.005 mg of Lumogen VSDs will redistribute between the aque- red, 8 mg of DOS, 4.5 mg of F127, and 1.2 ous and the organic phases to adapt to the mg of valinomycin in 3.0 mL of methanol membrane potential change. Because of to form a homogeneous solution. 0.2 mL the repartition kinetics, the response times of this solution was pipetted and injected are often relatively slow, typically hun- into 5 mL of deionized water (or cell cul- *Correspondence: Prof. Dr. E. Bakkera dreds of milliseconds. The fast-response ture) on a vortex with a spinning speed of E-mail: [email protected] VSDs behave mainly on the basis of elec- 1000 rpm. The resulting clear mixture was aDepartment of Inorganic and Analytical Chemistry trochromism (the Stark effect) and reori- blown with compressed air on the surface bDepartment of Biochemistry University of Geneva entation, regardless of any redistribution, for 30 min to remove methanol, giving a Quai Ernest-Ansermet 30, CH-1211 Geneva and thus are capable of achieving response clear particle suspension. Laureates: Junior Prizes, SCS Fall Meeting 2014 CHIMIA 2015, 69, No. 4 197

2.3 Instrumentation and Scheme 1. Represen­ Measurement tative illustration for The size of the nanospheres was mea- the VSD distribution + sured by dynamic light scattering (DLS) in the K nanosphere with a Zetasizer Nano ZS (Malvern Inc.) according to the phase boundary po- instrument. Fluorescence responses of the tential difference (∆Φ) nanospheres were measured with a fluores- cence spectrometer (Fluorolog3, Horiba Jobin Yvon) using disposable poly(methyl methacrylate) cuvettes with path length of 1 cm as sample container. The excita- tion wavelength was 480 nm. The desired analyte concentration in the nanosphere suspension was achieved by addition of ernedao+ () byrg the phase boundary potential which is close to the 488 nm laser used calculated volumes of stock solutions or K amounts of solid. difference ( ao),+ ()whichrg can be related to for fluorescence microscopy. An emission ∆ΦaoK + ()rg For transmission electron microscopic the analyte activityK (K+ in this case) in the peak around 595 nm that originated from (TEM) imaging of the nanospheres, the sampleaa+ao() qaccording()rg to Eqn. (1),symb where a R is the reference dye Lumogen red was also K aoK + ()rg K aa()q + suspension was dispersed onto a Formvar/ the gas constant,aa+ T() isq the absolute tempersymb- observed. a ∆Φ increases as the K concen- K + symb a Carbon film-coated TEM grid, counter- ature, F is Faraday’s constant, ao+ ()rg tration in the sample increases, forcing K + stained with uranyl acetate, dried in the and aa+ ()q are symbthe activity b of uncom- the VSDs to accumulate in the more polar aa0,aqK→+ org()q symb a ™ K + plexedΔΦ + K in the nanosphere symband in bthe aqueous region, and resulting in a decrease air and visualized using a FEI Tecnai G2 K 0,aq→ org symb b 0,aq→ org symb c Sphera transmission electron microscope. sample, and ΔΦ + is the standardaa+ () qion in the emissionsymb intensity a of the VSDs. ΔΦ K+ K symb c For confocal imaging, cells of the transfer potentialK for Ksymb+. b symb cCompared with previously reported nano- 0,aq→ org AX2(Ka) strain of Dictyostelium discoide- ΔΦ + spheres with more hydrophilic voltage ΔΦ K+ um expressing the ABD-GFP protein (con- K symb c sensitivesymb dye, b the relative intensity change 0,aq→ org 0,aq→ org RT aa+ ()ΔΦq + was smaller, i.e. the detection limit for this sisting of an actin binding domain fused K ΔΦ K+ ΔΦ = ΔΦ + + ln K (1) (1) symb c K 0,aq→ org RT aa+ ()q to a green fluorescent protein) were grown 0,aq→ org RT aaK + ()systemq was higher. This is likely caused by ΔΦ = ΔΦFa+ + ()org ln K (1) [16] ΔΦ = ΔΦ K + K+ ln (1) in HL5-C. Cells were detached, cen- K the higher lipophilicity of the VSD used in Fa+ ()org RT aaFa+ ()q K + ()org trifuged twice and resuspended in LoFlo, 0,aq→ org RT K K this work, which results in a significantly ΔΦ = ΔΦ + + ln (1) then plated on glass-bottom cell culture Note that theK same ∆Φ should also different standard ion transfer potential for Fa+ ()org Fa+ ()org RT aa+ ()q dishes. To these, 2 mL of either LoFlo or ­apply to VSDs, therefore, as ∆ΦK chang- 0,aq→the org VSD.RT K ΔΦ = ΔΦ + + ln (1) the potassium nanospheres suspended in es, so does the distribution of the VSD. K Fa+ ()org LoFlo were added. The particle-containing Fig. 1 shows the emission spectra of the 3.2 Choice ofK +Materials medium was replaced with LoFlo after ap- K+ selective nanosphere suspension with Since the fluorescence of the VSD proximately 20 minutes to remove unin- various KCl levels in the sample, covering depends on the polarity of the micro- gested extracellular particles. Image acqui- the normal intracellular K+ levels. Owing environment, the nanosphere materials sition was carried out with a laser confocal to the excellent selectivity of valinomycin choice becomes very important. Here, the microscope (Zeiss LSM780). to K+, the nanospheres gave no response to nanospheres were composed of a hydro- other common cations such as Na+, Mg2+, phobic nonpolar compound DOS and an Li+ and Ca2+ in this concentration window. amphiphilic block copolymer F127. The 3. Results and Discussion The excitation was chosen at 480 nm, nanospheres exhibited a long shelf-life of

3.1 Sensor Mechanism Fig. 1. Fluorescence For an established K+-selective ion spectra for the K+ optode containing a neutral chromoiono- 50 1.2 nanosphere with phore, ion exchanger (TFPB), and K+ iono- 45 different KCl back- phore, the response mechanism is based on 1 ground. Excitation: the partition equilibrium between H+ and 40 480 nm. Inset:

.u. 0.8 K+ at the aqueous-organic interface. Here, Calibration using the 0 normalized emission /a the VSDs doped ion sensors function on 35 I/I 0.6 intensity at 510 nm. I a similar but somewhat different basis. As 0 30 0.4 is the intensity with- shown in Scheme 1, the positively charged out addition of KCl. VSD can essentially exist in two different 25 0.2 micro-environments. The aqueous phase cence is more polar than the nanospheric or- 20 0 ganic phase. In a simplified manner, one -6 -5 -4 -3 -2 -1 012 could postulate that the role of H+ is now 15 log[K+] replaced by the positively charged VSDs. Fluor es 10 The VSD used here is more lipophilic com- pared with the one used in our previous re- 5 port, 3,3'-dibutylthiacarbocyanine iodide. Nevertheless, the core structure is similar, 0 giving lower emission intensity in the polar aqueous environment than in the organic 490 540 590 640 690 740 nanoparticle core. λ /nm The localization of the VSDs is gov- 198 CHIMIA 2015, 69, No. 4 Laureates: Junior Prizes, SCS Fall Meeting 2014

at least several months with a narrow size lar to mammalian phagocytes of the in- [1] X. Xie, E. Bakker, Anal. Bioanal. Chem. 2015, distribution in solution (polydispersity in- nate immune system in morphology and in press. dex, ca. 0.1) and small average diameter behavior.[20] The nanospheres were added [2] X. Xie, G. Mistlberger, E. Bakker, Anal. Chem. (<100 nm typically). to the cell culture medium containing D. 2013, 85, 9932. [3] J. M. Dubach, D. I. Harjes, H. A. Clark, Nano The same matrix material (DOS+F127) discoideum and incubated for ca. 20 min Lett. 2007, 7, 1827. has been used for conventional nano-opto- before monitoring by fluorescence confo- [4] K. Kurihara, M. Ohtsu, T. Yoshida, T. Abe, H. des and for ion releasing/up-taking nano- cal microscopy. As shown in Fig. 3, before Hisamoto, K. Suzuki, Anal. Chem. 1999, 71, spheres.[2,17] Fig. 2 shows a TEM image addition of the nanospheres, only slight red 3558. of the K+-selective nanospheres. Note that autofluorescence was observed. After ad- [5] X. Xie, M. Pawlak, M.-L. Tercier-Waeber, E. Bakker, Anal. Chem. 2012, 84, 3163. aggregation during the sample preparation dition of the nanospheres to the cell culture [6] I. H. A. Badr, M. E. Meyerhoff, J. Am. Chem. stage of the TEM experiment may influ- medium, the nanospheres are readily taken Soc. 2005, 127, 5318. ence the apparent size distribution. up and concentrated in the endosomes by [7] K. Seiler, W. Simon, Sensors Actuators B 1992, 6, 295.

Fig. 2. TEM image for the K+ sensitive nano- Fig. 3. Confocal fluorescence images ofD. discoideum without addition (left) and with addition spheres. (right) of K+ sensitive nanospheres into the cell culture medium. The green fluorescence comes from the green fluorescence proteins (GFP) localised at the actin cytoskeleton, while the red color is due to the emission of the reference dye Lumogen red within the nanospheres.

The use of DOS and F127 as matrix the cells, indicating the feasibility of ap- [8] H. Hisamoto, K. Suzuki, Trends Anal. Chem. ensured a relatively large polarity differ- plying such nanosensor to these phago- 1999, 18, 513. ence between the core and the surrounding cytes. The exact mechanism of the cellular [9] G. Mistlberger, G. A. Crespo, E. Bakker, Annu. aqueous phase. The polarity of the nano- uptake and a more quantitative analysis of Rev. Anal. Chem. 2014, 7, 483. + [10] E. Bakker, P. Bühlmann, E. Pretsch, Chem. Rev. sphere core could potentially be further the ion concentration, K in this case, are 1997, 97, 3083. decreased if a less polar surfactant than currently being studied in our groups. [11] X. Xie, J. Zhai, E. Bakker, Anal. Chem. 2014, F127 is used. 86, 2853. [12] X. Xie, J. Zhai, G. A. Crespo, E. Bakker, Anal. 3.3 Nanosphere Uptake by 4. Conclusion Chem. 2014, 86, 8770. [13] X. Xie, J. Zhai, E. Bakker, J. Am. Chem. Soc. Dictyostelium discoideum 2014, 136, 16465. It has been demonstrated previously VSDs have been recently applied in [14] P. R. Dragstent, W. W. Webb, Biochemistry that nanoscale ion sensors can be applied ion-selective optical nanosensors, making 1978, 17, 5228. to intracellular imaging with improved the sensors pH-independent. Here, a lipo- [15] A. S. Waggoner, Ann. Rev. Biophys. Bioeng. [18,19] 1979, 8, 47. characteristics. Nanospheres com- philic VSD was successfully used to pre- [16] K. M. Pang, E. Lee, D. A. Knecht, Curr. Biol. + posed of F127 and DOS are newly intro- pare K -sensitive nanospheres, resulting in 1998, 8, 405. duced research tools that have not yet been a sensor response range covering the intra- [17] X. Xie, E. Bakker, ACS Appl. Mater. Interfaces applied in intracellular analysis. cellular K+ level. The nanospheres can be 2014, 6, 2666. As a first attempt, the VSDs doped readily delivered into D. discoideum, mak- [18] M. Brasuel, R. Kopelman, T. J. Miller, R. Tjalkens, M. A. Philbert, Anal. Chem. 2001, 73, nanosensors were applied to the social ing them promising nanoscale ion imaging 2221. amoeba Dictyostelium discoideum in order tools for intracellular measurements. [19] J. M. Dubach, S. Das, A. Rosenzweig, H. A. to quantify the ion concentration changes Clark, PNAS 2009, 106, 16145. during the membrane trafficking of phago- Acknowledgements [20] P. Cosson, T. Soldati, Curr. Opin. Microbiol. cytosis and to understand its relevance to The authors thank the Swiss National 2008, 11, 271. host–pathogen interactions. D. discoideum Sciences Foundation (SNF) and the University is a professional phagocyte and very simi- of Geneva for financial support of this work. Received: January 23, 2015