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.