Luminescent TOP Nanosensors for Simultaneously Measuring

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Luminescent TOP Nanosensors for Simultaneously Measuring Temperature, Oxygen, and pH at a Single Excitation Wavelength Cui Wang, Sven Otto, Matthias Dorn, Katja Heinze, and Ute Resch-Genger Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 07 Jan 2019 Downloaded from http://pubs.acs.org on January 7, 2019

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Luminescent TOP Nanosensors for Simultaneously Measuring Temperature, Oxygen, and pH at a Single Excitation Wavelength Cui Wang,1,2 Sven Otto,3,4 Matthias Dorn,3 Katja Heinze,3,* and Ute Resch-Genger1,* Federal Institute for Materials Research and Testing (BAM), Richard-Willstätter-Str. 11, D-12489 Berlin, Germany Institute of Chemistry and Biochemistry, Free University of Berlin, Takustrasse 3, D-14195 Berlin, Germany 3) Institute of Inorganic Chemistry and Analytical Chemistry, Johannes Gutenberg University of Mainz, Duesbergweg 10-14, D-55128 Mainz, Germany 4) Graduate School Materials Science in Mainz, Staudingerweg 9, D-55128 Mainz, Germany Corresponding Author* Ute Resch-Genger: E-mail: [email protected]; phone, ++49(0)30-8104-1134; fax, ++49(0)308104-71134; Katja Heinze: E-mail: [email protected] KEYWORDS optical multianalyte nanosensors, chromium(III) complex, fluorescence, phosphorescence, lifetime, oxygen/pH/temperature biosensor 1) 2)

ABSTRACT: Two nanosensors for simultaneous optical measurements of the bioanalytically and biologically relevant analytes temperature (“T”), oxygen (“O”), and pH (“P”) have been designed. These “TOP” nanosensors are based on 100 nmsized silica-coated polystyrene nanoparticles (PS-NPs) doped with a near infrared emissive oxygen- and temperaturesensitive chromium(III) complex ([Cr(ddpd)2][BPh4]3, CrBPh4) and an inert reference dye (Nile Red, NR or 5,10,15,20tetrakis-(pentafluorophenyl) porphyrin, TFPP) and are covalently labeled with pH-sensitive fluorescein isothiocyanate (FITC). These emitters can be excited at the same wavelength and reveal spectrally distinguishable emission bands allowing for ratiometric intensity-based and time-resolved studies in the visible and near infrared wavelength region. Studies in PBS buffer solutions and in a model body liquid demonstrate the applicability of these nanosensors for the sensitive fluorescence readout of TOP simultaneously at the same position. Nondestructive chemical sensing with optical techniques length.13 pH - indicator Reference dye (Ref.) a) O2 / T - indicator has been increasingly used in the last years for detecting C F (BPh ) O and monitoring biologically and bioanalytically relevant O OH CH N N N N analytes.1-3 One of the most frequently employed readout NH N O O N O C F C F Cr N N HO CH technique is luminescence due to its multiparametric N HN N N N N N N nature, provided by the excitation and emission wavelength C C F S or NR FITC TFPP (region), luminescence intensity/quantum yield, emission CrBPh4 b-1) b-2) lifetime, and anisotropy. Moreover, luminescence 1,0 1,0 measurements are rapid, provide a high spatial resolution 0,8 0,8 in the nanometer range and single molecule sensitivity, can be performed with comparatively simple and inexpensive 0,6 0,6 instrumentation, and are suited for miniaturization, online 0,4 0,4 in-situ measurements as well as remote sensing.1,2,4,5 0,2 0,2 Luminescence sensing of optically inactive analytes like protons, metal ions, small organic molecules, and gases such 0,0 0,0 400 500 600 700 800 400 500 600 700 800 as oxygen requires sensor or probe molecules that change Wavelength [nm] Wavelength [nm] c) their optical properties selectively upon interaction with FITC TEOS, APTES the target analyte. Common strategies to realize spectral CrBPh4 , Ref. 15 h, RT and intensity changes in absorption/excitation and/or luminescence and luminescence lifetime are photoinduced PS(CrBPh4-Ref)-S-NH2 PS(CrBPh4-Ref)-S-F PS(CrBPh4-Ref)-NH2 PS-NH2 electron transfer (PET),6,7 photoinduced charge transfer Figure 1: a): Chemical structures of the luminescent (PCT),8 fluorescence resonance energy transfer (FRET),9 constituents of the dual and TOP nanosensors; b-1) and b-2): and collisional quenching of long lived triplet emitters.10 Absorption (dashed lines) and emission (solid lines) spectra of Particularly the latter is used for triplet oxygen. These the dyes used for sensor fabrication. CrBPh4 (λem = 740/778 sensor molecules are often incorporated into matrices like nm) and FITC (λem = 520 nm) were applied as O2- and pHorganic polymers or bound to carrier particles to produce responsive indicators and the ratiometric dual emission of sensor films or stimuli-responsive nanoparticles, thereby CrBPh4 I740/I778 provides T-sensitivity. As reference dyes, TFPP still enabling a direct interaction between the probe and the (λem = 663 nm) was chosen in b-1) and NR (λem = 596 nm) in banalyte.11-14 For luminescence readout, this simplifies the 2). All dyes can be excited at 440 or 435 nm (as indicated by combination with analyte-inert reference dyes for the black lines) and reveal spectrally distinguishable emission bands. c): Schematic illustration of the nanosensor fabrication ratiometric measurements, where the analyte signal is using 100 nm aminated polystyrene (PS-NH2) nanoparticles, referenced to that of the reference dye excited at the same that involves dye encapsulation, silanization (employing TEOS wavelength. This can render luminescence intensity = tetraethyl orthosilicate and APTES = (3measurements independent of fluctuations in the excitation light intensity, sensor concentration and optical path 6 5

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Another recently reported dual sensor for oxygen and pH relies on core-shell nanoparticles with an oxygen-sensitive core and a pH-responsive shell, that is read out with a frequency domain luminescence lifetime-based method.39 To the best of our knowledge, a single wavelength excitable luminescent TOP nanosensor has not yet been reported. In this study we present two novel luminescent multianalyte nanosensors operated with single-wavelength excitation, namely a dual sensor for O2 and pH, and a TOP sensor. As quite hydrophobic T- and O2-responsive sensor dye, we chose our previously studied near infrared (NIR)emissive chromium(III) complex [Cr(ddpd)2]3+,37 (CrBPh4 in Figure 1a)), with tetraphenylborate [BPh4]– counter anions). This complex, which can be excited at (430 – 450) nm, has a large ligand-field splitting. This results in an extremely large energy difference between the absorption maximum (435 nm) and the two emission peaks (740 and 778 nm) originating from two doublet states (2E/2T1) that are in thermal equilibrium. Its NIR emission is quenched by molecular oxygen via Dexter energy transfer21,22,40,41 and its dual emission (I740/I778) responses ratiometrically to temperature.26 This oxygen-independent self-referencing T-sensing overcomes one of the major experimental drawbacks of commonly employed triplet emitters featuring only a single emission band that responds to oxygen and temperature. As pH-sensitive fluorescent indicator, fluorescein-5-isothiocyanate (FITC) (see Figure 1a)) was selected, that reacts with amino functionalities and has a pKa in the bioanalytically relevant pH window. As inert reference dyes, 5,10,15,20-tetrakis(pentafluorophenyl) porphyrin (TFPP; Figure 1a)) and Nile Red (NR; Figure 1a)) were used. TFPP does not respond to changes in pO2 and pH, yet the T-dependence of its emission renders it unsuited for the construction of a TOP nanosensor. For the TOP nanosensor, T-, pO2- and pH-inert NR was used as reference. Pre-fabricated aminated polystyrene (PS-NH2) nanoparticles with high biocompatibility were chosen as carriers for these sensor constituents, which are commercially available in different sizes and show a high gas permeability, ensuring access of oxygen to the indicator dye inside the particle. Subsequently, the sensing behavior of single component model sensors and dual and multianalyte nanosensors were assessed in PBS buffer solutions (PBS = phosphate buffered saline) and in a model body liquid containing bovine serum albumin (BSA) using steady state and time-resolved fluorometry.

Among the parameters most frequently measured with chemical sensors are temperature T, oxygen pO2, and pH (“TOP”) because of their relevance for biological processes, diseases like cancer or inflammation, and corrosion processes in material sciences. Emitters typically used for optical measurements of pH are HPTS (8-hydroxypyrene1,3,6-trisulfonate),12 fluorescein and rhodamine derivatives,15,16 and boron dipyrromethene (BODIPY) dyes. There are also few examples for phosphorescent (and hence O2-sensitve) pH-responsive porphyrins.4,17,18 Oxygen sensing is commonly done with metalloporphyrins like the platinum(II) porphyrin complex such as PtTFPP,19 transition metal polypyridyl complexes such as [Ru(bpy)3]2+ or long lived polycyclic aromatic hydrocarbons like pyrene.3 More recent examples are the europium(III) complex Eu(HPhN)3(dpp),20 and the chromium(III) complex [Cr(ddpd)2]3+ (Figure 1a)).21,22 Typical temperature sensitive molecular luminophores23 are rhodamine B24 and certain iridium(III) complexes.25 Recently, also Eu(tta)3(dpbt)12 and [Cr(ddpd)2]3+ 26 have been applied for optical temperature measurements. Although a large toolbox of stimuli-responsive molecules for optically signaling changes in T, pO2, and pH exists utilizing PET, PCT, and FRET,23,26-31 there is still a considerable need for multianalyte sensors for the simultaneous measurement of these often related parameters e.g., for monitoring biological or biotechnological processes.32-35 This is mandatory to obtain information on changes in analyte concentration and/or physicochemical parameters at the same position. The realization of ratiometric TOP sensors requires analyteresponsive dyes with absorption and emission features, which enable single wavelength excitation and spectral discrimination of the luminescence bands. This can be only realized with fluorophores, that reveal a large energy difference between their absorption and emission bands. Beneficial are also sensor molecules, that respond to two different analytes simultaneously with spectrally distinguishable outputs. Rare examples are oxygen- and temperature-responsive platinum(II) and palladium(II) benzoporphyrins reported by the Klimant group36 and the [Cr(ddpd)2]3+ complex developed by us.21,22,26,37 Molecular TOP sensors have not been obtained yet. Multi-analyte sensors are commonly realized by doping polymer films or particles with dye combinations, with each dye responding to a single analyte. There are meanwhile a few reports on systems that can sense two or three of the TOP parameters simultaneously. For instance, dual sensors have been made that respond to pO2 and T,19,25,36,37 or to pO2 and pH.18,20,38 However, up to now, only very few examples have been demonstrated for sensing T, pO2, and pH simultaneously.12 Furthermore, most of these multianalyte sensors are based on planar thin films or layers, which are not applicable, e.g. for intracellular biological studies. An example for a nanoscale dual sensor are fluorophore-labeled micelles for intensity-based imaging of pH and oxygen concentration in cells.27 This system requires, however, two excitation wavelengths to excite the indicators and the reference dye.

Materials and Methods Materials. 100 nm-sized PS-NH2 particles were purchased from Micromod GmbH (Germany). Prior to use, they underwent three washing, centrifugation (Centrifuge 5415D from Eppendorf (Germany), 40 min at 16000 g for 100 nm-sized PSP), and resuspension (by ultrasonication) cycles. Tetraethyl orthosilicate (TEOS), 3aminopropyl)triethoxysilane (APTES), NaBPh4, TFPP, and FITC were purchased from Sigma-Aldrich and NR from Fluka Chemie GmbH (Germany), respectively. Synthesis of [Cr(ddpd)2][BPh4]3 (CrBPh4) 2


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[Cr(ddpd)2][BF4]3 (99.9 mg, 0.11 mmol), prepared according to the literature,21,22 was dissolved in CH3CN (6 mL). NaBPh4 (118.4 mg, 0.35 mmol) in CH3CN (8 mL) was added. After stirring for 30 min, deionized water was added until the precipitation was finished. After 12 h, [Cr(ddpd)2][BPh4]3 (CrBPh4) was collected as a fine yellow powder and washed with water (2  5 mL). Drying under reduced pressure gave CrBPh4 as fine yellow powder (159 mg, 89 %). Apart from the poor solubility of this salt in water and its smaller ionic conductivity in CH3CN (Ʌe = 205 S cm–1 M–1), its properties match those of the reported salts [Cr(ddpd)2][BF4]3 and [Cr(ddpd)2][PF6]3.21,22,41 The luminescence quantum yield of CrBPh4 in deaerated CH3CN amounts to Φ = 9.0% with a luminescence lifetime of τ = 640 µs. In O2-saturated CH3CN solution, these values drop to Φ = 0.9% and τ = 34.6 µs. Preparation of PS(CrBPh4-Ref)-NH2 (Ref = TFPP, NR) The preparation of the two nanosensors is illustrated in Figure 1-c) in step 1. The comparably hydrophobic salt CrBPh4 and the analyte-inert reference dyes TFPP or NR were encapsulated into 100 nm-sized PS-NH2 nanoparticles via a simple one-step staining procedure.42 The ratio of CrBPh4 to reference dye was chosen to be 10:1 in the staining procedure. 12 mg PS-NPs are dispersed in Milli-Q water to 2.5 mg/mL (4.8 mL). 90 µL CrBPh4 (5 mM in DMF) were mixed with 45 µL reference dye NR solution (1 mM NR in THF) and diluted with THF to give 800 µL dye solution. For the nanosensor with the reference dye TFPP the concentrations of both CrBPh4 and TFPP were increased by a factor of two. The spectroscopically determined actual loading concentrations are given in the Results and discussion section. After adding the dye solution to the PSNPs dispersion, the sample was shaken for 40 minutes. Shrinking of the particles was induced by adding 800 µL Milli-Q water, thereby sterically incorporating the dye molecules in the PS-NH2 particles. The particles were then centrifuged at 16,000 rcf for 60 minutes. The precipitate was washed three times with acetonitrile/water mixtures (volume ratios of 40/60, 30/70, and 20/80) and once with Milli-Q water to remove excess dye adsorbed onto the particle surface. PS-NH2 particles loaded with CrBPh4 and the reference dye (PS(CrBPh4-Ref)-NH2) were diluted to 10 mg/mL. Preparation of PS(CrBPh4-Ref)-S-NH2 (Ref = TFPP, NR) To trap the indicator and reference dyes inside the nanosensors, the particles were coated with a thin aminated silica shell using the ammonia-catalyzed Stöber process,43,44 see Figure 1-c), step 3). After adding 24 µL aqueous ammonia (25%) to the PS(CrBPh4-Ref)-NH2 dispersion, 24 µL tetraethyl orthosilicate (TEOS) with 5.0% (w/v) (3aminopropyl)triethoxysilane (APTES) were dissolved in 80 µL absolute ethanol and added slowly (10 µL every 10 minutes) to the stirred particle dispersion. The sample was shaken for 15 h. The particles were washed with ethanol/water mixtures (volume ratios of 40/60, 30/70, and 20/80) for three times and once with water. The resulting silica-coated aminated PS(CrBPh4-Ref)-S particles were dispersed in 1.2 mL PBS solution (0.1 M, pH 8.0), yielding a particle concentration of 10 mg/mL. Preparation of PS(CrBPh4-Ref)-S-F (Ref = TFPP, NR)

0.3 mg FITC was dissolved in 60 µL ethanol and added to the dispersion of the PS(CrBPh4-Ref)-S particles at pH 8.0. After stirring the reaction mixture for 3 hours, the particles were washed four times with ethanol/water mixtures (volume ratios of 40/60, 30/70, and twice 20/80, respectively) and once with water. The obtained PS(CrBPh4-Ref)-S-F particles (see Figure 1-c) were then dispersed in 1.2 mL MilliQ water yielding a particle concentration of 10 mg/mL. The resulting nanosensors have a diameter of around 130 nm as determined by Dynamic Light Scattering (DLS) with a Zetasizer Nano ZS from Malvern Panalytical (see Supporting Information, Figure-S2). Instrumentation Absorption measurements Absorption spectra of the dyes and nanosensor dispersions were obtained with a calibrated Cary 5000 spectrometer. The amount of encapsulated CrBPh4 and PS-NH2 bound FITC was obtained from the absorption spectra of particle samples in DMF (thereby dissolving the PS-NP) and the species’ molar absorption coefficients previously determined by absorption measurements of concentration series of the emitters dissolved in DMF (Supporting Information, Figure-S3 and Figure-S4). Luminescence spectra and lifetime measurements Luminescence spectra and decay kinetics were measured with a calibrated45 spectrofluorometer FSP 920 from Edinburgh Instruments. For the measurement of the emission spectra, a continuous xenon lamp was applied as excitation light source, while the time-resolved luminescence measurements were done with a µs xenon flashlamp and detection in a multi-channel scaling mode. All luminescence measurements were performed with aqueous nanosensor dispersions with particle concentrations of 1 mg/mL in 10 mm x 10 mm quartz cuvettes (Hellma) at room temperature if not otherwise stated. Excitation was typically at 435 nm or 440 nm. Determination of O2 partial pressure and pH The oxygen concentration was varied by purging the nanosensor dispersion with argon in a sealed cuvette. The oxygen partial pressure was determined with a commercial fiber-optic oxygen meter Firesting O2 from PyroScience and a solvent-resistant oxygen probe tip (OXSOLVPT). The pH values of the samples were determined with a calibrated microprocessor pH meter from Hanna Instruments.

Results and discussion Design of multianalyte nanosensors As shown in Figure 1 b-1) and b-2), the sensor constituents CrBPh4, FITC, TFPP, and NR (Figure 1a)) can be excited in the spectral window of (430 – 450 nm) and reveal clearly distinguishable emission bands centered at 778/740, 520, 663, and 596 nm, respectively, as required for ratiometric sensing. The design cornerstone for these ratiometric dual and multianalyte nanosensors is the extraordinarily large energy difference (absorption: 435 nm; emission: 740/778 nm; spectral window of 9475 cm-1 equaling here 305 nm) of the O2-sensitive chromium(III) complex [Cr(ddpd)2]3+ 21,22 compared with that of commonly employed O2-sensitive dyes. For example, red emissive benzoporphyrins with emission between about 740 and 800 nm, which are 3



typically excited at the Soret band (equaling the S0-S2 transition) at about 440 nm, have also less intense absorption bands between about 620 and 650 nm (the socalled Q bands, equaling the S0-S1 transition that reveals a vibronic fine structure). This narrows the spectral window for the combination with other luminophores to about 3000 cm-1 (equaling 150 nm) in average.36 This renders the CrIII emitter21,22 ideal for the combination with several stimuliresponsive luminophores with minimum spectral crosstalk like pH-sensitive dyes with fluorescence in the visible region and an additional reference dye. Furthermore, as previously stated, due to thermal equilibrium between the emissive 2E and 2T1 states of the [Cr(ddpd)2]3+ salts,26 this compound is one of the rare dual emissive molecules that can act as ratiometric self-referenced optical thermometer in the temperature range of –50 °C to 100°C.21,26 The preparation of the nanosensors using pre-fabricated PS-NH2 nanoparticles is summarized in Figure 1 c), using a simple one-step staining procedure (step 1)42,46,47 for the loading of the nanoparticles with the O2- and T-responsive chromium dye and the reference dyes TFPP or NR. As the prevention of the leaking of the sensor components is crucial especially for molecules that are only mechanically entrapped and not covalently bound to the particle matrix, we performed stability studies at different stages of the nanosensor fabrication. For sterically entrapped molecules, leaking can be encouraged for dyes that are not completely water-insoluble and can be further favored by the presence of e.g., traces of organic solvents, surfactants or proteins or for neutral indicators by protonation or coordination to a charged analyte. Water solubility is particularly an issue for the chromium(III) dye [Cr(ddpd)2]3+ with the [BF4]– counter anion. For this complex, we observed leaking from the PSNH2 nanoparticles over time. As the hydrophobicity of [Cr(ddpd)2]3+ can be fine-tuned by the choice of the counter anion, while preserving its favorable optical properties, the less hydrophilic [PF6]– salt and the sterically more demanding [BPh4]– salt (Cr(ddpd)2][PF6]3 (CrPF6)21; [Cr(ddpd)2][BPh4]3; CrBPh4) were synthesized and subsequently assessed. Spectroscopically monitored stability studies with [Cr(ddpd)2][PF6]3 (CrPF6)21 encapsulated in PS-NH2 nanoparticles in phosphatebuffered saline (PBS, 0.1 M, pH 8.0) revealed leaking of this slightly water-soluble complex from the PS-NH2 (Supporting Information, Figure-S5), that could be strongly reduced with the bulky anion [BPh4]–. Nevertheless, traces of this less hydrophilic complex could still be detected after storage of the (CrBPh4)-loaded PS-NH2 particles in PBS buffer at room temperature for 48 hours. In order to “seal” the nanosensors, the PS-NH2 particles were coated with a thin silica shell applying APTES in an ammonia-catalyzed Stöber-like procedure 43,44,48 (Figure 1c), step 2). This provides still good oxygen permeability and yields amino groups for further conjugation with amine-reactive dyes like FITC. Silica shell formation was confirmed via measurements of the size and zeta potential with dynamic light scattering (DLS; Supporting Information, Figure-S1). Stability studies with silica-sealed (CrBPh4)-loaded PS-NH2 particles in PBS over a period of two months demonstrated the high colloidal and chemical

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stability of these nanoparticles (Supporting Information, Figure-S5). Moreover, optically monitored stability studies in cell culture medium (PBS with 0.5% mass to volume percent (w/v) BSA) kept in the dark and illuminated at 435 nm (using the xenon lamp of the spectrofluorometer) revealed no changes confirming the mechanical, chemical, and photochemical stability of the nanosensors (Supporting Information, Figure-S6). With this proof at hand, finally, a pH-sensitive fluorescein was covalently bound to the surface of the sensor particles by reacting surface amino functionalities with the thiocyanate derivative FITC (Figure 1c), step 3).49,50 Sensing studies To demonstrate that the different components in the multianalyte nanosensors do not interfere with each other, single analyte nanosensors stained or labeled with a single stimulus-responsive dye were studied first in PBS, exemplarily using NR as reference dye. The sensing behavior of the O2-sensor particles was compared to that of CrBPh4 in acetonitrile (Supporting Information, Figure-S8) as this chromium(III) complex salt is only poorly water soluble. For the pH responsive nanosensors, a comparison to a pH-responsive fluorescein derivative of FITC (Supporting Information, Figure-S8) was done in aqueous solution in the pH-range of 4 to 9. The following nomenclature is subsequently used for the systems studied: a) single indicator nanosensors in PBS: PS(CrBPh4-NR)-SNH2 for O2- and T- sensing and PS-F for pH-sensing (Figure 2), b) multianalyte nanosensors in PBS: PS(CrBPh4-NR)-S-F for T-, O2-, and pH-sensing and PS(CrBPh4-TFPP)-S-F for O2and pH-sensing (Figure 3 and Figure 4) and c) multianalyte nanosensors in cell culture medium (Table 1). a) Single indicator nanosensors in PBS As shown in Figures 2 a-1) and a-2), for PS(CrBPh4-NR)-SNH2, the O2-induced decrease in the NIR emission intensity and luminescence lifetime of PS-NH2 encapsulated CrBPh4 (λmax = 778 nm) can be exploited for determining changes in pO2 from 0.3 hPa (Ar-saturated) to 214.5 hPa (airsaturated). The Stern-Volmer plots derived from emission intensity and lifetime measurements of the nanosensor PS(CrBPh4-NR)-S-NH2 reveal a smaller slope of 0.011 in comparison with the molecular sensor CrBPh4 in acetonitrile, which has a slope of the Stern-Volmer plot of 0.068. This is attributed to the different chemical environments of the O2 indicator, that could slightly affect the O2-sensitivity of the indicator. In the silica-coated nanosensor, access of O2 to the encapsulated CrBPh4 dye seems to be less facile as found for the Cr(III) complex dissolved in an organic solvent. Nevertheless, the O2 response of the nanosensor is still fast and is fully reversible as demonstrated by luminescence measurements of alternating cycles of purging nanosensor solutions with argon and their equilibration with air. A rise in temperature from 283 K to 343 K leads to an increase in the emission intensity ratio I740/I778 of PS(CrBPh4-NR)-S-NH2 revealing the inherent T-sensitivity of this system (Figure 2 a-3)), while expectedly the lifetime at 778 nm remains constant. The slopes of the Boltzmann plots obtained for CrBPh4 (Supporting Information, FigureS8-3)) and for PS(CrBPh4-NR)-S-NH2 are linear, 4


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was considered by calibrating the O2-responsive nanosensors at air-saturated atmosphere with a commercial O2 sensor, where the signal

substantiating a similar thermochromic behavior of both systems. The temperature rise concomitantly decreases the O2-solubility in the respective solvent, which results in a stronger emission at 778 nm. On the one hand, this effect a-1) PS(CrBPh4-NR)-S-NH2 in PBS: O2-sensing

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Figure 2: Sensing studies with the single components of the TOP nanosensors exploiting the parameters luminescence emission intensity and lifetime. a-1): O2-sensitivity of PS(CrBPh4-NR)-S-NH2 derived from luminescence intensity (λem = 778 nm) measurements and the respective Stern-Volmer plot Imax/I-1 vs. pO2; a-2): O2-sensitivity of PS(CrBPh4-NR)-S-NH2 obtained from luminescence lifetime studies (λexc. = 435 nm; λem. = 778 nm) and the respective Stern-Volmer plot Imax/I-1 vs. pO2; a-3): T-sensitivity of PS(CrBPh4-NR)-S-NH2 determined from the ratiometric dual emission I740/I778 and the respective Boltzmann plot ln(I740/I778) vs. T-1 measured in air saturated dispersion. Nanosensor PS(CrBPh4-NR)-S-NH2 was excited at 435 nm and the lifetimes were measured at 778 nm. a-4): Luminescence emission intensity-based pH-sensitivity of the single analyte nanosensor PS-S-F in PBS (0.1 M) and the respective calibration plots I520/I570-1 vs. pH values. The pKa value was obtained from a fit of the sigmoidal titration curve (pKa = 6.44 ± 0.01; R2 = 0.9981). Nanosensor PS-F was excited at 440 nm.

change, caused by O2-solubility, was expressed in the corresponding calibration parameter, namely the Boltzmann slope. On the other hand, the change in O2solubility with T can also be excluded by calibrating the sensor systems in argon-saturated atmosphere. After cooling to room temperature, the emission signals of CrBPh4 and PS(CrBPh4-NR)-S-NH2 are fully restored indicating the reversibility of T-sensing. Changing the pH from 8.7 to 3.8 is accompanied by a decreased emission intensity of PS-S-F in PBS at 520 nm (Figure 2 a-4)). The sigmoidal plot of the pH-dependent change in emission intensity yielded a pKa value of 6.4, which coincides with the pKa value of the respective FITC derivative in solution (Supporting Information, Figure-S84)). This good agreement between the molecular and the nanoparticular systems underlines the suitability of the chosen nanostructure of the nanosensor and the conjugation chemistry for pH sensing. Apparently, there are no steric constraints imposed on the PS-NH2-bound FITC molecules, since their protonation/deprotonation is as facile as that of the dissolved dye. The broad pH sensing range enables measurements of physiological pH values.

Adding aqueous NaOH solution to the PS-S-F particle dispersion restored the initial emission intensity at 520 nm, thereby confirming the full reversibility of the pH nanosensor. b) Multianalyte nanosensors in PBS The O2-, pH- and T-sensitivity of our dual and multianalyte nanosensors PS(CrBPh4-NR)-S-F and PS(CrBPh4-TFPP)-S-F was demonstrated by detecting changes in luminescence intensity and/or lifetime accompanying changes in T, pO2, and pH. The results are summarized in Figure 3 and Figure 4. Nanosensors PS(CrBPh4-NR)-S-F and PS(CrBPh4-TFPP)S-F generate spectrally distinguishable emission bands for 435 nm or 440 nm excitation. Increasing pO2 from 0.15 hPa to 204 hPa is signaled by a reduction of the emission intensity of CrBPh4 at 778 nm (Figure 3 b-1) and Figure 4 b-1)) and a concomitant shortening of the emission lifetime (Figure 3 b-2) and Figure 4 b-2)). Plotting the emission intensity or lifetime at 778 nm versus pO2 yields linear Stern-Volmer plots with a slope of 0.014 for nanosensor PS(CrBPh4-NR)-S-F. This demonstrates that neither the simultaneously encapsulated reference dye NR nor the pH5



Here, a change in pH from 8.9 to 4.7 results in a strongly decreased intensity of the green emission of fluorescein. The ratiometric calibration plots of the pH-sensitivity of both nanosensors reveal the same pKa value of around 6.4 as found for the single nanosensors PS-S-F and the molecular system as discussed above (Figure 2 a-4)). Adding concentrated aqueous NaOH solution to the dual and multianalyte nanosensors fully restored the initial luminescence intensity of the particle-bound fluorescein dye, thereby underlining the reversibility of their pH responsivity. The pH range covered by these nanosensors is well suited for measurements of physiologically relevant pH values as expected from the choice of the pH-sensitive dye. Nanosensor PS(CrBPh4-NR)-S-F reveals the expected Tdependent changes in the ratio of the luminescence intensities I740/I778 for rising T from 283 K to 343 K (Figure 3 b-4)). Plotting the logarithm of this quotient ln(I740/I778) versus the inverse temperature T-1 yields a linear Boltzmann-type behavior, which parallels the behavior of the molecular chromium(III) system in solution26 and that of the single analyte nanosensor PS(CrBPh4-NR)-S-NH2. The fully restored emission of the nanosensor in the T-range from 283 K to 343 K indicates its reversible performance (Supporting Information, Figure-S7)). The broad T- range renders this nanosensor fully appropriate for biological applications.

responsive fluorescein influence the O2-sensitivity of the system. Nanosensor PS(CrBPh4-TFPP)-S-F with TFPP as reference dye shows a linear Stern-Volmer plot in both emission- and lifetime-based measurements with basically the same slope of the Stern-Volmer plots (Figure 4 b-2) and Figure 4 b-3)) as nanosensor PS(CrBPh4-NR)-S-F. The pO2induced luminescence changes of both nanosensors are fully reversible. Consequently, both intensity and lifetimebased O2 sensing is feasible with both multianalyte nanosensors. The latter is generally preferred as timeresolved measurements eliminate many potential signal distortions and artefacts by minimizing background signals and excluding influences from changes in sensor molecule concentration and excitation light intensity.12 More importantly, the phosphorescence lifetime scale ranging from 170 µs to 680 µs ensures a high accuracy for O2sensing. Lowering the pH of the nanosensor PS(CrBPh4-NR)-S-F dispersion from 8.5 to 4.0 leads to the expected strong reduction of the fluorescein fluorescence at 520 nm (I520) (Figure 3 b-3)). Possible signal distortions from the partial spectral overlap of the fluorescence bands of FITC and NR can be eliminated by utilizing intensity ratios I520/I778, as the emission of CrBPh4 at 778 nm is inert against pH changes. For the nanosensor PS(CrBPh4-TFPP)-S-F, no spectral overlap is observed between the emission bands of the different emitters under 440 nm excitation (Figure 4 b-4)).

b-2) PS(CrBPh4-NR)-S-F in PBS: O2-sensing

b-1) PS(CrBPh4-NR)-S-F in PBS: O2-sensing 3

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Figure 3. Luminescence emission intensity and lifetime of the multianalyte nanosensor PS(CrBPh4-NR)-S-F in PBS accompanying changes in pO2, T, and pH. For nanosensor PS(Cr-NR)-S-F under 435 nm excitation: b-1): O2-sensitivity (778 nm) derived from luminescence intensity measurements and the respective Stern-Volmer plot Imax/I-1 vs. pO2; b-2): O2-sensitivity (778 nm) obtained from luminescence lifetime studies and the respective Stern-Volmer plot τmax/τ-1 vs. pO2; b-3): pH-sensitivity and the respective calibration plots I520/I570-1 vs. pH values; and b-4): T-sensitivity and the respective Boltzmann plot ln(I740/I778) vs. T-1.

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Figure 4. Luminescence emission intensity and lifetime of the multianalyte nanosensor PS(CrBPh4-TFPP)-S-F in PBS accompanying changes in pO2 and pH. For nanosensor PS(Cr-TFPP)-S-F under 440 nm excitation: b-1): O2-sensitivity (778 nm) derived from luminescence intensity measurements and the respective Stern-Volmer plot Imax/I-1 vs. pO2; b-2): O2sensitivity (778 nm) determined by luminescence lifetime measurements and b-3): Stern-Volmer plot based on lifetime measurements τmax/τ-1 vs. pO2; b-4): pH-sensitivity and the respective calibration plot I520/I570-1 vs. pH values.

The consistent sensing behavior of the monoanalyte nanosensors PS(CrBPh4-NR)-S-NH2 and PS-S-F, the multianalyte and dual nanosensors PS(CrBPh4-NR)-S-F and PS(CrBPh4-TFPP)-S-F and the respective molecular systems observed for all TOP parameters clearly demonstrates that the different components in the multianalyte nanosensors do not interfere with each other. c) Multianalyte nanosensors in cell culture media Aiming at bioanalytical applications of the dual and multianalyte nanosensors, we studied the response of these systems to pO2, pH, and T in cell culture medium (Supporting Information, Figure-S9). Table 1 summarizes the respective calibration parameters and their comparison with results from sensing studies in PBS. All relative standard deviations of the calibration parameters were derived from measurements of three independent nanosensor samples and every sample was independently calibrated twice. As shown in Table 1, both nanosensors exhibit the desired sensing behavior in PBS buffer and in cell culture medium. The lifetime-based Stern-Volmer slopes are generally slightly higher in PBS and in cell culture medium compared to the oxygen sensitivity derived from measurements of the emission intensity. In cell culture

medium, the emission-based and lifetime-based SternVolmer slopes are slightly lower than in PBS buffer pointing to a slightly reduced oxygen sensitivity. Interestingly, these differences are less pronounced for time-resolved measurements. One explanation may be the interaction of the nanoparticles with BSA molecules, acting as model for the most abundant serum protein, that may adsorb to the particle surface forming a protein corona. Such interactions can be prevented e.g., by modifying the surface of the nanosensors with a PEG shell. This has been demonstrated for other nanoparticle systems applied in biological environments where the formation of a protein corona is often undesired.51 The acidofluorometric behavior of particle-conjugated fluorescein in cell culture medium reveals pKa values of 6.5, which match excellently with the results of the previously detailed studies in PBS. The decreased ratiometric emission (I740/I778) with increasing T from 283 K to 343 K, which can be described with a linear Boltzmann behavior, shows the same slope in PBS and in the cell culture medium. Also, sensing of temperature T, pO2, and pH is fully reversible in both environments.

Nanosensors

Emission-based Stern-Volmer slope

Lifetime-based Stern-Volmer slope

pKa

Boltzmann slope

PS(CrBPh4-NR)-S-F in PBS

0.0139 ± 0.0024

0.0146 ± 0.0031

6.46 ± 0.14

-1546 ± 129

PS(CrBPh4-NR)-S-F in cell culture

0.0106 ± 0.0012

0.0118 ± 0.0014

6.57 ± 0.17

-1551 ± 141

PS(CrBPh4-TFPP)-S-F in PBS

0.0121 ± 0.0010

0.0143 ± 0.0012

6.47 ± 0.07

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7



PS(CrBPh4-TFPP)-S-F in cell culture

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0.0131 ± 0.0013

Page 8 of 10 6.53 ± 0.13

-

Table 1: Calibration parameters: Stern-Volmer slope for oxygen, pKa value for pH sensing, and Boltzmann slope for temperature sensing with the dual and multianalyte nanosensors in PBS buffer and in cell culture medium.

This work was supported by the Deutsche Forschungsgemeinschaft (GSC 266, Materials Science in Mainz, scholarship for S.O., HE 2778/10-1 and RE 1203/231). Funding from the internal university research funds of the Johannes Gutenberg University of Mainz is gratefully acknowledged. URG and CW acknowledge support from Dr. Bastian Rühle for the silica coating of the nanosensors.

Conclusion and Outlook In summary, by combining the NIR-emissive [Cr(ddpd)2][BPh4]3 complex CrBPh4 with its extremely large energy gap between the longest wavelength absorption and emission maxima with a pH-responsive fluorescein derivative (FITC) and an inert reference dye like Nile Red (NR) and 5,10,15,20-tetrakis-(pentafluorophenyl) porphyrin (TFPP), we developed nanosensors for simultaneously sensing temperature, O2 partial pressure, and pH. These novel TOP nanosensors (temperature, oxygen, pH) cover the biologically and physiologically relevant concentration ranges of these parameters/analytes with single wavelength excitation in PBS buffer and in a cell culture medium containing bovine serum albumin (BSA). The response of both nanosensors to all parameters is fully reversible and only minimally affected by the presence of BSA, the most common serum albumin. Moreover, comparative studies with nanosensors containing only a single type of stimuli-responsive molecule and with the respective molecular systems revealed that the different sensor components do not interfere with each other. Future research will include the testing of these nanosensors in cellular uptake studies and, after surface modification with targeted bioligands, eventually in in vivo experiments as previously done by some of us with other polystyrene nanoparticle reporters and nanosensors.46,47 Moreover, this concept of multianalyte sensing will be expanded to nanosensors derived from differently sized pre-manufactured biocompatible polymer particles and different stimuli-responsive dyes like fluorescent indicators for biologically and bioanalytically relevant metal ions.

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ASSOCIATED CONTENT Supporting Information Supporting Information Available: S1-S2: Size distribution and zeta potential of nanosensors; S3-S4: Quantification of dyes in/on nanosensors; S5-S7: Stability studies; S8-S9: Sensing studies of molecular indicator dyes and nanosensors in cell culture. The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author * Ute Resch-Genger: E-Mail: [email protected]; phone, ++49(0)30 8104-1134; fax, ++49(0)30-810471134. * Katja Heinze: E-Mail: [email protected].

ACKNOWLEDGMENT 8


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9



For Table of Contents Only PS(CrBPh4-TFPP)-S-F

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Luminescent TOP Nanosensors for Simultaneously Measuring

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