Oxygen-Unaffected Temperature Sensing in Air-Saturated Aqueous Dispersion with Oxygen-Sensitive Luminescent Metal Complexes Encapsulated in l‑Arginine-Type Silica Networks

Abstract

Bioimaging, sensing, and photonic applications of oxygen-sensitive luminophores such as near-infrared (NIR) emissive spin-flip complexes of earth-abundant chromium (Cr^III) and triplet metal-to-ligand charge transfer complexes like ruthenium (Ru^II), osmium (Os^II) or iridium (Ir^III) compounds in air require efficient strategies for their complete protection from luminescence quenching by oxygen (O₂). Here, we explored the applicability of our l-arginine-type sol–gel nanoparticle synthesis route developed for complete oxygen shielding of a molecular ruby-type luminophore [Cr­(ddpd)₂]­(BF₄)₃ (Crddpd) to a representatively chosen Ru^II complex [Ru­(phen)₃]­Cl₂ (Ruphen) and a Ru^II pyrene dyad [Ru­(phen)₂(phenpy)]­Cl₂ (Ruphenpy) with luminescence lifetimes of 1.1 μs and ∼50 μs in the absence of O₂, thus confirming its versatility. Temperature-dependent steady-state and time-resolved luminescence measurements with the Ru^II and Cr^III complexes in air- and argon (Ar)-saturated aqueous solution and encapsulated in silica nanoparticles (SiO₂ NPs) prepared by the l-arginine and the classical Stöber method demonstrated the preserved temperature sensitivity of these luminophores in both silica networks. These measurements highlight the importance of complete O₂ protection for reliable temperature sensing with O₂-sensitive luminophores in air. These findings, together with the enhanced photostability observed for l-arginine SiO₂ NPs-encapsulated Ruphen and Ruphenpy, can pave the road for many applications of O₂-sensitive systems in air in the life and material sciences.

1. Introduction

Luminescent complexes of transition metal ions such as Ru^II, Ir^III, Os^II, and Re^I with emission in the visible and near-infrared (NIR) regions, controlled by sophisticated ligand design, are frequently used as luminescent probes and reporters in sensing, bioimaging, materials science, and photonic applications. − Particularly, Ru^II compounds have been widely utilized as building blocks in sensors for contactless and minimally invasive measurements of environmental parameters such as pressure and oxygen (O₂), as well as temperature in the life and materials sciences and aerospace. − These applications exploit the responsivity of their photoluminescence (PL) quantum yields (PLQY), their PL intensities, and their relatively long PL lifetimes in the range of typically several hundred nanoseconds to changes in these parameters. − For applications such as time-gated luminescence, triplet–triplet annihilation upconversion (TTA-UC), and sensitizing solar cells as well as the determination of other environmental parameters or analytes, a pronounced O₂ sensitivity of PLQY, PL intensity, and PL decay kinetics or lifetime can present a disadvantage. This calls for concepts for the simultaneous determination of oxygen and temperature. Typical approaches are the combination of combining luminophores varying in O₂ and temperature sensitivity such as an O₂- sensitive Ru^II complex and a quantum dot − or efficient O₂ shielding. ,− For accurate and reliable sensor performance, commonly, complete O₂ protection is preferred to avoid signal-distorting interferences. Concepts for O₂ protection rely on preventing physical contact between the excited luminophore and O₂, which is required for Dexter-type energy transfer. This includes the synthetically challenging usage of bulky ligands or counterions, which, however, only partly hinders O₂ diffusion to the spin-flip emissive chromium center and is difficult to employ with charge-transfer emitters due to their excited state function extending over large parts of the ligand framework. On the other hand, luminophores can be encapsulated in matrices with a very low O₂ permeability, such as polyacrylonitrile, poly-α-pinene, or poly­(vinyl alcohol). − Alternatively, biocompatible silica nanomaterials and films have been utilized for O₂ protection. These silica materials were typically prepared by the well-established Stöber route. − For most matrix-embedded O₂-sensitive luminophores, the O₂ permeability and shielding efficiency of the matrix has not been quantified despite its impact on sensor performance. , This allows O₂ interference with the measurement of other parameters or analytes and can hamper comparability across different sensors, i.e., luminophores and matrices.

Complete O₂ protection of two O₂-sensitive luminescent complexes of earth-abundant Cr^III with lifetimes in the order of 1 ms has been recently achieved by us with amorphous nonporous silica nanoparticles (SiO₂ NPs) prepared by l-arginine mediated silica formation. In the present study, we explore the versatility and general applicability of our l-arginine silica-oxygen shielding approach for transition metal complexes such as a common Ru^II complex, here [Ru­(phen)₃]²⁺ (Ruphen), and the metal complex-organic chromophore dyad (Ruphenpy). The latter consists of a [Ru­(phen)₃]²⁺ complex covalently linked to a pyrene moiety (Figure ). This dyad was chosen because of its unusually long PL lifetime of about 50 μs in the absence of O_2, which is exceptionally long for a Ru^II complex − but can be explained by the intermediate population of the long-lived organic triplet state (Figure , middle). Further, we assessed the temperature sensitivity of the PL properties of both SiO₂ NP-encapsulated Ru^II compounds and the temperature-responsive Cr^III complex [Cr­(ddpd)₂]­(BF₄)₃ (Crddpd). The latter has previously been employed as a self-referenced ratiometric optical Boltzmann thermometer in the temperature range of 5 to 85 °C in air- and Ar-saturated solution. We now compared the results with those obtained for the corresponding molecular systems. The present metal complexes differ in the nature of the respective excited state (spin-flip, ³MLCT, ³π–π*), O₂ PL quenching dynamics, and temperature sensing mechanism. The two long-lived lowest-energy spin-flip states of the Cr^III complex (see middle right panel of Figure ), which allow ratiometric temperature sensing, are more shielded from the environment due to the metal-centered spin-flip nature of the excited state. These Cr^III complexes show O₂ PL quenching rate constants orders of magnitude below the diffusion limit. In contrast, the lowest excited triplet states of the Ru-based compounds, i.e., the ³MLCT state for Ruphen and a ³π–π* state for Ruphenpy, are quenched by O₂ at rate constants close to the diffusion limit, with most collisions with dissolved O₂ depopulating the excited states. ,− As the PL intensity and PL lifetime or decay kinetics are also the parameters exploited for temperature sensing, the presence of O₂ can be detrimental for reliable optical temperature measurements for luminophores such as the ruthenium chromophores, which is frequently underestimated.

1.

Overview of the luminophores, luminescence mechanism photophysics, and silica-encapsulation routes. Top: molecular structures of the Ru^II complexes Ruphen and Ruphenpy and the Cr^III complex Crddpd. Middle: simplified energy level scheme of Ruphen and Ruphenpy showing the photoexcitation of the Ru^II complex, intersystem crossing (ISC), and relaxation pathways (¹MLCT → ³MLCT/³(π–π*) states), along with subsequent intramolecular energy-transfer processes (FRET/iEnT), with the respective energies as well as the simplified energy level diagram of the Cr^III spin-allowed excitation (⁴A₂ → ⁴T₂; 435 nm = 2.85 eV), IC (internal conversion), VR (vibrational relaxation), ISC and NIR emission from the ²T₁/²E qmanifold (738 nm = 1.68 eV and 775 nm = 1.60 eV). Bottom: schematic presentation of the encapsulation of (a) Ruphenpy, (b) Ruphen, and (c) Crddpd, in silica nanoparticles (SiO₂ NPs) prepared by two sol–gel routes. Left: l-arginine-catalyzed approach in a biphasic system (l-arginine, 0.52 mmol; H₂O/cyclohexane = 15.3:1; TEOS, 24.63 mmol; 65 °C, 20 h) and right: Stöber process (TEOS, 0.55 mL; NH₃(aq) 25%, 0.5 mL; EtOH/H₂O = 57.5:1; r.t. (T = 23 ± 2 °C), 2 d, yielding Ruphenpy@ARGSiO ₂ , Ruphen@ARGSiO ₂ , Crddpd@ARGSiO ₂ , Ruphenpy@STOSiO ₂ , and Crddpd@STOSiO ₂ .

The aim of our spectroscopic studies is to confirm the general applicability of our l-arginine silica shielding concept for O₂ photosensitive metal complexes. This can pave the route to many new applications in air previously hampered by O₂ induced PL quenching. In addition, we reveal on highlighting the influence of O₂ on temperature sensing with O₂-sensitive ratiometric luminophores to underline the importance of quantifying the O₂ shielding sensitivity of matrices used for the design of sensor systems with O₂-responsive luminophores. This presents an important prerequisite for accurate and comparable sensor performance and needs to be accordingly considered.

2. Experimental Section

2.1. Materials

TEOS (≥99%), acetonitrile (ACN, 99.9%), cyclohexane (99.5%), and Ruphen ([Ru­(phen)₃]­Cl₂, 98%) were purchased from Sigma-Aldrich (Germany), while ethanol (EtOH, absolute, 99.9%) was purchased from Labsolute (Th. Geyer, Germany), l-arginine (≥98.5%) from Carl Roth GmbH (Germany), and ammonia (25%) from abcr GmbH (Germany). All chemicals, reagents, and solvents were used as received, unless otherwise stated. All aqueous solutions utilized for the syntheses of the SiO₂ NPs were prepared with Milli-Q-water (0.055 μS m^–1; Merck Milli-Q IQ 700). All materials required for the synthesis of the molecular O₂-sensitive luminophores Ruphenpy ([Ru­(phen)₂(phenpy)]­Cl₂) as well as Crddpd ([Cr­(ddpd)₂]­(BF₄)₃) and the synthesis procedures are given in the Supporting Information (Section S3. Particle Synthesis and Characterization).

2.2. Methods

Dynamic light scattering (DLS) and zeta potential (ζ-potential) measurements were performed using a Zetasizer Nano ZS (Malvern Panalytical, Germany) equipped with a 630 nm laser. Transmission electron microscopy (TEM) images were recorded on a F200S microscope (ThermoFisher Scientific, USA), while high-resolution TEM (HR-TEM), high-angle annular dark-field STEM (HAADF-STEM), and energy-dispersive X-ray (EDX) spectroscopy were carried out on a JEOL JEM-2800 S/TEM at the Electron Microscopy Facility at IST Austria. More details on the DLS and TEM measurements and data evaluation are provided in the Supporting Information.

PL spectra and PL decay curves used to determine the O₂ shielding efficiency of the two different silica networks were obtained with a calibrated FSP920 fluorescence spectrometer (Edinburgh Instruments, UK) at room temperature (r.t.; T = 22 ± 2 °C) and fitted using FAST software (Edinburgh Instruments, UK), employing X ² as the quality criterion for the fit evaluation for the Cr^III complex in solution and incorporated in silica NPs. For the measurement of the shorter PL decay times of Ruphen and Ruphenpy in solution at r.t., the lifetime spectrofluorometer FSP920 (Edinburgh Instruments, UK) was used. A detailed description of the fitting/analysis procedure used for the multiexponential PL decay kinetics of Ruphenpy@ARGSiO ₂ and Ruphenpy@STOSiO ₂ is provided in the Supporting Information (Section S4. Optical properties, subsection S4.1 Methods). All temperature-dependent PL spectra measurements were recorded with the spectrofluorometer FSP920 over a temperature range of 5–85 °C, using an equilibration time of 12 min for each temperature. The time interval between the temperature cycles did not exceed 20 min. The calculation of the thermometric performance parameters luminescence intensity ratio (LIR), relative sensitivity (S_r), and thermal resolution or temperature uncertainty (ΔT) is detailed in the Supporting Information (Section S5. Temperature sensing properties).

2.3. Synthesis of the Crddpd, Ruphen and Ruphenpy-Stained Core and Core/Shell SiO₂ NPs

Details on the syntheses of the SiO₂ NPs stained with Crddpd, Ruphen, and Ruphenpy and their structure-analytical and spectroscopic characterization are summarized in Figure and in the Supporting Information (Figures S4 and S5). For Ruphen, we solely focused on l-arginine-type SiO₂ NPs. The synthesis of Crddpd was previously reported. ,

3. Results and Discussion

Before exploring the general applicability of our previously developed complete oxygen shielding strategy relying on l-arginine silica networks to other transition metal complexes such as Ruphen and dyad Ruphenpy (Figure ), we first determined the PL decay kinetics of both Ru^II compounds, displaying broad MLCT-type emission bands with maxima around ∼600 nm upon 450 nm excitation in air-and in Ar-saturated solution to evaluate our O₂ removal procedure by flushing with Ar for 60 min. Time-resolved PL measurements (405 nm excitation) show monoexponential decays for Ruphen with lifetimes of ∼443 ns and ∼1 μs in air- and Ar-saturated water (Supporting Information, Figure S31a,b). The PL decay kinetics of Ruphenpy in aqueous solution are monoexponential with PL lifetimes of ∼1.6 μs and ∼57 μs in air- and Ar-saturated aqueous solution, respectively (Figure j). The long unquenched PL lifetime of Ruphenpy originates from a mainly pyrene-localized triplet state, populated by fast intramolecular Dexter-type energy transfer from the initially excited Ruphen unit in Ruphenpy (Figure , middle). , The good match of the PL lifetime data in Ar-saturated solution with the previously reported lifetimes of 1 and 52.6 μs confirms the suitability of our O₂ removal procedure. These PL lifetimes were subsequently used as reference values for Ru^II compounds, which are effectively protected from O₂ quenching.

2.

HR-TEM images of SiO₂ NPs prepared with the l-arginine approach (ARGSiO ₂ NPs) loaded with Ruphen (Ruphen@ARGSiO ₂ , top) and Ruphenpy (Ruphenpy@ARGSiO ₂ , bottom). Scale bars: 50 nm. (b,e) TEM particle size distributions of the Ruphen and the dyad Ruphenpy loaded l-arginine SiO₂ NPs derived from log–normal fits, yielding mean diameters of 26 ± 3 nm for Ruphen@ARGSiO ₂ and 22 ± 3 nm for Ruphenpy@ARGSiO ₂ . (c,f) Normalized PL spectra of Ruphen@ARGSiO ₂ and Ruphenpy@ARGSiO ₂ recorded under atmospheric (air-saturated, dark green) and anaerobic conditions (Ar-saturated, pink). The corresponding intensity ratios are I _Ar/I _air = 1.0 and 1.1. (g) HR-TEM image of SiO₂ NPs prepared with the Stöber approach (STOSiO ₂ NPs) loaded with Ruphenpy (Ruphenpy@STOSiO ₂ ). Scale bars: 50 nm. (h) TEM particle size distributions of Ruphenpy-loaded Stöber SiO₂ NPs derived from Gaussian fits yielding mean diameters of 24 ± 3 nm for Ruphenpy@STOSiO ₂ (i) Normalized PL spectra of Ruphenpy@STOSiO ₂ recorded under atmospheric (air-saturated, dark green) and anaerobic conditions (Ar-saturated, pink). The corresponding intensity ratios are I _Ar/I _air = 1.5. (j–l) PL decay kinetics of Ruphenpy (j) in water under atmospheric and anaerobic conditions (inset) and encapsulated in (k) l-arginine-type SiO₂ NPs, and (l) Stöber-type SiO₂ NPs in air- and Ar-saturated solution.

3.1. Assessing the Versatility of our SiO₂-Encapsulation Strategy

Next, we incorporated Ruphen and Ruphenpy into an amorphous, nonporous SiO₂ core and core/shell NPs using the respective chloride salts and the l-arginine-mediated biphasic sol–gel synthesis route, which had provided complete oxygen protection for the Cr^III complexes [Cr­(ddpd)₂]­(PF₆)₃ and [Cr­(ddpd)₂]­(BF₄)₃. We also prepared Crddpd-stained SiO₂ NPs for comparative studies on the luminescence sensing behavior of metal complex-stained SiO₂ NPs presented in a later section. The resulting metal complex-stained SiO₂ NPs were characterized by electron microscopy, DLS, and zeta potential measurements, revealing TEM average particle diameters of 26 ± 3 nm, 22 ± 3 nm, and 23 ± 3 nm for the Ruphen, Ruphenpy, and Crddpd-stained l-arginine-mediated SiO₂ NPs, respectively, along with a narrow size distribution, near-spherical morphology, and a smooth surface (Figure panels a,b,d,e,g,h and Figures S6a,S8a,S9a in the Supporting Information). The strongly negative zeta (ζ)-potentials of the Ruphen, Ruphenpy, and Crddpd-stained l-arginine SiO₂ NPs samples (−22.1 ± 1.2 mV, −22 ± 1.2 mV, and −23.8 ± 2.5 mV, respectively; Figures S12,S13 and S15 and Table S3 in the Supporting Information) indicate a high colloidal stability under the applied conditions. The unstained l-arginine SiO₂ particles exhibited an average diameter of 24 ± 1 nm and a ζ-potential of −21.3 ± 3.9 mV. Interestingly, incorporation of Ruphen, Ruphenpy, and Crddpd barely affects the size, morphology (shape/surface roughness), and the negative surface charge of the SiO₂ NPs. In addition, the negative ζ-potential values support the effective removal of free, positively charged Ru^II and Cr^III complexes via dialysis. In the present study, we did not determine pore-size distributions for all luminophore-loaded SiO₂ as N₂ sorption/BET analysis requires comparatively large amounts of dried material, which was not available for all stained nanoparticle samples. Moreover, the SiO₂ NPs prepared by the Stöber and l-arginine routes are amorphous, non templated SiO₂ nanomaterials and are not expected to contain ordered mesopores, as supported by our previous BET measurements of similarly prepared silica NPs.

In addition, dyad Ruphenpy was embedded into SiO₂ NPs using the conventional Stöber-type silica synthesis (Ruphenpy@STOSiO ₂ , d_TEM: 24.4 ± 2.9 nm) commonly used for the incorporation of such metal complexes into silica networks for comparison to literature systems , and to our previously reported Cr^III-stained Stöber-type SiO₂ NPs. Also, unstained Stöber SiO₂ NPs (US-STO@SiO ₂ , d_TEM: 24 ± 3 nm, ζ-potential of −32.1 ± 4.4 mV; synthesis scheme shown in the Supporting Information, Figure S1) were prepared following the same synthesis protocol to confirm that the particle size and size distribution of the SiO₂ NPs are unaffected by particle staining. The very negative ζ-potential of Ruphenpy@STOSiO ₂ of −36.3 ± 0.7 mV (Supporting Information, Figure S14 and Table S3) indicates a high colloidal stability and complete removal of free positively charged Ru^II complexes by particle purification via dialysis. The observed differences in ζ-potential of the SiO₂ NPs prepared by the l-arginine and Stöber method are attributed to the different surface chemistry resulting for the two sol–gel synthesis routes. Stöber SiO₂ particles are formed by ammonia-catalyzed hydrolysis and condensation of TEOS in EtOH/H₂O, whereas the l-arginine-derived particles are formed in a biphasic water/cyclohexane system using l-arginine as catalyst. These different reaction conditions can affect the number and accessibility of negatively charged silanolate groups at the particle surface.

Successful loading of the l-arginine and Stöber SiO₂ NPs with the Ru^II and Cr^III complexes was confirmed by ICP–MS measurements of dissolved luminophore-stained SiO₂ NPs provided in the Supporting Information (Tables S1 and S2). The number of incorporated luminophores was estimated from the Cr or Ru content determined by ICP–MS, assuming one metal center per complex. Thereby, the amount of complex was divided by the calculated number of NPs, derived from the particle mass concentration, TEM diameter, and silica density. The incorporation efficiency was determined by comparing the amount of detected complex after purification with the amount initially used for synthesis. EM combined with EDX (Supporting Information, section on electron microscopy (EM) measurements) was employed in addition but not used for the determination of luminophore loading concentrations as the Ru Lα line is difficult to distinguish in EDX due to its low energy and its close proximity to the Cl Kα line at about 2.62 keV. Determination of the encapsulation efficiency of the Ruphen, Ruphenpy, and Crddpd in the l-arginine SiO₂ NPs by ICP–MS (Table S2 in the Supporting Information) reveals about 2, 0.5, and 1 luminophores per SiO₂ NP on average. This corresponds to an overall loading (incorporation) efficiency of about 19%, 29%, and 64% for Ruphen@ARGSiO ₂ , Ruphenpy@ARGSiO ₂ , and Crddpd@ARGSiO ₂ , respectively. Successful loading of the Stöber SiO₂ NPs with Ruphenpy was also confirmed by ICP–MS measurements (Supporting Information, Tables S1 and S2), yielding on average about 5 complexes per SiO₂ NP. This equals a loading or incorporation efficiency of about 33%.

Next, the PL properties of the luminophore-stained SiO₂ NPs and the O₂ protection efficiency of the luminophores in the SiO₂ NPs were assessed and quantified by steady-state and time-resolved PL measurements in air and Ar-saturated dispersions at room temperature.

As detailed in the Supporting Information (Section 4.1 methods), the O₂ shielding efficiency of the different SiO₂ NPs was quantified by comparing the ratio of the steady state and time-resolved PL signals measured for the same samples under ambient air and after purging with argon (Ar-saturated dispersions), with the latter serving as benchmark values for the respective nonquenched luminophore in a deoxygenated environment. For samples exhibiting multiexponential decay, the shielding efficiency is calculated from the integrated areas of the PL decay curves rather than from individual lifetime components. The PL lifetimes of the molecular systems in Ar-saturated solution were employed as reference values for completely protected Ru^II compounds.

The emission spectra of the molecular systems and Ruphen@ARGSiO ₂ , Ruphenpy@ARGSiO ₂ , and Ruphenpy@STOSiO ₂ in air- and Ar-saturated solution are shown in Figure . Encapsulation in l-arginine and Stöber SiO₂ NPs results in slight spectral shifts of the emission bands of Ruphen and Ruphenpy from about 600 nm to about 590 nm (Supporting Information, Figure S38). This suggests small environment-induced changes in the energy landscape of the SiO₂-matrix encapsulated Ru^II compounds and increased ³MLCT energies by about 0.035 eV. The size of the PL intensity changes upon O₂ removal correlates with the O₂ sensitivity of Ruphen and Ruphenpy. Whereas a conventional Ru^II complex such as Ruphen reveals only a 2-fold enhancement (I _Ar/I _air ≈ 2) in Ar-saturated aqueous solution compared to air-saturated solution, the dyad shows a ∼20-fold higher luminescence intensity (I _Ar/I _air ≈ 20) upon O₂ removal. Encapsulation in l-arginine-type SiO₂ NPs effectively protects both complexes from O₂-induced PL quenching as revealed by the nearly identical PL intensities of Ruphen@ARGSiO ₂ and Ruphenpy@ARGSiO ₂ in air- and Ar-saturated solution (Figure , panels (c) and (f)), and the representatively shown PL decay curves closely match with the PL decay kinetics of Ruphenpy@ARGSiO ₂ in the presence and absence of O₂ (Figure k). From the measured PL intensity ratios of the molecular Ru^II compounds in Ar-saturated solution (I _Ar) and the encapsulated Ru^II compounds in air-saturated solution (I _air) of 1.01 and 1.1, the O₂ protection efficiency of the l-arginine-type silica network was calculated to 99.0% and 90.9% for Ruphen@ARGSiO ₂ and Ruphenpy@ARGSiO ₂ , respectively. In contrast, Stöber-type SiO₂ NPs provide only partial shielding of the Ru^II complexes from O₂ quenching, as reported in the literature for such silica networks. , For Ruphenpy@STOSiO ₂ , the intensity ratio I _Ar/I _air of 1.5 corresponds to an O₂ protection efficiency of 69.4%, confirming the previously observed reduced O₂ protection efficiency of this type of silica network for the Cr^III complex [Cr­(ddpd)₂]­(PF₆)₃ of 50%. Time-resolved PL measurements of air-saturated aqueous dispersions of Ruphen@ARGSiO ₂ reveal biexponential decay kinetics of encapsulated Ruphen and yield an average PL decay time of 3 μs. This value exceeds the previously reported lifetime of Ruphen of 1.1 μs in Ar-saturated water by a factor of almost three and implies complete O₂ shielding in the l-arginine-type SiO₂ NPs. The additional enhancement is tentatively attributed to the rigid silica matrix and the rigidification of Ruphen prolonging the PL lifetime. For time-resolved PL measurements of Ruphenpy in different environments, the time window to record the full PL decays had to be considerably expanded as O₂ removal and encapsulation into silica matrix led to an increase of the time window from about 20 μs in air-saturated water to about 1 ms for the free dyad in Ar-saturated solution and about 10 ms for Ruphenpy@ARGSiO ₂ in air (Figure , panels (j) and (k)). This reflects the strongly prolonged PL lifetimes under conditions where PL quenching by O₂ is prevented. As displayed in Figure (panel (k)) for Ruphenpy@ARGSiO ₂ , multiexponential PL decay kinetics are obtained (4-exponential reconvolution fit was applied) that indicate a distribution of emitting species or microenvironments rather than a single well-defined species/environment. The four-exponential fits present empirical descriptions of the heterogeneous decay kinetics. The different PL components are not assigned to specific molecules or environments within the SiO₂ NPs as they may arise from several effects. This includes different pyrene–metal complex separations, pyrene/Ru complex conformations, dye localization, aggregation state, and heterogeneous environments within the SiO₂ NPs. Therefore, no attempts were made to calculate O₂ protection efficiencies from PL lifetime measurements, and PL lifetime measurements were not used for temperature-sensing studies detailed in the following section. Instead, we compared the integrated areas under the PL decay curves measured in air and under Ar atmosphere to derive O₂ protection efficiencies from time-resolved PL measurements. This approach, which was also employed for lifetime-based temperature measurements, was later validated by comparison with PL intensity measurements and the results of fitted PL decay kinetics. The calculated Ar/air ratio of 0.99 derived from the almost identical PL decay profiles in the presence and absence of O₂ indicates complete protection from PL quenching by O₂. In contrast, for Ruphenpy encapsulated in Stöber-type SiO₂ NPs (Ruphenpy@STOSiO ₂ ), the integrated PL decay areas markedly differ between Ar- and air-saturated dispersions, yielding an Ar/air ratio of 1.9 (Figure , panel (l)). This corresponds to an O₂ shielding efficiency of about 50%. Please note that the O₂ shielding efficiency calculated from the integrated PL decay curves and from steady-state PL measurements is expected to yield values that closely match within the respective measurement uncertainties, but not identical values. The uncertainties of these measurements are estimated to be in the order of 5%.

As in our previous work, Ruphenpy-containing core–shell architectures were also prepared by l-arginine-mediated silica regrowth, yielding either core-loaded particles with an unstained shell (Ruphenpy@ARGSiO ₂ )@(US-ARGSiO ₂ ), or unstained cores with a Ruphenpy-containing shell (US-ARGSiO ₂ )@(Ruphenpy@ARGSiO ₂ ) (see Supporting Information for DLS: Figure S11; ζ-potential: Figure S16; TEM: Figures S26–S28). ICP–MS revealed a more efficient Ruphenpy incorporation during shell growth, with an average loading concentration of 27 complexes per particle for the shell-stained sample. Ar/air comparisons showed highly efficient O₂ shielding for these particles, with ratios in the measured PL intensity or integral PL decay of about 99% as derived from steady-state intensity and time-resolved decay-area analysis (Figure S30a,c). For the core-loaded particles coated with an unstained shell, the steady-state air/Ar signal ratio amounted to about 95% and 84% (Figure S30b,d) for the integral PL intensity and PL decay curves. Possible reasons for the lowered protection for (Ruphenpy@ARGSiO ₂ )@(US-ARGSiO ₂ ) could be the trapping of residual O₂ in the SiO₂ NPs during SiO₂ core formation or changes in the local luminophore environment caused by the additional shell-growth step.

3.2. Influence of l-Arginine SiO₂ NPs Encapsulation on the Photostability of the Ru^II Complexes

The molecular ruby Crddpd is characterized by an outstanding inherent photostability, such that decomposition issues are essentially absent even upon prolonged irradiation with intense light sources. , In contrast, tris­(diimine) complexes of Ru^II are prone to photodegradation. Mechanistically, this is caused by dissociation of the bidentate ligand upon thermal population of higher-lying metal-centered triplet states ³MC, see energy diagram in the right panel in Figure b. − As the resulting bis­(diimine) solvent ruthenium­(II) complexes do not show the characteristic ³MLCT emission after photosubstitution, the loss in PL intensity can be used to monitor complex photostability. − The photostability studies were performed with a beam-expanded blue cw laser to ensure well-defined homogeneous excitation at an intensity that does not permit two-photon absorption processes (447 nm, 0.5 W cm^–2, 20 °C, see Supporting Information for a description of the setup and experimental details). In these measurements, the ³MLCT emission at ∼600 nm was monitored for diluted aqueous Ru^II complex solutions as a function of illumination time. As first experiments with Ruphenpy in air-saturated solution indicated the formation of a new emitting species resulting from the reaction between Ruphenpy and reactive oxygen species produced by the quenching of the Ru^II-centered triplet state by O₂ (Figures S35–S37), all subsequent photostability measurements were performed in Ar-saturated solution under conditions that ensured a homogeneous light absorption and circumvention of an illumination-induced increase in temperature (the absorption at the laser excitation wavelength was always below 0.1, see Supporting Information for the corresponding UV/vis spectra). Under these conditions, the decrease in ³MLCT luminescence directly reflects the relative photodegradation quantum yields. CW laser illumination of Ruphen in aqueous solution for 110 min leads to a reduction in PL intensity by about 50%, indicating efficient photodecomposition. However, similar photostability studies with Ruphen encapsulated in l-arginine-type SiO₂ NPs, revealed a considerably enhanced photostability of Ruphen by a factor of more than five as reflected by a PL intensity decrease of only 10% under similar conditions (Figure a).

3.

(a) PL stability measurements of nonencapsulated (3 μM) and l-arginine SiO₂–NP-encapsulated Ruphen and Ruphenpy complexes in Ar-saturated aqueous solutions/dispersions under cw laser excitation at 447 nm (0.5 W cm^‑2) using the setup shown in Figure S32. The temporal evolution of the normalized emission intensity at 590 nm for Ruphen@ARGSiO ₂ and at 605 nm for nonencapsulated Ruphen, Ruphenpy, and encapsulated Ruphenpy@ARGSiO ₂ solutions/dispersions during illumination. (b) The simplified energy-level diagram of Ruphenpy, indicating the deactivation pathways and photodegradation following photoexcitation. The corresponding UV/vis absorption spectra are shown in the Supporting Information. The concentration of the nonencapsulated Ru^II compounds used as chloride salts was 3 μM.

Ruphenpy is even much less photostable than Ruphen as the efficiency of the photodegradation of Ru^II complexes by ligand dissociation frequently increases with the triplet lifetime. , As depicted in Figure b, the lowest triplet state of Ruphenpy is a long-lived pyrene-localized ³(π–π*) state, which is in a fast equilibrium with the emissive ³MLCT state at room temperature, that is the starting point for the thermal population of dissociative ³MC states. ,, Photostability studies with Ruphenpy in Ar-saturated solution and encapsulated in l-arginine NPs also revealed a beneficial effect of the silica network on dyad photostability. Under similar conditions as used for the photostability studies of Ruphen, the dyad shows 70% photodegradation (Figure a). The photodegradation was reduced by a factor of 2 (35% instead of 70%, see Figure a) and Figures S33 and 34 in the Supporting Information for Ruphenpy incorporated in l-arginine silica NPs. This is very encouraging, considering the extremely long lifetime of the Ru^II-dyad under these conditions. Overall, our findings agree well with photostability studies of well-explored [Ru­(bpy)₃]²⁺ (bpy = 2,2'-bipyridine) and related compounds incorporated into SiO₂ NPs. ,, A likely explanation for the significantly improved photostability is the rigid structure of the silica network, which considerably hampers elongation of the Ru–N bonds required for ligand dissociation and facilitates recoordination of dissociated ligands. These findings demonstrate that encapsulation in l-arginine-type SiO₂ NPs improves the photostability of photolabile Ru^II compounds, which, together with the strongly increased PL efficiency in air, improves and expands the application potential of such Ru^II complexes. Based on the excellent O₂ shielding efficiencies of Ruphen@ARGSiO ₂ and Ruphenpy@ARGSiO ₂ (shown in Figure ), we believe that ROS-induced decomposition, which has been observed for the unprotected dyad in homogeneous solution, see Supporting Information, Figure S36) only plays a minor role for Ruphenpy encapsulated in l-arginine-type SiO₂ NPs. Therefore, we regard the above-mentioned ligand loss as the only decomposition pathway for the encapsulated compounds.

3.3. Luminescence Temperature Sensing with Nonencapsulated and SiO₂-Embedded Ruphen and Ruphenpy in the Presence and Absence of Oxygen

Transition metal compounds such as Ru^II complexes are frequently used for contactless luminescence-based measurements of local temperature in air-saturated environments in the life and material sciences, thereby exploiting temperature-induced changes in PL intensity and lifetime, governed by the temperature dependence of PLQY in the respective environment. , To minimize PL quenching by O₂, which can interfere with temperature sensing, Ru^II compounds are typically encapsulated in O₂ shielding matrices. ,,, Literature data show that SiO₂-based matrices often do not provide efficient, i.e., complete O₂ shielding. In several sol–gel/ormosil systems, the reported Stern–Volmer constants indicate a substantial O₂ accessibility of the ruthenium luminophore to O₂ PL quenching. This makes these systems better suited for O₂ sensing than for O₂-independent thermometry. In contrast, Wencel et al. reported almost O₂-insensitive TEOS-based SiO₂ NPs, which exhibit PL intensity ratios of I ₀/I ₂₀ = 1.01, corresponding to a 99% emission retention at an O₂ concentration of 20% (oxygen fraction in the O₂/N₂ gas mixture applied to the aqueous nanoparticle suspension), while TEOS–MTEOS hybrid particles showed a higher O₂ permeability. Polymer matrices such as PVA and PCA were reported to effectively suppress O₂ cross-sensitivity. In these cases, the observed O₂ shielding was only qualitatively described rather than by Stern–Volmer constants.

However, the O₂ shielding efficiency of most matrices has not been quantified and is often not complete, i.e., quantitative. In addition, also a possible influence of the encapsulation chemistry and the matrix’s composition, structure, and rigidity on luminophore temperature sensitivity has to be considered. To explore the applicability of our SiO₂ shielding approach for PL thermometry with O₂-sensitive molecular compounds in air and to assess possible matrix effects arising from the different silica networks, we investigated the temperature dependence of the PL properties of free and SiO₂ NP-encapsulated Ruphen and Ruphenpy. Nanoparticles prepared by both the l-arginine and Stöber sol–gel routes were studied over the temperature range of 5–85 °C by steady-state and time-resolved PL spectroscopy in air- and Ar-saturated solutions. All measurements were performed in three successive heating–cooling cycles of thermally equilibrated samples. Deliberately, no measures were taken to ensure that the initial O₂ concentration present for the first cycles with air-saturated solutions was restored for the starting conditions of the lowest temperature of 5 °C. These experimental conditions were chosen to model those used for temperature sensing in many life sciences studies, in which the PL response of the O₂-sensitive luminophore is affected by temperature as well as by O₂ concentration, and to highlight the importance of complete O₂ shielding. Please note that the temperature-dependent solubility of O₂ in water and other environments leads to a steady decrease of O₂ concentration with increasing temperature, resulting in reduced PL quenching. Simultaneously, the increase in temperature favors a reduction of luminophore PLQY and PL lifetime. For temperatures ≥65 °C, O₂ is expected to have almost completely disappeared from solution. These opposite effects of temperature and temperature-dependent O₂ concentration affect the measured PL response of O₂-responsive luminescence thermometers, especially in the first heating cycle, while in the second and third heating cycles, the O₂ concentration is reduced compared to the first heating cycles as back diffusion of O₂ into solution is slow. Hence, the influence of O₂ sensitivity and O₂ shielding efficiency on PL temperature sensing is best reflected by the results of the first heating cycle and the comparison of air- and Ar-saturated solutions and l-arginine and Stöber type SiO₂ NPs. As highlighted in Figure for Ruphenpy, chosen for detailed investigation across different environments, the third heating cycle and the corresponding measurements of the nonencapsulated compound under an Ar atmosphere provide insights into the influence of O₂, the shielding efficiency of the SiO₂ matrix, and matrix effects on the dyad́s temperature sensitivity. The reversibility of the samples follows, e.g., from a comparison of the PL spectra and PL intensities of the O₂-protected systems. Temperature-dependent PL studies of less O₂- sensitive Ruphen embedded in l-arginine SiO₂ NPs, recorded over three consecutive heating cycles, are provided in the Supporting Information (Figures S56 and S57) and reveal similar trends.

4.

(a–d) Temperature-dependent PL spectra of the Ruphenpy complex in H₂O/air recorded over three consecutive heating–cooling cycles. The corresponding integrated emission areas for each cycle are provided in the right column. (e–h) Analogous data for H₂O/Ar. (i–l) Temperature-dependent PL spectra and integrated emission intensities of Ruphenpy@ARGSiO ₂ in aerated aqueous dispersion; (m–p) corresponding data for Ruphenpy@STOSiO ₂ . The integration limits were set from 500 to 800 nm. Aiming for a comparison of environment effects on the temperature dependence of the integral PL of the Ruphenpy complex in different environments, we chose a constant wavelength scale to assess the temperature-dependence of the integral PL of the Ruphenpy complex in different environments and not an energy scale. The equilibration time for each temperature was 12 min. After returning to 5 °C, a break of about 20 min was applied before starting the next heating–cooling cycle.

3.3.1. PL Intensity-Based Temperature Sensing

The temperature-dependent PL intensity measurements with the strongly O₂-responsive dyad Ruphenpy in different environments (Figure ) suggests two competing effects: thermally induced PL quenching, which decreases the emission intensity, and a temperature-induced decrease in dissolved O₂ concentration, which reduces O₂ quenching. The latter results in an increase in PL intensity. This follows from a comparison of the initial and finally reached PL intensities for the three sequentially performed measurement cycles for temperatures ≤65 °C.

The influence of O₂ is most pronounced for the first heating cycle for conditions, where Ruphenpy is not completely protected from O₂ quenching, i.e., for the dyad in air-saturated water and encapsulated in Stöber-type SiO₂ NPs and for temperatures between 5 °C and≤ 65 °C, where the solubility of O₂ is higher compared to temperatures ≥65 °C. Due to heating-induced O₂ depletion, the PL intensities steadily increase from cycle to cycle for temperatures between 5 °C and ≤65 °C and are already identical for the second and third heating cycles. For temperatures ≥65 °C, the PL intensities match across all heating cycles. On the contrary, under conditions where Ruphenpy is completely protected from O₂ quenching, i.e., for measurements under an Ar atmosphere or for Ruphenpy encapsulated in l-arginine-type SiO₂ NPs, the PL intensities do not vary between cycles. The minimal changes in initial PL intensity observed for the former system are ascribed to traces of O₂ introduced by back diffusion of O₂ due to the long measurement time of about 13 h for all three cycles.

The temperature-dependent changes in integral PL provided in the right panels of Figure , derived from the three heating cycles, also highlight the strong influence of O₂ on PL temperature sensing for conditions where the PL of Ruphenpy can be quenched by O₂. Especially promising for PL temperature sensing is the almost linear temperature dependence of the PL intensity of Ruphenpy@ARGSiO ₂ , see also next section. Overall, the generally observed matching PL intensities and PL spectra for temperatures ≥65 °C for all three heating cycles and the good match of the PL data at lower temperatures, e.g., at 5 °C reached for cycles 2 and 3 in Figure indicate a good thermal stability of the dyad and the dyad-stained SiO₂ NPs. In addition, the results, representatively displayed in Figure for Ruphenpy, clearly demonstrate the importance of separating temperature and oxygen-induced PL changes in PL intensity for PL thermometers with O₂-sensitive single-band luminescence.

To facilitate a comparison of the measured PL intensity-based temperature responses, the integrated PL in Figure d,h,l,p were normalized to the value at 5 °C for each individual heating cycle. The relative decrease between 5 and 85 °C was calculated using eq 11 (Supporting Information, Section S5.3). The normalized plots are shown in Figure S54e–h, and the extracted values are summarized in Table S15. These results confirm the strong influence of the surrounding medium on Ruphenpy’s intensity-based PL response. In air-saturated water, the first cycle shows an apparent intensity increase, corresponding to a relative change of −17%, while cycles 2 and 3 stabilize at 40–43%. In contrast, Ruphenpy@ARGSiO ₂ displays a highly reproducible response of 69–70% over all cycles. Ruphenpy@STOSiO ₂ shows a stronger but slightly less stable apparent decrease of 75–81%. The largest relative decrease is observed in Ar-saturated water, reaching 94–96%; however, the requirement for strict O₂ exclusion limits the practical applicability of the unprotected dyad under these conditions.

3.3.2. Lifetime-Based Temperature Sensing

For single-emission-band sensors lacking an internal reference, PL lifetime sensing is often preferred over PL intensity measurements since the PL intensity can vary with excitation power, collection efficiency, and luminophore concentration. However, in O₂-responsive systems, O₂ quenching affects the PL lifetime in the same way as PL intensity, implying that a PL lifetime readout is likewise susceptible to changes in dissolved O₂ concentration. Therefore, quantitative O₂ shielding is also essential for reliable lifetime-based PL thermometry. However, in more complex environments such as SiO₂ NPs, luminophores often reveal complex decay kinetics due to the heterogeneous luminophore microenvironment (see: PL decay curves of Ruphenpy embedded in l-arginine SiO₂ NPs shown in Figure k and in the Supporting Information, in Figures S59 and S60). For luminophores in matrices providing complete O₂ protection, such as l-arginine SiO₂ NPs, this can hamper simple PL lifetime sensing approaches, which commonly require monoexponential decay kinetics. For Ruphenpy@ARGSiO ₂ in aerated aqueous dispersion, we pursued two approaches. One approach relies on average PL lifetimes ⟨τ⟩ calculated from 4-exponential reconvolution of the fitted PL decay curves measured at different temperatures (Supporting Information, Section S4.1 methods subsection in Section 4. Optical properties). The results are displayed in Figure a; please note that this fitting approach presents only an empirical description of the PL decay shape, and we therefore did not attempt to assign the obtained lifetime components to luminophore molecules in a specific environment or under specific conditions. In addition, we explored the applicability of integral PL decay curves derived from the integration of the normalized PL decay curves measured at different temperatures. While ⟨τ⟩ primarily reports on the kinetic time scale, the integrated PL decay area is directly related to the total number of detected photons and correlates with PL QY for identical excitation and detection conditions. The results of this simpler, yet underexplored approach are shown in Figure b. As follows from Figure , both approaches yield closely matching trends and agree well across repeated heating cycles. Also, both approaches closely match the trend observed for the integral PL spectra shown in Figure (panel (l)). This is demonstrated in Figure c, which displays the linear correlation between the integral PL intensities and integral PL decay curves. This confirms the applicability of our integral PL decay curve approach.

5.

Lifetime-based temperature sensing with Ruphenpy@ARGSiO ₂ in air-saturated solution. (a) average decay time ⟨τ⟩ derived from a 4-exponential reconvolution fit. (b) Integral PL decay curves obtained by integrating the area below the normalized PL decay curves. (c) Correlation between the temperature dependence of the integral PL decay curves and the integral PL intensities measured for three heating cycles.

3.4. Luminescence Temperature Sensing with Dual Emissive Cr^III Complexes in the Presence and Absence of Oxygen

Especially attractive for contactless, minimally invasive PL temperature measurements are so-called Boltzmann thermometers, which rely on the temperature-dependent relative population ratio of two closely spaced, thermally equilibrated excited electronic states of a luminescent material. The energy difference (ΔE) between these spectrally distinguishable emissive states is commonly in the order of the thermal energy. The PL bands for these two close-lying states must be sufficiently small to allow for reliable discrimination. This provides the basis for ratiometric PL temperature measurements, directly probing the temperature-dependent population distribution of these excited states governed by the Boltzmann distribution, independent of fluctuations in the luminophore concentration, overall PL intensity and excitation intensity. This is often only feasible in a limited temperature operating range or can require special fitting models to exploit a higher energy gap for high relative sensitivities at appreciably low temperatures. Examples of dual emissive Boltzmann thermometers that span the visible and NIR spectral range and are highly susceptible to temperature variations are lanthanide-based nanoparticles and phosphors containing, e.g., Er^III, Nd^III, and Pr^III as well as phosphors relying on Cr^III in strong ligand fields. ,,− However, molecular species rarely display such behavior. Exceptions include Cr^III complexes such as the first generation of the molecular rubies, where earth-abundant Cr^III is in a very strong ligand field and exhibits dual NIR PL over a wide temperature range. ,

Cr^III complexes with different counteranions and varying solubility have been previously examined by us in environments varying in O₂ solubility and permeability including organic and aqueous solvents, polystyrene nanoparticles (PSNP), and SiO₂ NPs prepared by the l-arginine and Stöber sol–gel synthesis routes. ,, Temperature sensing with dual emissive Cr^III complexes has been demonstrated by us before in different environments varying in O₂ concentration or permeability, as well as composition, morphology, and rigidity, ,, yet not in silica networks. Hence, we also assessed the temperature sensing performance of Crddpd in air- and Ar-saturated solution as well as encapsulated in l-arginine- and Stöber-type SiO₂ NPs in air-saturated dispersion in three analogously performed heating cycles and evaluated the corresponding changes in integral PL intensity.

As shown in Figure a–d, repeated heating–cooling cycles in environments allowing for O₂ quenching of the Crddpd luminescence revealed similar trends of the measured PL intensities, as previously observed for Ruphenpy. For Cr^III complexes, which are not completely shielded from O₂, the PL intensity strongly changed from cycle to cycle, albeit with intensity trends different from those as observed for the Ru^II dyad (Figure ). However, the spectrally well-separated PL bands at 738 and 775 nm are similarly affected, rendering ratiometric temperature sensing with Cr^III-based systems O₂-independent. However, the O₂ concentration-dependent decrease in the intensities of both PL bands of Crddpd at temperatures ≤65 °C is likely to reduce the relative sensitivity of temperature measurements with such Cr^III complexes, as detailed in a subsequent section. For Crddpd protected from O₂ quenching, i.e., under Ar atmosphere or encapsulated in l-arginine type SiO₂ NPs, the cycle-dependent intensity variations are strongly reduced compared with unprotected Crddpd in air-saturated water, although small residual differences can still be observed. These remaining differences may originate from thermal-history-related effects, minor changes in the local microenvironment during repeated heating, or, in the case of Ar-saturated water, traces of O₂ introduced during the long measurement time. To further quantify the magnitude and reproducibility of the intensity-based temperature response, the integrated PL-intensity areas were normalized to the value obtained at 5 °C for each heating cycle, and the relative decrease between 5 and 85 °C was calculated (Figure S54a–d and Table S15). In air-saturated water, the relative decrease increases from 64% to 78% over three cycles. This confirms a cycle-dependent intensity response. In contrast, the behavior of Crddpd@ARGSiO ₂ is reproducible with a constant decrease of 56% in all cycles. Crddpd@STOSiO ₂ exhibits a lower first-cycle response of 49%, followed by higher values of 67–69% in cycles 2 and 3. The most pronounced temperature-induced PL quenching is observed in Ar-saturated water, with an 86–87% decrease.

6.

Temperature-dependent PL spectra of the Crddpd complex in H₂O/air recorded over three consecutive heating–cooling cycles, with the corresponding integrated PL for each cycle. (e–h) Analogous data for H₂O/Ar. (i–l) Temperature-dependent PL spectra and integrated emission areas of Crddpd@ARGSiO ₂ in aerated aqueous dispersion. (m–p) Corresponding spectra and integral emission intensities for Crddpd@STOSiO ₂ in air. The integration limits were set from 720 to 820 nm. Aiming for a comparison of environment effects on the temperature dependence of the integral PL of the Cr^III complex in different environments, we chose a constant wavelength scale to assess the temperature-dependence of the integral PL of the Cr^III complex in different environments and not an energy scale. The equilibration time for each temperature was 12 min. After returning to 5 °C, a break of about 20 min was applied before starting the next heating cycle.

The corresponding Boltzmann plots (slopes: H₂O/air: −883 ± 2 K; H₂O/Ar: −885 ± 2 K), yielding energy gaps (ΔE) of 614.5 ± 0.5 cm^–1, are shown in the SI (Figures S40 and S42; Tables S4 and S5). Encapsulation in silica networks slightly affects the temperature response and the energy gaps. Crddpd@ARGSiO ₂ displays a slope of −824 ± 8 K and an energy gap ΔE of 573 ± 6 cm^–1, and Crddpd@STOSiO ₂ a slope of −859 ± 8 K and an energy gap of 597 ± 6 cm^–1. The Boltzmann slope scales with −ΔE and the less negative slopes of Crddpd@ARGSiO ₂ and Crddpd@STOSiO ₂ indicate a smaller effective energy separation between the thermally coupled states governing the readout band ratio. This likely reflects an influence of the microenvironment within the silica particles originating from modified crystal fields, and interactions with the heterogeneous silica network acting as counteranions for the Cr^III complex.

3.5. Thermometric Performance Parameters Luminescence Intensity Ratio, Relative Sensitivity

For PL (nano)­thermometry, ratiometric approaches are widely applied that are less affected by luminophore concentration compared to thermal sensing based on PL intensity measurements. Examples include lanthanide NPs, that exploit the PL of lanthanide ions with thermally coupled energy levels separated by an energy gap in the range of 200 cm^–1 to 2000 cm^–1, such as the two green-emitting energy levels of Er³⁺, which are referred to as Boltzmann thermometers. The thermometric performance of PL nanothermometers such as lanthanide NPs and Cr^III- and transition metal ion-containing phosphors is commonly quantified by different thermometric parameters. ,,− This includes the relative thermal sensitivity (S_r), which correlates the change in PL intensity ratio (LIR) with a temperature change and is used to assess and compare the performance of different PL (nano)­thermometers. In addition, the thermal resolution or temperature uncertainty (δT) is used. δT represents the minimum statistically achievable temperature change that can be resolved. However, for molecular PL thermometers, commonly LIR, S_r, and δT values are barely reported. Moreover, it is important to note that the overall achievable precision of a luminescent (Boltzmann) thermometer is a compromise between the appreciable relative sensitivity S_r and the signal-to-noise ratio from both emission bands.

3.5.1. Thermometric Performance of Crddpd in Different Environments

For the calculation of the thermometric figures of merit S_r, LIR, and δT for dual emissive Crddpd in air- and Ar-saturated solution and encapsulated in l-arginine- and Stöber-type SiO₂ NPs in air-saturated dispersion, shown in Figure (top), data derived from triplicate measurements of the first heating cycle (Figure ) were used because the variability between the consecutive heating cycles was larger than the replicate-to-replicate variation within a single cycle. This also enabled a direct comparison with the values subsequently derived for Ruphenpy. First, each PL spectrum of the Cr^III complex was normalized at 736 nm, the maximum of the high-energy PL band and then divided into two spectral windows I ₁(690 – 744 nm) and I ₂(745–840 nm). The LIR data were then calculated as the ratio of the subsequently integrated areas of I ₁ and I ₂. S_r, and δT were calculated as detailed in the Supporting Information. LIR, S_r, and δT values calculated from data obtained for all three consecutive heating–cooling cycles are provided in the Supporting Information (Figure S55).

7.

Top: temperature dependence of (a) the PL intensity ratio (LIR), (b) the relative sensitivity (S_r), and (c) the thermal resolution (temperature uncertainty, δT) of Crddpd in air- and Ar-saturated aqueous solution and encapsulated in l-arginine- and Stöber-type SiO₂ NPs dispersed in air-saturated water. The LIR, S_r, and δT values were calculated from triplicate measurements of the first heating cycle (Figure ). Bottom: temperature dependence of (d) LIR, (e) S_r, and (f) δT of Ruphenpy in air- and Ar-saturated aqueous solution and encapsulated in l-arginine and Stöber-type SiO₂ NPs dispersed in air-saturated water. The LIR, S_r, and δT values were calculated from triplicate measurements of the first heating cycle (Figure ).

As shown in Figure (top), dual emissive Crddpd exhibits nearly identical LIR values in all environments, with only slightly increased error bars observed for the Stöber system at temperatures exceeding 30 °C. This indicates that the ratiometric calibration of Crddpd is largely independent of O₂, because both PL bands are similarly affected by O₂ quenching. However, O₂ quenching still reduces the absolute PL intensity. This can influence the practical thermometric performance through a lower signal-to-noise ratio, reduced sensitivity, and increased uncertainty of PL-based temperature measurements, even if the LIR calibration remains nearly unchanged. The temperature dependence of the LIR follows the expected Boltzmann-type behavior for two thermally coupled emissive states (see Supporting Information, Figures S40–S44). For Crddpd, the S_r values in aqueous solution in air and under Ar atmosphere and embedded in Stöber-type SiO₂ NPs in air closely match, reaching values of about 1.2% °C^–1 at 5 °C and decreasing to about 0.7% °C^–1 at 85 °C. For Crddpd encapsulated in l-arginine–type SiO₂ NPs, the S_r curve decreases from about 1.0% °C^–1 at 5 °C to about 0.6% °C^–1 at 85 °C, respectively. These values are in the lower-to-mid range reported for Cr^III and Ru^II luminescence thermometers at ambient or higher temperatures with the slight differences in S_r (S_r = ΔE/(k _B T ²) originating from the slightly different local crystal fields in the different environments of Crddpd. The reduction in S_r of Crddpd in the l-arginine type silica network could possibly point to changes in the vibrational freedom in this matrix. For Crddpd in air- and Ar-saturated aqueous solutions, the δT values remain very small over the entire temperature range assessed. The δT values of Crddpd embedded in SiO₂ NPs are slightly higher, especially for Stöber-type SiO₂ NPs, and increase with increasing temperature. Overall nonencapsulated and SiO₂ NP-encapsulated Crddpd perform very well as luminescent molecular or nanothermometers. Temperature-dependent time-resolved PL studies (Supporting Information, Section S5.2) reveal that especially Crddpd in l-arginine–type SiO₂ NPs shows the smallest response to thermal quenching in the temperature range, together with an excellent reproducibility (Supporting Information, Figure S50).

A comparison of the different encapsulated Crddpd-based PL thermometers assessed in this study to established solid-state PL Boltzmann thermometers relying on Cr^III in a strong ligand field, such as ruby (α-Al₂O₃/Cr³⁺) or AlB₄O₆N/Cr³⁺, , reveals a similar performance of all these systems in terms of S_r in the temperature range of 0 to 85 °C. This reflects the similar energy separation between the two lowest-energy doublet states ²T₁(²G) and ²E­(²G) of Cr^III in these systems. Ruby and AlB₄O₆N/Cr³⁺ show a good brightness, which is relevant for the overall achievable (statistical) precision of a luminescent Boltzmann thermometer, and only a limited influence of thermal quenching over a very wide temperature range as reflected by the long radiative decay times τ_rad of the ²E­(²G) state of Cr^III of about 6 and 30 ms, respectively. This is beneficial for a wide dynamic working range. ,, Moreover, with respect to the time scale of thermalization, nonradiative coupling between the ²T₁(²G) and ²E­(²G) state of Cr^III is very fast ,, and outperforms classical Boltzmann thermometers relying on the green-emitting excited levels of Er^III. However, the much smaller size of molecular rubies and derived NP systems is advantageous for temperature sensing, especially in the life sciences. In addition, the more covalent nature of the Cr–N bonds in the complexes strongly reduces interelectronic repulsion and shifts the spin-forbidden narrow-line PL in complexes such as Crddpd to longer wavelengths compared to the R line PL in ruby and AlB₄O₆N/Cr³⁺ (λ_em ≈ 694 or 683 nm, respectively).

3.5.2. Thermometric Performance of Ruphenpy in Different Environments

Subsequently, we determined the parameters LIR, S_r, and δT from the measured temperature-dependent PL data of free and SiO₂ NP-encapsulated Ruphenpy measured in air-saturated dispersion using the data from the first heating cycle (Figure , bottom row). The data derived from all three consecutive heating cycles are shown in the SI in Figure S55. We utilized a pseudoratiometric spectral-window analysis for the single-band emitter Ruphenpy following the example of other luminophores with noncoupled energy levels (single-band ratiometric approach) such as Ag₂S quantum dots that even exhibit temperature-induced spectral shifts and changes in spectral bandwidth. , First, the temperature-dependent PL spectra were normalized to the integrated area (integration limits were set from 500 to 800 nm, wavelength scale was used for integration) of each PL spectrum to enable consideration of changes in spectral shape. Next, the wavelength at which the normalized PL spectra cross was determined (a near-isothermal crossing region), and the PL spectra were normalized to the PL intensity at this near-isothermal crossing region average point, which served as the boundary for defining two spectral regions (I ₁ and I ₂) for this single-emissive system (Supporting Information, Figure S39). LIR values were then calculated as I ₁/I ₂, while S_r and δT values were determined according to the equations given in the Supporting Information (Section S5). As displayed in Figure (bottom), the LIR and S_r data derived for the Ruphenpy systems reveal the same trends independent of O₂ PL quenching or O₂ protection, yet the values differ. The highest LIR values were obtained for Ruphenpy@ARGSiO ₂ in air-saturated dispersion, and the lowest values for the nonencapsulated dyad in Ar-saturated aqueous solution. The LIR values derived for the free dyad and Ruphenpy@STOSiO ₂ in air-saturated water, where O₂ PL quenching is feasible, are nearly identical within the experimental uncertainty. The highest S_r values are found for the dyad in water/Ar with S_r reaching about 0.7% °C^1– at 5 °C and 0.4% °C^1– at 85 °C. These results agree well with the performance of other previously reported Ru^II thermometers such as [Ru­(dpp)₃]²⁺ and [Ru­(bpy)₃]²⁺ in solution (dpp = 4,7-diphenyl-1,10-phenanthroline). − For the other three Ruphenpy NP systems, the S_r values are very similar, and there is no significant difference between the S_r values obtained for the l-arginine- and Stöber-type SiO₂ NPs. This is tentatively ascribed to similar interactions between Ruphenpy and the silica matrix for both SiO₂ NPs. Interestingly, the comparison of the temperature dependence of S_r for Ruphen@ARGSiO ₂ and Ruphenpy@ARGSiO ₂ (Supporting Information, Figure S56) reveals consistently higher S_r values for Ruphen@ARGSiO _2. The δT values obtained for Ruphenpy in different environments closely match, except for water/air, where larger uncertainties were observed at 40 °C, 45 °C, and 55 °C. This is tentatively ascribed to O₂ escape from solution.

3.6. Stability and Repeatability Studies with Ruphenpy and Crddpd

Finally, we assessed the repeatability of optical temperature measurements with Ruphenpy and Crddpd in different environments of varying O₂ quenching protection, utilizing the data displayed in Figures and for different temperatures and three consecutive heating–cooling cycles. The results, obtained for Crddpd (a–d) and Ruphenpy (e–h) in different environments from the normalized integrated PL at 5 °C (red), 60 °C (blue), and 85 °C (green), are displayed in Figure . The blue, green, and red symbols refer to the integrated PL intensity measured at the same temperature in each cycle, while the values given in percentage provide measures for the consistency of these repeated measurements. A value close to 100% implies that the signal is highly reproducible from cycle to cycle, whereas lower percentages indicate larger fluctuations and therefore poorer stability/reversibility under the respective conditions, in our cases due to the temperature-dependent solubility of O₂ and its removal within the repeated measurement cycles. Using this description, with values of about 95–99%, the l-arginine SiO₂ encapsulated samples exhibit the best repeatability for both luminophores, whereas the nonencapsulated luminophores in H₂O/air show the largest variability, particularly at 5 °C. O₂ removal by flushing with Ar or encapsulation in Stöber SiO₂ improves the performance of both luminescence thermometers, especially for Ruphenpy. Overall, optical temperature measurements with O₂-sensitive luminophores can be performed with high repeatability only for efficient O₂ shielding. In addition, the data shown in Figure confirm the high thermal stability of our luminescence thermometers.

8.

Cycle-to-cycle changes of the normalized integral emission at 5 °C, 60 °C, and 85 °C derived for Crddpd (a–d) and Ruphenpy (e–h) in different environments: H₂O/air, H₂O/Ar, l-arginine–mediated SiO₂/air, and Stöber SiO₂/air. These data provide estimates for the good stability and reversibility of these luminescence thermometers.

Also, we performed a similar analysis as well as aging studies, representatively for Crddpd using time-resolved PL measurements (Supporting Information, Section S5.2). The results derived from steady state and time-resolved measurements show very similar temperature-dependent trends (Figures d,h,i,p and S50a–d) despite the different acquisition times for PL spectra and PL decay kinetics. A reversibility test performed representatively for Crddpd@STOSiO ₂ (three cycles, reoxygenation with pressed air, followed by an additional heating cycle) confirmed that the PL decay times resulting after reintroducing O₂ closely match the values measured in the first heating cycle (Supporting Information, Figure S51). Representative aging studies of Crddpd@STOSiO ₂ stored in air-saturated Milli-Q water (pH 6.9), monitored by time-resolved PL measurements, revealed a steady increase in the average PL lifetime and integral PL decay curve under atmospheric conditions already within 1 week (Supporting Information, Figure S52). This finding agrees well with the results of our previous studies with Cr^III compounds in different silica networks. This behavior is attributed to ongoing silica aging, most likely caused by the condensation of surface silanol groups, which can change the silica network/surface chemistry and thereby reduce the O₂ permeability of the Stöber- type SiO₂ NPs. This underlines the importance of silica surface chemistry for efficient O₂ shielding. In contrast, Crddpd@ARGSiO ₂ retains a stable oxygen-shielded PL behavior over at least 28 days under the applied storage and measurement conditions (Supporting Information, Figure S53).

4. Conclusion and Outlook

Incorporation of the representatively chosen Ru^II complex Ruphen and the long-lived Ru^II dyad Ruphenpy into silica nanoparticles (SiO₂ NPs) by the l-arginine mediated method (Ruphen@ARGSiO ₂ and Ruphenpy@ARGSiO ₂ ) and the classical ammonia-catalyzed Stöber method (Ruphenpy@STOSiO ₂ ) led to monodisperse Ruphen- and Ruphenpy-stained SiO₂ NPs with sizes of about 25 nm. The resulting PL properties in aqueous dispersion in air, observed for the former and latter type of silica network, verify the previously reported complete O₂ shielding of O₂-responsive luminescent Cr^III complexes by l-arginine- type silica networks. The observed partial protection from PL quenching by O₂ in Stöber-type SiO₂ NPs agrees well with the results reported by other research groups for Ru^II compounds , and by us for two molecular rubies. Remarkable differences in O₂ shielding efficiency of the l-arginine and Stöber-type silica networks underline the influence of the sol–gel synthesis route on the O₂ permeability of the resulting silica materials on silica matrix O₂ protection efficiency, which is attributed to different densities of the resulting SiO₂ NPs, together with a slightly different surface chemistry as supported by the different zeta potentials and stability studies with Stöber-type SiO₂ NPs. The broad applicability of our simple l-arginine-type silica shielding concept for the protection of O₂- sensitive luminophores from collisional PL quenching by O₂ can pave the road for many new applications of O₂- sensitive compounds in air in bioimaging, sensing, and energy conversion studies as well as in multiplexed or time-gated detection schemes and spectral shifting, that all require high PL efficiency. In addition, this encapsulation strategy can enhance the stability of photolabile compounds such as Ru^II complexes as demonstrated for Ruphen and Ruphenpy.

Temperature-dependent PL measurements of Crddpd and the two Ru^II complexes and the luminophores encapsulated in l-arginine- and Stöber-type SiO₂ NPs in air- and argon-saturated solutions demonstrate the almost completely preserved temperature sensitivity of all luminophores in both silica networks. In addition, our studies demonstrate the importance of complete O₂ protection for reliable and comparable temperature and analyte sensing with O₂-sensitive single wavelength emissive luminophores in air utilizing changes in PL intensity and PL decay kinetics or lifetime. For Boltzmann-type dual emissive Cr^III compounds, the influence of O₂ concentration on the reliability of optical temperature sensing is negligible, however, the signal intensities and hence the sensitivity of the unshielded Cr^III PL thermometer is slightly reduced. These findings highlight the importance of determining the matrix O₂ shielding efficiency to identify matrices and synthetic procedures, which provide optimum PL properties and brightness of O₂-sensitive luminophores, and to eliminate interferences from temperature-dependent O₂ solubility and to improve the comparability of different sensors with non-Boltzmann- type PL characteristics.

Overall, our results also underline the strong potential of molecular rubies such as Crddpd and encapsulated complexes such as Crddpd in l-arginine–type SiO₂ NPs or other nanoparticles for increasingly popular thermometry applications in the life sciences and bioimaging in the physiological relevant temperature range due to their optimally suited energy gap of ΔE ≈ 600 cm^–1 and the Cr^III PL in the first biological window (650 - 950 nm). A disadvantage is, however, the excitability of the Cr^III complexes at relatively short wavelengths ≤450 nm, resulting in a limited penetration depth of the exciting light, and favoring contributions from scattering and autofluorescence. Such signal distortions can be overcome by PL lifetime measurements and the usage of the demonstrated integral decay curve approach for temperature sensing. Important for temperature sensing is, however, to control quenching pathways of both spin-flip excited states of Cr^III, which could otherwise affect the accessible dynamic working range of this type of luminescent thermometer. For PL lifetime sensing with matrix-encapsulated luminophores, which often reveal complex PL decay kinetics, normalized integral PL decay curves could be exploited. This simple approach is independent of luminophore concentration and fluctuations of the excitation power densities contrary to PL intensity measurements and additionally avoids time-consuming and error-prone data fitting procedures of PL decay curves.

In the future, we will probe the O₂ permeability of silica networks prepared with different methods under different conditions with our Cr^III compounds to gain an in-depth understanding of the relevant synthetic parameters controlling the O₂ permeability of these networks. This will provide the basis to correlate silica network O₂ permeability and preparation method. Thereby, we will exploit the tunability of the dispersibility of these Cr^III complexes via the choice of the counteranion without affecting their optical properties. In addition, we will perform systematic long-term aging studies of Ru^II-loaded l-arginine and Stöber SiO₂ NPs to support future sensor applications and assess the possibility of PL lifetime tuning with the aid of differently synthesized silica networks for O₂-sensitive luminophores such as Cr^III and Ru^II complexes. Intended applications are the design of a set of nanoscale lifetime labels with matching absorption and emission spectra for lifetime multiplexing and encoding in the visible and NIR with single wavelength excitation.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmeasuresciau.6c00124.

• All data needed to support the conclusions in the article are presented in the manuscript and the Supporting Information (PDF)

V.O. performed the syntheses and characterization of the Cr^III and Ru^II complex-loaded silica nanoparticles, the optical studies, data preprocessing and analysis, designed several figures and helped in preparing the first draft of the manuscript. I.T. developed the different synthesis protocols of the unloaded and luminophore-loaded silica nanoparticles. C.H. helped with the analysis of the temperature dependent PL studies and the evaluation of the data, especially the calculation of the thermometric performance parameters, and considerably contributed to the presentation of the temperature-dependent PL data. A.C.W. synthesized the ruthenium dyad and carried out the photostability measurements, both under the supervision of C.K. E.M. synthesized the Cr^III complex under the guidance of K.H., based upon the ideas and ligand design of K.H. L.G. and M.R.R. performed the HR-TEM, HAADF-STEM, and EDX microstructural analyses. U.R.G. designed the study and the MS concept including figures, did most of the data interpretation and editing, and jointly prepared the first draft of the manuscript with V.O. and wrote the final manuscript. The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.