pH sensing and imaging in living cells based on fluorescence lifetime of carbon dot nanosensors

pH sensing and imaging in living cells based on fluorescence lifetime of carbon dot nanosensors Sergii Kalytchuk a,b,* , Tomaˇ s Malina a,b , Filip Mravec c , Kateˇ rina Pol´ akov´ a a , Luk´ aˇ s Zdraˇ zil a,b , ˇ Stˇ epan Kment a,b , Andrey L. Rogach d,e , Michal Otyepka a,e , Radek Zboˇ ril a,b,** a Regional Centre of Advanced Technologies and Materials, Czech Advanced Technology and Research Institute (CATRIN), Palacký University Olomouc, ˇ Slechtitelů 27, Olomouc, 783 71, Czech Republic b Nanotechnology Centre, Centre for Energy and Environmental Technologies, VSB–Technical University of Ostrava, 17. listopadu 2172/15, Ostrava, 708 00, Czech Republic c Materials Research Centre, Faculty of Chemistry, Brno University of Technology, Purkyˇ nova 464/118, Brno, 612 00, Czech Republic d Department of Materials Science and Engineering, and Centre for Functional Photonics (CFP), City University of Hong Kong, 83 Tat Chee Avenue, Hong Kong S.A.R., 999077, PR China e IT4Innovations, VSB–Technical University of Ostrava, 17. listopadu 2172/15, Ostrava, 708 00, Czech Republic ARTICLE INFO Keywords: Carbon dots Fluorescence pH nanosensor Intracellular sensing Fluorescence lifetime imaging microscopy ABSTRACT The pH value is one of the most frequently measured chemical parameters, yet developing nanometric sensors capable of accurately mapping pH distribution and dynamics with high spatial and temporal resolution remains a significant challenge. Such sensors are vital for advancing our understanding of numerous physiological and pathological processes. Nanoparticle-based sensors, commonly referred to as nanosensors, represent a promising class of optical sensors, with fluorescence lifetime-based probes offering superior sensitivity and quantitative reliability. However, existing pH nanosensors relying on fluorescence lifetime are challenging to synthesize and often suffer from poor biocompatibility, narrow pH response ranges, low stability, and calibration-dependent performance. Here, we overcome these limitations by introducing a water-dispersible pH nanosensor based on fluorescence lifetime of colloidal carbon dots (CDs) derived via a one-step reaction from a single precursor Rhodamine B. These CDs are biocompatible, non-toxic, and stable in highly acidic/basic conditions, which makes them well-suited for intracellular applications. The intrinsic fluorescence lifetime of these CDs exhibits a pseudolinear, self-referencing response across exceptionally broad pH range (1–11), driven by pH-induced transformations of their electronic structure occurring during protonation and deprotonation of CD surface. By applying micrometer-resolution, quantitative pH imaging via fluorescence lifetime imaging microscopy, we demonstrate how CDs are preferentially sequestered in lysosomes of human skin fibroblasts, enabling precise quantification of inhibitor-induced pH changes within these organelles. Our findings highlight a significant potential of the CD nanosensors for precise monitoring of lysosomal pH in living cells, offering broad utility in biomedical research and potential studies of pH-associated cellular dysfunction. 1. Introduction The pH value is a fundamental chemical parameter, which is frequently used across various applications and industries worldwide. Luminescent pH sensors have a wide range of applications, and should ideally enable real-time, non-invasive imaging of pH distribution with high spatial and temporal resolution, especially in those fields where precise pH monitoring is essential. In biomedical sciences, intracellular pH sensing is a vital diagnostic tool, as it plays a crucial role in various biological processes and serves as a key indicator for detecting and * Corresponding author. Regional Centre of Advanced Technologies and Materials, Czech Advanced Technology and Research Institute (CATRIN), Palacký University Olomouc, ˇ Slechtitelů 27, Olomouc, 783 71, Czech Republic. ** Corresponding author. Regional Centre of Advanced Technologies and Materials, Czech Advanced Technology and Research Institute (CATRIN), Palacký University Olomouc, ˇ Slechtitelů 27, Olomouc, 783 71, Czech Republic E-mail address: [email protected] (S. Kalytchuk). Contents lists available at ScienceDirect Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios https://doi.org/10.1016/j.bios.2025.118022 Received 14 July 2025; Received in revised form 17 September 2025; Accepted 22 September 2025 Biosensors and Bioelectronics 291 (2026) 118022 Available online 23 September 2025 0956-5663/© 2025 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ). monitoring cancer progression, as well as neurological and cardiovascular diseases (Cardone et al., 2005; Day et al., 2006; Perona and Serrano, 1988; S´ anchez-Arm´ ass et al., 2006; Vaughan-Jones et al., 2009). Other relevant areas of applications of pH sensors include environmental monitoring, food quality control, agriculture and industrial process control. Luminescent pH sensors offer advantages like high sensitivity, non-invasiveness, and the ability to operate under varying environmental conditions, making them in high demand for scientific, medical, and industrial purposes. In addition to conventional fluorimetric pH sensing, which relies on fluorescence microscopy of pH-responsive dyes, nanosensors represent a relatively new class of optical sensors which are based on colloidal nanoparticles. Colloidal carbon dots (CDs), with their size of less than 10 nm, were first introduced in 2006 (Sun et al., 2006) and emerged as strong contenders among fluorescent nanomaterials, rivaling semiconductor quantum dots and organic dyes due to their numerous advantages and expanding range of applications. CDs exhibit broadband optical absorption, high fluorescence efficiency, excellent dispersibility, outstanding biocompatibility, and significant chemical diversity (Ðorđevi´ c et al., 2022; Liu et al., 2024a, 2024b; Pelaz et al., 2017; Wang et al., 2025a; Yuan et al., 2023). Additionally, their low toxicity and facile, cheap synthesis from readily available organic precursors make them highly attractive for various fields. Thus, CDs have been widely explored for applications in optoelectronics (Wang et al., 2023; Yan et al., 2023; Zhang et al., 2023), photovoltaics (Kalytchuk et al., 2020; Zdraˇ zil et al., 2020), energy storage (Huang et al., 2024; Li et al., 2024), agriculture (Chaudhary et al., 2024; Chen et al., 2024), biomedicine (Ðorđevi´ c et al., 2022; Li et al., 2021; Sun et al., 2020; Wang et al., 2025b; Xiang et al., 2024; Xu et al., 2022; Zhu et al., 2022a), and photocatalysis (Morbiato et al., 2025; Zdraˇ zil et al., 2023a, 2024). More recently, they have attracted significant attention in sensing applications, where their bright fluorescence and chemically versatile surface functionalities are ideal for tailoring CDs toward specific sensing procedures (Crawford et al., 2022; E et al., 2023; Kalytchuk et al., 2017; Kalytchuk et al., 2021; Tiwari et al., 2023; Zdraˇ zil et al., 2023b). The most commonly used fluorescence pH sensing techniques are intensity-based (Huo et al., 2020; Liu et al., 2021; Shen et al., 2013; Wang et al., 2016a; Ye et al., 2019; Zhu et al., 2012, 2022b), ratiometric (Hou et al., 2017; Shangguan et al., 2016; Shi et al., 2012; Takahashi et al., 2018; Wang et al., 2016b), and colorimetric methods (Huo et al., 2023; Nawaz et al., 2023; Paek et al., 2014; Yang et al., 2019). However, fluorescence lifetime-based pH sensing (Ning et al., 2019; Orte et al., 2013; Rennick et al., 2022) offers several advantages over these approaches. Unlike intensity-based and ratiometric sensing, which rely on intensity measurements and require time-intensive calibration, fluorescence lifetime-based sensing is inherently self-referencing across a wide analyte concentration range. This enables quantitative analysis with a response unaffected by probe concentration, sample inhomogeneity, or excitation power fluctuations, thereby allowing high spatial resolution in fluorescence lifetime imaging. Despite extensive research on pH sensing, current fluorescence lifetime-based pH nanosensors are challenging to synthesize and they often suffer from bioincompatibility, limited pH response range, as well as uncertainties in responses calibration (see Table S1 for comparison) (Herrera-Ochoa et al., 2022; Huang et al., 2020; Linders et al., 2022; Orte et al., 2013). In the present work, we introduce a fluorescence lifetime-based pH nanosensor based on fluorescent CDs synthesized via one-step reaction using Rhodamine B (RhB) as a single precursor. This CD nanosensor features an exceptionally broad pH response range, high accuracy, and high relative sensitivity. We experimentally elucidate the underlying pH sensing mechanism, showing that protonation-deprotonation processes result in pH-induced transformations of electronic structure of CDs, which in turn modulate their optical properties. The CD-based nanosensor demonstrates several advantageous characteristics which are seldom observed collectively in other pH nanosensors, including water dispersibility, low cytotoxicity, excellent biocompatibility, superior photostability, and remarkable functional stability. Its in vitro performance is assessed through fluorescence lifetime imaging microscopy (FLIM), enabling high-resolution pH imaging of lysosomes in human skin fibroblasts. Our findings highlight the strong potential of CD-based nanosensors for applications in biological, biochemical, and medical fields, owing to their exceptionally broad pH-responsive range and capability to accurately detect and image acidic environments, such as lysosomes, within living cells. 2. Experimental section 2.1. Synthesis of water-dispersible RhB-CDs Water-dispersible RhB-CDs were synthesized via a one-step hydrothermal method. Briefly, 187.5 mg of RhB and 300 mg of NaOH were dissolved in 7.5 mL of water and sonicated for 15 min in an ultrasonic bath. The resulting suspension was subjected to hydrothermal treatment in a Teflon-lined stainless-steel autoclave at 220 ◦C for 8 h. After the reaction, the solution was filtered through a 200 nm Teflon syringe filter and subsequently dialyzed against deionized water for 24 h to remove residual small-molecule impurities. 2.2. In vitro FLIM measurements FLIM measurement of BJ cells incubated with RhB-CDs was performed using a MicroTime 200 time-resolved confocal fluorescence microscope system (PicoQuant, Germany), equipped with a pulsed diode laser LDH-P-C-470 (470 nm, 26.67 MHz; PicoQuant, Germany) as an excitation source. BJ cells were seeded into μ -Slide 8-well IbiTreat chamber (Ibidi, Germany) at a density of 5 ×10 4 cells/well and allowed to adhere overnight. Next day, cells were treated with 200 μ g/mL RhBCDs for 48 h. To elevate lysosomal pH, cells were pre-incubated with 20 nM bafilomycin A1 (a V-ATPase inhibitor) for 1 h prior to RhB-CDs exposure. Before imaging, the culture medium was replaced with warm phosphate-buffered saline (PBS, 0.1 M, pH 7.4). Fluorescence signals were collected through a long-pass filter (488LP, Chroma, USA) and detected using time-correlated single-photon counting (TCSPC) with SPCM-AQRH-WX-TR single-photon counting module (Excelitas, USA). FLIM images were captured at a resolution of 500 ×500 pixels and analyzed using SymphoTime64 software (PicoQuant, Germany) by fitting the decay traces to a two-exponential model. A reconstructed instrument response function (IRF) was generated from the global decay curve using the same software and used for FLIM reconvolution analysis. FLIM maps were visualized using an arbitrary color scale to represent fluorescence lifetime values in each pixel, with brightness indicating signal intensity. 3. Results and discussion 3.1. Synthesis and characterization of CDs derived from RhB The choice of precursors and synthesis methods plays a crucial role in determining the physicochemical properties of CDs, such as their size, degree of graphitization, and surface functionalization. Notably, certain structural features of the precursor molecules can be partially retained in the final nanoparticles, enabling a degree of rational design (Ðorđevi´ c et al., 2022). Rhodamine derivatives present themselves as promising candidates for engineering pH-sensitive CDs. In this study, green-emissive CDs (denoted as RhB-CDs) were synthesized via a one-step hydrothermal reaction in aqueous solution using Rhodamine B as a precursor and sodium hydroxide (NaOH) as a catalytic agent (Fig. 1a), following a modified literature protocol (Zhang et al., 2023). RhB, a member of the xanthene dye family, is fluorescent dye widely used biological imaging, dye lasers, and chemical sensing, owing to its highly conjugated structure comprising nitrogenand oxygen-containing aromatic rings. NaOH acts as a catalyst in the S. Kalytchuk et al. Biosensors and Bioelectronics 291 (2026) 118022 2 carbonization process by promoting the dehydration of organic species, thereby facilitating the formation of carbon-rich structures at relatively low temperatures (Minervini et al., 2022). The size and morphology of the RhB-CDs were characterized using transmission electron microscopy (TEM) and atomic force microscopy (AFM) (see SI for descriptions of experimental methods). A representative TEM image of the CDs is shown in Fig. 1b. A size histogram, derived from measurements of over 60 particles from multiple TEM images, shows that the CD diameters range from 4.0 to 7.8 nm, with an average size of 6.3 nm (Fig. 1b, inset). AFM image (Fig. 1c) shows that RhB-CDs are highly dispersible in water, remaining as individual particles upon dispersion. Line scans of six representative CDs (Fig. 1d) reveal particle heights ranging from 4.7 to 7.5 nm. X-ray photoelectron spectroscopy (XPS) was performed to analyze the elemental composition and surface chemical states of RhB-CDs. As shown in Fig. 1e, the survey XPS spectrum reveals 3 characteristic peaks corresponding to C 1s, O 1s, and N 1s, with atomic percentages of 57.5 %, 29.8 %, and 0.5 %, respectively. Deconvolution of the high-resolution C 1s spectrum (Fig. 1f) reveals both sp 2 -and sp 3 -hybridized carbon configurations, forming the primary framework of the RhB-CDs. The deconvoluted peaks further indicate the presence of C–O (~286 eV), C=O (~287 eV), and O=C–O (carboxyl) (~289 eV), confirming the functionalization of the RhB-CDs with carbonyl and carboxyl groups. Fourier-transform infrared spectroscopy (FTIR) analysis (Fig. 1g) further supports these findings. The broad absorption band in the 3200–3400 cm −1 region corresponds to O–H/N–H stretching, indicating hydroxyl or amine functionalities. Strong vibrational bands at 1600 cm −1 (C=C stretching), 1565 cm −1 (COO − asymmetric stretching), and 1390 cm −1 (COO − symmetric stretching) reinforce the XPS results, confirming the presence of aromatic, carbonyl, and carboxyl functional groups (Zdraˇ zil et al., 2023b). Optical properties of the RhB-CDs were characterized in dilute aqueous suspension at neutral pH (see SI for descriptions of experimental methods). The UV–Vis absorption spectrum of the RhB-CDs (Fig. 2a, cyan line) has two distinct regions at high-energy (230 ±1, 272 ±1, and 315 ±1 nm) and low-energy (454 ±1, and 482 ±1 nm) bands, attributed to π – π * transitions of C=C in aromatic sp 2 domains and surface-exposed groups and/or molecular fluorophores, respectively (Ragazzon et al., 2021; Xiong et al., 2018). The excitation spectrum of colloidal RhB-CDs spans from 230 to 500 nm, with a peak excitation at 489 ±1 nm (Fig. 2a, blue line). Upon excitation at 489 nm, the RhB-CDs exhibit a relatively narrow fluorescence spectrum (Fig. 2a, green line), with a full width at half maximum (fwhm) of 43 ±1 nm, centered at 513 ±1 nm. An absolute fluorescence quantum yield (QY) of RhB-CDs under 489 nm excitation is 56 ±3 %, surpassing that of the precursor RhB molecule in water (reference fluorescence QY =31 %). The excitation-emission map of RhB-CDs (Fig. 2b) illustrates excitation-independent emission, with the emission peak remaining constant across various excitation wavelengths. This suggests that the fluorescence of RhB-CDs originates from an excited state that is linked to molecular fluorophores (Zdraˇ zil et al., 2021). We have previously reported that the water-to-ice phase transition in CDs induced quenching of fluorescence originating from the molecular fluorophores attached to the surface of the CDs due to the formation of charge-separated CD core–shell trap states (Kalytchuk et al., 2021). Since RhB, a common fluorophore, was used as the sole precursor for the RhB-CD synthesis in this work, the same methodology was applied to investigate fluorescence centers in RhB-CDs. Upon freezing the aqueous RhB-CD dispersion, both steady-state (Fig. S1a) and time-resolved (Fig. S1b) fluorescence measurements revealed significant quenching – the integral fluorescence intensity decreased by more than 100-fold, while the fluorescence lifetime shortened from 4.0 ns to 0.9 ns. These results suggest that the fluorescence of RhB-CDs originates from the incorporated molecular fluorophore (Ehrat et al., 2017; Zdraˇ zil et al., 2021), possibly a Rhodamine derivative. This behavior indicates that the fluorescence of RhB-CDs is governed by uniform emissive states, making these CDs highly suitable as bright, excitation-independent luminescent probes. The spectrally and timeresolved fluorescence map of RhB-CDs in water at neutral pH (Fig. 2c) spans the whole spectral range of 400–750 nm and a time range of 0–15 ns, revealing a spectrally resolved fluorescence decay pattern. The fluorescence decay of RhB-CDs was analyzed using time-resolved fluorescence spectroscopy at the 513 nm emission maximum (Fig. 2d). A fluorescence lifetime of 4.0 ns was extracted using a stretched exponential fitting function (see SI for experimental details). For comparison, analogous characterizations were performed for parental RhB dissolved in water at neutral pH (Fig. 2e–h). The peak excitation and fluorescence spectra of RhB were redshifted by 64 ±1 nm, and the full width at half maximum (fwhm) was narrower by 3 ±1 nm compared to RhB-CDs (Fig. 2e). The excitation-emission map of RhB (Fig. 2f) displays a slightly different and redshifted excitation-emission pattern. The spectrally and time-resolved fluorescence map of RhB (Fig. 2g) reveals a noticeably faster fluorescence decay than that of RhB-CDs. An average fluorescence lifetime of 2.0 ns was extracted from the fluorescence decay of RhB at the emission maximum using a biexponential fitting function (Fig. 2h), which is half the value observed for RhB-CDs. The longer fluorescence lifetime of RhB-CDs compared to RhB allows for a better separation from background fluorescence (autofluorescence) in biological samples enhancing sensitivity and provides a broader dynamic range for sensing applications. All the observed differences in the optical properties of RhB-CDs compared to parental RhB confirm the successful conversion of this precursor into RhB-CDs during hydrothermal reaction, indicating effective synthesis and structural modification. 3.2. Implications of pH-dependent transient fluorescence of RhB-CDs for pH sensing Fluorescence-lifetime-based sensing is highly valued for its Fig. 1. Synthesis and structural characterization of RhB-CDs. (a) Schematics of the hydrothermal reaction used for the synthesis of RhB-CDs. (b) TEM image with a size histogram in the inset. (c, d) AFM topography images: (c) 2D heightmode, and (d) line scan profiles of six representative CDs. (e) XPS spectrum, with the inset showing the elemental composition. (f) High-resolution C 1s XPS spectrum. (g) FTIR spectrum. S. Kalytchuk et al. Biosensors and Bioelectronics 291 (2026) 118022 3 sensitivity and ability to provide quantitative, non-invasive, real-time monitoring, making it particularly useful for applications requiring rapid micro-environmental detection, such as live-cell imaging. To investigate the pH dependence of RhB-CD emission dynamics, spectrally and time-resolved fluorescence maps were acquired at three representative pH values (1, 7, and 11) (Fig. 3a–c). These transient fluorescence emission maps, covering the spectral range of 380–770 nm and time range of 0–15 ns, reveal a clear prolongation of fluorescence decay with increasing pH, highlighting the potential of RhB-CDs for fluorescence lifetime-based pH sensing and imaging. Additionally, normalized spectrally and time-resolved fluorescence maps were obtained at pH 1, pH 7, and pH 11 (Fig. S2a–c) to access the presence of multiple fluorescence centers contributing to pH sensitivity. These transient fluorescence emission maps, covering the spectral range of 480–610 nm and time range of 0–15 ns, demonstrate a consistent wavelength-dependent change in the fluorescence lifetime across pH levels (Fig. S2d), suggesting that fluorescence of RhB-CDs originates from the uniform emissive states. A systematic study of fluorescence lifetime variation across pH range of 1–11 was conducted, with time-resolved fluorescence data collected at the corresponding emission maximum (Fig. 3d). The color plot in Fig. 3d shows a monotonic increase in fluorescence decay time as pH rises. Each fluorescence decay curve was fitted using a stretched exponential model (alternatively, a biexponential function can be used; see SI for details), and fit quality was evaluated using weighted residuals and reduced χ 2 values. The standard deviation across three independent fluorescence lifetime measurements at different pH values averaged as 0.01 ns and did not exceed 0.03 ns. The extracted fluorescence lifetime values (Fig. 3e) show a steady increase from 2.59 to 4.54 ns as pH rises from 1 to 11. This pH dependence follows a fourth-order polynomial calibration curve, with an adjusted R 2 value of 0.99592: pH = − 111.11 +119.34 τ −47.63 τ 2+8.45 τ 3−0.54 τ 4(1) In this calibration, pH is directly correlated with fluorescence lifetime ( τ , ns). Aiming for sensitivity within physiologically relevant pH values, RhB-CDs demonstrate robust performance across a broad pH range of 1–11, encompassing and exceeding biologically significant conditions. This probe exhibits an absolute pseudo-linear sensitivity of 0.21 ns per pH unit and a maximum relative sensitivity of 9.52 % pH −1 at pH 2.74. A stretched-exponential fluorescence decay model is employed across the pH range, yielding a single lifetime parameter ( τ ) that can be directly converted to pH value using the established Fig. 2. Optical properties of RhB-CDs compared to parental RhB precursor. (a) Optical absorption, normalized fluorescence excitation (λ em =513 nm), and fluorescence emission (λ ex =489 nm) spectra in dilute aqueous solution at neutral pH. Inset shows a digital photograph of diluted RhB-CDs and under UV irradiation. (b) Excitation-emission color map. (c) Spectrally and time-resolved fluorescence emission. (d) Fluorescence decay collected at the emission maximum, with experimental data shown as symbols and the stretched exponential fit represented by a solid line. (e–h) Measurements analogous to (a–d) performed for parental RhB dissolved in water. Fig. 3. Demonstration of fluorescence lifetime pH sensing ability of RhB-CDs. (a–c) Spectrally and time-resolved emission color maps at pH 1, pH 7, and pH 11. (d) Normalized color plot of time-resolved fluorescence intensity across pH values from 1 to 11. (e) Corresponding extracted fluorescence lifetime versus pH between pH 1 and pH 11, highlighting pseudo-linear response. Data are represented as symbols, whereas solid line represents the polynomial fit, according to eq. (1); error bars represent the standard deviation from three measurements. (f) Normalized color map of time-resolved fluorescence intensity showing pH variation over time, induced by NaOH crystal dissolution and measured at 10-min intervals. (g) Corresponding pH time trace over a 10-h period, derived from fluorescence lifetime values. All decay curves in (d) and (f) were recorded at the maximum emission wavelength of RhB-CDs (λ em =513 nm). S. Kalytchuk et al. Biosensors and Bioelectronics 291 (2026) 118022 4 calibration curve. This model offers a distinct advantage over conventional semiconductor quantum dots, which typically exhibit multiexponential decay profiles that complicate lifetime-based sensing (Kalytchuk et al., 2017). Notably, RhB-CDs also show excellent chemical stability, with no degradation observed under strongly acidic or basic conditions, as the fluorescence lifetime at each pH remained stable for at least one month (Fig. S3). As a benchmark experiment, we evaluated the feasibility of RhB-CDs for long-term, real-time pH monitoring by recording their fluorescence decay profiles in a solution with dynamically changing pH, induced by the dissolution of NaOH crystal (see SI for experimental details). Fluorescence decay measurements were taken at 10-min intervals over a 10h period (Fig. 3f). The pH at each time point was calculated using the established calibration curve and fluorescence lifetime values extracted from the decay profiles, with resulting pH trend plotted over time (Fig. 3g). The final pH, measured independently using a calibrated pH meter (10.31), showed excellent agreement with the fluorescence lifetime-based estimate (10.38), validating the accuracy of the fluorescence lifetime-based method. This experiment underscores the strong potential of CD-based probes for precise, time-resolved pH sensing and imaging. A promising direction for future research would be the integration of fluorescent imaging with complementary sensing modalities. In this context, RhB-CDs can function as dual-mode probes, enabling both pH sensing/imaging and fluorescence imaging, offering a significant advantage over conventional luminescent nanomaterials. 3.3. Stability assessment For fluorescent nanomaterials to be effective in sensing applications, they must demonstrate high stability. Accurate pH sensing requires probes with a suitable detection range, high photostability and thermal stability, excellent spatial and detection resolution, and reliable performance under varying environmental conditions. To evaluate the potential of RhB-CDs as luminescent pH probes for diverse applications, we examined several critical stability-related parameters listed above. The photostability of the RhB-CDs was assessed by exposing them to a pulsed laser excitation for an extended duration. Fig. 4a represents fluorescence decay data recorded every 15 min for 72 h under continuous photoexcitation. The variation in the fluorescence lifetime over time (Fig. 4b) confirms excellent photostability of RhB-CDs throughout the 72-h period. This finding is highly encouraging, demonstrating the feasibility of CD nanosensors for long-term pH monitoring. Moreover, fluorescence QY and lifetime measurements demonstrate that RhB-CDs show high storage stability: samples stored in darkness for up to 20 months exhibited no detectable changes in these parameters relative to their original values (Fig. S4). High reproducibility of the RhB-CD synthesis was demonstrated by time-resolved fluorescence studies in ultrapure water, where the original and newly prepared batches exhibited nearly identical fluorescence lifetimes of 3.76 ns and 3.77 ns, respectively (Fig. S5). The thermal stability of the RhB-CDs was also assessed over biologically relevant temperature range (0–42 ◦C). Fig. 4c displays fluorescence decay data recorded at 3 ◦C increments, with the corresponding fluorescence lifetime variation shown in Fig. 4d. The RhB-CDs maintain excellent thermal stability between 0 and 36 ◦C, though a slight increase in fluorescence decay (0.15–0.2 ns) is observed at temperatures above 36 ◦C. The influence of ionic strength on fluorescence lifetime of RhBCDs was evaluated using KCl solutions of varying concentrations. Fig. 4e shows the fluorescence decay profiles, while Fig. 4f reveals that that fluorescence lifetime remains unaffected even at a high ionic strength of 500 mM KCl – well above the typical physiological level of ~100 mM. To better represent biologically relevant conditions, we assessed the stability of RhB-CDs in PBS supplemented with fetal bovine serum (FBS), accounting for potential protein interactions (protein corona formation), and under macromolecular crowding conditions using polyethylene glycol (PEG). The fluorescence lifetime of RhB-CDs in FBS-supplemented PBS (4.21 ns) was slightly longer than in ultrapure water (3.76 ns), attributable to the response of RhB-CDs to the higher pH of the FBSsupplemented PBS, and remained stable over 7 days of storage (Fig. S6). For crowding studies, PEGs of two molecular weights (PEG, 2000 and PEG 35000) were tested at two concentrations (1 and 5 wt%). At 1 wt%, neither PEG 2000 or PEG 35000 induced any noticeable changes in fluorescence lifetime (3.80 and 3.77 ns, respectively) compared to ultrapure water, with stability maintained for at least 7 days (Fig. S7–S8). At higher concentrations (5 wt%), an increase in fluorescence lifetime was observed, as anticipated due to high viscosity of densely crowded solutions, along with deviations over 7 days of storage (Fig. S9–S10). This environmental insensitivity is the key advantage, as it eliminates the need for environment-specific fluorescence lifetime calibration, ensuring that the pH responsiveness of RhB-CDs remains consistent Fig. 4. Evaluation of photo-, thermo-, and ionic strength stability of RhB-CDs. (a) Normalized color plot of the fluorescence decay recorded every 15 min over 72 h under continuous excitation with a pulsed laser. (b) Corresponding photostability of the fluorescence lifetime over 72-h period. (c) Normalized color plot of the fluorescence decay across a temperature range from 0 to 42 ◦C. (d) Corresponding thermal stability of fluorescence lifetimes between 0 and 42 ◦C. (e) Normalized color plot of the fluorescence decay in KCl solutions with concentrations ranging from 0 to 4.6 M. (f) Stability of the fluorescence lifetime as a function of the ionic strength for samples in aqueous KCl solutions with concentrations from 0 to 4.6 M. All decay curves were recorded at the maximum fluorescence emission of RhB-CDs (λ em =513 nm). Greenand red-shaded regions serve as visual guides to indicate the ranges where the fluorescence lifetime remains stable or begins to deviate, respectively. S. Kalytchuk et al. Biosensors and Bioelectronics 291 (2026) 118022 5 regardless of the surrounding medium. Overall, RhB-CDs demonstrate exceptional stability across multiple critical parameters, underscoring their robustness and reliability for long-term high-precision pH sensing applications. 3.4. Transformation of the electronic structure of RhB-CDs induced by pH To gain deeper insight into the emission behavior of RhB-CDs under dynamically varying pH conditions, we performed a series of targeted experiments. First, we correlated the radiative ( τ r −1 ) and nonradiative ( τ nr −1 ) recombination rates of RhB-CDs with their fluorescence QY at different pH levels. The radiative rate was determined from the fluorescence QY and the measured recombination rate ( τ −1 ) using the equation τ r −1 =QY × τ −1 , while the nonradiative relaxation rate was calculated as τ nr −1 = τ −1 – τ r −1 . The fluorescence QY of RhB-CDs at various pH levels was directly measured using an absolute method, revealing a more than 30-fold increase from 2.7 % at pH 1–84.9 % at pH 11 (Fig. 5a). The radiative and nonradiative recombination rates, derived from time-resolved fluorescence measurements, are plotted as functions of pH in Fig. 5b. A drastic 18-fold exponential increase of the radiative recombination rate (from 0.11 ×10 6 to 1.89 ×10 6 s −1 ) is accompanied by an 11-fold exponential drop of the nonradiative rate (from 3.76 ×10 6 to 0.34 ×10 6 s −1 ) when pH increases from 1 to 11 (see Table S2–S3 for details). A pH-dependent crossover of the radiative and nonradiative rates occurs at pH 6.3, where the fluorescence QY reaches 50 %. These results suggest that the pH-induced changes in the emission dynamics of RhB-CDs are driven by transformations of their electronic structure, affecting both radiative and nonradiative relaxation pathways. To further confirm this, we measured the absorption and FTIR spectra of RhB-CDs as a function of pH, along with fluorescence excitationemission mapping. Fig. 5c illustrates the absorption spectra of RhBCDs in aqueous solution at varying pH levels. A significant transformation of the absorption spectrum is observed as the pH changes from 1 to 11. At pH 1, the first highest occupied molecular orbital – lowest unoccupied molecular orbital (HOMO-LUMO) transition is located at 437 nm, accompanied by a weak shoulder extending to ~580 nm. As the pH increases to 3, the intensity of the 437 nm peak decreases, while a new peak emerges at 476 nm, becoming dominant at pH 5 and 7. With a further increase in pH, this peak disappears, and at pH 9–13, the absorption spectra are dominated by a main peak at 491 nm and a less intense peak at 453 nm. Notably, the absorption spectra exhibit different behavior with increasing pH: the intensity of the 453 nm peak remains unchanged, whereas the 491 nm peak increases with pH. Such alterations of the absorption spectra of RhB-CDs should be related to the protonation/deprotonation processes of their surface, occurring at low/ high pH levels, respectively. To better understand these processes, Fourier-transform infrared spectroscopy (FTIR) (Fig. 5d) was employed to investigate the structural and surface chemistry variations of RhB-CDs across different pH values. Notable spectral changes were observed in the 1710 cm −1 and 1565/ 1390 cm −1 regions, corresponding to protonated (-COOH) and deprotonated (-COO - ) carboxyl groups, respectively. At low pH (pH 1), a prominent peak at 1700 cm −1 indicates that carboxyl groups remain protonated (-COOH). As the pH increases beyond 3–4, approaching the pKa value of the aromatic carboxylic acid moiety in RhB, this peak diminishes, while bands at 1565 cm −1 (asymmetric COO − stretching) and 1390 cm −1 (symmetric COO − stretching) emerge. This shift confirms deprotonation of carboxyl groups to their carboxylate (COO − ) form in RhB-CDs (Hol´ a et al., 2020). The fluorescence excitation-emission maps of RhB-CDs (Fig. 5e–k) exhibit a pH-dependent transformation consistent with changes observed in the absorption spectra. At pH 1 (Fig. 5e), the maximum excitation occurs at 436 nm, with a broad emission spanning 475–670 nm. As the pH increases to 3 (Fig. 5f), a new emission band emerges with excitation at ~490 nm, becoming dominant at pH 5 (Fig. 5g). Concurrently, the emission associated with 436 nm excitation diminishes and disappears by pH 7 (Fig. 5h). At higher pH values (pH 9–13; Fig. 5i–k), the fluorescence emission narrows down (490–590 nm) compared to strongly acidic conditions, with a redshifted excitation range and a maximum excitation at 489 nm. These findings demonstrate that the electronic structure of RhB-CDs undergoes significant pH-induced transformations, directly influencing their optical properties. The observed changes in fluorescence QY, radiative and nonradiative recombination rates, and absorption spectra are attributed to protonation and deprotonation of CD surface, as confirmed by the FTIR analysis. These results provide valuable insight into the pH responsivity of RhB-CDs. Fig. 5. pH-induced transformation of the electronic structure of RhB-CDs. (a) Fluorescence QY and fluorescence lifetime as functions of pH. The experimental data are represented by symbols, while the solid lines serve as visual guides. (b) Radiative ( τ r ) and nonradiative ( τ nr ) recombination rates plotted against pH. The experimental data are shown as symbols, while the solid lines represent exponential fits (see SI for the details). (c) Absorption spectra as a function of pH, presented as line plots with a constant offset. (d) FTIR spectrum at neutral pH (pH 7) compared to FTIR spectra at acidic (pH 1) and basic (pH 11) conditions. (e–k) Fluorescence excitation-emission color maps at selected pH values. S. Kalytchuk et al. Biosensors and Bioelectronics 291 (2026) 118022 6 3.5. In vitro pH sensing and imaging using FLIM with RhB-CDs To evaluate the potential of RhB-CDs as probes for pH sensing and imaging in biological systems, BJ cells, a model of healthy human skin fibroblasts, were incubated with RhB-CDs at 37 ◦C for 48 h (see SI for experimental details). Efficient cellular internalization of RhB-CDs is confirmed by the presence of strong green fluorescence, observed exclusively in cells treated with 200 μ g/mL RhB-CDs (Fig. S11). Consistent with previous studies demonstrating the low toxicity of various types of CDs (Kalytchuk et al., 2017; Malina et al., 2019), RhB-CD-treated cells show no morphological signs of damage, indicating good biocompatibility (Fig. S11). Potential cytotoxicity was further assessed using live-dead assay after 48 h of incubation with RhB-CDs (see SI for experimental details). No significant difference in cell viability is observed between RhB-CD-treated and control groups; under all tested conditions, viability remains above 96 % (Fig. 6a), confirming excellent biocompatibility of RhB-CDs and supporting their suitability for pH sensing in live cells. To further investigate the intracellular localization of RhB-CDs following cellular uptake, confocal microscopy was employed in a colocalization experiment using LysoTracker, a lysosome-specific fluorescent dye excited at λ ex =594 nm (see SI for experimental details). Clear co-localization of RhB-CDs with lysosomes is observed, indicated by the merged punctuate pattern of green fluorescence from RhB-CDs overlapping with the red LysoTracker signal (Fig. 6b). No green fluorescence is detected in the untreated control sample (Fig. 6b). The observed lysosomal sequestration suggests that RhB-CDs are internalized via the endocytic pathway and subsequently accumulate in acidic vesicular organelles, consistent with previously reported internalization mechanisms for CDs (Malina et al., 2019). Lysosomal localization is further confirmed by a 2.5D Z-stack image of a single BJ cell co-stained with RhB-CDs and LysoTracker (Fig. S12). Given the lysosomal localization of RhB-CDs, we investigated their application for real-time pH sensing in live cells using FLIM. During FLIM measurements on the live BJ cells, the fluorescence decay curves of RhB-CDs were consistently acquired at each pixel with high repeatability and well-described by biexponential decay model across the entire image. The fluorescence lifetime at each pixel was extracted from this model (see Experimental Section for details), enabling the generation of fluorescence lifetime maps. Crucially, the observed fluorescence lifetime signal is attributed specifically to RhB-CDs, as control (untreated) cells exhibit negligible fluorescence (Fig. S13a). In contrast, treated cells show bright, punctuate fluorescence localized within intracellular vesicles (Fig. 6c, Fig. S13b). The pH of these vesicles was estimated from the fluorescence lifetimes of internalized RhB-CDs and corresponded to approximately pH 4, consistent with the known acidic environment of lysosomes. This low pH is essential for the activity of their degradative hydrolases and proteases and is maintained by the vacuolar H + -ATPase (v-ATPase), an ATP-driven proton pump, in concert with various ion channels (Lawrence and Zoncu, 2019). To test whether RhB-CDs could detect changes in lysosomal pH, cells were treated with bafilomycin A1 (Baf. A1), a well-characterized inhibitor of V-ATPase Fig. 6. Micrometer resolution in vitro pH imaging via FLIM using RhB-CDs. (a) Cell viability assessment of BJ cells after 48 h incubation with RhB-CDs at 100 and 200 μ g/mL. NC: negative control, PC: positive control. Error bars represent the standard deviation from three independent experiments. (b) Fluorescence images of BJ cells stained with a lysosomal marker (LysoTracker) alone, and co-stained with RhB-CDs and LysoTracker. Images were acquired selectively using green and red detector channels, with merged images of both detector channels shown. (c) FLIM image of BJ cells incubated with RhB-CDs (200 μ g/ml) for 48 h. (d–e) Highresolution FLIM images of lysosomes in BJ cells incubated with RhB-CDs (200 μ g/ml) for 48 h: either in the absence (d) or presence (e) of the V-ATPase inhibitor bafilomycin A1 (Baf. A1). FLIM maps are displayed using an arbitrary color scale representing average fluorescence lifetimes, with brightness indicating signal intensity. Additional color bars are included to indicate the corresponding pH values, calculated from fluorescence lifetimes using the calibration curve. (f) Lysosomal pH changes following inhibitor treatment. pH values were calculated from fluorescence lifetimes using calibration eq. (1), based on 10 randomly selected lysosomes from each condition shown in (d) and (e). Mean pH values are shown as bars. S. Kalytchuk et al. Biosensors and Bioelectronics 291 (2026) 118022 7 (Malina et al., 2025). In Baf. A1-treated cells (Fig. 6e), RhB-CDs report a significant increase in lysosomal pH compared to untreated cells (Fig. 6d), with an average elevation of 3.1 pH units (Fig. 6f, Fig. S14, and Table S4), indicating impaired lysosomal acidification. Additionally, an increased number of vesicular structures is observed in these cells, consistent with Baf. A1’s known ability to block autophagosome-lysosome fusion, leading to the accumulation of undigested material within lysosomes (Mauvezin and Neufeld, 2015). Collectively, these findings demonstrate that RhB-CDs function as effective nanosensors capable of reporting lysosomal pH changes via fluorescence lifetime, offering a robust tool for monitoring pH dynamics in live cells. This is particularly relevant, as lysosomal pH dysregulation is implicated in numerous cellular processes and disease states (Cao et al., 2021; Malina et al., 2025). 4. Conclusion In this study, we report the use of the water-dispersible pH nanosensor based on RhB-CDs derived from a single precursor Rhodamine B, exhibiting a fluorescence lifetime highly sensitive to pH in exceptionally broad range. Transient fluorescence spectroscopy was employed to examine the carrier dynamics of RhB-CDs across the broad pH range of 1–11, revealing a pseudo-linear self-referencing response under various conditions and enabling precise and sensitive fluorescence lifetimebased pH sensing and imaging. The pH-induced transformations of the electronic structure of RhB-CDs, driven by protonation and deprotonation of their surface, were confirmed experimentally and found to significantly affect their optical properties. RhB-CDs demonstrated excellent photostability under continuous photoexcitation for at least 72 h, maintained functionality in high ionic strength environments (up to 500 mM KCl), showed consistent performance across a temperature from 0 to 36 ◦C, and achieved a maximum sensitivity of 9.52 % per pH unit, ensuring reliable operation in diverse environments and making them highly suitable for biomedical applications. Using these RhB-CD nanosensors, we achieved high-resolution, quantitative pH imaging in human skin fibroblasts via FLIM. Notably, the CD nanosensors selectively accumulated in lysosomes and enabled determination of physiologically relevant pH values within these organelles. Given the critical role of lysosomal acidification in key biological processes such as autophagy, inflammation, and neurodegeneration, these findings highlight high potential of RhB-CDs for studying pH-associated cellular dysfunctions. Future studies will focus on imaging of additional physiological processes, including starvation-induced autophagy, inflammation, and cancer-associated lysosomal remodeling, using diseaserelevant models. The RhB-CDs offer an exceptionally broad pHresponsive range, high sensitivity, strong photostability, and excellent biocompatibility, making them versatile and high-performing pH nanoprobes that surpass previously reported fluorescence lifetime-based sensors. CRediT authorship contribution statement Sergii Kalytchuk: Writing – original draft, Visualization, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization. Tomaˇ s Malina: Writing – original draft, Methodology, Investigation, Conceptualization. Filip Mravec: Investigation, Formal analysis. Kateˇ rina Pol´ akov´ a: Investigation. Luk´ aˇ s Zdraˇ zil: Formal analysis. ˇ Stˇ epan Kment: Supervision. Andrey L. Rogach: Writing – review & editing, Supervision, Funding acquisition. Michal Otyepka: Supervision, Funding acquisition. Radek Zboˇ ril: Supervision, Funding acquisition. Declaration of generative AI and AI-assisted technologies in the writing process During the preparation of this work, the authors used ChatGPT (version 4.0) for grammar checking. The authors subsequently reviewed and edited the content carefully and take full responsibility for the final version of the manuscript. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements We acknowledge financial support by the ERDF/ESF project TECHSCALE (No. CZ.02.01.01/00/22_008/0004587), the European Union under the REFRESH – Research Excellence For Region Sustainability and High-tech Industries project number CZ.10.03.01/00/ 22_003/0000048 via the Operational Programme Just Transition, and the project “Experimental and theoretical studies of near-infraredemitting and chiral carbon dot luminophores” from the Moravian-Silesian Region, contract No. 00734/2023/RRC. SKa, KP and LZ acknowledge the support of the Czech Science Foundation (GACR), project No. 25-16657S. Authors also gratefully acknowledge the help of Dr. Petr, Dr. Opletalov´ a, and Ms. Str´ ask´ a with material’s characterization. Appendix A. 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