A Near-Infrared Excited FRET-Based Fluorescent Sensor Using Core–Shell Upconversion Nanoparticles Functionalized with Rhodamine B Derivative for Highly Selective Detection of Hg²⁺

A Near-Infrared Excited FRET-Based Fluorescent Sensor Using Core–Shell Upconversion Nanoparticles Functionalized with Rhodamine B Derivative for Highly Selective Detection of Hg²⁺ | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A Near-Infrared Excited FRET-Based Fluorescent Sensor Using Core–Shell Upconversion Nanoparticles Functionalized with Rhodamine B Derivative for Highly Selective Detection of Hg²⁺ Yanmei Zhang, Rui Jiang, Hongze Yang, Cheng Zhou, Mengxi Sun, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8629365/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Apr, 2026 Read the published version in Microchimica Acta → Version 1 posted 11 You are reading this latest preprint version Abstract A near-infrared (NIR)-excitable ratiometric fluorescent nanosensor, CS-UCNP@mSiO₂–RBPA, was developed for the selective and sensitive detection of Hg²⁺. The probe consists of core–shell NaYF₄:Yb,Er upconversion nanoparticles (UCNPs) coated with mesoporous silica and functionalized with a rhodamine B-derived chemosensor (RBPA). Upon 980 nm excitation, the UCNPs emit green (~ 543 nm) and red (~ 658 nm) luminescence. In the presence of Hg²⁺, RBPA undergoes spirolactam ring-opening, generating a yellow-emitting species that accepts energy from the UCNP donor via Förster resonance energy transfer (FRET). This results in a concentration-dependent decrease in green emission and a concomitant increase in yellow fluorescence at ~ 584 nm, while the red emission serves as an internal reference. The sensor exhibits excellent selectivity for Hg²⁺ over other common metal ions and operates effectively at neutral pH. A linear response was observed in the Hg²⁺ concentration range of 0–70 µM by UV–vis absorption, and a remarkably low limit of detection of 14 nM was achieved by ratiometric fluorescence (I₅₈₄/I₆₅₈). The combination of NIR excitation, ratiometric output, and high specificity makes this nanoplatform promising for environmental monitoring and potential biological applications. Upconversion nanoparticles Ratiometric fluorescence sensing Förster resonance energy transfer Mercury(II) detection Near-infrared excitation Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction In recent years, heavy metal pollution has intensified dramatically due to anthropogenic activities, posing severe threats to ecosystems and human health 1 – 4 . Among toxic heavy metals, mercury(II) ion (Hg²⁺) is particularly hazardous. Although it can originate from natural processes such as rock weathering, volcanic eruptions, and vegetation release, the majority of environmental Hg²⁺ stems from industrial discharges, coal combustion, and mining operations. Upon entering the human body, Hg²⁺ exhibits a strong affinity for thiol-containing biomolecules—including cysteine, homocysteine, and reduced glutathione—disrupting their physiological functions. Critically, Hg²⁺ can cross the blood–brain barrier, leading to neurotoxicity characterized by symptoms such as headache, insomnia, depression, memory impairment, and, in severe cases, coma or epilepsy. Moreover, chronic exposure causes irreversible damage to the liver and kidneys 5 – 7 . Consequently, the development of reliable methods for the quantitative detection of trace Hg²⁺ is of paramount importance for environmental monitoring and public health protection. Conventional analytical techniques for Hg²⁺ detection—including atomic absorption spectrometry (AAS) 8 , inductively coupled plasma mass spectrometry (ICP-MS) 9 , and UV–vis spectrophotometry 10 —offer high sensitivity. However, they suffer from significant drawbacks: expensive instrumentation, complex sample pretreatment, skilled operators, and lengthy analysis times. These limitations hinder on-site or real-time monitoring. Therefore, there is an urgent need for rapid, cost-effective, and user-friendly sensing platforms capable of visual or instrumental Hg²⁺ detection. Fluorescent probes have emerged as promising alternatives due to their high sensitivity, operational simplicity, low cost, and potential for real-time response. Among various fluorophores, rhodamine-based derivatives are especially attractive owing to their excellent photostability, large molar absorptivity, high fluorescence quantum yield, and reversible ring-opening/closure behavior in response to specific analytes. For instance, Zhang et al. developed a rhodamine–anthracene hybrid probe that enables colorimetric and fluorescent detection of Hg²⁺ within 30 seconds with a visible color change 11 . Roy et al. reported a dual-responsive rhodamine probe for simultaneous detection of Hg²⁺ and Al³⁺ in aqueous–organic media 12 . Erdemir et al. designed a rhodamine–isophorone conjugate with a large Stokes shift to minimize background interference, achieving selective sensing of Hg²⁺ and Cu²⁺ even in complex matrices such as fungicide formulations 13 . Zhu et al. further enhanced selectivity by incorporating sulfur-functionalized rhodamine units, enabling sensitive Hg²⁺ detection in environmental water samples 14 . Despite these advances, challenges remain in improving selectivity against competing metal ions, reducing cytotoxicity for biological applications, and enabling excitation in the near-infrared (NIR) window to avoid autofluorescence and enhance tissue penetration. Lanthanide-doped upconversion nanoparticles (UCNPs) offer a compelling solution to these limitations. These nanomaterials absorb low-energy NIR photons (typically at 980 nm) and emit higher-energy visible light through anti-Stokes processes. Their unique advantages—including sharp emission bands, long luminescence lifetimes, exceptional photostability, negligible autofluorescence, and deep tissue penetration—make them ideal candidates for biosensing and environmental analysis 15 , 16 . In particular, core–shell structured UCNPs (CS-UCNPs) significantly suppress surface-related quenching, thereby enhancing luminescence intensity and quantum yield. When integrated with responsive organic dyes via fluorescence resonance energy transfer (FRET), UCNPs serve as efficient energy donors for ratiometric or turn-on/turn-off sensing. Several studies have demonstrated the utility of UCNP–rhodamine hybrids: Tang et al. constructed a UCNP@SiO₂-based FRET probe for Fe³⁺ detection 17 ; Wang et al. developed a rhodamine-functionalized UCNP system for Hg²⁺ sensing 18 ; Xu et al. employed core–shell UCNPs with rhodamine B hydrazide for intracellular Cu²⁺ imaging 19 ; while others have extended this strategy to detect CO 20 and glutathione 21 . Herein, we report a novel NIR-excitable fluorescent nanosensor, CS-UCNP@mSiO₂–RBPA, fabricated by conjugating a rhodamine B-derived chemosensor (RBPA) onto amino-functionalized mesoporous silica-coated core–shell upconversion nanoparticles. The core–shell architecture enhances luminescence efficiency by passivating surface defects, while the mesoporous silica shell provides abundant anchoring sites for RBPA and improves colloidal stability. In the absence of Hg²⁺, RBPA remains in its colorless, non-fluorescent spirolactam form. Upon Hg²⁺ binding, the spirolactam ring opens, generating a magenta-colored species with strong absorption overlapping the green emission (∼543 nm) of the UCNP donor. This spectral overlap triggers efficient FRET, resulting in a ratiometric fluorescence response: the green emission decreases while a new yellow emission (∼584 nm) from RBPA increases, with the red emission (∼658 nm) serving as an internal reference. This design enables highly selective, sensitive, and visually discernible detection of trace Hg²⁺ under NIR excitation—a feature particularly advantageous for minimizing background interference in complex environmental or biological samples. The preparation and sensing mechanism of the probe are illustrated in Scheme 1 . 2. Experimental Section Materials All reagents and solvents were used as received without further purification. YCl₃·6H₂O (99.99%), YbCl₃·6H₂O (99.99%), ErCl₃·6H₂O (99.99%), 4-hydroxybenzaldehyde (99%), (3-aminopropyl)triethoxysilane (APTES, 99%), trifluoroacetic acid (TFA, 99%), oleic acid (OA, 90%), 1-octadecene (ODE, 90%), and tetraethyl orthosilicate (TEOS, 98%) were purchased from Aladdin Biochemical Technology Co., Ltd. Rhodamine B hydrazide (97%) was obtained from Zhengzhou Alpha Chemical Co., Ltd. Ammonium fluoride (NH₄F, 96%) was supplied by Shanghai Trial Sihewei Chemical Co., Ltd. Sodium hydroxide (NaOH, 96%) was acquired from Xilong Scientific Co., Ltd. Hexadecyltrimethylammonium bromide (CTAB, 99%) was purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. Cyclohexane (AR, ≥ 99.5%), anhydrous ethanol (AR, ≥ 99.7%), methanol (AR, ≥ 99.5%), and hydrochloric acid (AR, 36–38%) were obtained from China National Pharmaceutical Group Chemical Reagent Co., Ltd. Deionized water (18.2 MΩ·cm) was used for all aqueous solutions. Standard aqueous solutions (1 mM) of K⁺, Na⁺, Ca²⁺, Mg²⁺, Cu²⁺, Zn²⁺, Co²⁺, Ni²⁺, Cd²⁺, Hg²⁺, Ba²⁺, Fe³⁺, Al³⁺, Cr³⁺, Mn²⁺, and Pb²⁺ were prepared from their corresponding chloride or nitrate salts. Characterization Transmission electron microscopy (TEM) images were acquired using a Talos L120C microscope (Thermo Fisher Scientific, USA) by drop-casting nanoparticle suspensions onto copper grids. X-ray diffraction (XRD) patterns were recorded on a Rigaku MiniFlex 600 diffractometer (Rigaku Corporation, Japan) with Cu Kα radiation (λ = 1.5406 Å). Fourier-transform infrared (FT-IR) spectra were collected on a Nicolet iS10 spectrometer (Thermo Fisher Scientific, USA) in the range of 400–4000 cm⁻¹. UV–vis absorption spectra were measured using an Agilent 8453 spectrophotometer (Agilent Technologies, USA). Upconversion fluorescence (UCF) spectra were recorded on an F97XP fluorescence spectrophotometer (Shanghai Lengguang Technology Co., Ltd., China) under 980 nm laser diode excitation (power density: ~1.0 W/cm²). ¹H NMR spectra were obtained on a JNM-ECZ401S spectrometer (JEOL Ltd., Japan) at 400 MHz using deuterated solvents. Synthesis of β-NaYF₄:Yb,Er UCNPs (UCNPs) β-NaYF₄:Yb,Er upconversion nanoparticles were synthesized via a thermal decomposition method. LnCl₃·6H₂O precursors (total 1 mmol; Y:Yb:Er molar ratio = 80:18:2) were dissolved in 10 mL methanol and transferred to a 250 mL three-neck flask. Oleic acid (6 mL) and 1-octadecene (15 mL) were added, and the mixture was heated to 90°C under vigorous stirring to remove methanol until a clear solution formed. The temperature was then raised to 150°C and held for 30 min under N₂ atmosphere. After cooling to 50°C, a methanolic solution containing NaOH (2.5 mmol) and NH₄F (4 mmol) in 15 mL methanol was added dropwise under stirring (400 rpm). The mixture was heated again to 90°C to evaporate residual methanol, then ramped to 315°C and maintained for 60 min under N₂. After cooling to room temperature, the product was precipitated by adding ethanol, centrifuged at 8000 rpm for 8 min, and washed twice with cyclohexane/ethanol (1:1 v/v). The final UCNPs were dispersed in 10 mL cyclohexane for further use. Synthesis of Core–Shell UCNPs (CS-UCNPs) A NaYF₄ inert shell was grown epitaxially on the β-NaYF₄:Yb,Er core. Briefly, 10 mL of the as-prepared UCNP cyclohexane dispersion (~ 10 mg/mL) was mixed with a solution of YCl₃·6H₂O (1 mmol) in 10 mL methanol. The mixture was processed identically to the core synthesis, except that NaOH/NH₄F were replaced by the preformed UCNP dispersion as the seed. The resulting CS-UCNPs were purified and stored in cyclohexane. Synthesis of Mesoporous Silica-Coated CS-UCNPs (CS-UCNP@mSiO₂) A microemulsion method was employed for silica coating. CTAB (0.2 g) was dissolved in 20 mL water, followed by addition of 5 mL CS-UCNP cyclohexane solution (10 mg/mL). The mixture was stirred vigorously at 60°C until cyclohexane evaporated, yielding a transparent aqueous dispersion. Ethanol (3 mL), water (10 mL), and NaOH (150 µL of 0.1 M) were added, and the mixture was sonicated for 15 min. TEOS (220 µL) was then injected dropwise, and the reaction was refluxed at 70°C for 2 h. The product was collected by centrifugation, washed twice with ethanol, and dried at 60°C overnight. Synthesis of Amino-Functionalized CS-UCNP@mSiO₂ (CS-UCNP@mSiO₂–NH₂) The CTAB template was removed by refluxing the CS-UCNP@mSiO₂ in 30 mL of 0.1 M HCl in ethanol at 70°C for 24 h. The solid was centrifuged, washed with ethanol, and dried at 80°C. For amino functionalization, 100 mg of the template-free material was dispersed in 15 mL anhydrous ethanol, and APTES (150 µL) was added under N₂. The mixture was refluxed at 70°C for 24 h, then cooled, centrifuged, washed twice with ethanol, and dried under vacuum. Synthesis of Rhodamine B-Based Probe (RBPA) RBPA was synthesized via condensation of rhodamine B hydrazide (RBH, 1 mmol) with 4-hydroxybenzaldehyde (1.5 mmol) in 10 mL methanol. After heating to 60°C, three drops of TFA were added as catalyst, and the mixture was refluxed for 24 h. The crude product was purified by column chromatography on silica gel using ethyl acetate/petroleum ether (1:4, v/v) as eluent. The purified yellow solid named RBPA was dried under vacuum and characterized by 13 C NMR and 1 H NMR (yield: ~68%; see Figure S1 ). Conjugation of RBPA to Nanoprobe (CS-UCNP@mSiO₂–RBPA) CS-UCNP@mSiO₂–NH₂ (50 mg) was dispersed in 10 mL anhydrous ethanol and sonicated for 15 min. RBPA (50 mg) was added, and the mixture was refluxed at 70°C for 24 h under N₂ to enable amide bond formation between surface –NH₂ groups and the carboxylic acid (or aldehyde/hydrazide-derived imine) of RBPA. The final product was isolated by centrifugation, washed thoroughly with ethanol to remove unbound dye, and dried under vacuum. 3. Results and Discussion 3.1 Characterization of the Nanoprobe CS-UCNP@mSiO₂–RBPA The as-synthesized nanomaterials were systematically characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), and Fourier-transform infrared (FT-IR) spectroscopy. As shown in Fig. 1 a, the bare upconversion nanoparticles (UCNPs) exhibited a uniform spherical morphology and excellent dispersion in cyclohexane, with an average diameter of approximately 36 nm. Following epitaxial growth of an inert NaYF₄ shell, the resulting core–shell UCNPs (CS-UCNPs) maintained good monodispersity and colloidal stability in the same solvent (Fig. 1 b), while the average particle size increased to ཞ42 nm. This core–shell architecture led to a pronounced enhancement in luminescence intensity under 980 nm excitation, as illustrated in Fig. 1 g. This improvement is attributed to the effective passivation of surface defects that otherwise facilitate non-radiative relaxation pathways 15 . Both UCNP and CS-UCNP displayed characteristic Er³⁺ emission bands at ཞ544 nm (⁴S₃/₂ → ⁴I₁₅/₂) and ཞ660 nm (⁴F₉/₂ → ⁴I₁₅/₂) 16 . Subsequent coating with a mesoporous silica (mSiO₂) layer yielded the CS-UCNP@mSiO₂ nanostructure, which showed good dispersibility in absolute ethanol. TEM imaging in Fig. 1 h clearly reveals a well-defined silica shell uniformly encapsulating the CS-UCNP core. Amino functionalization of the silica surface to produce CS-UCNP@mSiO₂(NH₂) did not induce any noticeable morphological alterations, and the characteristic Er³⁺ emission bands remained unchanged, confirming the structural integrity and optical robustness of the nanoplatform throughout the sequential surface modifications. XRD analysis confirmed that the crystal phase of the synthesized UCNP matched well with the hexagonal standard reference (JCPDS No. 16–0334) 17 – 18 , with no impurity peaks detected, indicating high phase purity (Figure S2 ). A slight reduction in diffraction peak intensity was observed for CS-UCNP@mSiO₂–RBPA compared to the bare UCNP and CS-UCNP, likely due to the incorporation of the organic RBPA molecules within the mesopores, which partially disrupts the long-range order of the inorganic framework. FT-IR spectroscopy provided further evidence for successful stepwise functionalization. The appearance of characteristic absorption bands at 554 cm⁻¹ (Si–O bending) and 1082 cm⁻¹ (Si–O–Si asymmetric stretching) confirmed the formation of the silica shell 19 – 20 . The disappearance of C–H stretching vibrations at 2910 and 2980 cm⁻¹ in the CS-UCNP@mSiO₂ spectrum indicated effective removal of residual cetyltrimethylammonium bromide (CTAB) surfactant after silanization 21 . Finally, the FT-IR spectrum of CS-UCNP@mSiO₂–RBPA (Figure S3 ) exhibited new peaks at 1398, 1445, and 1590 cm⁻¹, assigned to C = C stretching vibrations of aromatic rings, along with a band at 1690 cm⁻¹ corresponding to C = N stretching 22 . These features unambiguously verify the successful conjugation of the rhodamine-based probe RBPA onto the amino-functionalized mesoporous silica surface. 3.2 Selectivity and Anti-Interference Performance of the RBPA Chemosensor toward Hg²⁺ The selectivity of the RBPA chemosensor for Hg²⁺ was evaluated by monitoring its UV–vis absorption response upon exposure to a panel of metal ions. As shown in Fig. 2 a, RBPA in anhydrous ethanol exhibited negligible absorbance above 500 nm, consistent with its closed spirolactam structure, which suppresses the conjugated chromophore system 23 . Upon addition of Hg²⁺, a pronounced absorption band emerged at 558 nm, accompanied by a distinct color change from colorless to magenta (Fig. 2 c). This spectral shift is attributed to Hg²⁺-induced ring opening of the spirolactam moiety, resulting in the formation of a delocalized xanthene-based fluorophore (see proposed mechanism in Figure S4 ). In contrast, the introduction of other common metal ions—including K⁺, Na⁺, Ca²⁺, Mg²⁺, Cu²⁺, Mn²⁺, Co²⁺, Ni²⁺, Cd²⁺, Ba²⁺, Zn²⁺, and Pb²⁺—elicited no significant changes in the absorption spectrum. Slight absorbance increases were observed only with Fe³⁺, Al³⁺, and Cr³⁺, likely due to their high charge density and partial interaction with the probe; however, the resulting color development was markedly weaker than that induced by Hg²⁺. To further assess the anti-interference capability of RBPA, competitive experiments were conducted by treating RBPA with equimolar mixtures of Hg²⁺ and each interfering ion. As illustrated in Fig. 2 b, the absorbance at 558 nm remained essentially unchanged in all coexistence scenarios, demonstrating that the presence of competing metal ions does not compromise the sensor’s response to Hg²⁺. These results confirm that RBPA exhibits excellent specificity and robustness for Hg²⁺ detection even in complex ionic matrices. 3.3 Optimization of Reaction Conditions The influence of pH on the RBPA–Hg²⁺ recognition process was systematically investigated over the range of pH 3–10 (Fig. 3 a). The absorbance of the RBPA–Hg²⁺ complex was highest between pH 4 and 7, with a maximum at pH 4. However, this peak absorbance does not necessarily correspond to optimal sensing performance. Notably, in the absence of Hg²⁺, RBPA itself displayed measurable background absorbance in mildly acidic conditions (pH 4–5), attributable to protonation-induced partial ring opening of the spirolactam. This leads to a non-specific signal that could interfere with accurate Hg²⁺ quantification. At pH < 4, the absorbance of the RBPA–Hg²⁺ complex decreased significantly, likely due to competitive protonation of the nitrogen and carbonyl oxygen atoms in the receptor site, which diminishes its affinity for Hg²⁺. Conversely, under alkaline conditions (pH > 7), the absorbance declined sharply and approached baseline levels. This is ascribed to the hydrolysis of Hg²⁺, which forms insoluble HgO or hydroxo complexes (e.g., Hg(OH)₂), thereby reducing the concentration of free Hg²⁺ available for binding 24 . Considering both signal intensity and background interference, neutral pH (~ 7) was selected as the optimal condition for Hg²⁺ detection. At this pH, RBPA remains predominantly in its non-absorbing closed form in the absence of Hg²⁺, while still enabling efficient Hg²⁺-triggered ring opening and a strong analytical signal. This balance ensures high sensitivity, low false-positive rates, and reliable performance in practical applications. The kinetics of the RBPA–Hg²⁺ interaction were examined by recording time-resolved UV–vis absorption spectra (Fig. 3 b). The absorbance at 558 nm reached a stable plateau within seconds after Hg²⁺ addition, with no further significant change observed over longer periods. This rapid response indicates that the coordination-driven ring-opening reaction occurs almost instantaneously, enabling real-time visual or spectroscopic detection of Hg²⁺ without the need for extended incubation. 3.4 Response Sensitivity of the RBPA Chemosensor toward Hg²⁺ To assess the sensitivity of RBPA for Hg²⁺ detection, UV–vis absorption spectra were recorded upon titration of RBPA with increasing concentrations of Hg²⁺ (0–70 µM) under the optimized conditions (pH ≈ 7, room temperature). As shown in Fig. 3 e, a gradual and concentration-dependent increase in absorbance at 558 nm was observed, accompanied by a visible color transition from colorless to magenta (Fig. 3 g), confirming the progressive formation of the ring-opened RBPA–Hg²⁺ complex. A calibration curve was constructed by plotting the absorbance at 558 nm against Hg²⁺ concentration (Fig. 3 f). The linear regression yielded the equation: A = 0.2117 C + 0.2557 where A is the absorbance and C is the Hg²⁺ concentration (in µM). The high correlation coefficient ( R 2 = 0.9956) indicates excellent linearity over the tested range. The limit of detection (LOD) was calculated using the standard formula 25 – 26 : LOD = k/ 3 σ ​ where σ is the standard deviation of 11 replicate blank measurements (RBPA without Hg²⁺), and k is the slope of the calibration curve (0.2117 µM⁻¹). This analysis gave an LOD of 0.227 µM, demonstrating that RBPA enables sensitive detection of Hg²⁺ at sub-micromolar levels. The molar absorptivity ( ε ) of the RBPA–Hg²⁺ complex was determined using the Beer–Lambert law ( A = εbc ), where b = 1 cm is the path length and c is the Hg²⁺ concentration. Based on the absorbance data in Fig. 3 c, ε was calculated to be 3.26 × 10⁴ M⁻¹ cm⁻¹, reflecting a strong absorption cross-section suitable for optical sensing. Finally, the binding stoichiometry between RBPA and Hg²⁺ was investigated using the method of continuous variation (Job’s plot; Fig. 3 d). The maximum absorbance occurred at a mole fraction of 0.5, confirming a 1:1 binding ratio between RBPA and Hg²⁺ in the resulting complex. These results demonstrate that the RBPA-based probe exhibits high sensitivity, strong chromogenic response, well-defined stoichiometry, and a low detection limit—key attributes for its potential application in environmental monitoring and biological sensing of mercury ions. 3.5 Ratiometric Fluorescence Response of CS-UCNP@mSiO₂–RBPA to Hg²⁺ To enable sensitive and self-referenced detection of Hg²⁺ under near-infrared (NIR) excitation, we constructed a core–shell upconversion nanoprobe, denoted CS-UCNP@mSiO₂–RBPA, by conjugating the RBPA receptor onto silica-coated upconversion nanoparticles (UCNPs). Upon 980 nm laser irradiation, the bare probe exhibited characteristic green emissions at ~ 543 nm and red emissions at ~ 655 nm, originating from the 4S 3/2 ​ → 4I 15/2​ and 4F 9/2​ → 4I 15/2​ transitions of Er³⁺ dopants, respectively (Fig. 4 a). Upon addition of Hg²⁺, a new yellow emission band centered at ~ 584 nm emerged and intensified progressively with increasing Hg²⁺ concentration (Fig. 4 b). This band is attributed to the ring-opened form of RBPA, which becomes fluorescent upon binding Hg²⁺ and subsequently accepts energy from the UCNP via Förster resonance energy transfer (FRET) or radiative reabsorption. The appearance of this Hg²⁺-dependent yellow emission provides a built-in signaling channel distinct from the intrinsic UCNP emissions. Quantitative analysis revealed that the intensity of the green emission at 543 nm remained relatively stable (Fig. 4 c), while the yellow emission at 584 nm increased linearly with Hg²⁺ concentration (Fig. 4 d), confirming the selective activation of the RBPA reporter unit. To minimize environmental fluctuations and enhance reliability, ratiometric measurements were employed. As shown in Fig. 4 e, the intensity ratio of green-to-red fluorescence ( I 543 ​/ I 655 ​) showed minimal variation, validating the red emission as a stable internal reference. In contrast, the yellow-to-red intensity ratio ( I 584 ​/ I 655 ​) exhibited a strong, concentration-dependent increase (Fig. 4 f), offering a robust ratiometric signal for quantitative Hg²⁺ detection. 3.6 Detection Limit and Comparative Performance The limit of detection (LOD) for the CS-UCNP@mSiO₂–RBPA nanoprobe was determined based on the linear relationship between the ratiometric signal ( I 584 ​/ I 655 ​) and Hg²⁺ concentration, yielding an exceptionally low LOD of 14 nM (0.014 µM). As summarized in Table 1 , this performance surpasses or rivals that of most recently reported fluorescent or colorimetric nanoprobes for Hg²⁺. Table 1 Comparison of other reported different types of nanoprobe and their detection limits for Hg 2+ ion with the CS-UCNP@SiO 2 -RBPA. Probes Media Test Method Sensitivity/µM LOD/µM References CdTe-MSA water UV-Vis 0.2-6 0.05 27 CsPbBr 3 nano-crystals water Fluorescence 0.37–7.5 0.229 28 Eu-DHTADPA ethanol Fluorescence 2–20 40 29 FA-PDA FONs water Fluorescence 0–18 0.18 30 N-CDs water Fluorescence 0.014-50 0.014 31 RBLS ethanol, water Fluorescence 0–20 0.26 32 Rh-PP-Rh ethanol, water Fluorescence 0–5 334 33 Si-CQDsRhB HEPES buffer solution Fluorescence 0–80 0.6 34 TPH water Fluorescence 0–5 16 35 Yellow emissive carbon dots (YCDs) water Fluorescence 0.4-1.0 16.8 36 CS-UCNP @mSiO 2 -RBPA ethanol ethanol UV-Vis Fluorescence 0–70 0–70 0.227 0.014 This work This work 4. Conclusion In this work, we successfully designed and fabricated a silica-coated, amino-functionalized upconversion luminescent nanoprobe, CS-UCNP@mSiO₂–RBPA, for the highly sensitive and selective detection of trace Hg²⁺ ions. The core–shell UCNPs serve as efficient nanotransducers, converting near-infrared (NIR) excitation at 980 nm into visible upconverted emissions. The rhodamine-based chemosensor RBPA was covalently anchored to the silica shell, acting as a Hg²⁺-responsive signaling unit that remains non-fluorescent in its closed spirolactam form but undergoes ring-opening upon binding Hg²⁺, thereby generating a strong yellow emission at 584 nm. Upon addition of Hg²⁺, the probe solution exhibited a distinct colorimetric response—changing from colorless to magenta—and, under 980 nm irradiation, displayed a progressive decrease in green upconversion fluorescence (543 nm) accompanied by the emergence and intensification of a new yellow emission band (584 nm). This spectral evolution confirms effective energy transfer from the UCNP donor to the ring-opened RBPA acceptor, triggered specifically by Hg²⁺ coordination. By employing a ratiometric fluorescence strategy based on the intensity ratios I 584 ​/ I 655​ and I 543 ​/ I 655 ​, we achieved reliable and self-calibrated quantification of Hg²⁺, minimizing interference from environmental fluctuations or probe concentration variations. The sensor demonstrated a remarkably low limit of detection of 14 nM, outperforming many existing fluorescent probes. Moreover, the use of NIR excitation eliminates background autofluorescence and enables potential application in complex or turbid media, including biological and environmental samples. Declarations Declaration of Competing Interest The authors declare no competing financial interest. CRediT authorship contribution statement Yanmei Zhang : Data curation, Formal analysis, Investigation, Writing-original draft. Rui Jiang: Formal analysis, Investigation. Hongze Yang : Investigation, Formal analysis. Cheng Zhou: Data curation, Formal analysis. Mengxi Sun: Data curation, Methodology. Hong Chen: Investigation, Formal analysis. Donghai Lin: Funding acquisition, Methodology, Writing-review & editing. Li ming Huang: Writing-review & editing. Guosong Chen : Conceptualization, Project administration, Funding acquisition, Supervision, Writing-review & editing. Data Availability Data will be made available on request. ORCID Donghai Lin: 0000-0003-4073-4132 Funding Declaration This work was supported by the National Major Scientific Research Instrument Development Project of China (Grant No. 22327809) and the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning (SIHL), the Gaoyuan Discipline of Shanghai—Materials Science and Engineering (Grant No. A30NH221903), and the Shanghai Polytechnic University–Drexel University Joint Research Center for Optoelectronics and Sensing. Additional support was provided by the Science Fund for Distinguished Young Scholars of Fujian Province (Grant No. 2019J06027) and the Postdoctoral Fund of Foshan (Grant No. BKS206140). Appendix A. Supplementary data Supplementary data related to this article can be found online at https://doi.org/xxx.xxx/xxx.xxx. References Chen, S.; Gao, Y. T.; Wang, C. K.; Gu, H. L.; Sun, M. K.; Dang, Y. H.; Ai, S. W. Heavy metal pollution status, children health risk assessment and source apportionment in farmland soils in a typical polluted area, Northwest China. Stochastic Environmental Research and Risk Assessment 2024, 38 (6), 2383-2395, DOI: 10.1007/s00477-024-02685-4. Asiminicesei, D. M.; Fertu, D. I.; Gavrilescu, M. Impact of Heavy Metal Pollution in the Environment on the Metabolic Profile of Medicinal Plants and Their Therapeutic Potential. Plants (Basel, Switzerland) 2024, 13 (6), DOI: 10.3390/plants13060913. Naik, S.; Pradhan, U.; Karthikeyan, P.; Begum, M.; Panda, U. 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D.; Ferraro, F.; Ibarguen Becerra, C.; Chamorro, Y.; Maldonado, A. F. Coordination of Mercury(II) in Water Promoted over Hydrolysis in Solvated Clusters [Hg(H 2 O) 1-6 ](aq) 2+ : Insights from Relativistic Effects and Free Energy Analysis. J Phys Chem A 2023, 127 (39), 8032-8049, DOI: 10.1021/acs.jpca.3c02927. Lin, D.; Tang, T.; Jed Harrison, D.; Lee, W. E.; Jemere, A. B. A regenerating ultrasensitive electrochemical impedance immunosensor for the detection of adenovirus. Biosens Bioelectron 2015, 68 , 129-134, DOI: 10.1016/j.bios.2014.12.032. Yin, S. Y.; Wang, J. K.; Li, Y.; Wu, T. X.; Song, L. Y.; Zhu, Y. B.; Chen, Y. Z.; Cheng, K.; Zhang, J.; Ma, X. Z.; Donghai, L.; Chen, G. S. Macroscopically Oriented Magnetic Core-regularized Nanomaterials for Glucose Biosensors Assisted by Self-sacrificial Label. Electroanal 2021, 33 (10), 2216-2225, DOI: 10.1002/elan.202100231. Mohamed, A.; A., A. F. I.; A., A. T. Y.; Meryem, H.; Sarra, B. 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Shenna, C.; Yunping, H.; Ronghui, L.; Yanxu, L.; Jinxia, L.; Lina, G. N-doped carbon dots as the multifunctional fluorescent probe for mercury ion, glutathione and pH detection. Nanotechnology 2022, 34 (12), 125501, DOI: 10.1088/1361-6528/acade7. Jie, H.; Kaiyue, L.; Jiaxin, T.; Haiyan, W.; Chun, K. A rhodamine NIR probe for naked eye detection of mercury ions and its application. Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy 2023, 306 , 123553. Saleh, H. A.; Ferruh, L.; Sinan, B.; Haydar, K.; Mahmut, T. A Novel Rhodamine-Phenolphthalein Architecture for Selective Mercury Ion Detection in Aqueous Media. ChemPlusChem 2024, 89 (5), e202300649, DOI: 10.1002/cplu.202300649. Sen, L.; Lilei, Z.; Shiyu, L.; Siyao, Y.; Guoping, W. Selective detection of mercury ions via single and dual signals by silicon-doped carbon quantum dots. New Journal of Chemistry 2023, (30), 14242-14248, DOI: 10.1039/D3NJ02521G. Yanfeng, S.; Bingxu, L.; Zhifeng, W.; Yanhao, Z.; Zhibin, Z.; Xu, Z.; Fulin, L. Highly selective fluorescent probe for detecting mercury ions in water. RSC Advances 2023, 13 (28), 19091-19095, DOI: 10.1039/D3RA02791K. Yunqi, H.; Ying, S.; Tingting, L.; Yuzhuo, T.; Miaomiao, T.; Fang, C. Controllable synthesis of yellow emissive carbon dots by mild heating process and their utility as fluorescent test paper for detection of mercury(II) ions assistants by smartphone. Journal of Environmental Chemical Engineering 2023, 11 (3), 109863, DOI: 10.1016/j.jece.2023.109863. Additional Declarations No competing interests reported. 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particle size distribution of (e) UCNP and (f) CS-UCNP; luminescence effect of (g) UCNP and (f) CS-UCNP under \u0026nbsp;excitation with a 980 nm light source.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8629365/v1/65ed599b169227abe5f3797b.png"},{"id":101308426,"identity":"182f7f57-96d0-417b-9252-dbce82c722b5","added_by":"auto","created_at":"2026-01-28 10:29:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":156790,"visible":true,"origin":"","legend":"\u003cp\u003e(a) UV-visible absorption spectra of RBPA following the addition of 50 μM metal ions (K\u003csup\u003e+\u003c/sup\u003e, Na\u003csup\u003e+\u003c/sup\u003e, Ca\u003csup\u003e2+\u003c/sup\u003e, Mg\u003csup\u003e2+\u003c/sup\u003e, Cu\u003csup\u003e2+\u003c/sup\u003e, Co\u003csup\u003e2+\u003c/sup\u003e, Ni\u003csup\u003e2+\u003c/sup\u003e, Pb\u003csup\u003e2+\u003c/sup\u003e, Hg\u003csup\u003e2+\u003c/sup\u003e, Mn\u003csup\u003e2+\u003c/sup\u003e, Ba\u003csup\u003e2+\u003c/sup\u003e, Cd\u003csup\u003e2+\u003c/sup\u003e, Zn\u003csup\u003e2+\u003c/sup\u003e, Al\u003csup\u003e3+\u003c/sup\u003e, Fe\u003csup\u003e3+\u003c/sup\u003e and Cr\u003csup\u003e3+\u003c/sup\u003e); (b) test results of RBPA's anti-interference performance; (c) chromogenic effect of RBPA in the presence of various metal ions\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8629365/v1/7269e7b4a3fe661a28a978a4.png"},{"id":101308424,"identity":"d0db1a00-1c75-48c3-9943-53bdde213880","added_by":"auto","created_at":"2026-01-28 10:29:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":246264,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Absorbance of RBPA-Hg\u003csup\u003e2+\u003c/sup\u003e as a function of pH and (b) time; (c) molar absorbance and (d) molar ratio test spectrum of RBPA (e) absorption spectra of the reaction products formed between Hg\u003csup\u003e2+\u003c/sup\u003e with RBPA at varying concentrations; (f) calibration curve correlating the absorbance at 558 nm with the concentration of Hg\u003csup\u003e2+\u003c/sup\u003e; (g) color development effect of RBPA in response to different concentrations of Hg\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8629365/v1/c622e22cbac147e74721118f.png"},{"id":101308430,"identity":"c6e4e139-5900-4f39-b9d7-fc0c9c41b060","added_by":"auto","created_at":"2026-01-28 10:29:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":157492,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence spectrum of (a) CS-UCNP@SiO\u003csub\u003e2\u003c/sub\u003e-RBPA (b) newly observed yellow emission spectrum under 980 nm light source irradiation varies with Hg\u003csup\u003e2+\u003c/sup\u003e concentration; relationship between the intensity of (c) 543 nm green fluorescence and (d) 584 nm yellow fluorescence in relation to Hg\u003csup\u003e2+\u003c/sup\u003e concentration; intensity ratio of (e) green to red fluorescence and (f) yellow to red fluorescence in relation to Hg\u003csup\u003e2+\u003c/sup\u003e concentration.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8629365/v1/34b106bb96a708d36171d386.png"},{"id":108438041,"identity":"1eccbbdd-10a6-435b-bc97-65177d4ba5ed","added_by":"auto","created_at":"2026-05-04 16:05:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1572077,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8629365/v1/86dbaa64-f656-483e-89f4-abd96a342ed3.pdf"},{"id":101308422,"identity":"fc400229-0c5c-4c16-9205-eb5521bc0300","added_by":"auto","created_at":"2026-01-28 10:29:08","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2808320,"visible":true,"origin":"","legend":"","description":"","filename":"SupprotingInformation.doc","url":"https://assets-eu.researchsquare.com/files/rs-8629365/v1/26889fe2f54fedf8fe3f7cdd.doc"},{"id":101308428,"identity":"c286beda-a5b0-410f-bd66-6caed2bfa059","added_by":"auto","created_at":"2026-01-28 10:29:10","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":400897,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicAbstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-8629365/v1/b1d98091947913741f5ea430.docx"},{"id":101308427,"identity":"e147aed2-a1bc-40aa-a85e-bc6f830545ee","added_by":"auto","created_at":"2026-01-28 10:29:10","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":386830,"visible":true,"origin":"","legend":"","description":"","filename":"Scheme1.tif","url":"https://assets-eu.researchsquare.com/files/rs-8629365/v1/88bd38add8a5ad959a2e0708.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eA Near-Infrared Excited FRET-Based Fluorescent Sensor Using Core–Shell Upconversion Nanoparticles Functionalized with Rhodamine B Derivative for Highly Selective Detection of Hg²⁺\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIn recent years, heavy metal pollution has intensified dramatically due to anthropogenic activities, posing severe threats to ecosystems and human health\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Among toxic heavy metals, mercury(II) ion (Hg\u0026sup2;⁺) is particularly hazardous. Although it can originate from natural processes such as rock weathering, volcanic eruptions, and vegetation release, the majority of environmental Hg\u0026sup2;⁺ stems from industrial discharges, coal combustion, and mining operations. Upon entering the human body, Hg\u0026sup2;⁺ exhibits a strong affinity for thiol-containing biomolecules\u0026mdash;including cysteine, homocysteine, and reduced glutathione\u0026mdash;disrupting their physiological functions. Critically, Hg\u0026sup2;⁺ can cross the blood\u0026ndash;brain barrier, leading to neurotoxicity characterized by symptoms such as headache, insomnia, depression, memory impairment, and, in severe cases, coma or epilepsy. Moreover, chronic exposure causes irreversible damage to the liver and kidneys\u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Consequently, the development of reliable methods for the quantitative detection of trace Hg\u0026sup2;⁺ is of paramount importance for environmental monitoring and public health protection. Conventional analytical techniques for Hg\u0026sup2;⁺ detection\u0026mdash;including atomic absorption spectrometry (AAS)\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, inductively coupled plasma mass spectrometry (ICP-MS)\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, and UV\u0026ndash;vis spectrophotometry\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e\u0026mdash;offer high sensitivity. However, they suffer from significant drawbacks: expensive instrumentation, complex sample pretreatment, skilled operators, and lengthy analysis times. These limitations hinder on-site or real-time monitoring. Therefore, there is an urgent need for rapid, cost-effective, and user-friendly sensing platforms capable of visual or instrumental Hg\u0026sup2;⁺ detection.\u003c/p\u003e \u003cp\u003eFluorescent probes have emerged as promising alternatives due to their high sensitivity, operational simplicity, low cost, and potential for real-time response. Among various fluorophores, rhodamine-based derivatives are especially attractive owing to their excellent photostability, large molar absorptivity, high fluorescence quantum yield, and reversible ring-opening/closure behavior in response to specific analytes. For instance, Zhang et al. developed a rhodamine\u0026ndash;anthracene hybrid probe that enables colorimetric and fluorescent detection of Hg\u0026sup2;⁺ within 30 seconds with a visible color change\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Roy et al. reported a dual-responsive rhodamine probe for simultaneous detection of Hg\u0026sup2;⁺ and Al\u0026sup3;⁺ in aqueous\u0026ndash;organic media\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Erdemir et al. designed a rhodamine\u0026ndash;isophorone conjugate with a large Stokes shift to minimize background interference, achieving selective sensing of Hg\u0026sup2;⁺ and Cu\u0026sup2;⁺ even in complex matrices such as fungicide formulations\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Zhu et al. further enhanced selectivity by incorporating sulfur-functionalized rhodamine units, enabling sensitive Hg\u0026sup2;⁺ detection in environmental water samples\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Despite these advances, challenges remain in improving selectivity against competing metal ions, reducing cytotoxicity for biological applications, and enabling excitation in the near-infrared (NIR) window to avoid autofluorescence and enhance tissue penetration.\u003c/p\u003e \u003cp\u003eLanthanide-doped upconversion nanoparticles (UCNPs) offer a compelling solution to these limitations. These nanomaterials absorb low-energy NIR photons (typically at 980 nm) and emit higher-energy visible light through anti-Stokes processes. Their unique advantages\u0026mdash;including sharp emission bands, long luminescence lifetimes, exceptional photostability, negligible autofluorescence, and deep tissue penetration\u0026mdash;make them ideal candidates for biosensing and environmental analysis\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In particular, core\u0026ndash;shell structured UCNPs (CS-UCNPs) significantly suppress surface-related quenching, thereby enhancing luminescence intensity and quantum yield. When integrated with responsive organic dyes via fluorescence resonance energy transfer (FRET), UCNPs serve as efficient energy donors for ratiometric or turn-on/turn-off sensing. Several studies have demonstrated the utility of UCNP\u0026ndash;rhodamine hybrids: Tang et al. constructed a UCNP@SiO₂-based FRET probe for Fe\u0026sup3;⁺ detection\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e; Wang et al. developed a rhodamine-functionalized UCNP system for Hg\u0026sup2;⁺ sensing\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e; Xu et al. employed core\u0026ndash;shell UCNPs with rhodamine B hydrazide for intracellular Cu\u0026sup2;⁺ imaging\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e; while others have extended this strategy to detect CO\u003csup\u003e20\u003c/sup\u003e and glutathione\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHerein, we report a novel NIR-excitable fluorescent nanosensor, CS-UCNP@mSiO₂\u0026ndash;RBPA, fabricated by conjugating a rhodamine B-derived chemosensor (RBPA) onto amino-functionalized mesoporous silica-coated core\u0026ndash;shell upconversion nanoparticles. The core\u0026ndash;shell architecture enhances luminescence efficiency by passivating surface defects, while the mesoporous silica shell provides abundant anchoring sites for RBPA and improves colloidal stability. In the absence of Hg\u0026sup2;⁺, RBPA remains in its colorless, non-fluorescent spirolactam form. Upon Hg\u0026sup2;⁺ binding, the spirolactam ring opens, generating a magenta-colored species with strong absorption overlapping the green emission (\u0026sim;543 nm) of the UCNP donor. This spectral overlap triggers efficient FRET, resulting in a ratiometric fluorescence response: the green emission decreases while a new yellow emission (\u0026sim;584 nm) from RBPA increases, with the red emission (\u0026sim;658 nm) serving as an internal reference. This design enables highly selective, sensitive, and visually discernible detection of trace Hg\u0026sup2;⁺ under NIR excitation\u0026mdash;a feature particularly advantageous for minimizing background interference in complex environmental or biological samples. The preparation and sensing mechanism of the probe are illustrated in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Experimental Section","content":"\u003cp\u003e \u003cb\u003eMaterials\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAll reagents and solvents were used as received without further purification. YCl₃\u0026middot;6H₂O (99.99%), YbCl₃\u0026middot;6H₂O (99.99%), ErCl₃\u0026middot;6H₂O (99.99%), 4-hydroxybenzaldehyde (99%), (3-aminopropyl)triethoxysilane (APTES, 99%), trifluoroacetic acid (TFA, 99%), oleic acid (OA, 90%), 1-octadecene (ODE, 90%), and tetraethyl orthosilicate (TEOS, 98%) were purchased from Aladdin Biochemical Technology Co., Ltd. Rhodamine B hydrazide (97%) was obtained from Zhengzhou Alpha Chemical Co., Ltd. Ammonium fluoride (NH₄F, 96%) was supplied by Shanghai Trial Sihewei Chemical Co., Ltd. Sodium hydroxide (NaOH, 96%) was acquired from Xilong Scientific Co., Ltd. Hexadecyltrimethylammonium bromide (CTAB, 99%) was purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. Cyclohexane (AR, \u0026ge;\u0026thinsp;99.5%), anhydrous ethanol (AR, \u0026ge;\u0026thinsp;99.7%), methanol (AR, \u0026ge;\u0026thinsp;99.5%), and hydrochloric acid (AR, 36\u0026ndash;38%) were obtained from China National Pharmaceutical Group Chemical Reagent Co., Ltd. Deionized water (18.2 MΩ\u0026middot;cm) was used for all aqueous solutions. Standard aqueous solutions (1 mM) of K⁺, Na⁺, Ca\u0026sup2;⁺, Mg\u0026sup2;⁺, Cu\u0026sup2;⁺, Zn\u0026sup2;⁺, Co\u0026sup2;⁺, Ni\u0026sup2;⁺, Cd\u0026sup2;⁺, Hg\u0026sup2;⁺, Ba\u0026sup2;⁺, Fe\u0026sup3;⁺, Al\u0026sup3;⁺, Cr\u0026sup3;⁺, Mn\u0026sup2;⁺, and Pb\u0026sup2;⁺ were prepared from their corresponding chloride or nitrate salts.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCharacterization\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTransmission electron microscopy (TEM) images were acquired using a Talos L120C microscope (Thermo Fisher Scientific, USA) by drop-casting nanoparticle suspensions onto copper grids. X-ray diffraction (XRD) patterns were recorded on a Rigaku MiniFlex 600 diffractometer (Rigaku Corporation, Japan) with Cu Kα radiation (λ\u0026thinsp;=\u0026thinsp;1.5406 \u0026Aring;). Fourier-transform infrared (FT-IR) spectra were collected on a Nicolet iS10 spectrometer (Thermo Fisher Scientific, USA) in the range of 400\u0026ndash;4000 cm⁻\u0026sup1;. UV\u0026ndash;vis absorption spectra were measured using an Agilent 8453 spectrophotometer (Agilent Technologies, USA). Upconversion fluorescence (UCF) spectra were recorded on an F97XP fluorescence spectrophotometer (Shanghai Lengguang Technology Co., Ltd., China) under 980 nm laser diode excitation (power density: ~1.0 W/cm\u0026sup2;). \u0026sup1;H NMR spectra were obtained on a JNM-ECZ401S spectrometer (JEOL Ltd., Japan) at 400 MHz using deuterated solvents.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSynthesis of β-NaYF₄:Yb,Er UCNPs (UCNPs)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eβ-NaYF₄:Yb,Er upconversion nanoparticles were synthesized via a thermal decomposition method. LnCl₃\u0026middot;6H₂O precursors (total 1 mmol; Y:Yb:Er molar ratio\u0026thinsp;=\u0026thinsp;80:18:2) were dissolved in 10 mL methanol and transferred to a 250 mL three-neck flask. Oleic acid (6 mL) and 1-octadecene (15 mL) were added, and the mixture was heated to 90\u0026deg;C under vigorous stirring to remove methanol until a clear solution formed. The temperature was then raised to 150\u0026deg;C and held for 30 min under N₂ atmosphere. After cooling to 50\u0026deg;C, a methanolic solution containing NaOH (2.5 mmol) and NH₄F (4 mmol) in 15 mL methanol was added dropwise under stirring (400 rpm). The mixture was heated again to 90\u0026deg;C to evaporate residual methanol, then ramped to 315\u0026deg;C and maintained for 60 min under N₂. After cooling to room temperature, the product was precipitated by adding ethanol, centrifuged at 8000 rpm for 8 min, and washed twice with cyclohexane/ethanol (1:1 v/v). The final UCNPs were dispersed in 10 mL cyclohexane for further use.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSynthesis of Core\u0026ndash;Shell UCNPs (CS-UCNPs)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA NaYF₄ inert shell was grown epitaxially on the β-NaYF₄:Yb,Er core. Briefly, 10 mL of the as-prepared UCNP cyclohexane dispersion (~\u0026thinsp;10 mg/mL) was mixed with a solution of YCl₃\u0026middot;6H₂O (1 mmol) in 10 mL methanol. The mixture was processed identically to the core synthesis, except that NaOH/NH₄F were replaced by the preformed UCNP dispersion as the seed. The resulting CS-UCNPs were purified and stored in cyclohexane.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSynthesis of Mesoporous Silica-Coated CS-UCNPs (CS-UCNP@mSiO₂)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA microemulsion method was employed for silica coating. CTAB (0.2 g) was dissolved in 20 mL water, followed by addition of 5 mL CS-UCNP cyclohexane solution (10 mg/mL). The mixture was stirred vigorously at 60\u0026deg;C until cyclohexane evaporated, yielding a transparent aqueous dispersion. Ethanol (3 mL), water (10 mL), and NaOH (150 \u0026micro;L of 0.1 M) were added, and the mixture was sonicated for 15 min. TEOS (220 \u0026micro;L) was then injected dropwise, and the reaction was refluxed at 70\u0026deg;C for 2 h. The product was collected by centrifugation, washed twice with ethanol, and dried at 60\u0026deg;C overnight.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSynthesis of Amino-Functionalized CS-UCNP@mSiO₂ (CS-UCNP@mSiO₂\u0026ndash;NH₂)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe CTAB template was removed by refluxing the CS-UCNP@mSiO₂ in 30 mL of 0.1 M HCl in ethanol at 70\u0026deg;C for 24 h. The solid was centrifuged, washed with ethanol, and dried at 80\u0026deg;C. For amino functionalization, 100 mg of the template-free material was dispersed in 15 mL anhydrous ethanol, and APTES (150 \u0026micro;L) was added under N₂. The mixture was refluxed at 70\u0026deg;C for 24 h, then cooled, centrifuged, washed twice with ethanol, and dried under vacuum.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSynthesis of Rhodamine B-Based Probe (RBPA)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eRBPA was synthesized via condensation of rhodamine B hydrazide (RBH, 1 mmol) with 4-hydroxybenzaldehyde (1.5 mmol) in 10 mL methanol. After heating to 60\u0026deg;C, three drops of TFA were added as catalyst, and the mixture was refluxed for 24 h. The crude product was purified by column chromatography on silica gel using ethyl acetate/petroleum ether (1:4, v/v) as eluent. The purified yellow solid named RBPA was dried under vacuum and characterized by \u003csup\u003e13\u003c/sup\u003eC NMR and \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR (yield: ~68%; see Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eConjugation of RBPA to Nanoprobe (CS-UCNP@mSiO₂\u0026ndash;RBPA)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCS-UCNP@mSiO₂\u0026ndash;NH₂ (50 mg) was dispersed in 10 mL anhydrous ethanol and sonicated for 15 min. RBPA (50 mg) was added, and the mixture was refluxed at 70\u0026deg;C for 24 h under N₂ to enable amide bond formation between surface \u0026ndash;NH₂ groups and the carboxylic acid (or aldehyde/hydrazide-derived imine) of RBPA. The final product was isolated by centrifugation, washed thoroughly with ethanol to remove unbound dye, and dried under vacuum.\u003c/p\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Characterization of the Nanoprobe CS-UCNP@mSiO₂\u0026ndash;RBPA\u003c/h2\u003e \u003cp\u003eThe as-synthesized nanomaterials were systematically characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), and Fourier-transform infrared (FT-IR) spectroscopy. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, the bare upconversion nanoparticles (UCNPs) exhibited a uniform spherical morphology and excellent dispersion in cyclohexane, with an average diameter of approximately 36 nm. Following epitaxial growth of an inert NaYF₄ shell, the resulting core\u0026ndash;shell UCNPs (CS-UCNPs) maintained good monodispersity and colloidal stability in the same solvent (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), while the average particle size increased to ཞ42 nm. This core\u0026ndash;shell architecture led to a pronounced enhancement in luminescence intensity under 980 nm excitation, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg. This improvement is attributed to the effective passivation of surface defects that otherwise facilitate non-radiative relaxation pathways\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Both UCNP and CS-UCNP displayed characteristic Er\u0026sup3;⁺ emission bands at ཞ544 nm (⁴S₃/₂ \u0026rarr; ⁴I₁₅/₂) and ཞ660 nm (⁴F₉/₂ \u0026rarr; ⁴I₁₅/₂)\u003csup\u003e16\u003c/sup\u003e. Subsequent coating with a mesoporous silica (mSiO₂) layer yielded the CS-UCNP@mSiO₂ nanostructure, which showed good dispersibility in absolute ethanol. TEM imaging in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh clearly reveals a well-defined silica shell uniformly encapsulating the CS-UCNP core. Amino functionalization of the silica surface to produce CS-UCNP@mSiO₂(NH₂) did not induce any noticeable morphological alterations, and the characteristic Er\u0026sup3;⁺ emission bands remained unchanged, confirming the structural integrity and optical robustness of the nanoplatform throughout the sequential surface modifications.\u003c/p\u003e \u003cp\u003eXRD analysis confirmed that the crystal phase of the synthesized UCNP matched well with the hexagonal standard reference (JCPDS No. 16\u0026ndash;0334)\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, with no impurity peaks detected, indicating high phase purity (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). A slight reduction in diffraction peak intensity was observed for CS-UCNP@mSiO₂\u0026ndash;RBPA compared to the bare UCNP and CS-UCNP, likely due to the incorporation of the organic RBPA molecules within the mesopores, which partially disrupts the long-range order of the inorganic framework. FT-IR spectroscopy provided further evidence for successful stepwise functionalization. The appearance of characteristic absorption bands at 554 cm⁻\u0026sup1; (Si\u0026ndash;O bending) and 1082 cm⁻\u0026sup1; (Si\u0026ndash;O\u0026ndash;Si asymmetric stretching) confirmed the formation of the silica shell\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. The disappearance of C\u0026ndash;H stretching vibrations at 2910 and 2980 cm⁻\u0026sup1; in the CS-UCNP@mSiO₂ spectrum indicated effective removal of residual cetyltrimethylammonium bromide (CTAB) surfactant after silanization\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Finally, the FT-IR spectrum of CS-UCNP@mSiO₂\u0026ndash;RBPA (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e) exhibited new peaks at 1398, 1445, and 1590 cm⁻\u0026sup1;, assigned to C\u0026thinsp;=\u0026thinsp;C stretching vibrations of aromatic rings, along with a band at 1690 cm⁻\u0026sup1; corresponding to C\u0026thinsp;=\u0026thinsp;N stretching\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. These features unambiguously verify the successful conjugation of the rhodamine-based probe RBPA onto the amino-functionalized mesoporous silica surface.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Selectivity and Anti-Interference Performance of the RBPA Chemosensor toward Hg\u0026sup2;⁺\u003c/h2\u003e \u003cp\u003eThe selectivity of the RBPA chemosensor for Hg\u0026sup2;⁺ was evaluated by monitoring its UV\u0026ndash;vis absorption response upon exposure to a panel of metal ions. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, RBPA in anhydrous ethanol exhibited negligible absorbance above 500 nm, consistent with its closed spirolactam structure, which suppresses the conjugated chromophore system\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Upon addition of Hg\u0026sup2;⁺, a pronounced absorption band emerged at 558 nm, accompanied by a distinct color change from colorless to magenta (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). This spectral shift is attributed to Hg\u0026sup2;⁺-induced ring opening of the spirolactam moiety, resulting in the formation of a delocalized xanthene-based fluorophore (see proposed mechanism in Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). In contrast, the introduction of other common metal ions\u0026mdash;including K⁺, Na⁺, Ca\u0026sup2;⁺, Mg\u0026sup2;⁺, Cu\u0026sup2;⁺, Mn\u0026sup2;⁺, Co\u0026sup2;⁺, Ni\u0026sup2;⁺, Cd\u0026sup2;⁺, Ba\u0026sup2;⁺, Zn\u0026sup2;⁺, and Pb\u0026sup2;⁺\u0026mdash;elicited no significant changes in the absorption spectrum. Slight absorbance increases were observed only with Fe\u0026sup3;⁺, Al\u0026sup3;⁺, and Cr\u0026sup3;⁺, likely due to their high charge density and partial interaction with the probe; however, the resulting color development was markedly weaker than that induced by Hg\u0026sup2;⁺. To further assess the anti-interference capability of RBPA, competitive experiments were conducted by treating RBPA with equimolar mixtures of Hg\u0026sup2;⁺ and each interfering ion. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, the absorbance at 558 nm remained essentially unchanged in all coexistence scenarios, demonstrating that the presence of competing metal ions does not compromise the sensor\u0026rsquo;s response to Hg\u0026sup2;⁺. These results confirm that RBPA exhibits excellent specificity and robustness for Hg\u0026sup2;⁺ detection even in complex ionic matrices.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Optimization of Reaction Conditions\u003c/h2\u003e \u003cp\u003eThe influence of pH on the RBPA\u0026ndash;Hg\u0026sup2;⁺ recognition process was systematically investigated over the range of pH 3\u0026ndash;10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The absorbance of the RBPA\u0026ndash;Hg\u0026sup2;⁺ complex was highest between pH 4 and 7, with a maximum at pH 4. However, this peak absorbance does not necessarily correspond to optimal sensing performance. Notably, in the absence of Hg\u0026sup2;⁺, RBPA itself displayed measurable background absorbance in mildly acidic conditions (pH 4\u0026ndash;5), attributable to protonation-induced partial ring opening of the spirolactam. This leads to a non-specific signal that could interfere with accurate Hg\u0026sup2;⁺ quantification. At pH\u0026thinsp;\u0026lt;\u0026thinsp;4, the absorbance of the RBPA\u0026ndash;Hg\u0026sup2;⁺ complex decreased significantly, likely due to competitive protonation of the nitrogen and carbonyl oxygen atoms in the receptor site, which diminishes its affinity for Hg\u0026sup2;⁺. Conversely, under alkaline conditions (pH\u0026thinsp;\u0026gt;\u0026thinsp;7), the absorbance declined sharply and approached baseline levels. This is ascribed to the hydrolysis of Hg\u0026sup2;⁺, which forms insoluble HgO or hydroxo complexes (e.g., Hg(OH)₂), thereby reducing the concentration of free Hg\u0026sup2;⁺ available for binding\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Considering both signal intensity and background interference, neutral pH (~\u0026thinsp;7) was selected as the optimal condition for Hg\u0026sup2;⁺ detection. At this pH, RBPA remains predominantly in its non-absorbing closed form in the absence of Hg\u0026sup2;⁺, while still enabling efficient Hg\u0026sup2;⁺-triggered ring opening and a strong analytical signal. This balance ensures high sensitivity, low false-positive rates, and reliable performance in practical applications.\u003c/p\u003e \u003cp\u003eThe kinetics of the RBPA\u0026ndash;Hg\u0026sup2;⁺ interaction were examined by recording time-resolved UV\u0026ndash;vis absorption spectra (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). The absorbance at 558 nm reached a stable plateau within seconds after Hg\u0026sup2;⁺ addition, with no further significant change observed over longer periods. This rapid response indicates that the coordination-driven ring-opening reaction occurs almost instantaneously, enabling real-time visual or spectroscopic detection of Hg\u0026sup2;⁺ without the need for extended incubation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Response Sensitivity of the RBPA Chemosensor toward Hg\u0026sup2;⁺\u003c/h2\u003e \u003cp\u003eTo assess the sensitivity of RBPA for Hg\u0026sup2;⁺ detection, UV\u0026ndash;vis absorption spectra were recorded upon titration of RBPA with increasing concentrations of Hg\u0026sup2;⁺ (0\u0026ndash;70 \u0026micro;M) under the optimized conditions (pH\u0026thinsp;\u0026asymp;\u0026thinsp;7, room temperature). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee, a gradual and concentration-dependent increase in absorbance at 558 nm was observed, accompanied by a visible color transition from colorless to magenta (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg), confirming the progressive formation of the ring-opened RBPA\u0026ndash;Hg\u0026sup2;⁺ complex. A calibration curve was constructed by plotting the absorbance at 558 nm against Hg\u0026sup2;⁺ concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). The linear regression yielded the equation:\u003c/p\u003e \u003cp\u003e \u003cem\u003eA\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.2117\u003cem\u003eC\u003c/em\u003e\u0026thinsp;+\u0026thinsp;0.2557\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003eA\u003c/em\u003e is the absorbance and \u003cem\u003eC\u003c/em\u003e is the Hg\u0026sup2;⁺ concentration (in \u0026micro;M). The high correlation coefficient (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9956) indicates excellent linearity over the tested range.\u003c/p\u003e \u003cp\u003eThe limit of detection (LOD) was calculated using the standard formula\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e:\u003c/p\u003e \u003cp\u003eLOD\u0026thinsp;=\u0026thinsp;\u003cem\u003ek/\u003c/em\u003e3\u003cem\u003eσ\u003c/em\u003e​\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003eσ\u003c/em\u003e is the standard deviation of 11 replicate blank measurements (RBPA without Hg\u0026sup2;⁺), and \u003cem\u003ek\u003c/em\u003e is the slope of the calibration curve (0.2117 \u0026micro;M⁻\u0026sup1;). This analysis gave an LOD of 0.227 \u0026micro;M, demonstrating that RBPA enables sensitive detection of Hg\u0026sup2;⁺ at sub-micromolar levels. The molar absorptivity (\u003cem\u003eε\u003c/em\u003e) of the RBPA\u0026ndash;Hg\u0026sup2;⁺ complex was determined using the Beer\u0026ndash;Lambert law (\u003cem\u003eA\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u003cem\u003eεbc\u003c/em\u003e), where \u003cem\u003eb\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1 cm is the path length and \u003cem\u003ec\u003c/em\u003e is the Hg\u0026sup2;⁺ concentration. Based on the absorbance data in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, \u003cem\u003eε\u003c/em\u003e was calculated to be 3.26 \u0026times; 10⁴ M⁻\u0026sup1; cm⁻\u0026sup1;, reflecting a strong absorption cross-section suitable for optical sensing. Finally, the binding stoichiometry between RBPA and Hg\u0026sup2;⁺ was investigated using the method of continuous variation (Job\u0026rsquo;s plot; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). The maximum absorbance occurred at a mole fraction of 0.5, confirming a 1:1 binding ratio between RBPA and Hg\u0026sup2;⁺ in the resulting complex. These results demonstrate that the RBPA-based probe exhibits high sensitivity, strong chromogenic response, well-defined stoichiometry, and a low detection limit\u0026mdash;key attributes for its potential application in environmental monitoring and biological sensing of mercury ions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Ratiometric Fluorescence Response of CS-UCNP@mSiO₂\u0026ndash;RBPA to Hg\u0026sup2;⁺\u003c/h2\u003e \u003cp\u003eTo enable sensitive and self-referenced detection of Hg\u0026sup2;⁺ under near-infrared (NIR) excitation, we constructed a core\u0026ndash;shell upconversion nanoprobe, denoted CS-UCNP@mSiO₂\u0026ndash;RBPA, by conjugating the RBPA receptor onto silica-coated upconversion nanoparticles (UCNPs). Upon 980 nm laser irradiation, the bare probe exhibited characteristic green emissions at ~\u0026thinsp;543 nm and red emissions at ~\u0026thinsp;655 nm, originating from the 4S\u003csub\u003e3/2\u003c/sub\u003e​ \u0026rarr; 4I\u003csub\u003e15/2​\u003c/sub\u003e and 4F\u003csub\u003e9/2​\u003c/sub\u003e \u0026rarr; 4I\u003csub\u003e15/2​\u003c/sub\u003e transitions of Er\u0026sup3;⁺ dopants, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Upon addition of Hg\u0026sup2;⁺, a new yellow emission band centered at ~\u0026thinsp;584 nm emerged and intensified progressively with increasing Hg\u0026sup2;⁺ concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). This band is attributed to the ring-opened form of RBPA, which becomes fluorescent upon binding Hg\u0026sup2;⁺ and subsequently accepts energy from the UCNP via F\u0026ouml;rster resonance energy transfer (FRET) or radiative reabsorption. The appearance of this Hg\u0026sup2;⁺-dependent yellow emission provides a built-in signaling channel distinct from the intrinsic UCNP emissions. Quantitative analysis revealed that the intensity of the green emission at 543 nm remained relatively stable (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec), while the yellow emission at 584 nm increased linearly with Hg\u0026sup2;⁺ concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed), confirming the selective activation of the RBPA reporter unit. To minimize environmental fluctuations and enhance reliability, ratiometric measurements were employed. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee, the intensity ratio of green-to-red fluorescence (\u003cem\u003eI\u003c/em\u003e\u003csub\u003e543\u003c/sub\u003e​/\u003cem\u003eI\u003c/em\u003e\u003csub\u003e655\u003c/sub\u003e​) showed minimal variation, validating the red emission as a stable internal reference. In contrast, the yellow-to-red intensity ratio (\u003cem\u003eI\u003c/em\u003e\u003csub\u003e584\u003c/sub\u003e​/\u003cem\u003eI\u003c/em\u003e\u003csub\u003e655\u003c/sub\u003e​) exhibited a strong, concentration-dependent increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef), offering a robust ratiometric signal for quantitative Hg\u0026sup2;⁺ detection.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Detection Limit and Comparative Performance\u003c/h2\u003e \u003cp\u003eThe limit of detection (LOD) for the CS-UCNP@mSiO₂\u0026ndash;RBPA nanoprobe was determined based on the linear relationship between the ratiometric signal (\u003cem\u003eI\u003c/em\u003e\u003csub\u003e584\u003c/sub\u003e​/\u003cem\u003eI\u003c/em\u003e\u003csub\u003e655\u003c/sub\u003e​) and Hg\u0026sup2;⁺ concentration, yielding an exceptionally low LOD of 14 nM (0.014 \u0026micro;M). As summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, this performance surpasses or rivals that of most recently reported fluorescent or colorimetric nanoprobes for Hg\u0026sup2;⁺.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of other reported different types of nanoprobe and their detection limits for Hg\u003csup\u003e2+\u003c/sup\u003e ion with the CS-UCNP@SiO\u003csub\u003e2\u003c/sub\u003e -RBPA.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProbes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedia\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTest Method\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSensitivity/\u0026micro;M\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eLOD/\u0026micro;M\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eReferences\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCdTe-MSA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ewater\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eUV-Vis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.2-6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e\u003csup\u003e27\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPbBr\u003csub\u003e3\u003c/sub\u003e nano-crystals\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ewater\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFluorescence\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.37\u0026ndash;7.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.229\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e\u003csup\u003e28\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEu-DHTADPA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eethanol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFluorescence\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u0026ndash;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e\u003csup\u003e29\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFA-PDA FONs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ewater\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFluorescence\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026ndash;18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e\u003csup\u003e30\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN-CDs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ewater\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFluorescence\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.014-50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e\u003csup\u003e31\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRBLS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eethanol, water\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFluorescence\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026ndash;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e\u003csup\u003e32\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRh-PP-Rh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eethanol, water\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFluorescence\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026ndash;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e334\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e\u003csup\u003e33\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSi-CQDsRhB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHEPES buffer solution\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFluorescence\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026ndash;80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e\u003csup\u003e34\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTPH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ewater\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFluorescence\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026ndash;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e\u003csup\u003e35\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYellow emissive carbon dots (YCDs)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ewater\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFluorescence\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.4-1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e\u003csup\u003e36\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCS-UCNP\u003c/p\u003e \u003cp\u003e@mSiO\u003csub\u003e2\u003c/sub\u003e-RBPA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eethanol\u003c/p\u003e \u003cp\u003eethanol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eUV-Vis Fluorescence\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u0026ndash;70\u003c/p\u003e \u003cp\u003e0\u0026ndash;70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.227\u003c/p\u003e \u003cp\u003e0.014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eThis work\u003c/p\u003e \u003cp\u003eThis work\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn this work, we successfully designed and fabricated a silica-coated, amino-functionalized upconversion luminescent nanoprobe, CS-UCNP@mSiO₂\u0026ndash;RBPA, for the highly sensitive and selective detection of trace Hg\u0026sup2;⁺ ions. The core\u0026ndash;shell UCNPs serve as efficient nanotransducers, converting near-infrared (NIR) excitation at 980 nm into visible upconverted emissions. The rhodamine-based chemosensor RBPA was covalently anchored to the silica shell, acting as a Hg\u0026sup2;⁺-responsive signaling unit that remains non-fluorescent in its closed spirolactam form but undergoes ring-opening upon binding Hg\u0026sup2;⁺, thereby generating a strong yellow emission at 584 nm. Upon addition of Hg\u0026sup2;⁺, the probe solution exhibited a distinct colorimetric response\u0026mdash;changing from colorless to magenta\u0026mdash;and, under 980 nm irradiation, displayed a progressive decrease in green upconversion fluorescence (543 nm) accompanied by the emergence and intensification of a new yellow emission band (584 nm). This spectral evolution confirms effective energy transfer from the UCNP donor to the ring-opened RBPA acceptor, triggered specifically by Hg\u0026sup2;⁺ coordination. By employing a ratiometric fluorescence strategy based on the intensity ratios \u003cem\u003eI\u003c/em\u003e\u003csub\u003e584\u003c/sub\u003e​/\u003cem\u003eI\u003c/em\u003e\u003csub\u003e655​\u003c/sub\u003e and \u003cem\u003eI\u003c/em\u003e\u003csub\u003e543\u003c/sub\u003e​/\u003cem\u003eI\u003c/em\u003e\u003csub\u003e655\u003c/sub\u003e​, we achieved reliable and self-calibrated quantification of Hg\u0026sup2;⁺, minimizing interference from environmental fluctuations or probe concentration variations. The sensor demonstrated a remarkably low limit of detection of 14 nM, outperforming many existing fluorescent probes. Moreover, the use of NIR excitation eliminates background autofluorescence and enables potential application in complex or turbid media, including biological and environmental samples.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYanmei Zhang\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eData curation, Formal analysis, Investigation, Writing-original draft. \u003cstrong\u003eRui Jiang:\u003c/strong\u003e Formal analysis, Investigation. \u003cstrong\u003eHongze Yang\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Investigation, Formal analysis. \u003cstrong\u003eCheng Zhou:\u0026nbsp;\u003c/strong\u003eData curation, Formal analysis. \u003cstrong\u003eMengxi Sun:\u0026nbsp;\u003c/strong\u003eData curation, Methodology. \u003cstrong\u003eHong Chen:\u003c/strong\u003e Investigation, Formal analysis. \u003cstrong\u003eDonghai Lin:\u0026nbsp;\u003c/strong\u003eFunding acquisition, Methodology, Writing-review \u0026amp; editing.\u003cstrong\u003e\u0026nbsp;Li\u003c/strong\u003e\u003cstrong\u003eming\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eHuang:\u003c/strong\u003e Writing-review \u0026amp; editing. \u003cstrong\u003eGuosong Chen\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eConceptualization, Project administration, Funding acquisition, Supervision, Writing-review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eORCID\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDonghai Lin: 0000-0003-4073-4132\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Major Scientific Research Instrument Development Project of China (Grant No. 22327809) and the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning (SIHL), the Gaoyuan Discipline of Shanghai—Materials Science and Engineering (Grant No. A30NH221903), and the Shanghai Polytechnic University–Drexel University Joint Research Center for Optoelectronics and Sensing. Additional support was provided by the Science Fund for Distinguished Young Scholars of Fujian Province (Grant No. 2019J06027) and the Postdoctoral Fund of Foshan (Grant No. BKS206140).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAppendix A. Supplementary data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary data related to this article can be found online at https://doi.org/xxx.xxx/xxx.xxx.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChen, S.; Gao, Y. T.; Wang, C. K.; Gu, H. L.; Sun, M. K.; Dang, Y. H.; Ai, S. W. Heavy metal pollution status, children health risk assessment and source apportionment in farmland soils in a typical polluted area, Northwest China. \u003cem\u003eStochastic Environmental Research and Risk Assessment \u003c/em\u003e\u003cstrong\u003e2024,\u003c/strong\u003e \u003cem\u003e38\u003c/em\u003e (6), 2383-2395, DOI: 10.1007/s00477-024-02685-4.\u003c/li\u003e\n\u003cli\u003eAsiminicesei, D. M.; Fertu, D. 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Highly selective fluorescent probe for detecting mercury ions in water. \u003cem\u003eRSC Advances \u003c/em\u003e\u003cstrong\u003e2023,\u003c/strong\u003e \u003cem\u003e13\u003c/em\u003e (28), 19091-19095, DOI: 10.1039/D3RA02791K.\u003c/li\u003e\n\u003cli\u003eYunqi, H.; Ying, S.; Tingting, L.; Yuzhuo, T.; Miaomiao, T.; Fang, C. Controllable synthesis of yellow emissive carbon dots by mild heating process and their utility as fluorescent test paper for detection of mercury(II) ions assistants by smartphone. \u003cem\u003eJournal of Environmental Chemical Engineering \u003c/em\u003e\u003cstrong\u003e2023,\u003c/strong\u003e\u003cem\u003e11\u003c/em\u003e (3), 109863, DOI: 10.1016/j.jece.2023.109863.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microchimica-acta","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"miac","sideBox":"Learn more about [Microchimica Acta](https://link.springer.com/journal/604)","snPcode":"604","submissionUrl":"https://submission.springernature.com/new-submission/604/3","title":"Microchimica Acta","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Upconversion nanoparticles, Ratiometric fluorescence sensing, Förster resonance energy transfer, Mercury(II) detection, Near-infrared excitation","lastPublishedDoi":"10.21203/rs.3.rs-8629365/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8629365/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA near-infrared (NIR)-excitable ratiometric fluorescent nanosensor, CS-UCNP@mSiO₂\u0026ndash;RBPA, was developed for the selective and sensitive detection of Hg\u0026sup2;⁺. The probe consists of core\u0026ndash;shell NaYF₄:Yb,Er upconversion nanoparticles (UCNPs) coated with mesoporous silica and functionalized with a rhodamine B-derived chemosensor (RBPA). Upon 980 nm excitation, the UCNPs emit green (~\u0026thinsp;543 nm) and red (~\u0026thinsp;658 nm) luminescence. In the presence of Hg\u0026sup2;⁺, RBPA undergoes spirolactam ring-opening, generating a yellow-emitting species that accepts energy from the UCNP donor via F\u0026ouml;rster resonance energy transfer (FRET). This results in a concentration-dependent decrease in green emission and a concomitant increase in yellow fluorescence at ~\u0026thinsp;584 nm, while the red emission serves as an internal reference. The sensor exhibits excellent selectivity for Hg\u0026sup2;⁺ over other common metal ions and operates effectively at neutral pH. A linear response was observed in the Hg\u0026sup2;⁺ concentration range of 0\u0026ndash;70 \u0026micro;M by UV\u0026ndash;vis absorption, and a remarkably low limit of detection of 14 nM was achieved by ratiometric fluorescence (I₅₈₄/I₆₅₈). The combination of NIR excitation, ratiometric output, and high specificity makes this nanoplatform promising for environmental monitoring and potential biological applications.\u003c/p\u003e","manuscriptTitle":"A Near-Infrared Excited FRET-Based Fluorescent Sensor Using Core–Shell Upconversion Nanoparticles Functionalized with Rhodamine B Derivative for Highly Selective Detection of Hg²⁺","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-28 10:28:29","doi":"10.21203/rs.3.rs-8629365/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-15T13:32:08+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-04T12:32:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-04T05:40:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-28T08:35:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"312390360676654543640593320600710504723","date":"2026-01-28T01:09:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"30863453122157928412470530675527738625","date":"2026-01-27T09:38:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"74244705277803410537228452033208851409","date":"2026-01-27T03:48:56+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-27T02:20:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-26T01:28:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-26T01:27:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microchimica Acta","date":"2026-01-18T05:41:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microchimica-acta","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"miac","sideBox":"Learn more about [Microchimica Acta](https://link.springer.com/journal/604)","snPcode":"604","submissionUrl":"https://submission.springernature.com/new-submission/604/3","title":"Microchimica Acta","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7861539d-b6c2-46d6-97f4-c1ba5a2c34b3","owner":[],"postedDate":"January 28th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T16:05:39+00:00","versionOfRecord":{"articleIdentity":"rs-8629365","link":"https://doi.org/10.1007/s00604-026-08050-2","journal":{"identity":"microchimica-acta","isVorOnly":false,"title":"Microchimica Acta"},"publishedOn":"2026-04-27 15:58:21","publishedOnDateReadable":"April 27th, 2026"},"versionCreatedAt":"2026-01-28 10:28:29","video":"","vorDoi":"10.1007/s00604-026-08050-2","vorDoiUrl":"https://doi.org/10.1007/s00604-026-08050-2","workflowStages":[]},"version":"v1","identity":"rs-8629365","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8629365","identity":"rs-8629365","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}