Construction of a platform based on biocompatible near-infrared Ag₂S quantum dots for detection of copper ions in real samples and cellular environments

2.1. Materials

DPA (98%) and silver nitrate (AgNO₃, 99%) were procured from Jiuding Chemical (Shanghai) and Sinopharm Chemical Reagent Co., Ltd., respectively. River water, tap water, and serum samples were collected from the Xiangjiang River, Hunan Normal University campus, and its affiliated hospital in Changsha, China. Detailed specifications of all characterization instruments have been provided in the Supporting Information.

2.2. Synthesis of Ag₂S QDs

Ag₂S QDs were synthesized following a modified version of our previously reported procedure [29]. Briefly, DPA and AgNO₃ were dissolved in deionized water at a molar ratio of 5:4 and stirred for 20 min to obtain a homogeneous solution. The resulting mixture was then subjected to microwave-assisted heating for 10 min, during which the color of the reaction solution gradually changed from colorless to dark brown, indicating the formation of Ag₂S QDs. The obtained product was purified using a 0.22 μm ultrafiltration membrane and stored at 4°C for subsequent use.

2.3. General procedure for the detection of Cu²⁺

Ag₂S QDs (600 μL) were mixed with PBS buffer (300 μL, 0.1 M, pH 6.0), spiked with Cu²⁺, incubated at 40°C for 30 s, and fluorescence measured (λex=405 nm) with 695 nm emission quantified.

2.4. Evaluation in real samples

Tap water and river water samples were filtered through 0.22 μm membranes prior to use. Subsequently, Cu²⁺ recovery experiments were performed in tap water, river water, and serum samples following the standard procedure described above.

2.5. Cytotoxicity and imaging

Cytocompatibility of Ag₂S QDs was assessed using a CCK-8 assay under gradient exposure conditions. HeLa and 293T cells (7000/well) were seeded in 96-well plates and incubated with QD-containing medium at 37°C/5% CO₂ for 24 h. Following the addition of the CCK-8 reagent, absorbance was measured at 450 nm (reference 650 nm) using a microplate reader, with viability normalized by inter-well calibration.

HeLa cells were seeded in confocal culture dishes and incubated at 37^oC for 12 h to allow complete cell adhesion. Subsequently, 100 μL of Ag₂S QDs aqueous solution was added to the medium, and the cells were further incubated for 2 h. Thereafter, Cu²⁺ solution was introduced and incubated for 30 min, followed by three washes with PBS (0.1 M, pH 7.4). Fluorescence imaging was then performed using confocal microscopy.

3.1. Design and preparation of Ag₂S QDs

As illustrated in Scheme 1, a water-soluble and biocompatible near-infrared Ag₂S QD platform was developed based on modifications of our previous work. This platform employs DPA as both the sulfur source and stabilizing agent, together with AgNO₃ as the silver precursor, and was synthesized via a microwave-assisted method. To achieve Ag₂S QDs with optimal fluorescence properties, the effects of the sulfur source, reactant ratio, and microwave-assisted reaction time were systematically investigated. As shown in Figure 1(a), only the DPA ligand successfully promoted the formation of Ag₂S QDs, likely due to its dual function as both a protective agent and an in situ S^2- during synthesis. As revealed in Figure 1(b) and Figure S1, the stoichiometric ratio between silver precursors and DPA ([Ag⁺]/[DPA]) was the key factor governing Ag₂S QDs formation. Interestingly, distinct fluorescence emission was observed only when the [Ag⁺]/[DPA] ratio exceeded 1, accompanied by a color change of the solution to reddish-brown. This phenomenon can be attributed to the excess Ag⁺ facilitating the cleavage of the C–S bond in DPA, induced by silver sulfide clusters, thereby releasing S²⁻ ions and enabling the formation of stable, ultrasmall Ag₂S QDs under microwave-assisted conditions [30]. In contrast, an excessive amount of DPA led to non-radiative Ag-based assemblies via multidentate carboxyl coordination and argentophilic interfacial coupling, which suppressed metal-to-ligand charge transfer and promoted the growth of non-fluorescent Ag–S oligomers [31]. Furthermore, the microwave-assisted reaction time was identified as another critical factor affecting the structural and optical properties of the Ag₂S QDs. As shown in Figure 1(c) and Figure S1, prolonging the microwave-assisted reaction time resulted in the gradual disappearance of larger particles, suggesting that extended microwave time promotes more complete precursor conversion and yields a narrower particle size distribution. The fluorescence intensity reached its maximum at 10 min and slightly decreased thereafter, possibly due to partial dissociation of surface ligands and enhanced nonradiative energy dissipation at prolonged reaction times. Therefore, the optimal synthesis conditions for Ag₂S QD were determined to be a microwave time of 10 min and an R[AgNO₃/DPA] ratio of 5/4.

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Scheme 1.

Schematic of Ag₂S QDs synthesis and Cu²⁺ detection.

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Figure 1.

(a) Fluorescence of Ag₂S QDs with different ligands (9 min, [Ag⁺]/ligand = 5:4, λex = 405 nm). (b) Effect of [Ag⁺]/DPA ratio (9 min, λex = 405 nm). (c) Effect of microwave time ([Ag⁺]/DPA = 5:4, λex = 405 nm). (d) UV–Vis absorption spectrum. (e) Excitation-dependent emission (λex = 405–510 nm). (f) Photostability with/without 405 nm irradiation. (d–f obtained under identical conditions: [Ag⁺]/DPA = 5:4, 10 min).

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The fluorescence properties of QDs, a key determinant of their sensing performance, were systematically characterized through absorption/emission profiling and photobleaching resistance analysis (Figures 1d-f). The excitation-dependent fluorescence response indicated optimal emission at λex=405 nm (Figure 1e), while a large Stokes shift of 290 nm facilitated phonon-assisted anti-Stokes emission, effectively minimizing Förster resonance energy transfer (FRET) interference and self-absorption artifacts. Notably, the photostability evaluation under continuous 405 nm irradiation for 1 h confirmed the excellent durability of the QDs, thereby establishing a solid foundation for their subsequent application in monitoring Cu²⁺ within biological systems.

To further investigate the morphology of the synthesized QDs, transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM) were utilized for characterization. As illustrated in Figure 2(a), the Ag₂S QDs synthesized in aqueous phase exhibit uniformly dispersed spherical nanostructures with an average diameter of 2.5 ± 0.1 nm, indicating excellent colloidal stability and homogeneous nucleation. Furthermore, HRTEM image (Figure 2b) reveals distinct lattice fringes with an interplanar spacing of 0.234 nm, corresponding to the (103) plane of monoclinic α-Ag₂S. These results collectively confirm the formation of phase-pure, highly crystalline Ag₂S QDs with well-defined structural integrity.

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Figure 2.

(a) TEM image and (b) HRTEM image of Ag₂S QDs. (c) Ag 3d, (d) S 2p, and (e) C 1s XPS spectrum of Ag₂S QDs. (f) FT-IR spectra of the DPA and Ag₂S QDs.

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Furthermore, the chemical composition and surface functionalization of Ag₂S QDs were comprehensively characterized using X-ray photoelectron spectroscopy (XPS) and fourier transform infrared (FT-IR) analyses. As evidenced by the survey spectrum (Figure S2), the QDs primarily contain Ag, S, C, N, and O elements. High-resolution XPS spectra provide detailed insights into the chemical states of these elements. Specifically: (i) The Ag 3d spectrum exhibits two peaks at 367.8 eV (3d₅/₂) and 373.8 eV (3d₃/₂), with a spin-orbit splitting of 6.0 eV (Figure 2c), characteristic of Ag⁺ species in α-Ag₂S crystalline phase [29,32]. (ii) The S 2p₃/₂ peak centered at 162.0 eV confirms the presence of sulfide (S²⁻) rather than elemental sulfur, verifying successful coordination with Ag (Figure 2d). (iii) The C 1s spectrum (Figure 2e) deconvolutes into three peaks at 284.6 eV (CH₃), 286.2 eV (CH), and 287.3 eV (COO⁻), where the oxygenated species play a crucial role in electrostatic stabilization and steric protection of the QDs. Complementary FT-IR analysis (Figure 2f) further confirms the surface chemistry, displaying characteristic bands at 1670 cm⁻¹ (N-H bending), 1448 cm⁻¹ (symmetric COO⁻ stretching), while notably lacking the S–H stretching vibration (2534 cm⁻¹). The absence of this S–H signal, in combination with the XPS S 2p data, evidences the formation of thiolate-Ag covalent bonds (Ag–SR), thereby excluding the presence of unbound DPA ligands [33,34]. Collectively, these results establish a synergistic surface stabilization mechanism, integrating electrostatic repulsion (COO⁻), hydrogen bonding (NH₂), and covalent anchoring (Ag–S). This multi-interaction framework endows the Ag₂S QDs with outstanding aqueous stability and robust biofunctionalization capability, enabling their effective application in serum copper quantification and bioimaging studies.

3.2. Optimization of conditions for detecting Cu²⁺

As shown in Figure S3(a), the synthesized Ag₂S QDs function as a highly sensitive fluorescence sensing platform for Cu²⁺ detection, exhibiting a pronounced decrease in emission intensity upon Cu²⁺ addition. To optimize the sensing performance, the effects of temperature, pH, and reaction time on Cu²⁺ detection were systematically investigated. As depicted in Figure S3(b), the optimal quenching temperature for Ag₂S QDs was determined to be 40°C, which can be attributed to the enhanced kinetic interactions between Cu²⁺ and the surface ligands of Ag₂S QDs (e.g., carboxyl and amino groups) at moderately elevated temperatures. This acceleration in surface binding facilitates efficient Cu²⁺ coordination and promotes fluorescence quenching within 30 s. However, further temperature increases lead to partial ligand dissociation, resulting in diminished fluorescence intensity. Similarly, as shown in Figure S3(c), the maximum fluorescence response was observed at pH 6.0, which can be ascribed to the reduced surface defect density and improved photoluminescence of the QDs under mildly acidic conditions. In addition, this pH environment maintains charge balance and enhances the synergistic coordination between Cu²⁺ and surface functional groups (−COO⁻ and −NH₂), forming stable complexes that facilitate efficient nonradiative energy transfer and quenching. Finally, based on the time-dependent fluorescence response (Figure S3d), the optimal detection time was determined to be 30 s, ensuring rapid and reproducible Cu²⁺ sensing under the optimized conditions.

Moreover, the stability of the Ag₂S QDs sensor is of critical importance for their practical applications. The storage stability, batch-to-batch reproducibility, and photostability of Ag₂S QDs were systematically evaluated. The results indicate that the fluorescence performance of the Ag₂S QDs exhibited only negligible changes even after storage at 4°C for 4 days (Figure S4a). Moreover, the fluorescence characteristics remained highly consistent across different batches (Figure S4b), and the QDs demonstrated excellent photostability (Figure S4c). This remarkable stability can be attributed to their superior water solubility and good dispersibility. Overall, these findings confirm that the Ag₂S QDs possess outstanding stability and reproducibility, providing a reliable foundation for stable and highly sensitive Cu²⁺ detection, while also highlighting their great potential for practical analytical and bioimaging applications.

3.3. Analysis of Cu²⁺

To explore the performance of Ag₂S QDs in detecting Cu²⁺, the Cu²⁺-dependent fluorescence quenching behavior of the Ag₂S QDs was systematically investigated. As demonstrated in Figure 3(a), a gradual decrease in fluorescence intensity was observed over a Cu²⁺ concentration range of 0–20 μM. A linear response was established within the 0.1–12 μM range (R² = 0.988, Figure 3b), described by the equation F = −57.16[Cu²⁺] + 1133.1. Based on the standard 3σ/slope and 10σ/slope methods, the limit of detection (LOD) and limit of quantification (LOQ) were determined to be 21 nM and 70 nM, respectively, which are well below the U.S. EPA guideline for Cu²⁺ in drinking water (20 μM = 1.3 ppm). Notably, the constructed Ag₂S QD sensor exhibits superior performance compared to most previously reported sensors in terms of LOD, response time, and application scope (see Table S1). These results indicate that the constructed sensor possesses sufficient sensitivity, and this outstanding sub-nanomolar sensitivity, combined with rapid response kinetics, underscores the tremendous potential of Ag₂S QDs for real-time monitoring of copper speciation dynamics in complex aqueous environments.

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Figure 3.

(a) Fluorescence spectra of Ag₂S QDs with varying Cu²⁺ concentrations. (b) Fluorescence intensity vs. Cu²⁺ concentration. (c) Selectivity of Ag₂S QDs for Cu²⁺ (20 μM) against interferences (50 μM), except for bovine serum albumin (Alb, 100 μg mL^-1).

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To evaluate the selectivity of Ag₂S QDs toward Cu²⁺, a series of comparative experiments was performed under identical conditions using various potentially interfering ions. The fluorescence quenching efficiency (QE) was calculated according to Eq. (1).

As depicted in Figure 3(c), the QE of Ag₂S QDs was systematically evaluated upon the addition of various substances, including Cys, Glu, DA, Al³⁺, Ca²⁺, Co²⁺, Fe³⁺, Fe²⁺, Mg²⁺, Mn²⁺, Na⁺, Zn²⁺, EDTA, GSH, Hg²⁺, Ni²⁺, Pb²⁺, Alb, His, and Cu²⁺. The experimental results demonstrate that only Cu²⁺ induces significant fluorescence quenching, whereas all other tested species exert negligible effects, even at concentrations 2.5-fold higher than that of Cu²⁺. The outcome fully substantiates the excellent selectivity of Ag₂S QDs for detecting Cu²⁺, indicating that its application in complex real samples is highly potential.

To further elucidate the interaction mechanism between Ag₂S QDs and Cu²⁺, we studied the absorption and emission spectra of Ag₂S QDs alongside the absorption spectrum of Cu²⁺. The results indicate that the excitation and emission bands of Ag₂S QDs exhibit negligible spectral overlap with the characteristic absorption band of Cu²⁺ (Figures 4a and b). This spectral decoupling effectively rules out FRET and inner filter effect (IFE) as quenching mechanisms, both of which require substantial spectral overlap for non-radiative energy transfer processes. Therefore, Cu²⁺-induced fluorescence quenching of Ag₂S QDs does not proceed via these photophysical pathways. FT-IR analysis (Figure 4c) further reveals that the characteristic peaks at 2931 cm⁻¹ and 2853 cm⁻¹ correspond to the symmetric and asymmetric C-H stretching vibrations of DPA. Notably, upon Cu²⁺ incorporation, the carboxylate (COO⁻) stretching vibration at 1448 cm⁻¹ and C–H vibrational signatures completely disappear, accompanied by substantial attenuation of the N–H bending vibration at 1670 cm⁻¹ compared with pristine Ag₂S QDs. These spectral changes strongly indicate that Cu²⁺ coordinates with the carboxyl and amino functional groups on the surface of Ag₂S QDs, leading to a reorganization of the surface ligand structure and resulting in effective fluorescence quenching [35]. Furthermore, the concentrations of silver and copper ions in the supernatant before and after Cu²⁺ addition were analyzed by inductively coupled plasma-optical emission spectroscopy (ICP-OES) (Figure 4d). The results showed that the Cu²⁺ concentration decreased by approximately 96%, while the Ag⁺ concentration remained nearly unchanged. This finding further confirms that the fluorescence quenching mechanism primarily arises from the coordination interaction between Cu²⁺ and the surface functional groups of Ag₂S QDs, rather than from an ion-exchange process.

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Figure 4.

(a) Absorption and emission spectra of Ag₂S QDs. (b) Fluorescence spectrum of Ag₂S QDs and UV–vis absorption spectrum of Cu²⁺. (c) FT-IR spectra of the Ag₂S QDs and Ag₂S QDs + Cu²⁺. (d) Comparison of changes in element content in the supernatant after the addition of Cu²⁺.

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3.4. Actual sample analysis

To systematically assess the practical sensing performance of the Ag₂S QDs sensor, multiple real samples were employed for validation. Municipal tap water, river water, and human serum, representing complex matrices with potential interfering species, were selected to evaluate detection robustness under challenging conditions. Different concentrations of Cu²⁺ standard solutions were spiked into these samples (Table 1) to perform recovery experiments, thereby assessing the accuracy and anti-interference capability of the sensor. Statistical results showed that the recovery rates in tap water, river water, and human serum ranged from 92.25% to 122%, indicating that the sensing system maintained excellent linearity and quantitative detection performance even in complex sample matrices. Notably, the Ag₂S QDs sensor exhibited outstanding selectivity, sensitivity, and stability across various complex matrices, further confirming its feasibility and applicability for Cu²⁺ determination in real environmental and biological samples.

3.5. Biological assessment

The cytotoxicity of Ag₂S QDs, a critical factor for their biomedical applicability, was systematically evaluated using HeLa and 293T cells (Figure S5). The results demonstrated excellent biocompatibility, with cell viability exceeding 85% even after 24 h exposure to high QD concentrations. To further explore the bioimaging potential of Ag₂S QDs, intracellular Cu²⁺ monitoring was performed. Confocal fluorescence microscopy imaging (Figure 5a) revealed distinct near-infrared emission in control cells labeled with Ag₂S QDs, whereas cells treated with exogenous Cu²⁺ exhibited near-complete fluorescence quenching (Figures 5b-c). These results confirm that the platform can achieve real-time tracking of Cu²⁺ in living cells without interference from biological autofluorescence, providing a robust approach for long-term live-cell studies of metal ion homeostasis.

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Figure 5.

Fluorescence imaging of HeLa cells incubated with (a) Ag₂S QDs, (b) Ag₂S QDs + Cu²⁺ (10 μM), (c) Ag₂S QDs + Cu²⁺ (20 μM). λex =488 nm, scale bar: 25 μm.

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