A fast-responding dual-mode probe for colorimetric and turn-on fluorescence sensing of mercury(II) in water and living cells

Abstract

A mercury-sensitive probe (Cy-M), synthesized in two simple steps, provides excellent selectivity and low detection limits with fast response times. The probe's dual-mode sensing enables naked-eye detection of Hg²⁺ on portable test strips and turn-on fluorescence imaging of intracellular Hg²⁺ in living cells.

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The mercury ion (Hg²⁺) is a highly toxic heavy metal ion that poses severe risks to both biological and environmental systems.¹ It accumulates in living organisms, causing various disorders and irreversible damage to the endocrine, hepatic, renal, and cerebrovascular systems.^2,3 As a non-biodegradable ion, Hg²⁺ biomagnifies through the food chain, meaning that even low concentrations can pose significant threats to human health.^4,5 While traditional analytical techniques offer sensitive detection of Hg²⁺, they are often cost-prohibitive and require specialized expertise.^6–10 Consequently, there is a critical need to develop simple and rapid probes for Hg²⁺ detection to safeguard environmental and public health.^11–16

Organic chromophore-based sensors have garnered significant attention for Hg²⁺ detection owing to their high sensitivity,¹⁷ ease of use, low cost, and real-time monitoring capabilities.^18,19 Typically, these sensors produce noticeable color and fluorescence changes observable by the naked eye, offering a rapid and intuitive detection method.^20,21 However, many existing probes have limitations. For instance, some colorimetric sensors based on naphthalene or coumarin display low-contrast color changes confined to the ultraviolet region, hindering naked-eye detection.²² Additionally, other probes rely on fluorescence quenching or color fading, signals that can be easily overlooked.²³ A fast response time is critical for the real-time monitoring of Hg²⁺,^24,25 and an ideal sensor should combine a rapid, visible color change with a strong turn-on fluorescence signal.

Herein, we report a merocyanine-based dual-mode probe (Cy-M) for the detection of Hg²⁺. This probe incorporates a phenyl thiobenzoate moiety as the specific recognition unit. In its initial state, the probe appears yellow due to the presence of the thioester group. Upon interaction with Hg²⁺, the removal of the phenyl thiobenzoate moiety triggers a molecular rearrangement, resulting in a distinct color change from yellow to red and a strong turn-on fluorescence signal at 555 nm (Scheme 1). The probe exhibits a rapid response time (45 s), a low detection limit of 97 nM, and high selectivity. It has been successfully applied to the quantification of Hg²⁺ in environmental water samples with good recovery rates. Furthermore, the prominent color change facilitates the naked-eye detection of Hg²⁺ on test strips. Exhibiting low cytotoxicity and a clear turn-on fluorescence response to Hg²⁺, Cy-M is also a promising tool for imaging intracellular Hg²⁺.

Scheme 1 (A) Sensing mechanism of Cy-M for mercury ions (Hg²⁺). (B) Applications of Cy-M in the detection of Hg²⁺ in water, on test strips, and within living cells. Scale bars: 150 μm.

Cy-M was synthesized as outlined in Fig. 1. First, the quaternization of 2,3,3-trimethyl-3H-indole with iodopropane yielded compound 1.²⁶ Subsequently, a condensation reaction between compound 1 and 4-hydroxybenzaldehyde produced the intermediate Cy–OH. To confer high sensitivity and specificity towards Hg²⁺, the phenyl thiobenzoate recognition moiety was then introduced into Cy–OH to yield the final probe, Cy-M. All compounds were characterized by ¹H NMR, ¹³C NMR, mass spectrometry (MS) and infrared spectroscopy (Fig. S1–S11).

Fig. 1 The synthetic route of Cy-M.

We first investigated the responsive properties of the Cy-M probe in a PBS solution (pH 7.4, 0.5% DMSO). Cy-M exhibits a major absorption peak at 385 nm and a broad weak emission around 500 nm, similar to common organic solvents (Fig. S12). Upon addition of Hg²⁺, a significant redshift in the absorption peak (from 388 nm to 525 nm) is observed; meanwhile, the color change is easily distinguishable with the naked eye after addition (Fig. 2A, inset). As Hg²⁺ concentration increases, the absorption peak at 388 nm decreases, while the peak at 525 nm increases. The fluorescence properties of Cy-M were also studied, showing a significant enhancement in the fluorescence peak at 555 nm upon addition of Hg²⁺ (Fig. 2B).

Fig. 2 (A) UV-vis absorption and (B) fluorescence (FL) spectra of 50 μM Cy-M in the absence and presence of 50 μM Hg²⁺ in PBS solution (pH 7.4, 0.5% DMSO). The data were acquired at 24 °C with excitation at 525 nm. (C) Job's plot and the stoichiometric ratio of Cy-M and Hg²⁺ obtained by plotting the fluorescence differences at 555 nm against the mole fraction of Hg²⁺ ([Cy-M] + [Hg²⁺] = 20 μM). (D) Fluorescence spectra of 50 μM Cy-M upon addition of different concentrations of Hg²⁺ (0–50 μM) in PBS solution. (E) Linear calibration curve between fluorescence intensity at 555 nm and Hg²⁺ concentrations. σ = 9.95 × 10⁻⁴. (F) Time-dependent changes of fluorescence intensity at 555 nm of 50 μM Cy-M in the presence of 50 μM Hg²⁺ in PBS solution n = 3.

To determine the binding stoichiometry, the fluorescence intensity at 555 nm was plotted as a function of the Hg²⁺ mole fraction (Fig. 2C and S13). The resulting Job's plot reached its maximum at a mole fraction of 0.5. This observation is indicative of a 1 : 1 stoichiometric ratio between Cy-M and Hg²⁺. A strong linear relationship between absorbance and fluorescence intensity and Hg²⁺ concentration was observed over the range from 2.5 to 50 μM (Fig. 2D, E and Fig. S14). The linear portions of the fluorescence intensity plots were fitted, and the limit of detection (LOD) was calculated using the 3σ/k method.²⁷ The LOD of Cy-M towards Hg²⁺ was estimated at 97 nM. From the fluorescence spectral data, the binding constant of Cy-M was found to be 4.12 × 10⁴ M⁻¹ (Fig. S15), indicating high selectivity for Hg²⁺.

We next investigated the time-dependent fluorescent response of Cy-M in the presence of Hg²⁺ to assess its detection potential. In the presence of Hg²⁺, within the initial 40 s, the fluorescence of Cy-M exhibited a rapid increase to reach peak levels by 45 s (Fig. 2F and S16). This response was significantly faster than those of most recently reported Hg²⁺ probes (Table S1). These results demonstrate that Cy-M is a promising colorimetric and fluorescent probe for rapid, naked-eye detection of Hg²⁺, which holds significant importance in practical applications.

The specificity of Cy-M was investigated by introducing various competing metal ions. As shown in Fig. 3A and S17, only Hg²⁺ induced significant changes in the absorption and fluorescence spectra of Cy-M, whereas other common metal ions caused negligible changes. The anti-interference capacity of Cy-M was subsequently assessed by evaluating its response to 50 μM Hg²⁺ in the presence of various interfering metal ions at 500 μM. Measurements of both the absorption and fluorescence spectra demonstrated the excellent selectivity and robustness of Cy-M, as the spectral changes induced by Hg²⁺ were nearly identical despite the coexistence of other ions (Fig. 3B, C, S18 and S19). Consistent with the spectral data, a clear color change from light yellow to red was observed only in the presence of Hg²⁺ (Fig. 3D). These results demonstrate the excellent selectivity and interference resistance of Cy-M in Hg²⁺ detection.

Fig. 3 (A) Fluorescence spectra of 50 μM Cy-M in the presence of 50 μM Hg²⁺ or other metal ions in PBS solution. (B) Fluorescence spectra of 50 μM Cy-M in PBS solution upon the simultaneous addition of both 50 μM Hg²⁺ and 500 μM other metal ions. (C) Fluorescence intensities of Cy-M at 555 nm in the presence of Hg²⁺ (red bar), other metal ions (green bar), and the simultaneous presence of Hg²⁺ with other metal ions (blue bar). (D) Color changes of Cy-M in the presence of Hg²⁺ or other metal ions in PBS solution.

We propose a sensing mechanism, outlined in Scheme 1, to elucidate the probe's response to Hg²⁺. The process is initiated by the strong affinity of the C=S group for Hg²⁺, leading to coordination and subsequent desulfurization of the phenyl thiobenzoate moiety. This is followed by a π-conjugated rearrangement that results in significant shifts in the absorption and fluorescence spectra, which is supported by electrostatic potential analysis (Fig. S20). This rearrangement is pH dependent. With a measured pK_a of 6.44 for Cy–OH, the conversion to Cy=O is favored under neutral and alkaline conditions (Fig. S21). After the reaction, the product exists in equilibrium between two tautomeric forms: the enol (Cy–OH) and the keto (Cy=O). This mechanism was confirmed by mass spectrometry (MS) and ¹H NMR spectroscopy. For instance, after Cy-M was treated with Hg²⁺, the original peak for Cy-M at m/z = 442.1868 ([M]⁺) disappeared and a new peak appeared at m/z = 306.1860 ([M]⁺), which represents the release of Cy–OH or Cy=O (Fig. S22). The proposed mechanism was verified by ¹H NMR spectroscopy (Fig. 4A). Upon addition of excess Hg²⁺ to Cy-M, the complete disappearance of the phenyl thiobenzoate proton signals (d in spectrum I) confirmed the cleavage of the reactive group. Concurrently, the backbone protons (a′, b′, and c′ in spectrum II) exhibited a significant upfield shift, consistent with the formation of an electron-rich phenol. The resulting spectrum of the reaction mixture (III) is dominated by signals nearly identical to those of an authentic Cy–OH sample (II), corroborating the main product's structure. Furthermore, additional multiples are observed at δ 6.7 and 7.1 ppm in spectrum III, which are assigned to the cleaved benzoyl byproduct.

Fig. 4 (A) ¹H NMR spectra of (I) Cy-M, (II) Cy–OH, and (III) Cy-M in the presence of excess Hg²⁺, recorded in DMSO-d₆. (B) Calculated HOMO and LUMO energy levels and their corresponding frontier molecular orbital distributions for Cy-M, Cy–OH, and Cy=O.

To investigate the formation of enolic (Cy–OH) and keto (Cy=O) species from Cy-M upon Hg²⁺ treatment, UV-vis spectra were recorded in PBS solution. As shown in Fig. S23, the enolic species exhibits a peak at 425 nm, while the keto form displays a red-shifted absorption band near 525 nm. As previously described, the addition of Hg²⁺ to the probe results in a new absorption band at 525 nm, corresponding to the appearance of ketone species.

To further understand the observed spectral redshift, we performed theoretical calculations using time-dependent density functional theory (TD-DFT). As shown in Fig. 4B, the calculations reveal that the desulfurization and molecular rearrangement significantly reduce the HOMO–LUMO energy gap from 3.38 eV (Cy-M) and 3.02 eV (Cy–OH) to 2.86 eV (Cy=O). This narrower energy gap, attributed to enhanced electron redistribution and extended π-conjugation in the Cy=O backbone, explains the redshift in the absorption spectrum upon interaction with Hg²⁺.

To evaluate the practical applicability of Cy-M, we first investigated its stability. The probe demonstrated high stability under storage conditions (room temperature and refrigeration) (Fig. S24) and various pH values²⁸ (Fig. S25). Additionally, it exhibited excellent resistance to photodegradation after one hour of UV light exposure (Fig. S26), underscoring its potential for real-world use. We performed a quantitative analysis of Hg²⁺ in various real-world water samples, including tap water, deionized water, PBS, and local river water. Known concentrations of Hg²⁺ were spiked into these samples, and the fluorescence response of Cy-M (50 μM) was recorded. The results, summarized in Table S2 and Fig. S27 and S28, show excellent recovery rates, demonstrating that the probe can accurately and reliably quantify Hg²⁺ in complex matrices. These findings highlight the potential of Cy-M for environmental monitoring and other practical applications.

For convenient, on-site detection, test strips were prepared by impregnating strips with a Cy-M solution.²⁹ As shown in Fig. 5A, upon the addition of a Hg²⁺-containing solution, the test strips exhibited a distinct color change from amber to light yellow and bright fluorescence under UV light. We then employed the prepared Cy-M-based test strips for the quantitative analysis of Hg²⁺. The results (Fig. 5B) demonstrated that the strips could differentiate Hg²⁺ concentrations via both visible colorimetric and fluorescence changes. Based on the 3σ/k method, the LODs were determined to be 1.21 μM for the naked-eye mode and 1.48 μM for the fluorescence mode (Fig. 5C and D). These results validate the feasibility of Cy-M for on-site detection. In contrast, no significant changes were observed for solutions containing other metal ions. This result demonstrates the potential of Cy-M for use as a portable, selective and naked-eye sensor for rapid screening of Hg²⁺.

Fig. 5 (A) Photographs of the test strips with 50 μM Hg²⁺, Pb²⁺, Ca²⁺, and Mg²⁺, and a PBS control under natural light (top) and 365 nm UV irradiation (bottom). (B) Photographs of the test strips with different concentrations (0–50 μM) of Hg²⁺ under natural light (top) and 365 nm UV irradiation (bottom). (C) Quantitative analysis of fluorescence intensity from the natural light group. σ = 1.30. (D) Quantitative analysis of fluorescence intensity from the UV light group. σ = 1.26.

The potential of Cy-M for biological applications was then investigated. First, its subcellular localization was examined. As shown in Fig. 6A, the green fluorescence of Cy-M showed strong colocalization with the red fluorescence of a mitochondrial tracker, yielding a high Pearson's correlation coefficient (PCC) of 0.94.³⁰ This indicates that Cy-M predominantly accumulates within the mitochondria after cellular uptake. Furthermore, a CCK-8 assay was performed to evaluate the cytotoxicity of Cy-M.³¹ As shown in Fig. 6B, Cy-M exhibited low cytotoxicity towards 4T1 cells, even at concentrations up to 100 μM, indicating good biocompatibility. For fluorescence imaging, 4T1 cells were incubated with Cy-M alone or co-incubated with different concentrations of Hg²⁺. Cells treated only with Cy-M (50 μM) exhibited weak fluorescence. In contrast, a significantly enhanced green fluorescence was observed after co-incubation with Hg²⁺ (0–50 μM) in a concentration-dependent manner (Fig. 6C). The LOD for Hg²⁺ in this cellular context was calculated to be 0.27 μM (Fig. 6D). These results suggest that Cy-M possesses good cell membrane permeability and can respond to intracellular Hg²⁺ with a turn-on fluorescence signal, validating its potential as a promising bioimaging probe.

Fig. 6 (A) Colocalization experiments of Cy-M in 4T1 cells. Scale bars = 75 μm. (B) Cell cytotoxicity of Cy-M (0–100 μM). (C) Fluorescence images of the 50 μM Cy-M probe in the presence of different concentrations of Hg²⁺. (D) Quantitative analysis of fluorescence intensity from C. σ = 0.15. Scale bars = 150 μm.

Conclusions

In conclusion, we have developed a dual-mode colorimetric and fluorescent probe, Cy-M, for the rapid and selective detection of Hg²⁺. Key features of the probe include a fast response time (45 s), a low detection limit (97 nM), and a distinct color change from yellow to red that allows for naked-eye detection. The practical utility of Cy-M was demonstrated by its successful application in quantifying Hg²⁺ in environmental water samples and its use in portable test strips for on-site screening. Furthermore, due to its low cytotoxicity and effective turn-on fluorescence response to intracellular Hg²⁺, Cy-M proved to be a capable tool for bioimaging in living cells. Taken together, the combination of these features makes Cy-M a robust and versatile platform for Hg²⁺ detection across environmental and biological contexts.

Author contributions

Yanxin Wu: sample preparation, data curation, formal analysis, writing – original draft, and writing – review & editing. Huiyuan Wang: sample preparation and data curation. Kai Wei: conceptualization and methodology. Chendong Ji: funding acquisition, supervision, project administration, and writing – review & editing. Meizhen Yin: funding acquisition, supervision, project administration, and writing – review & editing.

Conflicts of interest

There are no conflicts to declare.

Data availability

The data supporting this article have been included as part of the supplementary information (SI). Supplementary information: experimental procedure, ¹H NMR spectrum, infrared spectrum, absorption spectra, fluorescence spectra, etc. See DOI: https://doi.org/10.1039/d5an00908a.

All relevant data that support the findings of this study are available from the corresponding author upon reasonable request.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (grant numbers: 52130309, W2412081 and 22578023).

References

1. D. Dai, J. Yang, Y. Wang and Y. W. Yang, Adv. Funct. Mater., 2020, 31, 2006168.
2. K. A. Nath, A. J. Croatt, S. Likely, T. W. Behrens and D. Warden, Kidney Int., 1996, 50, 1032–1043.
3. A. A. Pinheiro, A. Thakuri, S. Saha, M. Banerjee and A. Chatterjee, Analyst, 2025, 150, 3208–3216.
4. X.-R. Bai, Y. Zeng, X.-D. Zhou, X.-H. Wang, A.-G. Shen and J.-M. Hu, Anal. Chem., 2017, 89, 10335–10342.
5. Z. Cheng, J. Wei, L. Gu, L. Zou, T. Wang, L. Chen, Y. Li, Y. Yang and P. Li, J. Hazard. Mater., 2022, 431, 128606.
6. O. R. Marinho, M. J. A. Lima and B. F. Reis, Talanta, 2020, 206, 120207.
7. C. Zheng, L. Hu, X. Hou, B. He and G. Jiang, Anal. Chem., 2018, 90, 3683–3691.
8. K. Debsharma, S. Dey, B. Dutta and S. Dey, Analyst, 2025, 150, 2725–2775.
9. X. Luan, D. Zhu, P. He, P. Han and G. Wei, Environ. Sci.: Nano, 2023, 10, 2767–2777.
10. J. Feng, L. Shi, D. Chang, C. Dong and S. Shuang, Chem. Eng. J., 2024, 490, 151839.
11. K. Wei, H. Wang, C. Ji and M. Yin, Chin. Sci. Bull., 2020, 66, 1614–1627.
12. X. Zheng, W. Cheng, C. Ji, J. Zhang and M. Yin, Rev. Anal. Chem., 2020, 39, 231–246.
13. J. Raina, G. Kaur and I. Singh, Talanta, 2024, 277, 126372.
14. H. Li, J. Wang, H. Kim, X. Peng and J. Yoon, Angew. Chem., Int. Ed., 2024, 63, e202311764.
15. Z. Wu, T. Lu, Y. Hu, J. Mao, X. Hu and J. Qiu, Coord. Chem. Rev., 2025, 537, 216711.
16. H. Chen, Y. Li, Z. Wang, D. Wang, L. Feng, S. Li, C. Wu and H. Wang, Analyst, 2024, 149, 1784–1790.
17. S. S. M. Ameen, K. M. Omer, A. Bedair, M. Hamed and F. R. Mansour, Mater. Today Chem., 2025, 45, 102686.
18. Y. Yu, C. Liu, B. Tian, X. Cai, H. Zhu, P. Jia, Z. Li, X. Zhang, W. Sheng and B. Zhu, Dyes Pigm., 2020, 177, 108290.
19. L. Shi, F. Jia, L. Wang, M. Jalalah, M. S. Al-Assiri, T. Gao, F. A. Harraz and G. Li, Sens. Actuators, B, 2021, 326, 128976.
20. A. Tarai, Y. Li, B. Liu, D. Zhang, J. Li, W. Yan, J. Zhang, J. Qu and Z. Yang, Coord. Chem. Rev., 2021, 445, 214070.
21. H. Kim, Y. J. Jung and J. K. Lee, Polym. Chem., 2021, 12, 970–974.
22. C. Zhang, H. Zhang, M. Li, Y. Zhou, G. Zhang, L. Shi, Q. Yao, S. Shuang and C. Dong, Talanta, 2019, 197, 218–224.
23. P. Singh and P. Sharma, J. Photochem. Photobiol., A, 2021, 408, 113096.
24. M. Su, C. Liu, Y. Zhang, X. Rong, X. Wang, X. Li, K. Wang, H. Zhu and B. Zhu, Anal. Chim. Acta, 2022, 1230, 340337.
25. L. Tang, L. Zhou, X. Yan, K. Zhong, X. Gao and J. Li, J. Photochem. Photobiol., A, 2020, 38, 112160.
26. T. Jing and L. Yan, Talanta, 2017, 170, 185–192.
27. H. Geng, Z. Li, Q. Liu, Q. Yang, H. Jia, Q. Chen, A. Zhou and W. He, Dalton Trans., 2022, 51, 11693–11702.
28. Q. Yang, J. Zhang, Y. Tang, Y. Ju, X. Gao, C. Chu, H. Jia and W. He, Chem. Sci., 2025, 16, 10842–10851.
29. S. Zhang, G. Fu, W. Yi, J. Zhou, W. Liang, W. Zhao, T. Zhang, G. Zhang, J. Zhang, H. Li, H. Yan, B. Wu, S. Fu, Y. Liu, Y. Xia, J. Wang, J. Qiu and S. Xu, Chem. Eng. J., 2024, 500, 157035.
30. C. Liu, M. Zhang, H. Geng, P. Zhang, Z. Zheng, Y. Zhou and W. He, Appl. Catal., B, 2021, 295, 120317.
31. L. Wang, Y. Liu, W. Li, X. Jiang, Y. Ji, X. Wu, L. Xu, Y. Qiu, K. Zhao, T. Wei, Y. Li, Y. Zhao and C. Chen, Nano Lett., 2011, 11, 772–780.

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