Nguyen Khoa Hiena,
Trinh Thi Giao Chaua,
Nguyen Dinh Luyenb,
Quan V. Vo
c,
Mai Van Bayd,
Son Tung Ngoef,
Pham Cam Nam
*g and
Duong Tuan Quang*b
aMientrung Institute for Scientific Research, Vietnam National Museum of Nature, Vietnam Academy of Science and Technology, Hue 49000, Vietnam
bDepartment of Chemistry, Hue University, Hue 49000, Vietnam. E-mail: duongtuanquang@dhsphue.edu.vn
cFaculty of Chemical Technology-Environment, The University of Danang - University of Technology and Education, 48 Cao Thang, Danang 50000, Vietnam
dThe University of Danang, University of Science and Education, Danang 50000, Vietnam
eLaboratory of Biophysics, Institute for Advanced Study in Technology, Ton Duc Thang University, Ho Chi Minh City 72915, Vietnam
fFaculty of Pharmacy, Ton Duc Thang University, Ho Chi Minh City 72915, Vietnam
gThe University of Danang, University of Science and Technology, Danang 50000, Vietnam. E-mail: pcnam@dut.udn.vn
First published on 13th June 2025
A novel fluorescent sensor, Hg(DST)2, was developed for the selective detection of biological thiols, including glutathione (GSH), cysteine (Cys), and homocysteine (Hcy), in fully aqueous solutions at pH 7.2. The sensor exhibited significant fluorescence quenching upon coordination with Hg2+, which was reversibly restored in the presence of thiols due to the formation of thermodynamically favored Hg-thiol complexes. The OFF–ON fluorescence mechanism of the sensor was elucidated using DFT calculations. Fluorescence titration experiments revealed a strong linear correlation (R2 ≈ 0.998) between fluorescence intensity and thiol concentrations within the ranges of 0.34–8.00 μM for GSH, 0.47–10.00 μM for Cys, and 0.26–8.00 μM for Hcy, with corresponding limits of detection (LOD) of 0.34, 0.47, and 0.26 μM, respectively. The sensor demonstrated high selectivity toward thiols in the presence of common amino acids, metal ions, and anions, with interference from Ag+, Cu2+, Co2+, and Ni2+ mitigated using 1,10-phenanthroline (PHEN). Owing to its high sensitivity, selectivity, and water solubility, Hg(DST)2 represents a promising tool for thiol quantification in biological and environmental matrices.
Glutathione is a tripeptide made up of three amino acids: L-glutamate, L-cysteine, and L-glycine. It is the most abundant non-protein thiol in cells, with intracellular concentrations ranging from 1 to 15 mM, primarily in its reduced form.6 GSH is one of the body's most powerful antioxidants, protecting cells from oxidative damage by neutralizing free radicals.6,7 It plays a critical role in detoxification, particularly in the liver, where it helps eliminate harmful substances and convert them into less toxic forms.8 Additionally, GSH is essential for maintaining and regulating the immune system.6 It is also believed to be associated with diseases, including cancer, stroke, heart disease, pancreatic and kidney disorders, diabetes, Alzheimer's, Parkinson's, gastritis, peptic ulcers, and atherosclerosis diseases.6,7
Homocysteine (Hcy) is a metabolic intermediate formed during the catabolism of methionine in the one-carbon metabolism cycle. It participates in methylation processes, methionine regeneration, and cysteine biosynthesis, with its homeostasis maintained through pathways that either regenerate methionine or convert it to cysteine.9 Typical blood Hcy levels range from 5 to 13 μM.10 Elevated Hcy levels, a condition known as hyperhomocysteinemia, are linked to cardiovascular, neurological, and bone-related disorders, as well as fertility issues, including recurrent miscarriages and infertility in both genders.11–14
Given the important roles of Cys, GSH, and Hcy, various methods for their determination have been developed, including high-performance liquid chromatography,1,15 gas chromatography-mass spectrometry,16 electrochemistry,17 and UV-Vis absorption and fluorescence spectroscopy.18–20 Among these, fluorescent sensors have garnered significant interest from scientists due to their high sensitivity, simple analysis methods, and ability to monitor in living cells.21–24
Some recently reported fluorescent sensors for biothiol detection based on metal ion complexes are summarized in Table 1. Most reported fluorescent sensors do not selectively detect individual biothiols because Cys, GSH, and Hcy have similar structures.25 Nevertheless, this limitation does not diminish the efforts to develop new fluorescent sensors for biothiols. This is because, in practice, there are cases where only the concentration change of a specific biothiol related to a particular disease is of interest, such as monitoring glutathione levels in the liver to assess liver function or monitoring homocysteine levels in the blood to evaluate the risk of cardiovascular diseases and stroke.26–28 Additionally, different biothiols are distributed differently in various body parts. For example, the concentrations of cysteine and glutathione are highest in tissues, particularly in the liver, while homocysteine is usually undetectable in tissues and is primarily found in plasma.29–31 Notably, fluorescent sensors often interact reversibly with biothiols, which is very useful for monitoring real-time changes in biothiol concentrations, and providing detailed information about pathological conditions.10
Complexes of metal ions | Detectable biothiols | LOD (μM) | Solvent/pH | The influence of other thiols | Ref. |
---|---|---|---|---|---|
Cu2+ | GSH | 0.16 | HEPES/DMSO (95/5, v/v), pH 7.4 | Affected by Cys and Hcy | 32 |
Cu2+ | GSH | 0.80 | DMF/HEPES (3/7, v/v), pH 7.4 | Cannot detect individual thiols separately | 33 |
Cys | 1.00 | ||||
Hcy | 1.50 | ||||
Cu2+ | GSH | 0.30 | Ethanol/HEPES (1/1, v/v), pH 7.4 | Cannot detect individual thiols separately | 19 |
Cys | |||||
Hcy | |||||
Cu2+ | Cys | 0.17 | CH3CN/HEPES (1/1, v/v), pH 7.4 | - Cannot detect individual thiols separately | 34 |
Hcy | 0.25 | - The influence of GSH has not been reported | |||
Cu2+ | GSH | 0.20 | CH3OH/HEPES (9/1, v/v), pH 7.2 | The influence of Cys and Hcy has not been reported | 35 |
Cu2+ | GSH | 0.44 | DMF/HEPES (7![]() ![]() |
Cannot detect individual thiols separately | 36 |
Cys | 0.96 | ||||
Hcy | 0.68 | ||||
Cu2+ | Cys | 0.015 | CH3CN/HEPES (7/3, v/v), pH 7.4 | The influence of GSH and Hcy has not been reported | 37 |
Hg2+ | Cys | 0.20 | Ethanol/HEPES (1![]() ![]() |
Affected by GSH and Hcy | 20 |
Hg2+ | Cys | 0.016 | CH3CN | The influence of GSH and Hcy has not been reported | 38 |
Ag+ | GSH | 0.208 | Dioxane/Tris–HClO4 (3/7, v/v), pH 7.4 | Cannot detect individual thiols separately | 39 |
Cys | 0.089 | ||||
Hcy | 0.174 | ||||
Fe3+ | Cys | 0.45 | Water (1% DMSO) pH: 2–11 | Not affected by GSH and Hcy | 40 |
In this study, we report a fluorescent sensor (Hg(DST)2) that can detect the real-time concentrations of Cys, GSH, and Hcy, based on the complex of a fluorescent compound DST with Hg2+ ions. The Hg(DST)2 complex interacts reversibly with Cys, GSH, and Hcy, releasing DST, accompanied by a fluorescence signal change from OFF to ON at 445 nm. This process can be reversed at least five times by alternating the addition of Hg2+ and biothiols.
Experimental results in Fig. 2 showed that DST exhibited a fluorescence spectrum with a maximum wavelength at 445 nm, with an excitation wavelength at 368 nm. DST reacted with Hg2+ in a 2:
1 molar ratio, accompanied by approximately 95% fluorescence quenching. The Stern–Volmer plot for the fluorescence quenching of DST by Hg2+ (Fig. S4†) showed that the relationship between the ratio F0/F and the concentration Q of Hg2+ (where F0 and F were the fluorescence intensities of the DST solution in the absence and presence of Hg2+ at concentration Q, respectively) was not linear but exhibited an upward (positive) deviation. This suggested that both static and dynamic quenching mechanisms were responsible for the observed decrease in fluorescence intensity.49,50
The stable structure of the complex between DST and Hg2+ in a 2:
1 molar ratio was determined at the DFT/PBE0/Lanl2dz and is presented in Fig. 3. The calculation results show that the Hg(DST)2 complex is stabilized by two Hg⋯S interactions, with a contact distance of 2.81 Å, which is significantly smaller than the van der Waals radius sum of Hg and S atoms, which is 3.35 Å. The Hg⋯S⋯Hg bond angle is nearly linear, with a value of 179.3°.51 The two DST molecules in the complex are not in the same plane but lie in two parallel planes (considering only the π-conjugated system plane).
The results of the study on the singlet electron transition process from the ground state S0 to the excited state S1 (Fig. 4) indicated that in the DST compound, this process was determined by the electron transition from HOMO to LUMO (oscillator strength f = 1.2441). Meanwhile, in the Hg(DST)2 complex, this process was determined by the electron transition from HOMO − 1 to LUMO (f = 0.5515). Therefore, in the excited state, the Hg(DST)2 complex underwent a photoinduced electron transfer (PET) process with the transfer of an electron from HOMO to HOMO − 1, leading to the absence of an excited electron transition from LUMO to HOMO − 1 (which would normally result in energy emission in the form of fluorescence).51–54 Instead, there was an internal conversion process from LUMO to HOMO, resulting in the quenching of fluorescence in the Hg(DST)2 complex.
![]() | ||
Fig. 4 Diagram illustrating the singlet electron transition processes in the excited state for DST and Hg(DST)2. |
Hg2+ + 2DST = Hg(DST)2 | (1) |
![]() | (2) |
If the initial concentration of DST is denoted as CL, the initial fluorescence intensity of the DST solution as F0, and the fluorescence intensity of the DST solution at equilibrium as F, then let x = F/F0.The concentration of Hg2+ used is denoted as y = CM. At equilibrium, the concentrations are expressed as follows:
![]() | (3) |
![]() | (4) |
![]() | (5) |
Substituting into eqn (2) yields (6) as follows:
![]() | (6) |
![]() | (7) |
![]() | (8) |
![]() | (9) |
The fluorescence titration results of the DST solution (CL = 20 μM) with Hg2+ (concentrations of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 μM) and the nonlinear curve fitted to eqn (8) are presented in Fig. 5. The P value obtained from this process is 0.01006 ± 0.00201. From eqn (9), the calculated stability constant βex is 2.458 μM−1 (or 100.395 μM−1, equivalent to 1012.395 M−1).
According to the calculations above, the logβexof the Hg(DST)2 complex is 12.395, much smaller than the log
βex of the
complexes, which are 41.58, 43.57, and 39.4, respectively. Therefore, theoretically, GSH, Cys, and Hcy can react with Hg(DST)2 to form corresponding complexes and release free DST, altering the fluorescence intensity of the solution. In other words, Hg(DST)2 can be used as a fluorescence sensor to detect GSH, Cys, and Hcy. One noteworthy point is that since the log
βex values of the
complexes are approximately similar, it is unlikely that Hg(DST)2 can selectively detect each thiol in the GSH, Cys, and Hcy group.
Indeed, the experimental results on the fluorescence titration of the Hg(DST)2 sensor solution by the thiols GSH, Cys, and Hcy, as presented in Fig. 6, are entirely consistent with the aforementioned observations. All three thiols – GSH, Cys, and Hcy – react with Hg(DST)2, releasing DST and leading to an increase in the fluorescence intensity of the solution (The images showing the fluorescence color changes of the Hg(DST)2 solution in the presence of biothiols, taken inside the fluorescence spectrophotometer chamber, are presented in Fig. S5†). When the concentration of the thiols approaches approximately twice the concentration of Hg(DST)2 used, as well as beyond this point, the fluorescence intensity of the solution remains nearly unchanged. This indicates that the complexes formed between Hg2+ and GSH, Cys, and Hcy are predominantly in the forms, as previously noted.
The survey results indicate that the Hg(DST)2 complex sensor can quantitatively detect GSH, Cys, and Hcy, as evidenced by the excellent linear relationship between the fluorescence intensity of the solution and the concentrations of GSH, Cys, and Hcy within a certain range. Accordingly, the linear ranges for determining GSH, Cys, and Hcy using the Hg(DST)2 sensor are 0.34–8.00 μM, 0.47–10.00 μM, and 0.26–8.0 μM, respectively, with corresponding linear correlation coefficients (R) of approximately 0.998. Among these, the values of 0.34, 0.47, and 0.26 μM represent the detection limits of GSH, Cys, and Hcy, respectively, using the Hg(DST)2 sensor, as determined from the calibration curve equation at low concentrations (see Fig. S6 and Table. S1 of the ESI†).62 Compared to some fluorescent sensors for thiol detection based on metal ion complexes reported in recent times (Table 1), the Hg(DST)2 sensor had a comparable LOD for thiols but had the advantage of operating in a fully aqueous environment.
The influence of amino acids on the use of the Hg(DST)2 sensor for thiol detection was also investigated and presented in Fig. 7. The results showed that, except for Lys (lysine), which altered the fluorescence spectrum of the Hg(DST)2 sensor solution, the other amino acids, including Ala (alanine), Arg (arginine), Asp (aspartic acid), Glu (glutamic acid), Gly (glycine), His (histidine), Ile (isoleucine), Leu (leucine), Met (methionine), Ser (serine), Thr (threonine), Trp (tryptophan), Tyr (tyrosine), and Val (valine), had little to no effect on the fluorescence spectrum of the Hg(DST)2 sensor solution. These results indicated that the Hg(DST)2 sensor could detect the thiols GSH, Cys, and Hcy in the presence of the above amino acids, except for Lys. Additionally, the investigation results showed that the Hg(DST)2 sensor was unable to detect individual thiols within the GSH, Cys, and Hcy groups separately. This finding was consistent with previous reports on fluorescent sensors for thiol detection based on metal ion complexes. However, this limitation did not diminish the applicability of such sensors, as thiols typically do not coexist in equal concentrations in real samples. For instance, in human whole blood samples, GSH is significantly higher than other thiols (sometimes up to 1 mM), whereas, in human plasma samples, Cys is much more abundant than other thiols (sometimes reaching up to 250 μM).63–65
The use of the Hg(DST)2 complex fluorescent sensor for detecting the thiols GSH, Cys, and Hcy was also investigated for its susceptibility to interference by various ions, including alkali metal ions (Na+, K+), alkaline earth metal ions (Ca2+, Ba2+, Mg2+), transition metal ions (Ag+, Cu2+, Co2+, Ni2+, Mn2+, Fe2+, Fe3+, Cr3+), other metal ions (Zn2+, Pb2+, Cd2+, Al3+), as well as common anions such as sulfate (SO42−), carbonate (CO32−), chloride (Cl−), bromide (Br−), iodide (I−), and cyanide (CN−). The results, presented in Fig. 8, S7, and S8† (in the ESI†), demonstrated that, except for Ag+, Cu2+, Co2+, and Ni2+, the presence of the other ions did not affect the thiol detection method using the Hg(DST)2 sensor, as evidenced by the absence of significant fluorescence intensity changes in (Hg(DST)2 + thiol) solutions. The interference caused by Cu2+, Co2+, and Ni2+ could be eliminated by the complexing agent 1,10-phenanthroline (PHEN). Meanwhile, a suitable complexing agent to mitigate the interference of Ag+ has not yet been identified. However, since Ag+ is not a biological metal, its interference in thiol detection in biological samples is not a major concern.
The reaction time between the Hg(DST)2 complex and the biothiols GSH, Cys, and Hcy was almost instantaneous. After 1 minute, the fluorescence intensity of the solution had nearly stabilized. The sensing mechanism is summarized in Scheme 1.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5ra02268a |
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