Abstract
A probe based on 2-(2′-hydroxyphenyl) benzothiazole (HBT) and thiophosphate has been synthesized and used for the ratiometric detection of Hg2+. The probe was designed in such a way that the excited state intramolecular proton transfer (ESIPT) of the HBT moiety get blocked. The probe exhibited a strong fluorescence enhancement upon addition of Hg2+ while showing almost no response to other cations in CH3CN/HEPES buffer solution. The probe exhibited fast selectivity towards Hg2+ and could be completed in 1 min. Fluorescence imaging experiments of Hg2+ in living TE-1 cells demonstrated its value of practical applications in biological systems.
Similar content being viewed by others
Explore related subjects
Discover the latest articles, news and stories from top researchers in related subjects.Avoid common mistakes on your manuscript.
Introduction
The design and development of fluorescent probes for the detection of heavy and transition metals are significant due to their vital role in biological and environmental application in recent years [1–4]. Mercury is one of the most prevalent toxic metals in both the environment and biological system [5, 6]. Even at a very low concentration, the mercuric ion (Hg2+) which combines with both inorganic and organic ligands, can readily penetrate through biological membranes. Mercury can cause serious and irreversible DNA damage, mitosis impairment and nervous system defects [7–9]. Therefore, it is of great importance to develop advanced methods for detecting mercury ions in biological system and natural environment. Until recently, many excellent works of Hg2+ sensing by synthesized fluorescent probes have been reported and investigated [10–12].
Among these works been reported, many fluorescent probes were based on single emission intensity change [13–15]. However, changes in the emission intensity at a single wave length being the only detection signal, such turn-on probes tend to be affected by the variations in the sample and probe environment, illumination intensity or instrumental efficiency [16]. Ratiometric probes can eliminate most of these interferences through simultaneous recording ratio signals of two emission intensities at different wavelengths, which provided a built-in correction for the environmental effects [17, 18]. From this point of view, 2-(2′-hydroxyphenyl) benzothiazole (HBT) is very familiar because of its intramolecularly hydrogen-bonded property, which exhibits excited state intramolecular proton transfer (ESIPT) [19, 20]. There were a number of reactive probes reported based on the HBT moiety for the selective detection of different analytes via “protection-deprotection” sequence [21–23]. According to the strong thiophilic affinity of Hg2+, many chemodosimeters contained an “S″ group [7, 24, 25]. Thus, along with the leaving of HgS, the “protection-deprotection” reaction is accomplished, as well as the resulting ESIPT modulated fluorescence off–on response.
Herein, we present a simple and new fluorescent probe BTP for the detection of Hg2+ based on ESIPT mechanism. Probe BTP contained an “P = S″ group, and as expected, it exhibited a nonreversible, highly selective and sensitive recognition toward Hg2+ over other examined metal ions in CH3CN/HEPES (10 mM, pH = 7.4, 1:4, v/v) solution. Additionally, according to the fluorescence imaging experiments of Hg2+ ions in living TE-1 cells, BTP could be used for detecting Hg2+ in biological samples.
Experimental Section
Apparatus
Fluorescence spectra were recorded on the F-7000 FL Spectrophotometer (Hitachi, Japan), and the excitation and emission wavelength band passes were both set at 5.0 nm. 1H and 13C NMR spectra were recorded using a Bruker DTX-400 spectrometer. Samples were dissolved in CDCl3 and placed in 5 mm NMR tubes, TMS was used as internal reference. ESI mass spectra were carried out on an HPLC Q-Exactive HR-MS spectrometer (Thermo, USA) by using methanol as mobile phase. Fluorescence images experiments were carried out with a Zeiss-Axio Observer D1 inverted fluorescence microscope.
Materials
All chemicals reagents were used as received from commercial sources without further purification. Solvents for chemical synthesis and analysis were purified according to standard procedures. Deionized water was used throughout the experiment. Chloride salts of metal ions (Li+, K+, Na+, Ca2+, Mg2+, Ba2+, Zn2+, Fe2+, Mn2+, Cu2+, Co2+, Ni2+, Cd2+, Cr3+, Hg2+, Al3+) and the nitrate salts of Ag+, Pb2+ and Fe3+ ions were prepared as 10.00 mM in water solution.
Synthesis of Probe BTP
The synthetic routine of probe BTP is outlined in Scheme 1. HBT was synthesized by a similar way described in a reported method [26]. Dimethylthiophosphinoyl chloride (105 μL, 1 mmol) in 20 mL dichloromethane was added to a mixture of HBT (136 mg, 0.60 mmol) and triethylamine (138 μL, 1 mmol) in 20 mL dichloromethane. The reaction mixture was stirred at room temperature for 4 h, and concentrated under reduced pressure. The crude product was purified by column chromatography (silica gel, hexane/ethyl acetate =2/1) to afford BTP (124 mg, 65 %) as a white powder. 31P NMR (162 MHz, CDCl3): δ = 96.16 ppm. 1H NMR (400 MHz, CDCl3, ppm) δ: 2.11 (d, 3 H, J = 4 Hz), 2.13 (d, 3 H, J = 4 Hz),7.35 (t, 1 H, J = 8 Hz), 7.50 (m, 3 H), 7.88 (d, 1 H, J = 8 Hz), 7.96 (d, 1 H, J = 8 Hz), 8.13 (d, 1 H, J = 8 Hz), 8.33 (d, 1 H, J = 4 Hz); 13C NMR (100 MHz, CDCl3, ppm) δ: 23.88, 24.61, 121.40, 121.69, 121.74, 123.31, 125.21, 125.38, 125.84, 125.89, 126.41, 130.81, 131.47, 135.54, 148.80, 148.89, 152.63, 162.45; HR-MS m/z: Calcd for C15H15NOPS2 + ([M + H+]+) 320.0333, found 320.0315 [M + H+]+, 342.0129 [M + Na+]+.
Results and Analysis
Probe BTP was dissolved in CH3CN to make a 1 mM stock solution. Then the stock solution was further diluted to require concentration for measurement.
Fluorescence Spectral Responses of BTP
As is well known, the HBT and its derivatives produced the ESIPT tautomers (the keto forms), which showed fluorescence more powerfully at longer wavelengths compared to the phenol forms upon irradiation. The selectivity of BTP was observed in the fluorescence emission profile of BTP (10 μM) in a CH3CN/HEPES (10 mM, pH = 7.4, 1:4, v/v) solution with appropriate amounts of metal ions (Fig. 1. inset). Probe BTP alone displayed an emission band centered at 377 nm, when excited at 310 nm. Upon addition of 10 eq. Hg2+, the emission at 377 nm decreased, and a significant enhancement at 470 nm emerged quickly. This indicated that the chemical reaction between Hg2+ and the receptor (thiophosphinated phenolic) started at this minimum concentration and thus the ESIPT properties of HBT were demasked (Scheme 2). The fluorescence intensity ratio (F470/F377) of probe BTP toward different metal ions was recorded in Fig. 1, which exhibited a prominent enhancement of the fluorescence ratio (F470/F377) in the presence of 10 eq. Hg2+. In the meantime, no response could be observed upon the addition of the same amount of other ions. This strongly suggested that BTP can serve as a high sensitivity for Hg2+.
Furthermore, as shown in Fig. 2, the ratiometric fluorescence signal response of probe BTP toward Hg2+ in the presence of various coexistent anions such as NO3 −, NO2 −, Cl−, Br−, PO4 3−, SO4 2− and CO3 2−, which revealed that all the tested anions have little interference on the detecting of Hg2+. It was also investigated that the competitive experiment also confirmed that the background metal ions showed very low interference with the detection of Hg2+ (Fig. S5), only Cu2+ has posed a negligible effect on the fluorescence response of BTP for Hg2+, it may due to the quenching effect of the paramagnetic Cu2+ [27]. Therefore, these results suggested that probe BTP has a high selectivity for Hg2+ in the presence of these tested foreign metal ions and anions.
As shown in Fig. 3, the time dependence of the response of BTP to Hg2+ ions was investigated. It can be seen that the fluorescence intensity ratio signal of the BTP with Hg2+ ion increased for a few seconds, and leveled off as the time continues, while the fluorescence intensity of blank solution (only BTP, 10 μM) showed almost unchanged at the same conditions. The time-dependent change plot demonstrated the reaction could complete in about 1 min, which indicated the probe BTP had a fast response for Hg2+. Therefore, a 1 min reaction time was selected in subsequent experiments in order to make the metal ions chelate with the sensors sufficiently.
To further investigate the interaction between Hg2+ and probe BTP, a fluorescence titration experiment was carried out. The fluorescence spectra of BTP (10 μM) exposed to CH3CN/HEPES (10 mM, pH = 7.4, 1:4, v/v) solution containing different concentrations of Hg2+ were then recorded at an excitation wavelength of 310 nm (Fig. 4). Upon treatment with increasing concentrations of Hg2+, the fluorescence intensity ratio (F470/F377) gradually increased, and reached saturation when the amount of Hg2+ was more than 12 μM (Fig. S6). Moreover, a linear relationship was found between the fluorescence intensity ratio (F470/F377) and the Hg2+ concentration from 4 to 12 μM (Fig. S7), the detection limit (3σ/slope) of probe BTP for the determination of Hg2+ was found to be 12 nM [28, 29]. These results demonstrated that probe BTP could detect Hg2+ quantitatively.
For practical applicability, the proper pH condition of this new probe for Hg2+ detection was also evaluated. We investigated the fluorescence properties of probe BTP and that probe BTP with Hg2+ (10 eq.) under different pH values, respectively (Fig. 5). Probe BTP was pH insensitive, and its ratiometric fluorescence response (F470/F377) was quite weak from pH 4 to 9. However, the fluorescent sensing toward Hg2+ was obviously affected by the change of pH values. Ratiometric fluorescence response (F470/F377) reached its maximum and kept constant around biologically relevant pH 4 to 9, indicating that its potential for application in biological systems.
Mechanism
In addition, the KI-adding experiments were conducted to examine the reversibility of this reaction as shown in Fig. S8. When excess KI (2 eq. of Hg2+) was added to the BTP (10 μM) and Hg2+ (100 μM) in CH3CN/HEPES (10 mM, 1:4, v/v) solution, the fluorescence intensity at 470 nm almost unchanged, indicating that the coordination of BTP with Hg2+ was chemically nonreversible.
According to the previous reported work [30–32], we proposed that the strong fluorescence enhancement was attributed to the deprotection of dimethyl- thiophosphinoyl group and concurrent generation of HBT. The observed change in presence of Hg2+ may arise from the HBT moiety which was released from BTP with the leaving of HgS. In order to verify this speculation, the reaction products of probe BTP and Hg2+ were subjected to ESI-HRMS spectrum. A major ion peak was founded at m/z = 228.0469 (Fig. S9), corresponding to the resulting HBT ([M + H]+), clearly confirmed the proposed mechanism as shown in Scheme 2.
Bioimaging Applications of Probe BTP in TE-1 Cells
We further investigated the practical application of BTP in biological systems [33, 34]. Fluorescent imaging inside TE-1 cells was monitored by fluorescence microscopy. As shown in Fig. 6, very weak fluorescence of BTP inside the living TE-1 cells was observed (Fig. 6b). After washing with water twice, 30 μM of Hg2+ were then supplemented to the cells. After incubated at 37 °C for another 30 min, a significant increase in the fluorescence from the intracellular area was observed (Fig. 6d). A bright field transmission image of cells with BTP and BTP with Hg2+ confirmed that the cells were viable throughout the imaging experiments (Fig. 6a and c). Therefore, these results demonstrated that probe BTP was cell membrane permeable and capable of fluorescence imaging of Hg2+ in biological samples.
Conclusion
In summary, a new ESIPT-based ratiometric fluorescence probe BTP for Hg2+ was prepared and reported. Probe BTP exhibited highly selective binding with Hg2+ over other metal ions and various coexistent anions in CH3CN/HEPES (10 mM, 1:4, v/v) solution. Moreover, the preliminary experimental results demonstrated that probe BTP could be used for detecting Hg2+ in biological samples.
Reference
Quang DT, Kim JS (2010) Fluoro- and chromogenic- chemodosimeters for heavy metal ion detection in solution and biospecimens. Chem Rev 110:6280–6301
Du JJ, Hu MM, Fan JL, Peng XJ (2012) Fluorescent chemo-dosimeters using “mild” chemical events for the detection of small anions and cations in biological and environmental media. Chem Soc Rev 41:4511–4535
Formica M, Fusi V, Giorgi L, Micheloni M (2012) New fluorescent chemosensors for metal ions in solution. Coord Chem Rev 256:170–192
Yang Y, Zhao Q, Feng W, Li F (2013) Luminescent chemodosimeters for bioimaging. Chem Rev 113:192–270
Harris HH, Pickering IJ, George GN (2003) The chemical form of mercury in fish. Science 301:1203
Clarkson TW, Magos L, Myers GJ (2003) The toxicology of mercury-current exposures and clinical manifestations. N Engl J Med 49:1731–1737
Liu W, Xu L, Zhang H, You J, Zhang X, Sheng R, Li H, Wu S, Wang P (2009) Dithiolane linked thiorhodamine dimer for Hg2+ recognition in living cells. Org Biomol Chem 7:660–664
Huang HJ, Xu Y, Qian X (2009) A rhodamine-based Hg2+ sensor with high selectivity and sensitivity in aqueous solution: a NS2-containing receptor. J Org Chem 74:2167–2170
Boening DW (2000) Ecological effects, transport, and fate of mercury: a general review. Chemosphere 40:1335–1351
D. Zhang, M. Li, M. Wang, J. H. Wang, X. Yang, Y. Ye, Y. F. Zhao. A rhodamine- phosphonate off-on fluorescent sensor for Hg2+ in natural water and its application in live cell imaging. Sens Actuator B 2013; 177: 997–1002. [11] R. Han, X. Yang, D. Zhang, M. Fan, Y. Ye, Y. F. Zhao. A bis(rhodamine)-based highly sensitive and selective fluorescent chemosensor for Hg(II) in aqueous media. New J. Chem., 2012; 36: 1961–1965.
Huang JH, Xu YF, Qian XH (2009) A rhodamine-based Hg2+ sensor with high selectivity and sensitivity in aqueous solution: a NS2-containing receptor. J Org Chem 74:2167–2170
Shi W, Ma HM (2008) Rhodamine B thiolactone: a simple chemosensor for Hg2+ in aqueous media. Chem Commun 16:1856–1858
Yan FY, Cao DL, Wang M, Yang N, Yu QH, Dai LF (2012) A new rhodamine-based “off-on” fluorescent chemosensor for Hg (II) ion and its application in imaging Hg (II) in living cells. J Fluoresc 22:1249–1256
Wang HH, Xue L, Yu CL, Qian YY, Jiang H (2011) Rhodamine-based fluorescent sensor for mercury in buffer solution and living cells. Dyes Pigments 91:350–355
Im HG, Kim HY, Chang SK (2014) Dual signaling of Hg2+ ions by selective cleavage of thiophosphinated rhodol. Sensors Actuators B 191:854–859
Fan JL, Sun W, Hu MM, Cao JF, Cheng GH, Dong HJ, Song KD, Liu YC, Sun SG, Peng XJ (2012) An ICT-based ratiometric probe for hydrazine and its application in live cells. Chem Commun 48:8117–8119
Lee MH, Kim HJ, Yoon S, Park N, Kim JS (2008) Metal ion induced FRET off-on in tren/dansyl-appended rhodamine. Org Lett 10:213–216
Lin Y, Lin W, Xie Y, Chen B, Song J (2011) Development of a ratiometric fluorescent sensor for ratiometric imaging of endogenously produced nitric oxide in macrophage cells. Chem Commun 47:9372–9374
Zhang J, Guo W (2014) A new fluorescent probe for gasotransmitter H2S: high sensitivity, excellent selectivity, and a significant fluorescence off–on response. Chem Commun 50:4214–4217
Xu Z, Xu L, Zhou J, Xu Y, Zhu W, Qian X (2012) A highly selective fluorescent probe for fast detection of hydrogen sulfide in aqueous solution and living cells. Chem Commun 48:10871–10873
Goswami S, Manna A, Mondala M, Sarkar D (2014) Cascade reaction-based rapid and ratiometric detection of H2S/S2 in the presence of bio-thiols with live cell imaging: demasking of ESIPT approach. RSC Adv 4:62639–62643
Goswami S, Das S, Aich K, Pakhira B, Panja S, Mukherjee SK, Sarkar S (2013) A Chemodosimeter for the ratiometric detection of hydrazine based on return of ESIPT and its application in live-cell imaging. Org Lett 15:5412–5415
Tang Y, Lee D, Wang J, Li G, Yu J, Lin W, Yoon J (2015) Development of fluorescent probes based on protection-deprotection of the key functional groups for biological imaging. Chem Soc Rev 44:5003–5015
Wanichacheva N, Setthakarn K, Prapawattanapol N, Hanmeng O, Lee VS, Grudpan K (2012) Rhodamine B-based “turn-on” fluorescent and colorimetric chemosensors for highly sensitive and selective detection of mercury (II) ions. J Lumin 132:35–40
Lee MH, Wu JS, Lee JW, Jung JH, Kim JS (2007) Highly sensitive and selective chemosensor for Hg2+ based on the rhodamine fluorophore. Org Lett 9:2501–2504
Zhou J, Shi R, Liu J, Wang R, Xu Y, Qian X (2015) An ESIPT-based fluorescent probe for sensitive detection of hydrazine in aqueous solution. Org Biomol Chem 13:5344–5348
Wang H-H, Xue L, Yu C-L, Qian Y-Y, Jiang H (2011) Rhodamine-based fluorescent sensor for mercury in buffer solution and living cells. Dyes Pigments 91:350–355
Zhou Y, Zhang J, Zhang L, Zhang Q, Ma T, Niu J (2013) A rhodamine-based fluorescent enhancement chemosensor for the detection of Cr3+ in aqueous media. Dyes Pigments 97:148–154
Liu D, Pang T, Ma K, Jiang W, Bao X (2014) A new highly sensitive and selective fluorescence chemosensor for Cr3+ based on rhodamine B and a 4,13-diaza-18-crown 6-ether conjugate. RSC Adv 4:2563–2567
Liu B, Wang H, Wang T, Bao Y, Du F, Tian J, Li Q, Bai R (2012) A new ratiometric ESIPT sensor for detection of palladium species in aqueous solution. Chem Commun 48:2867–2869
Chen S, Hou P, Wang J, Song X (2012) A highly sulfite-selective ratiometric fluorescent probe based on ESIPT. RSC Adv 2:10869–10873
Goswami S, Maity S, Maity AC, Das AK, Pakhira B, Khanra K, Bhattacharyya N, Sarkar S (2015) ESIPT based Hg2+ and fluoride chemosensor for sensitive and selective ‘turn on’ red signal and cell imaging. RSC Adv 5:5735–5740
Zhao Y, Sun Y, Lv X, Liu Y, Chen M, Wei G (2010) Rhodamine-based chemosensor for Hg2+ in aqueous solution with a broad pH range and its application in live cell imaging. Org Biomol Chem 8:4143–4147
Wang C, Zhang D, Huang X, Ding P, Wang Z, Zhao Y, Ye Y (2014) A ratiometric fluorescent chemosensor for Hg2+ based on FRET and its application in living cells. Sensors Actuators B Chem 198:33–40
Acknowledgments
This work was supported by the National Science Foundation of China (Nos. 21305031), Scientific and Technological Project of Henan Province of China (152102110135 and 162102310352), Science-Technology Foundation for Outstanding Young Scientists of Henan Academy of Agricultural Sciences (Grant no. 2016YQ22).
Author information
Authors and Affiliations
Corresponding authors
Electronic Supplementary Material
ESM 1
(DOC 1.16 mb)
Rights and permissions
About this article
Cite this article
Zhang, D., Liu, J., Yin, H. et al. A Turn on ESIPT Probe for Rapid and Ratiometric Fluorogenic Detection of Hg2+ and its Application in Live-Cell Imaging. J Fluoresc 26, 1367–1372 (2016). https://doi.org/10.1007/s10895-016-1826-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10895-016-1826-z