Biosensors and Bioelectronics 63 (2015) 513–518
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An ESIPT based fluorescent probe for highly selective and ratiometric detection of periodate Chusen Huang n, Ti Jia, Congjun Yu, Amin Zhang, Nengqin Jia n The Education Ministry Key Laboratory of Resource Chemistry and Shanghai Key Laboratory of Rare Earth Functional Materials, Department of Chemistry, College of Life and Environmental Sciences, Shanghai Normal University, 100 Guilin Road, Shanghai 200234, China
art ic l e i nf o
a b s t r a c t
Article history: Received 16 May 2014 Received in revised form 23 July 2014 Accepted 4 August 2014 Available online 8 August 2014
Periodate is widely used in organic and bioorganic chemistry, and also related to food and environmental safety. To best of our knowledge, there is no efficient tools reported for simultaneously quantifying periodate with high accuracy and discriminating periodate from other forms of iodine. We have synthesized, characterized and applied a first ratiometric fluorescent probe (PDS-2) for simultaneous monitoring of changes of periodate based on the excited-state intramolecular proton transfer mechanism. This PDS-2 based fluorescent technique may enable for a better understanding of periodate related biological and chemical processes. Also, it is an efficient tool for public health, food safety and environmental protection. & 2014 Elsevier B.V. All rights reserved.
Keywords: Periodate Fluorescent probe Ratiometric detection Excited-state intramolecular proton transfer
1. Introduction Periodate is usually found as salts with potassium (e.g. KIO4) or sodium (e.g. NaIO4). In neutral solution, periodate can cleave carbon–carbon bonds when two carbon atoms bear hydroxyl groups (vicinal diols), creating two aldehydes and/or ketones groups (Fatiadi, 1974). This property is widely applied in organic synthesis (Binder et al., 2008; Outram et al., 2002) and commonly utilized in molecular biochemistry (Badalassi et al., 2000; Wahler and Reymond, 2001a,b), as well as its contribution to instrumental methods of micro-analysis (Lin et al., 1998, 2001, 2013; Vlessidis and Evmiridis, 2009). For example, sodium periodate was used to oxidize diols to afford aldehyde which was in situ trapped with stabilized ylides. This developed one-pot synthetic procedure provides access to a variety of useful natural products (Dunlap et al., 2002; Outram et al., 2002). In addition, sodium periodate was also used to oxidize cellulose and create a biocompatible and biodegradable compound that can be used as suture, as a scaffold for tissue engineering, or for drug delivery (Varma and Kulkarni, 2002). For a good understanding of the roles, sodium periodate is often introduced to open saccharide rings between vicinal diols leaving two reactive aldehyde groups, which can be easily modified by primary amine-containing molecules and surfaces in physiological conditions (Amore et al., 2013). This process was n
Corresponding authors. E-mail addresses: [email protected] (C. Huang), [email protected] (N. Jia).
http://dx.doi.org/10.1016/j.bios.2014.08.005 0956-5663/& 2014 Elsevier B.V. All rights reserved.
commonly used to label saccharides with fluorescent probe and other detectable tags such as biotin. Compared to DNA, 3ʹ-termini of RNA (ribose has vicinal diols) can be selectively labeled with detectable tags (Willkomm and Hartmann, 2008) through above process. Periodate oxidation based micro-analysis methods were also developed for detection of a variety of analytes including hydrogen peroxide (Lin et al., 1998, 2001), catechins (Lin and Yamada, 2001), ethylene glycol (Evmiridis, 1989) etc. The periodate–H2O2 chemiluminescence (CL) system was further used for detection of sodium dodecyl benzenesulfonate (Guan et al., 2014). Another significance of periodate was its application in enzyme assay. A periodate-coupled fluorogenic assay was developed for screening hydrolases in a high-throughput format (Badalassi et al., 2000; Goddard and Reymond, 2004; Nyfeler et al., 2003; Wahler and Reymond, 2001a,b). Some coumarin-based fluorescent substrates were synthesized, after the hydrolases hydrolyze the substrates to produce vicinal-diols-containing products, which were oxidized by periodate to release the fluorescent umbelliferone catalyzed by bovine serum albumin (BSA). Then the type and concentration of hydrolases could be recorded by fluorescence intensity changes of umbelliferone. Besides its special effect on organic and bioorganic chemistry, periodate is also related to food and environmental safety because of the conversion of periodate to some other iodine species that were considered as potentially hazardous contaminant (Hariri et al., 2013; Edmonds and Morita, 1998; von Gunten, 2003). Finally, some iodine species is also essential component for biomolecule such as thyroid hormones. Some literatures reported that the periodate and iodate were
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related to goiter and hypothyroidism as well as hyperthyroidism (Greer et al., 1968; Larsen et al., 1981). Hence, development of methods for simultaneously monitoring periodate as well as highly discriminating periodate from other forms of iodine is of considerable significance, not only for a good understand of the roles of periodate in biological and chemical processes, but also for public health, food safety and environmental protection. Several approaches for periodate detection were available, including electrochemical methods (Chatraei and Zare, 2013; Salimi et al., 2007; Shamsipur et al., 2013) and UV–vis spectrophotometry (Afkhami et al., 2001; Afkhami and Zarei, 2001; Benvidi et al., 2012; Ensafi and Chamjangali, 2003; Ni and Wang, 2007). To best of our knowledge, there is no efficient tool reported for discriminating periodate from other forms of iodine species and simultaneous monitoring of concentration changes of periodate with high accuracy. Only a few efforts come from fluorescence based techniques for quantifying periodate (Badalassi et al., 2000; Ensafi and Bagherian Dehaghi, 2000). Fluorescent probe based technology with the advantage of high selectivity and sensitivity, spatiotemporal resolution and visibility has been viewed as a powerful and versatile toolbox in the field of physiology and molecular biology, environmental monitoring and clinical diagnosis (Chan et al., 2012; de Silva et al., 1997; Li et al., 2013; Stennett et al., 2014). Thus, fluorescent probes based technique is good choice for simultaneous detection of periodate. However, recently few reported fluorescent probes (Badalassi et al., 2000; Ensafi and Bagherian Dehaghi, 2000) for detecting periodate were mainly on the fluorescence intensity changes, which might be interfered by fluorescence self-quenching, large background signal and some complex test systems. Such obstacles can be overcome by a ratiometric detection mechanism. Excited-state intramolecular proton transfer (ESIPT) is a significantly important phenomenon that is widely used for ratiometric detection in biological systems. Generally, enol–keto phototautomerism takes place in the excited state of ESIPT molecule which leads to initial normal form (NF*) and phototautomer form (PT*) (Li et al., 2011). Hence, dual emission signals could be observed (Fig. S1). And the intensity ratio of this two emission bands will make the ratiometric fluorescence detection possible. Many ESIPT based fluorescent probes have been constructed for ratiometric sensing of anion (Hu et al., 2010), cation (Cui et al., 2013), enzyme activity (Kim et al., 2009), and signaling molecule (Murale et al., 2013) etc. (Wu et al., 2011). Herein, we present a first ratiometric fluorescent probe for selective detection of periodate based on ESIPT mechanism.
Fluorescence spectra were recorded using a Hitachi fluorescence spectrophotometer (F-7000). Samples for absorption and fluorescence measurements were contained in 1 cm 1 cm quartz cuvettes (3.5 mL volume). All cell images were taken by a Leica TCS SP5 II Confocal Laser Scanning Microscope. 2.2. Synthesis 2.2.1. Synthesis of 2-(2-(2-(2.2-dimethyl-1.3-dioxolan-4-yl)ethoxy)3-methoxyphenyl)benzo[d]thiazole (PDS-1) To a solution of HMBT (405 mg, 1.576 mmol) and K2CO3 (432 mg, 3.15 mmol) in anhydrous DMF (10 mL), compound C (404 mmg, 1.578 mmol) was added. The reaction mixture was continued to stirring for about 12 h under dark. After TLC shows material HMBT disappeared, the reaction mixture was concentrated under reduced pressure. The obtained crude product was purified by chromatography on silica gel (petrol ether:EtOAc, from 30:1 to 10:1, v/v) to give a pale white solid (401 mg, 66% yield). 1H NMR (400 MHz, CDCl3) δ 8.11 (d, J ¼8.0 Hz, 1H), 8.07 (d, J ¼8.0 Hz, 2H), 7.95 (d, J¼ 8.0 Hz, 1H), 7.51 (t, J ¼8.0 Hz, 1H), 7.40 (t, J ¼8.0 Hz, 1H), 7.20 (t, J¼ 8.0 Hz, 1H), 7.03 (d, J¼ 8.0 Hz, 1H), 4.51–4.44 (m, 1H), 4.30–4.18 (m, 1H), 3.92 (s, 3H), 3.72 (t, J ¼7.6, 1H), 2.33–2.18 (m, 2H), 1.47 (s, 3H), 1.42 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 162.89, 152.98, 152.24, 146.49, 136.14, 127.43, 126.04, 124.99, 124.36, 123.01, 121.36, 120.92, 114.17, 108.65, 73.74, 70.18, 69.72, 55.96, 34.35, 27.01, 25.83; HRMS (ES þ ): calc. for C21H24NO4S [MþH] þ 386.1426, found 386.1423. 2.2.2. Synthesis of 4-(2-(benzo[d]thiazol-2-yl)-6-methoxyphenoxy) butane-1.2-diol (PDS-2) A solution of PDS-1 (385 mg, 1 mmol) in 80% aqueous acetic acid (5 mL) was stirred at room temperature for 12 h. TLC shows the material PDS-1 disappeared, then the reaction mixture was concentrated under reduced pressure. The obtained crude product was purified by chromatography on silica gel (petrol ether:EtOAc, from 2:1 to 1:1, v/v) to afford a colorless syrup which gradually became a pale white solid (306 mg, 90% yield). 1H NMR (400 MHz, CDCl3) δ 8.12 (d, J ¼8.4 Hz, 1H), 7.98 (d, J¼ 8.0 Hz, 1H), 7.93 (d, J¼ 8.0 Hz, 1H), 7.50 (t, J ¼7.6 Hz, 1H), 7.39 (t, J ¼7.6 Hz, 1H), 7.20 (t, J¼ 8.0 Hz, 1H), 7.01 (d, J ¼8.0 Hz, 1H), 4.28–4.19 (m, 3H), 3.91 (s, 3H), 3.81–3.78 (m, 1H), 3.69–3.64 (m, 1H), 2.07–2.02 (m, 2H); 13C NMR (100 MHz, CDCl3) δ163.04, 152.82, 152.33, 146.17, 135.87, 127.47, 126.19, 125.16, 124.66, 123.03, 121.41, 121.30, 114.12, 70.77, 70.14, 66.58, 56.03, 33.28; HRMS (ES þ ): calc. for C18H20NO4S [MþH] þ 346.1113, found 346.1111.
2. Experimental
3. Results and discussion
2.1. Chemicals and apparatus
3.1. Design strategy
All chemical reagents and solvents were purchased from commercial sources and used without further purification except for N,N-dimethylformamide (DMF) which was purified according to the handbook (see supporting information). Thin-layer chromatography (TLC) was performed on silica gel plates. Column chromatography was performed using silica gel (Hailang, Qingdao) 200–300 mesh. 1H and 13C NMR spectra were recorded employing a Bruker AV-400 spectrometer with chemical shifts expressed in parts per million (in deuteriochloroform, Me4Si as internal standard). Electrospray ionization (ESI) mass spectrometry was performed in a HP 1100 LC–MS spectrometer. Glass fiber membrane was purchased from Shanghai Jiening Biotechnology companies. All pH measurements were made with a pH-Meter PHB-4 (INESA Scientific Instrument Co., Ltd.). Absorption spectra were recorded using a Hitachi UV–visible spectrophotometer (U-3900).
Our investigation begins with designing an active probe for selective interaction with periodate. Since the well-known reaction mechanism that periodate can selectively breaks vicinal diols into aldehyde or ketone, a vicinal diols group was introduced as a specific receptor for periodate. Then an ESIPT fluorophore (2(benzo[d]thiazol-2-yl)-6-methoxyphenol (HMBT), Scheme S1) was taken as the reporter group to construct target probe (PDS-2, Scheme 1). With the advantage of ESIPT fluorophore which involves initial normal form (NF*) and phototautomer form (PT*), HMBT also exhibits dual maximum emission at 406 and 462 nm, respectively (Fig. S1). The intensity ratio of this two emission bands will make the ratiometric fluorescence detection possible. Many HMBT based fluorescent probes have been constructed for sensing anion (Hu et al., 2010), cation (Cui et al., 2013), enzyme activity (Kim et al., 2009), and signaling molecule (Murale et al.,
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Scheme 1. Design strategy of PDS-2 for selective detection of periodate (IO4-) in aqueous solution.
2013) etc. (Wu et al., 2011). In specific, through modification of hydroxyl group of HMBT with butane-1.2.4-triol to block ESIPT and exclusively results in short-wavelength. After target fluorescent probe was selectively oxidized by periodate, HMBT will be released from the formed oxidative product via a fast β-elimination catalyzed by bovine serum albumin (BSA, Scheme 1). This detection mechanism was explored by liquid chromatography–mass spectrometry (LC–MS) analysis (Fig. S2). An absorption peak at about 9.306 min can be attributed to PDS-2 according to the mass result, then this peak disappeared after PDS-2 was treated with sodium periodate. Meanwhile, a new absorption peak at about 12.793 min appeared, which can be attributed to product HMBT according to the mass results. The results were similar as reported literature (Badalassi et al., 2000). This sensing system will ensure a selective and quantitative detection of periodate through the fluorescence intensity ratio related to enol and keto forms of HMBT. In order to demonstrate this design, a control probe (PDS1) with the vicinal diols protected by acetone was also synthesized (Scheme S1). 3.2. Fluorescence sensing behavior of PDS-2 toward periodate Next, we investigated the fluorescence sensing behavior of PDS-2 toward periodate. Initially, pH titration was conducted in
water. As shown in Fig. S3, no difference in fluorescence of PDS-2 was observed over the pH range of 6–10. Thus, a PBS buffer (pH 7.4, containing 1% DMSO) was chosen as the test medium. Then a catalytic amount of BSA (2 mg/ml) and 1 mM CTAB were also added to present the final test system. As displayed in the absorption spectrum (Fig. S4), after 250 μM of periodate was added into PBS buffer containing 5 μM of PDS-2, maximum absorption at 308 nm decreased readily with a subsequent increase in absorption at 350 nm. An isosbestic point appears at 330 nm. Accordingly, the fluorescence emission at 406 nm decreased with concomitant ingrowth of fluorescence emission at 462 nm under the excitation wavelength at 339 nm (Fig. 1A, Fig. S5). Meanwhile, fluorescence changes of the test solution is also visible (from blue to green, Fig. 1A, inset) with UV lamp irradiation (365 nm). The fluorescence intensity ratio between maximum emission at 406 and 462 nm was also recorded in Fig. 1B. We can observe that the intensity ratio changed quickly within 15 min and then reached a plateau. This result demonstrates that the reaction between periodate and PDS-2 was nearly completed within 15 min. In the following assay, 20 min was taken for the detection time. Control probe PDS-1 (5 μM) was also applied for detection of periodate. As shown in Fig. S6, negligible changes in the fluorescence signal were observed in the presence of 250 μM of periodate
Fig. 1. (A) Time-dependent changes in fluorescence emission of PDS-2 (5 μM) with addition of IO4- (250 μM) in PBS buffer (pH 7.4, containing 1% DMSO, 2 mg/ml BSA, 1 mM CTAB). (B) A plot of the fluorescence intensity changes (based on the peak heights at the maxima) depending on time. λex ¼ 339 nm. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
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Fig. 2. (A) Changes in fluorescence emission (or (B) emission at 406 and 462 nm respectively) of PDS-2 (5 μM) with increasing addition of IO4- (from 0 to 300 μM) in PBS buffer (pH 7.4, containing 1% DMSO, 2 mg/ml BSA, 1 mM CTAB) after 20 min. (C) A plot of fluorescence intensity ratio changes (based on the peak heights at the maxima, 406 and 462 nm respectively) depending on IO4- concentration. Error bar represents s.d. (D) Linear relationship between fluorescence intensity ratio changes and concentration of periodate over the range from 30 to 100 μM in PBS buffer (R2 ¼ 0.99433). λex ¼ 339 nm. Error bar represents s.d.
even the reaction time extended to 20 min. Thus, the vicinal diols group of the probe was needed for its selective interaction with periodate. 3.3. Quantitative detection of periodate with PDS-2 Then, in order to test the sensitivity of PDS-2 for periodate, titration experiments were carried out. The changes in fluorescence emission were recorded upon gradual addition of sodium periodate (from 0 to 300 μM). As suggested in Fig. 2A and B, the fluorescence intensity at 406 nm decreased accompanying the increase in
emission wavelength at 462 nm. After the amount of periodate increased to 150 μM, there were no significant fluorescence changes. Hence, about 150 μM of periodate was needed for completing the reaction in the presence of 5 μM of PDS-2 within 20 min. In addition, the fluorescence ratio of emission at 462 nm and 406 nm (F462/F406) also increased with increasing addition of periodate (Fig. 2C). From the concentration-dependent fluorescence ratio intensity changes, the detection limit of PDS-2 for periodate was about 26 μM (see Fig. S7 about detailed information). Through a nonlinear fitting, F462/F406 and concentration of periodate X0) X)P was fitted to a formula (Y¼A1 þ(A2 A1)/(1þ10(lg( ), Fig. S8).
Fig. 3. (A) Changes in fluorescence emission of PDS-2 (5 μM) with addition of IO4- (250 μM) and other interfering reagents (250 μM) after 20 min in PBS buffer (pH 7.4, containing 1% DMSO, 2 mg/ml BSA, 1 mM CTAB). (B) Fluorescence intensity ratio changes (based on the peak heights at the maxima, 406 and 462 nm respectively) with addition of IO4- (250 μM) and other interfering reagents (250 μM) after 20 min. λex ¼ 339 nm. Error bar represents s.d. Inset: fluorescence color changes with addition of IO4(250 μM) and other interfering reagents (250 μM) in PBS after 20 min buffer under a hand-held UV lamp irradiation (365 nm). 1: probe only, 2: sodium hypochlorite (ClO-), 3: hydrogen peroxide(H2O2), 4: potassium nitrate, 5: sodium oxalate, 6: sodium periodate, 7: sodium borate, 8: sodium citrate, 9: sodium fluoride, 10: sodium iodide, and 11: sodium iodate.
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Then after a mathematic manipulation by original software, a linear relationship between changes of F462/F406 and the concentration of periodate changes from 30 μM to 100 μM (Fig. 2D) was obtained (please see Fig. S8 about the detailed manipulation and discussion in the supporting information). Thus, this linear curve makes quantitative detection of periodate very convenient over the 30– 110 μM concentration range. The reported electrochemical methods for detection of periodate have made a good progress in a lower detection limit and wider linear range (for example, the detection limit from electrochemical methods for periodate is 0.4 μM) (Salimi et al., 2008, 2006). However, PDS-2 can be applied in physiological condition without lower-pH solution (pH 2.0 for electrochemical methods) present, which may enable for a live-cell imaging of periodate in living cells. Meanwhile, the ratiometric detection with PDS-2 can be used for simultaneously quantifying periodate with high accuracy. 3.4. Selectivity of PDS-2 for periodate To test other analytes could interfere with this detection, fluorescence response of PDS-2 for other analytes were also studied. As demonstrated in Fig. 3A, there was no significant fluorescence changes in the presence of sodium hypochlorite, hydrogen peroxide (H2O2), potassium nitrate, sodium oxalate, sodium borate, sodium citrate, sodium fluoride, sodium iodide and sodium iodate. In contrast, a red shift of about 56 nm can be observed after addition of periodate. This result also can be recorded by changes of fluorescence ratio intensity (Fig. 3B). Meanwhile, the fluorescence changes were visible through naked eyes under a UV lamp irradiation (365 nm). Only periodate promotes blue fluorescence change to green fluorescence, whereas other analytes have no interference (Fig. 3B, inset). All these results demonstrated that PDS-2 was a highly selective fluorescent probe for periodate. Specifically, PDS-2 can be used for discriminating different periodate from other forms of iodine species such as iodate ion and iodate (Fig. S9, Fig. 3B).
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3.5. Cellular imaging experiment For further application in biological samples, low cell cytotoxicity of the probe is very important (Wu et al., 2013, 2012, 2014), especially for live cell imaging. Herein, cell cytotoxicity of PDS-2 was studied by using a standard methyl thiazolyl tetrazolium (MTT) assay according to above reported literatures (Wu et al., 2013, 2012, 2014). Over the concentration range between 0 and 500 μM, PDS-2 and control probe (PDS-1) exhibited no cell cytotoxicity (Fig. S10A) when the incubation time was 6 h. After the incubation time extends to 24 h, PDS-2 has no remarkable influence on cell viability at the concentration of 5 μM (Fig. S10B). Thus 5 μM of PDS-2 was taken in the following assay. Then we performed ratiometric fluorescence imaging of periodate in live HeLa cells. 5 μM of PDS-2 was loaded into live HeLa cells and incubating for 20 min. After washed with PBS for three times, the fluorescence images were taken with confocal microscopy. A strong blue emission (410–440 nm) but no green fluorescence signal (450–480 nm) was observed from the intracellular zone. When the cells were firstly treated with PDS-2, then further incubated with periodate (100 μM), the intensity of blue fluorescence signal decreased and a strong green fluorescence was observed (Fig. 4). Furthermore, control experiments with PDS-1 were also conducted in live cells. As shown in Fig. S11, PDS-1stained cells loaded with 100 μM of periodate only give rise to blue fluorescence signal. These studies revealed that PDS-2 can be applicable in ratiometric detection of periodate in biological samples and a useful tool for further investigation of the effect of periodate on biological samples. It can be also potentially useful for exploring the effect of iodine species on goiter and hypothyroidism as well as hyperthyroidism. As HMBT was a typical ESIPT fluorophore that exhibits solidstate fluorescence, a test paper of PDS-2 was prepared for detection of periodate. The glass fiber membrane was cut into strips (1 cm in width), and then they were immersed in 50 μM of PDS-2 in PBS solution (pH 7.4, containing 1% DMSO, 2 mg/ml BSA, 1 mM CTAB) for 60 min to absorb PDS-2, and then dried at 37 °C overnight to afford the test papers. After the test papers were
Fig 4. Fluorescence images of periodate with PDS-2 in live cells. (A) Fluorescence of cells treated with PDS-2 (5 μM) for 20 min. (B) Fluorescence of cells treated with PDS-2 (5 μM), then washed with PBS (pH 7.4, containing 1% DMSO, 2 mg/ml BSA) for three times and followed by addition of sodium periodate (100 μM) and incubating for another 20 min. The experiment was conducted on Leica TCS SP5 II confocal laser scanning microscopy using HC PLAPO 63 oil objective (NA: 1.40), with excitation by UV laser (405 nm). 410–440 nm (blue channel) and 450–480 nm (green channel) emission light was collected, respectively. Scale bar: 100 μm. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
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treated with solutions containing different reagents, respectively, we can see that only sodium periodate induced an orange fluorescence under a hand-held UV lamp irradiation (365 nm), while other reagents make no fluorescence changes on the test papers (Fig. S12). Because solid samples were easier for transport, this test paper based technique is convenient for detection through the visible solid-state fluorescence changes and may be used in monitoring periodate in waters and organic reaction liquid.
4. Conclusions We have designed and synthesized a highly selective and ratiometric fluorescent probe (PDS-2) for simultaneous detection of periodate. By using this probe, we have developed a ratiometric approach based on ESIPT mechanism for monitoring changes of periodate in aqueous solution for the first time. Through difference in the fluorescence ratio of emission intensity at 406 and 462 nm, periodate can be discriminated from other forms of iodine species and quantified. Furthermore, this ratiometric method may be further developed for screening hydrolases which can produce products with vicinal diols in high-throughput format. Compared to fluorescence intensity based platform for screening hydrolases (Badalassi et al., 2000), PDS-2 may present a ratiometric platform for measuring changes in hydrolases concentration with high accuracy. Additionally, live-cell fluorescence imaging with PDS-2 can be further used for studying effect of periodate on live cells. It can be also potentially useful for exploring the roles of periodate in biological samples and iodine species on goiter and hypothyroidism as well as hyperthyroidism. Meanwhile, solid-state fluorescence property of PDS-2 makes it more convenient for monitoring periodate in water by using the PDS-2 based test papers. In conclusion, this PDS-2 based fluorescent technique may enable for a better understanding of periodate related biological and chemical processes. Also, it is an efficient tool for public health, food safety and environmental protection.
Acknowledgment We thank National Natural Science Foundation of China (Grants 21302125 and 21373138), Doctoral Fund of Ministry of Education of China (Grant no. 20133127120005) Program for Shanghai Sci. & Tech. Committee (Grants 13ZR1458800 and 12JC1407200), Program for Cultivation of Young Teacher of Shanghai University, The Innovative Program of Shanghai Normal University (Grant no. SK201331), Science and Technology Innovation Foundation for College Students (Grant no. 2012 þ10270þ 115) for the financial support.
Appendix A. supporting information Supporting information associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios. 2014.08.005.
References Afkhami, A., Zarei, A.R., 2001. Talanta 53, 815–821. Afkhami, A., Madrakian, T., Zarei, A.R., 2001. Anal. Sci. 17, 1199–1202. Amore, A., Wals, K., Koekoek, E., Hoppes, R., Toebes, M., Schumacher, T.N.M., Rodenko, B., Ovaa, H., 2013. ChemBioChem 14, 123–131. Badalassi, F., Wahler, D., Klein, G., Crotti, P., Reymond, J.-L., 2000. Angew. Chem. 112, 4233–4236. Benvidi, A., Heidari, F., Tabaraki, R., Mazloum-Ardakani, M., 2012. J. Anal. Chem 67, 661–668. Binder, C.M., Dixon, D.D., Almaraz, E., Tius, M.A., Singaram, B., 2008. Tetrahedron Lett. 49, 2764–2767. Chan, J., Dodani, S.C., Chang, C.J., 2012. Nat. Chem. 4, 973–984. Chatraei, F., Zare, H.R., 2013. Mater. Sci. Eng. B 33, 721–726. Cui, L., Zhu, W., Xu, Y., Qian, X., 2013. Anal. Chim. Acta 786, 139–145. Dunlap, N.K., Mergo, W., Jones, J.M., Carrick, J.D., 2002. Tetrahedron Lett. 43, 3923– 3925. Edmonds, J.S., Morita, M., 1998. Pure Appl. Chem. 70, 1567–1584. Ensafi, A.A., Bagherian Dehaghi, G., 2000. Anal. Sci. 16, 61–64. Ensafi, A.A., Chamjangali, M.A., 2003. Spectrochim. Acta A 59, 2897–2903. Evmiridis, N.P., 1989. Talanta 36, 357–362. Fatiadi, A.J., 1974. Synthesis 1974, 229–272. Goddard, J.-P., Reymond, J.-L., 2004. Trends Biotechnol. 22, 363–370. Greer, M.A., Grimm, Y., Studer, H., 1968. Endocrinology 83, 1193–1198. Guan, W., Zhou, W., Han, D., Zhang, M., Lu, C., Lin, J.-M., 2014. Talanta 120, 268–273. Hariri, S., Tam, M.C., Lee, D., Hileeto, D., Moayed, A.A., Bizheva, K., 2013. J. Biomed. Opt. 18, 026017. Hu, R., Feng, J., Hu, D., Wang, S., Li, S., Li, Y., Yang, G., 2010. Angew. Chem. Int. Ed. 49, 4915–4918. Kim, T.-I., Kang, H.J., Han, G., Chung, S.J., Kim, Y., 2009. Chem. Commun., 5895–5897. Larsen, P.R., Silva, J.E., Kaplan, M.M., 1981. Endocrine Rev. 2, 87–102. Li, H., Fan, J., Peng, X., 2013. Chem. Soc. Rev. 42, 7943–7962. Li, H., Niu, L., Xu, X., Zhang, S., Gao, F., 2011. J. Fluorescence 21, 1721–1728. Lin, J.-M., Yamada, M., 2001. Chromatography 22, 57–68. Lin, J.-M., Arakawa, H., Yamada, M., 1998. Anal. Chim. Acta 371, 171–176. Lin, J.-M., Sato, K., Yamada, M., 2001. Microchem. J. 69, 73–80. Lin, Z., Chen, H., Lin, J.-M., 2013. Analyst 138, 5182–5193. Murale, D.P., Kim, H., Choi, W.S., Churchill, D.G., 2013. Org. Lett. 15, 3946–3949. Ni, Y., Wang, Y., 2007. Microchem. J. 86, 216–226. Nyfeler, E., Grognux, J., Wahler, D., Reymond, J.-L., 2003. Helv. Chim. Acta 86, 2919– 2927. Outram, H.S., Raw, S.A., Taylor, R.J.K., 2002. Tetrahedron Lett. 43, 6185–6187. Salimi, A., Noorbakhash, A., Karonian, F.S., 2006. Int. J. Electrochem. Sci. 1, 435–445. Salimi, A., Noorbakhsh, A., Ghadermarzi, M., 2007. Sensor. Actuators B—Chem. 123, 530–537. Salimi, A., Kavosi, B., Babaei, A., Hallaj, R., 2008. Anal. Chim. Acta 618, 43–53. Shamsipur, M., Roushani, M., Mansouri, G., 2013. J. Chin. Chem. Soc. 60, 171–178. de Silva, A.P., Gunaratne, H.Q.N., Gunnlaugsson, T., Huxley, A.J.M., McCoy, C.P., Rademacher, J.T., Rice, T.E., 1997. Chem. Rev. 97, 1515–1566. Stennett, E.M.S., Ciuba, M.A., Levitus, M., 2014. Chem. Soc. Rev. 43, 1057–1075. Varma, A.J., Kulkarni, M.P., 2002. Polym. Degrad. Stabil. 77, 25–27. Vlessidis, A.G., Evmiridis, N.P., 2009. Anal. Chim. Acta 652, 85–127. von Gunten, U., 2003. Water Res. 37, 1469–1487. Wahler, D., Reymond, J.-L., 2001a. Curr. Opin. Biotechnol. 12, 535–544. Wahler, D., Reymond, J.-L., 2001b. Curr. Opin. Chem. Biol. 5, 152–158. Willkomm, D.K., Hartmann, R.K., 2008. In: Hartmann, R.K., Bindereif, A., Schön, A., Westhof, E. (Eds.), Handbook of RNA Biochemistry. Wiley-VCH Verlag GmbH, pp. 86–94. Wu, J., Chen, Q., Liu, W., Lin, J.-M., 2013. Lab Chip 13, 1948–1954. Wu, J., Liu, W., Ge, J., Zhang, H., Wang, P., 2011. Chem. Soc. Rev. 40, 3483–3495. Wu, J., Chen, Q., Liu, W., Zhang, Y., Lin, J.-M., 2012. Lab Chip 12, 3474–3480. Wu, J., Li, H., Chen, Q., Lin, X., Liu, W., Lin, J.-M., 2014. RSC Adv. 4, 24929–24934.
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An ESIPT based fluorescent probe for highly selective and ratiometric detection of periodate Chusen Huang n, Ti Jia, Congjun Yu, Amin Zhang, Nengqin Jia n The Education Ministry Key Laboratory of Resource Chemistry and Shanghai Key Laboratory of Rare Earth Functional Materials, Department of Chemistry, College of Life and Environmental Sciences, Shanghai Normal University, 100 Guilin Road, Shanghai 200234, China
art ic l e i nf o
a b s t r a c t
Article history: Received 16 May 2014 Received in revised form 23 July 2014 Accepted 4 August 2014 Available online 8 August 2014
Periodate is widely used in organic and bioorganic chemistry, and also related to food and environmental safety. To best of our knowledge, there is no efficient tools reported for simultaneously quantifying periodate with high accuracy and discriminating periodate from other forms of iodine. We have synthesized, characterized and applied a first ratiometric fluorescent probe (PDS-2) for simultaneous monitoring of changes of periodate based on the excited-state intramolecular proton transfer mechanism. This PDS-2 based fluorescent technique may enable for a better understanding of periodate related biological and chemical processes. Also, it is an efficient tool for public health, food safety and environmental protection. & 2014 Elsevier B.V. All rights reserved.
Keywords: Periodate Fluorescent probe Ratiometric detection Excited-state intramolecular proton transfer
1. Introduction Periodate is usually found as salts with potassium (e.g. KIO4) or sodium (e.g. NaIO4). In neutral solution, periodate can cleave carbon–carbon bonds when two carbon atoms bear hydroxyl groups (vicinal diols), creating two aldehydes and/or ketones groups (Fatiadi, 1974). This property is widely applied in organic synthesis (Binder et al., 2008; Outram et al., 2002) and commonly utilized in molecular biochemistry (Badalassi et al., 2000; Wahler and Reymond, 2001a,b), as well as its contribution to instrumental methods of micro-analysis (Lin et al., 1998, 2001, 2013; Vlessidis and Evmiridis, 2009). For example, sodium periodate was used to oxidize diols to afford aldehyde which was in situ trapped with stabilized ylides. This developed one-pot synthetic procedure provides access to a variety of useful natural products (Dunlap et al., 2002; Outram et al., 2002). In addition, sodium periodate was also used to oxidize cellulose and create a biocompatible and biodegradable compound that can be used as suture, as a scaffold for tissue engineering, or for drug delivery (Varma and Kulkarni, 2002). For a good understanding of the roles, sodium periodate is often introduced to open saccharide rings between vicinal diols leaving two reactive aldehyde groups, which can be easily modified by primary amine-containing molecules and surfaces in physiological conditions (Amore et al., 2013). This process was n
Corresponding authors. E-mail addresses: [email protected] (C. Huang), [email protected] (N. Jia).
http://dx.doi.org/10.1016/j.bios.2014.08.005 0956-5663/& 2014 Elsevier B.V. All rights reserved.
commonly used to label saccharides with fluorescent probe and other detectable tags such as biotin. Compared to DNA, 3ʹ-termini of RNA (ribose has vicinal diols) can be selectively labeled with detectable tags (Willkomm and Hartmann, 2008) through above process. Periodate oxidation based micro-analysis methods were also developed for detection of a variety of analytes including hydrogen peroxide (Lin et al., 1998, 2001), catechins (Lin and Yamada, 2001), ethylene glycol (Evmiridis, 1989) etc. The periodate–H2O2 chemiluminescence (CL) system was further used for detection of sodium dodecyl benzenesulfonate (Guan et al., 2014). Another significance of periodate was its application in enzyme assay. A periodate-coupled fluorogenic assay was developed for screening hydrolases in a high-throughput format (Badalassi et al., 2000; Goddard and Reymond, 2004; Nyfeler et al., 2003; Wahler and Reymond, 2001a,b). Some coumarin-based fluorescent substrates were synthesized, after the hydrolases hydrolyze the substrates to produce vicinal-diols-containing products, which were oxidized by periodate to release the fluorescent umbelliferone catalyzed by bovine serum albumin (BSA). Then the type and concentration of hydrolases could be recorded by fluorescence intensity changes of umbelliferone. Besides its special effect on organic and bioorganic chemistry, periodate is also related to food and environmental safety because of the conversion of periodate to some other iodine species that were considered as potentially hazardous contaminant (Hariri et al., 2013; Edmonds and Morita, 1998; von Gunten, 2003). Finally, some iodine species is also essential component for biomolecule such as thyroid hormones. Some literatures reported that the periodate and iodate were
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related to goiter and hypothyroidism as well as hyperthyroidism (Greer et al., 1968; Larsen et al., 1981). Hence, development of methods for simultaneously monitoring periodate as well as highly discriminating periodate from other forms of iodine is of considerable significance, not only for a good understand of the roles of periodate in biological and chemical processes, but also for public health, food safety and environmental protection. Several approaches for periodate detection were available, including electrochemical methods (Chatraei and Zare, 2013; Salimi et al., 2007; Shamsipur et al., 2013) and UV–vis spectrophotometry (Afkhami et al., 2001; Afkhami and Zarei, 2001; Benvidi et al., 2012; Ensafi and Chamjangali, 2003; Ni and Wang, 2007). To best of our knowledge, there is no efficient tool reported for discriminating periodate from other forms of iodine species and simultaneous monitoring of concentration changes of periodate with high accuracy. Only a few efforts come from fluorescence based techniques for quantifying periodate (Badalassi et al., 2000; Ensafi and Bagherian Dehaghi, 2000). Fluorescent probe based technology with the advantage of high selectivity and sensitivity, spatiotemporal resolution and visibility has been viewed as a powerful and versatile toolbox in the field of physiology and molecular biology, environmental monitoring and clinical diagnosis (Chan et al., 2012; de Silva et al., 1997; Li et al., 2013; Stennett et al., 2014). Thus, fluorescent probes based technique is good choice for simultaneous detection of periodate. However, recently few reported fluorescent probes (Badalassi et al., 2000; Ensafi and Bagherian Dehaghi, 2000) for detecting periodate were mainly on the fluorescence intensity changes, which might be interfered by fluorescence self-quenching, large background signal and some complex test systems. Such obstacles can be overcome by a ratiometric detection mechanism. Excited-state intramolecular proton transfer (ESIPT) is a significantly important phenomenon that is widely used for ratiometric detection in biological systems. Generally, enol–keto phototautomerism takes place in the excited state of ESIPT molecule which leads to initial normal form (NF*) and phototautomer form (PT*) (Li et al., 2011). Hence, dual emission signals could be observed (Fig. S1). And the intensity ratio of this two emission bands will make the ratiometric fluorescence detection possible. Many ESIPT based fluorescent probes have been constructed for ratiometric sensing of anion (Hu et al., 2010), cation (Cui et al., 2013), enzyme activity (Kim et al., 2009), and signaling molecule (Murale et al., 2013) etc. (Wu et al., 2011). Herein, we present a first ratiometric fluorescent probe for selective detection of periodate based on ESIPT mechanism.
Fluorescence spectra were recorded using a Hitachi fluorescence spectrophotometer (F-7000). Samples for absorption and fluorescence measurements were contained in 1 cm 1 cm quartz cuvettes (3.5 mL volume). All cell images were taken by a Leica TCS SP5 II Confocal Laser Scanning Microscope. 2.2. Synthesis 2.2.1. Synthesis of 2-(2-(2-(2.2-dimethyl-1.3-dioxolan-4-yl)ethoxy)3-methoxyphenyl)benzo[d]thiazole (PDS-1) To a solution of HMBT (405 mg, 1.576 mmol) and K2CO3 (432 mg, 3.15 mmol) in anhydrous DMF (10 mL), compound C (404 mmg, 1.578 mmol) was added. The reaction mixture was continued to stirring for about 12 h under dark. After TLC shows material HMBT disappeared, the reaction mixture was concentrated under reduced pressure. The obtained crude product was purified by chromatography on silica gel (petrol ether:EtOAc, from 30:1 to 10:1, v/v) to give a pale white solid (401 mg, 66% yield). 1H NMR (400 MHz, CDCl3) δ 8.11 (d, J ¼8.0 Hz, 1H), 8.07 (d, J ¼8.0 Hz, 2H), 7.95 (d, J¼ 8.0 Hz, 1H), 7.51 (t, J ¼8.0 Hz, 1H), 7.40 (t, J ¼8.0 Hz, 1H), 7.20 (t, J¼ 8.0 Hz, 1H), 7.03 (d, J¼ 8.0 Hz, 1H), 4.51–4.44 (m, 1H), 4.30–4.18 (m, 1H), 3.92 (s, 3H), 3.72 (t, J ¼7.6, 1H), 2.33–2.18 (m, 2H), 1.47 (s, 3H), 1.42 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 162.89, 152.98, 152.24, 146.49, 136.14, 127.43, 126.04, 124.99, 124.36, 123.01, 121.36, 120.92, 114.17, 108.65, 73.74, 70.18, 69.72, 55.96, 34.35, 27.01, 25.83; HRMS (ES þ ): calc. for C21H24NO4S [MþH] þ 386.1426, found 386.1423. 2.2.2. Synthesis of 4-(2-(benzo[d]thiazol-2-yl)-6-methoxyphenoxy) butane-1.2-diol (PDS-2) A solution of PDS-1 (385 mg, 1 mmol) in 80% aqueous acetic acid (5 mL) was stirred at room temperature for 12 h. TLC shows the material PDS-1 disappeared, then the reaction mixture was concentrated under reduced pressure. The obtained crude product was purified by chromatography on silica gel (petrol ether:EtOAc, from 2:1 to 1:1, v/v) to afford a colorless syrup which gradually became a pale white solid (306 mg, 90% yield). 1H NMR (400 MHz, CDCl3) δ 8.12 (d, J ¼8.4 Hz, 1H), 7.98 (d, J¼ 8.0 Hz, 1H), 7.93 (d, J¼ 8.0 Hz, 1H), 7.50 (t, J ¼7.6 Hz, 1H), 7.39 (t, J ¼7.6 Hz, 1H), 7.20 (t, J¼ 8.0 Hz, 1H), 7.01 (d, J ¼8.0 Hz, 1H), 4.28–4.19 (m, 3H), 3.91 (s, 3H), 3.81–3.78 (m, 1H), 3.69–3.64 (m, 1H), 2.07–2.02 (m, 2H); 13C NMR (100 MHz, CDCl3) δ163.04, 152.82, 152.33, 146.17, 135.87, 127.47, 126.19, 125.16, 124.66, 123.03, 121.41, 121.30, 114.12, 70.77, 70.14, 66.58, 56.03, 33.28; HRMS (ES þ ): calc. for C18H20NO4S [MþH] þ 346.1113, found 346.1111.
2. Experimental
3. Results and discussion
2.1. Chemicals and apparatus
3.1. Design strategy
All chemical reagents and solvents were purchased from commercial sources and used without further purification except for N,N-dimethylformamide (DMF) which was purified according to the handbook (see supporting information). Thin-layer chromatography (TLC) was performed on silica gel plates. Column chromatography was performed using silica gel (Hailang, Qingdao) 200–300 mesh. 1H and 13C NMR spectra were recorded employing a Bruker AV-400 spectrometer with chemical shifts expressed in parts per million (in deuteriochloroform, Me4Si as internal standard). Electrospray ionization (ESI) mass spectrometry was performed in a HP 1100 LC–MS spectrometer. Glass fiber membrane was purchased from Shanghai Jiening Biotechnology companies. All pH measurements were made with a pH-Meter PHB-4 (INESA Scientific Instrument Co., Ltd.). Absorption spectra were recorded using a Hitachi UV–visible spectrophotometer (U-3900).
Our investigation begins with designing an active probe for selective interaction with periodate. Since the well-known reaction mechanism that periodate can selectively breaks vicinal diols into aldehyde or ketone, a vicinal diols group was introduced as a specific receptor for periodate. Then an ESIPT fluorophore (2(benzo[d]thiazol-2-yl)-6-methoxyphenol (HMBT), Scheme S1) was taken as the reporter group to construct target probe (PDS-2, Scheme 1). With the advantage of ESIPT fluorophore which involves initial normal form (NF*) and phototautomer form (PT*), HMBT also exhibits dual maximum emission at 406 and 462 nm, respectively (Fig. S1). The intensity ratio of this two emission bands will make the ratiometric fluorescence detection possible. Many HMBT based fluorescent probes have been constructed for sensing anion (Hu et al., 2010), cation (Cui et al., 2013), enzyme activity (Kim et al., 2009), and signaling molecule (Murale et al.,
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Scheme 1. Design strategy of PDS-2 for selective detection of periodate (IO4-) in aqueous solution.
2013) etc. (Wu et al., 2011). In specific, through modification of hydroxyl group of HMBT with butane-1.2.4-triol to block ESIPT and exclusively results in short-wavelength. After target fluorescent probe was selectively oxidized by periodate, HMBT will be released from the formed oxidative product via a fast β-elimination catalyzed by bovine serum albumin (BSA, Scheme 1). This detection mechanism was explored by liquid chromatography–mass spectrometry (LC–MS) analysis (Fig. S2). An absorption peak at about 9.306 min can be attributed to PDS-2 according to the mass result, then this peak disappeared after PDS-2 was treated with sodium periodate. Meanwhile, a new absorption peak at about 12.793 min appeared, which can be attributed to product HMBT according to the mass results. The results were similar as reported literature (Badalassi et al., 2000). This sensing system will ensure a selective and quantitative detection of periodate through the fluorescence intensity ratio related to enol and keto forms of HMBT. In order to demonstrate this design, a control probe (PDS1) with the vicinal diols protected by acetone was also synthesized (Scheme S1). 3.2. Fluorescence sensing behavior of PDS-2 toward periodate Next, we investigated the fluorescence sensing behavior of PDS-2 toward periodate. Initially, pH titration was conducted in
water. As shown in Fig. S3, no difference in fluorescence of PDS-2 was observed over the pH range of 6–10. Thus, a PBS buffer (pH 7.4, containing 1% DMSO) was chosen as the test medium. Then a catalytic amount of BSA (2 mg/ml) and 1 mM CTAB were also added to present the final test system. As displayed in the absorption spectrum (Fig. S4), after 250 μM of periodate was added into PBS buffer containing 5 μM of PDS-2, maximum absorption at 308 nm decreased readily with a subsequent increase in absorption at 350 nm. An isosbestic point appears at 330 nm. Accordingly, the fluorescence emission at 406 nm decreased with concomitant ingrowth of fluorescence emission at 462 nm under the excitation wavelength at 339 nm (Fig. 1A, Fig. S5). Meanwhile, fluorescence changes of the test solution is also visible (from blue to green, Fig. 1A, inset) with UV lamp irradiation (365 nm). The fluorescence intensity ratio between maximum emission at 406 and 462 nm was also recorded in Fig. 1B. We can observe that the intensity ratio changed quickly within 15 min and then reached a plateau. This result demonstrates that the reaction between periodate and PDS-2 was nearly completed within 15 min. In the following assay, 20 min was taken for the detection time. Control probe PDS-1 (5 μM) was also applied for detection of periodate. As shown in Fig. S6, negligible changes in the fluorescence signal were observed in the presence of 250 μM of periodate
Fig. 1. (A) Time-dependent changes in fluorescence emission of PDS-2 (5 μM) with addition of IO4- (250 μM) in PBS buffer (pH 7.4, containing 1% DMSO, 2 mg/ml BSA, 1 mM CTAB). (B) A plot of the fluorescence intensity changes (based on the peak heights at the maxima) depending on time. λex ¼ 339 nm. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
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Fig. 2. (A) Changes in fluorescence emission (or (B) emission at 406 and 462 nm respectively) of PDS-2 (5 μM) with increasing addition of IO4- (from 0 to 300 μM) in PBS buffer (pH 7.4, containing 1% DMSO, 2 mg/ml BSA, 1 mM CTAB) after 20 min. (C) A plot of fluorescence intensity ratio changes (based on the peak heights at the maxima, 406 and 462 nm respectively) depending on IO4- concentration. Error bar represents s.d. (D) Linear relationship between fluorescence intensity ratio changes and concentration of periodate over the range from 30 to 100 μM in PBS buffer (R2 ¼ 0.99433). λex ¼ 339 nm. Error bar represents s.d.
even the reaction time extended to 20 min. Thus, the vicinal diols group of the probe was needed for its selective interaction with periodate. 3.3. Quantitative detection of periodate with PDS-2 Then, in order to test the sensitivity of PDS-2 for periodate, titration experiments were carried out. The changes in fluorescence emission were recorded upon gradual addition of sodium periodate (from 0 to 300 μM). As suggested in Fig. 2A and B, the fluorescence intensity at 406 nm decreased accompanying the increase in
emission wavelength at 462 nm. After the amount of periodate increased to 150 μM, there were no significant fluorescence changes. Hence, about 150 μM of periodate was needed for completing the reaction in the presence of 5 μM of PDS-2 within 20 min. In addition, the fluorescence ratio of emission at 462 nm and 406 nm (F462/F406) also increased with increasing addition of periodate (Fig. 2C). From the concentration-dependent fluorescence ratio intensity changes, the detection limit of PDS-2 for periodate was about 26 μM (see Fig. S7 about detailed information). Through a nonlinear fitting, F462/F406 and concentration of periodate X0) X)P was fitted to a formula (Y¼A1 þ(A2 A1)/(1þ10(lg( ), Fig. S8).
Fig. 3. (A) Changes in fluorescence emission of PDS-2 (5 μM) with addition of IO4- (250 μM) and other interfering reagents (250 μM) after 20 min in PBS buffer (pH 7.4, containing 1% DMSO, 2 mg/ml BSA, 1 mM CTAB). (B) Fluorescence intensity ratio changes (based on the peak heights at the maxima, 406 and 462 nm respectively) with addition of IO4- (250 μM) and other interfering reagents (250 μM) after 20 min. λex ¼ 339 nm. Error bar represents s.d. Inset: fluorescence color changes with addition of IO4(250 μM) and other interfering reagents (250 μM) in PBS after 20 min buffer under a hand-held UV lamp irradiation (365 nm). 1: probe only, 2: sodium hypochlorite (ClO-), 3: hydrogen peroxide(H2O2), 4: potassium nitrate, 5: sodium oxalate, 6: sodium periodate, 7: sodium borate, 8: sodium citrate, 9: sodium fluoride, 10: sodium iodide, and 11: sodium iodate.
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Then after a mathematic manipulation by original software, a linear relationship between changes of F462/F406 and the concentration of periodate changes from 30 μM to 100 μM (Fig. 2D) was obtained (please see Fig. S8 about the detailed manipulation and discussion in the supporting information). Thus, this linear curve makes quantitative detection of periodate very convenient over the 30– 110 μM concentration range. The reported electrochemical methods for detection of periodate have made a good progress in a lower detection limit and wider linear range (for example, the detection limit from electrochemical methods for periodate is 0.4 μM) (Salimi et al., 2008, 2006). However, PDS-2 can be applied in physiological condition without lower-pH solution (pH 2.0 for electrochemical methods) present, which may enable for a live-cell imaging of periodate in living cells. Meanwhile, the ratiometric detection with PDS-2 can be used for simultaneously quantifying periodate with high accuracy. 3.4. Selectivity of PDS-2 for periodate To test other analytes could interfere with this detection, fluorescence response of PDS-2 for other analytes were also studied. As demonstrated in Fig. 3A, there was no significant fluorescence changes in the presence of sodium hypochlorite, hydrogen peroxide (H2O2), potassium nitrate, sodium oxalate, sodium borate, sodium citrate, sodium fluoride, sodium iodide and sodium iodate. In contrast, a red shift of about 56 nm can be observed after addition of periodate. This result also can be recorded by changes of fluorescence ratio intensity (Fig. 3B). Meanwhile, the fluorescence changes were visible through naked eyes under a UV lamp irradiation (365 nm). Only periodate promotes blue fluorescence change to green fluorescence, whereas other analytes have no interference (Fig. 3B, inset). All these results demonstrated that PDS-2 was a highly selective fluorescent probe for periodate. Specifically, PDS-2 can be used for discriminating different periodate from other forms of iodine species such as iodate ion and iodate (Fig. S9, Fig. 3B).
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3.5. Cellular imaging experiment For further application in biological samples, low cell cytotoxicity of the probe is very important (Wu et al., 2013, 2012, 2014), especially for live cell imaging. Herein, cell cytotoxicity of PDS-2 was studied by using a standard methyl thiazolyl tetrazolium (MTT) assay according to above reported literatures (Wu et al., 2013, 2012, 2014). Over the concentration range between 0 and 500 μM, PDS-2 and control probe (PDS-1) exhibited no cell cytotoxicity (Fig. S10A) when the incubation time was 6 h. After the incubation time extends to 24 h, PDS-2 has no remarkable influence on cell viability at the concentration of 5 μM (Fig. S10B). Thus 5 μM of PDS-2 was taken in the following assay. Then we performed ratiometric fluorescence imaging of periodate in live HeLa cells. 5 μM of PDS-2 was loaded into live HeLa cells and incubating for 20 min. After washed with PBS for three times, the fluorescence images were taken with confocal microscopy. A strong blue emission (410–440 nm) but no green fluorescence signal (450–480 nm) was observed from the intracellular zone. When the cells were firstly treated with PDS-2, then further incubated with periodate (100 μM), the intensity of blue fluorescence signal decreased and a strong green fluorescence was observed (Fig. 4). Furthermore, control experiments with PDS-1 were also conducted in live cells. As shown in Fig. S11, PDS-1stained cells loaded with 100 μM of periodate only give rise to blue fluorescence signal. These studies revealed that PDS-2 can be applicable in ratiometric detection of periodate in biological samples and a useful tool for further investigation of the effect of periodate on biological samples. It can be also potentially useful for exploring the effect of iodine species on goiter and hypothyroidism as well as hyperthyroidism. As HMBT was a typical ESIPT fluorophore that exhibits solidstate fluorescence, a test paper of PDS-2 was prepared for detection of periodate. The glass fiber membrane was cut into strips (1 cm in width), and then they were immersed in 50 μM of PDS-2 in PBS solution (pH 7.4, containing 1% DMSO, 2 mg/ml BSA, 1 mM CTAB) for 60 min to absorb PDS-2, and then dried at 37 °C overnight to afford the test papers. After the test papers were
Fig 4. Fluorescence images of periodate with PDS-2 in live cells. (A) Fluorescence of cells treated with PDS-2 (5 μM) for 20 min. (B) Fluorescence of cells treated with PDS-2 (5 μM), then washed with PBS (pH 7.4, containing 1% DMSO, 2 mg/ml BSA) for three times and followed by addition of sodium periodate (100 μM) and incubating for another 20 min. The experiment was conducted on Leica TCS SP5 II confocal laser scanning microscopy using HC PLAPO 63 oil objective (NA: 1.40), with excitation by UV laser (405 nm). 410–440 nm (blue channel) and 450–480 nm (green channel) emission light was collected, respectively. Scale bar: 100 μm. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
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treated with solutions containing different reagents, respectively, we can see that only sodium periodate induced an orange fluorescence under a hand-held UV lamp irradiation (365 nm), while other reagents make no fluorescence changes on the test papers (Fig. S12). Because solid samples were easier for transport, this test paper based technique is convenient for detection through the visible solid-state fluorescence changes and may be used in monitoring periodate in waters and organic reaction liquid.
4. Conclusions We have designed and synthesized a highly selective and ratiometric fluorescent probe (PDS-2) for simultaneous detection of periodate. By using this probe, we have developed a ratiometric approach based on ESIPT mechanism for monitoring changes of periodate in aqueous solution for the first time. Through difference in the fluorescence ratio of emission intensity at 406 and 462 nm, periodate can be discriminated from other forms of iodine species and quantified. Furthermore, this ratiometric method may be further developed for screening hydrolases which can produce products with vicinal diols in high-throughput format. Compared to fluorescence intensity based platform for screening hydrolases (Badalassi et al., 2000), PDS-2 may present a ratiometric platform for measuring changes in hydrolases concentration with high accuracy. Additionally, live-cell fluorescence imaging with PDS-2 can be further used for studying effect of periodate on live cells. It can be also potentially useful for exploring the roles of periodate in biological samples and iodine species on goiter and hypothyroidism as well as hyperthyroidism. Meanwhile, solid-state fluorescence property of PDS-2 makes it more convenient for monitoring periodate in water by using the PDS-2 based test papers. In conclusion, this PDS-2 based fluorescent technique may enable for a better understanding of periodate related biological and chemical processes. Also, it is an efficient tool for public health, food safety and environmental protection.
Acknowledgment We thank National Natural Science Foundation of China (Grants 21302125 and 21373138), Doctoral Fund of Ministry of Education of China (Grant no. 20133127120005) Program for Shanghai Sci. & Tech. Committee (Grants 13ZR1458800 and 12JC1407200), Program for Cultivation of Young Teacher of Shanghai University, The Innovative Program of Shanghai Normal University (Grant no. SK201331), Science and Technology Innovation Foundation for College Students (Grant no. 2012 þ10270þ 115) for the financial support.
Appendix A. supporting information Supporting information associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios. 2014.08.005.
References Afkhami, A., Zarei, A.R., 2001. Talanta 53, 815–821. Afkhami, A., Madrakian, T., Zarei, A.R., 2001. Anal. Sci. 17, 1199–1202. Amore, A., Wals, K., Koekoek, E., Hoppes, R., Toebes, M., Schumacher, T.N.M., Rodenko, B., Ovaa, H., 2013. ChemBioChem 14, 123–131. Badalassi, F., Wahler, D., Klein, G., Crotti, P., Reymond, J.-L., 2000. Angew. Chem. 112, 4233–4236. Benvidi, A., Heidari, F., Tabaraki, R., Mazloum-Ardakani, M., 2012. J. Anal. Chem 67, 661–668. Binder, C.M., Dixon, D.D., Almaraz, E., Tius, M.A., Singaram, B., 2008. Tetrahedron Lett. 49, 2764–2767. Chan, J., Dodani, S.C., Chang, C.J., 2012. Nat. Chem. 4, 973–984. Chatraei, F., Zare, H.R., 2013. Mater. Sci. Eng. B 33, 721–726. Cui, L., Zhu, W., Xu, Y., Qian, X., 2013. Anal. Chim. Acta 786, 139–145. Dunlap, N.K., Mergo, W., Jones, J.M., Carrick, J.D., 2002. Tetrahedron Lett. 43, 3923– 3925. Edmonds, J.S., Morita, M., 1998. Pure Appl. Chem. 70, 1567–1584. Ensafi, A.A., Bagherian Dehaghi, G., 2000. Anal. Sci. 16, 61–64. Ensafi, A.A., Chamjangali, M.A., 2003. Spectrochim. Acta A 59, 2897–2903. Evmiridis, N.P., 1989. Talanta 36, 357–362. Fatiadi, A.J., 1974. Synthesis 1974, 229–272. Goddard, J.-P., Reymond, J.-L., 2004. Trends Biotechnol. 22, 363–370. Greer, M.A., Grimm, Y., Studer, H., 1968. Endocrinology 83, 1193–1198. Guan, W., Zhou, W., Han, D., Zhang, M., Lu, C., Lin, J.-M., 2014. Talanta 120, 268–273. Hariri, S., Tam, M.C., Lee, D., Hileeto, D., Moayed, A.A., Bizheva, K., 2013. J. Biomed. Opt. 18, 026017. Hu, R., Feng, J., Hu, D., Wang, S., Li, S., Li, Y., Yang, G., 2010. Angew. Chem. Int. Ed. 49, 4915–4918. Kim, T.-I., Kang, H.J., Han, G., Chung, S.J., Kim, Y., 2009. Chem. Commun., 5895–5897. Larsen, P.R., Silva, J.E., Kaplan, M.M., 1981. Endocrine Rev. 2, 87–102. Li, H., Fan, J., Peng, X., 2013. Chem. Soc. Rev. 42, 7943–7962. Li, H., Niu, L., Xu, X., Zhang, S., Gao, F., 2011. J. Fluorescence 21, 1721–1728. Lin, J.-M., Yamada, M., 2001. Chromatography 22, 57–68. Lin, J.-M., Arakawa, H., Yamada, M., 1998. Anal. Chim. Acta 371, 171–176. Lin, J.-M., Sato, K., Yamada, M., 2001. Microchem. J. 69, 73–80. Lin, Z., Chen, H., Lin, J.-M., 2013. Analyst 138, 5182–5193. Murale, D.P., Kim, H., Choi, W.S., Churchill, D.G., 2013. Org. Lett. 15, 3946–3949. Ni, Y., Wang, Y., 2007. Microchem. J. 86, 216–226. Nyfeler, E., Grognux, J., Wahler, D., Reymond, J.-L., 2003. Helv. Chim. Acta 86, 2919– 2927. Outram, H.S., Raw, S.A., Taylor, R.J.K., 2002. Tetrahedron Lett. 43, 6185–6187. Salimi, A., Noorbakhash, A., Karonian, F.S., 2006. Int. J. Electrochem. Sci. 1, 435–445. Salimi, A., Noorbakhsh, A., Ghadermarzi, M., 2007. Sensor. Actuators B—Chem. 123, 530–537. Salimi, A., Kavosi, B., Babaei, A., Hallaj, R., 2008. Anal. Chim. Acta 618, 43–53. Shamsipur, M., Roushani, M., Mansouri, G., 2013. J. Chin. Chem. Soc. 60, 171–178. de Silva, A.P., Gunaratne, H.Q.N., Gunnlaugsson, T., Huxley, A.J.M., McCoy, C.P., Rademacher, J.T., Rice, T.E., 1997. Chem. Rev. 97, 1515–1566. Stennett, E.M.S., Ciuba, M.A., Levitus, M., 2014. Chem. Soc. Rev. 43, 1057–1075. Varma, A.J., Kulkarni, M.P., 2002. Polym. Degrad. Stabil. 77, 25–27. Vlessidis, A.G., Evmiridis, N.P., 2009. Anal. Chim. Acta 652, 85–127. von Gunten, U., 2003. Water Res. 37, 1469–1487. Wahler, D., Reymond, J.-L., 2001a. Curr. Opin. Biotechnol. 12, 535–544. Wahler, D., Reymond, J.-L., 2001b. Curr. Opin. Chem. Biol. 5, 152–158. Willkomm, D.K., Hartmann, R.K., 2008. In: Hartmann, R.K., Bindereif, A., Schön, A., Westhof, E. (Eds.), Handbook of RNA Biochemistry. Wiley-VCH Verlag GmbH, pp. 86–94. Wu, J., Chen, Q., Liu, W., Lin, J.-M., 2013. Lab Chip 13, 1948–1954. Wu, J., Liu, W., Ge, J., Zhang, H., Wang, P., 2011. Chem. Soc. Rev. 40, 3483–3495. Wu, J., Chen, Q., Liu, W., Zhang, Y., Lin, J.-M., 2012. Lab Chip 12, 3474–3480. Wu, J., Li, H., Chen, Q., Lin, X., Liu, W., Lin, J.-M., 2014. RSC Adv. 4, 24929–24934.
