Analytica Chimica Acta 812 (2014) 145–151

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A ratiometric fluorescent probe for iron(III) and its application for detection of iron(III) in human blood serum Lingliang Long a,∗ , Liping Zhou a , Lin Wang a , Suci Meng a , Aihua Gong b , Chi Zhang a,∗ a Functional Molecular Materials Research Centre, Scientific Research Academy & School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang, Jiangsu 212013, PR China b School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang, Jiangsu 212013, PR China

h i g h l i g h t s

g r a p h i c a l

a b s t r a c t

• A fluorescence ratiometric probe for Fe3+ has been developed.

• The probe was developed based on the Fe3+ -mediated deprotection of acetal reaction. • Upon treatment with Fe3+ , fluorescence ratio (I522 /I390 ) of the probe displayed significant changes. • The probe can be used to monitor Fe3+ level in human blood serum.

a r t i c l e

i n f o

Article history: Received 2 September 2013 Received in revised form 9 December 2013 Accepted 17 December 2013 Available online 28 December 2013 Keywords: Fluorescent probes Fluorescence Dyes/pigments Ratiometric Iron

a b s t r a c t A simple and versatile ratiometric fluorescent Fe3+ detecting system, probe 1, was rationally developed based on the Fe3+ -mediated deprotection of acetal reaction. Notably, this reaction was firstly employed to design fluorescent Fe3+ probe. Upon treatment with Fe3+ , probe 1 showed ratiometric response, with the fluorescence spectra displaying significant red shift (up to 132 nm) and the emission ratio value (I522 /I390 ) exhibiting approximately 2362-fold enhancement. In addition, the probe is highly sensitive (with the detection limit of 0.12 ␮M) and highly selective to Fe3+ over other biologically relevant metal ions. The sensing reaction product of the probe with Fe3+ was confirmed by NMR spectra and mass spectrometry. TD-DFT calculation has demonstrated that the ratiometric response of probe 1 to Fe3+ is due to the regulation of intramolecular charge transfer (ICT) efficiency. Moreover, the practical utility in fluorescence detection of Fe3+ in human blood serum was also conducted, and probe 1 represents the first ratiometric fluorescent probe that can be used to monitor Fe3+ level in human blood serum. Finally, probe 1 was further employed in living cell imaging with pancreatic cancer cells, in which it displayed low cytotoxicity, satisfactory cell permeability, and selective ratiometric response to Fe3+ . © 2013 Elsevier B.V. All rights reserved.

1. Introduction Iron is a trace element that is vital for life. It serves as a cofactor for many proteins and enzymes, which are required for oxygen and energy metabolism, as well as for several other essential processes [1]. However, in human iron metabolism, iron uptake, trafficking,

∗ Corresponding authors. Tel.: +86 511 88797815; fax: +86 511 88797815. E-mail addresses: [email protected] (L. Long), [email protected] (C. Zhang). 0003-2670/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aca.2013.12.024

export, and utilization are strictly regulated; the abnormal iron levels may result in serious adverse effects and diseases [2–4]. For example, iron deficiency is the most common cause of anemia in the world [5]. In addition, the iron deficiency may be the sign of a gastrointestinal malignancy [6]. Conversely, iron overload can induce severe cell damage and organ dysfunction by irregular production of reactive oxygen species (ROS) [7]. Recent research suggests that high levels of iron within the body have been associated with several serious diseases, such as cancer [8], hepatitis [9], and neurodegenerative diseases including Alzheimer’s disease and Parkinson’s disease [10]. Consequently, the iron plays essential

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roles in both healthy and diseased states of the living system. And thus, the monitoring of iron levels in the human body is critically important. In blood serum, the iron is bound to the iron transport protein, transferrin, in ferric (Fe3+ ) form. The transport of iron throughout the body by transferrin is central to iron homeostasis [2–4]. The content of serum iron is considered to be an important index for iron status (deficiency or overload) [11–13]. Thus, a number of methods, including colorimetry [14,15], spectrophotometry [16], resonance ionization isotope dilution mass spectrometry [17], atomic absorption spectrometry [18], and inductively coupled plasma mass spectrometry (ICP-MS) [19], have been devoted to detection of Fe3+ in blood serum. Compared with these methods, the fluorescence sensing is most attractive, because it offers apparent advantages in terms of high sensitivity, fast response time, and technical simplicity [20]. So far, numerous fluorescent probes have been developed for detection of Fe3+ [21–44], but unfortunately, only limited examples, such as calcein [45,46], Fl-DFO [47,48], and NBD-DFO [49], have been utilized to detection of Fe3+ in blood serum. Furthermore, these probes respond to Fe3+ in blood serum with changes only in fluorescent intensity. A major limitation of intensity-based probes is that the fluorescent signals are prone to be disturbed in quantitative detection by factors of excitation intensity, emission collection efficiency, probe distribution and the environment conditions [50]. By contrast, ratiometric fluorescent probes can eliminate most or all ambiguities by employing the ratio of two emissions at different wavelengths [51]. As far as we know, ratiometric fluorescent probes for Fe3+ are rare [40–44], and none of them has been exploited in detection of Fe3+ in blood serum probably due to the low selectivity or sensitivity. Herein, we rationally developed a simple and versatile ratiometric fluorescent probe for Fe3+ , and the probe have been successfully applied for detection of Fe3+ levels in human blood serum. 2. Experimental 2.1. Reagents Unless otherwise stated, all reagents were purchased from commercial suppliers and used without further purification. Solvents were purified and dried by standard methods prior to use. Twice-distilled water was used throughout all experiments. The solutions of various cations were prepared from NaCl, KCl, CaCl2 , MgCl2 ·6H2 O, Ba(NO3 )2 , Zn(NO3 )2 ·6H2 O, CdCl2 ·2.5H2 O, CuCl2 ·2H2 O, HgCl2 , CoCl2 ·6H2 O, MnSO4 ·H2 O, FeCl2 ·4H2 O, NiCl2 ·6H2 O, CuCl, AgNO3 , Pb(NO3 )2 , and Fe(NO3 )3 ·9H2 O. Thin layer chromatography (TLC) analyses were performed on silica gel plates and column chromatography was conducted over silica gel (mesh 200–300), both of which were obtained from the Qingdao Ocean Chemicals. 2.2. Apparatus Melting points of compounds were measured on a Beijing Taike XT-4 microscopy melting point apparatus, and all melting points were uncorrected. Mass spectra were recorded on a LXQ Spectrometer (Thermo Scientific) operating on electrospray ionization (ESI). 1 H and 13 C nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance 400 spectrometer operating at 400 MHz and 100 MHz, respectively. Elemental (C, H, N) analysis was carried out using Flash EA 1112 analyzer. Electronic absorption spectra were obtained on a SHIMADZU UV-2450 spectrometer. Fluorescence spectra were measured on a Photon Technology International (PTI) Quantamaster fluorometer with 2 nm excitation and emission slit widths. Cells imaging were performed with an

inverted fluorescence microscope (Carl Zeiss, Axio Observer A1). All pH measurements were performed with a pH-3c digital pHmeter (Shanghai ShengCi Device Works, Shanghai, China) with a combined glass–calomel electrode. 2.3. Synthetic procedure 2.3.1. Synthesis of 4-(1H-phenanthro[9,10-d]imidazol-2-yl)benzaldehyde (2) The compound 2 was synthesized according to a reported procedure [52]. A solution of 9,10-phenanthroquinone (277 mg, 1.33 mmol), terephthalaldehyde (536 mg, 4 mmol) and ammonium acetate (2 g, 25.9 mmol) in acetic acid (10 mL) was heated at 100 ◦ C for 50 min. The hot solution was cooled to room temperature, and the resulting precipitate was collected by vacuum filtration in a Buchner funnel (with a medium speed qualitative filter paper). Then, the precipitate was washed with acetate acid, dilute sodium hydrogen carbonate solution, and water. After dried under vacuum, the precipitate was further purified by column chromatography on silica gel (eluent: acetone) to afford the probe 1 as yellow solid (327 mg, 76.2%). Mp: >300 ◦ C. 1 H NMR (DMSO-d6 , 400 MHz), ı (ppm): 7.67 (m, 2H), 7.77 (m, 2H), 8.14 (d, J = 8.4 Hz, 2H), 8.53 (d, J = 8.4 Hz, 2H), 8.57 (d, J = 7.6 Hz, 1H), 8.63 (d, J = 7.6 Hz, 1H), 8.86 (d, J = 8.4 Hz, 1H), 8.90 (d, J = 8.4 Hz, 1H), 10.10 (s, 1H). MS (ESI) m/z 323.1 [M+H]+ . 2.3.2. Synthesis of probe 1 A mixture of compound 2 (50 mg, 0.155 mmol), trimethyl orthoformate (0.5 mL), absolute methanol (10 mL), and NH4 Cl (8 mg, 0.149 mmol) was heated to 80 ◦ C, and the mixture was refluxed overnight under N2 atmosphere. After the reaction mixture cooled to room temperature, the resulting precipitate was collected by vacuum filtration in a Buchner funnel (with a medium speed qualitative filter paper). The crude product was further purified by column chromatography on neutral alumina (petroleum ether: acetone = 12:1, v/v) to afford compound 1 as white solid (47.5 mg, 83.1%). Mp: 202–206 ◦ C. 1 H NMR (DMSO-d6 , 400 MHz), ı (ppm): 3.31 (s, 6H), 5.50 (s, 1H), 7.62 (m, 4H), 7.74 (m, 2H), 8.34 (d, J = 8.4 Hz, 2H), 8.59 (m, 2H), 8.87 (m, 2H). 13 C NMR (DMSO-d6 , 100 MHz), ı (ppm): 53.11, 102.85, 122.47, 124.22, 124.58, 125.86, 126.40, 127.60, 128.19, 130.86, 137.50, 139.56, 149.25. MS (ESI) m/z 369.2 [M+H]+ . Elemental analysis calcd (%) for C24 H20 N2 O2 : C 78.24, H 5.47, N 7.60; found C 78.12, H 5.49, N 7.58. 2.4. Measurement procedures A stock solution of probe 1 was prepared at 2.5 × 10−4 M in acetone, and a stock solution of various cations (1 × 10−2 M) was prepared by dissolving an appropriate amount of cations in H2 O. The test solution of the probe 1 (5 ␮M) in 20 mM potassium phosphate buffer/acetone (pH 7.0, 1:4 (v/v)) was prepared by placing 0.1 mL of the probe 1 stock solution, 3.9 mL acetone, and an appropriate aliquot of each cation stock into a 5 mL volumetric flask, and then diluting the solution to 5 mL with 20 mM potassium phosphate buffer (pH = 7.0). The resulting solution was shaken well and incubated at room temperature for 30 min before recording the fluorescence and UV–vis absorption spectra. 2.5. Detection of the Fe3+ in human blood serum For determination of Fe3+ in human blood serum, the serum was firstly treated with trichloroacetic acid (TCA) to release Fe3+ from protein according to a reported procedure [53]. 4 mL of 20% TCA was added to 4 mL serum, and then the mixture was stirred and heated to 90 ◦ C for 15 min. After cooling, the mixture was sonicated for 2 min. The protein precipitate was removed by centrifugation

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at 10,000 rpm for 10 min. Upon adjusted the pH value to 7.0, the supernatant was used for the Fe3+ assay. Aliquots of the above deproteinized serum sample (0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, or 1 mL) were added to 4 mL probe 1 solution (6.25 ␮M, in acetone) in a 5 mL volumetric flask, and then diluting the solution to 5 mL with 20 mM potassium phosphate buffer (pH = 7.0). After the resulting solution incubated at room temperature for 30 min, the emission ratios at 522 and 390 nm (I522 /I390 ) (ex = 360 nm) were recorded. The unknown amount of Fe3+ in the serum sample was estimated by using the standard addition method with Fe(NO3 )3 (5 × 10−3 M stock) as the standard. Different volumes of the Fe3+ stock (0, 1, 2, 3, 4 or 5 ␮L) was added directly to a mixture of 4 mL probe 1 solution (6.25 ␮M, in acetone) and 0.5 mL deproteinized serum in a 5 mL volumetric flask, and then diluting the mixture to 5 mL with 20 mM potassium phosphate buffer (pH = 7.0). The resulting solution was shaken well. After 30 min, the emission ratios at 522 and 390 nm (I522 /I390 ) (ex = 360 nm) were recorded. 2.6. Cell culture and fluorescence imaging Pancreatic cancer cells were seeded in a 24-well plate in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum for 24 h. Before the experiments, the cells were washed with phosphate buffered saline (PBS) buffer, and then the cells were incubated with Fe(NO3 )3 (0 or 50 ␮M) in the culture medium for 30 min at 37 ◦ C. After washing with PBS three times to remove the remaining Fe3+ ions, the cells were further incubated with probe 1 (1 ␮M) for 30 min at 37 ◦ C. Subsequently, the fluorescence images were acquired with a fluorescent microscope (Carl Zeiss, Axio Observer A1). Excitation wavelength of laser was 365 nm, and emissions were centered at 445 ± 10 nm and 530 ± 10 nm. 3. Results and discussion 3.1. Design of the ratiometric probe for Fe3+ In the past few years, the fluorescent Fe3+ probes were often designed by carrying a fluorophore linked to a particular Fe3+ binding moiety. However, because of the paramagnetic nature of Fe3+ , most of the probes undergo fluorescence quenching [21–30]. Although some probes with fluorescence turn on signal have also been developed [31–39], the design of ratiometric fluorescent probe for Fe3+ based on this approach remains a challenging task. Recently, analyte specifically promoted reactions have been widely employed to devise fluorescent probes [54]. Therefore, we

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turn our attention to design the ratiometric probe based on a Fe3+ promoted specific reaction. In organic synthesis, aldehyde groups are frequently protected as acetals, which can be rapidly deprotected by Fe3+ at room temperature (Fig. 1a) [55]. We reasoned that this deprotection reaction can be used to design ratiometric fluorescent Fe3+ probe. It is worth noting that this reaction has not been previously employed in fluorescent Fe3+ probe design. Thus, we rationally developed compound 1 as the novel ratiometric fluorescent probe for Fe3+ (Fig. 1b). Compound 1 is composed of a 1H-phenanthro[9,10-d]imidazole dye and an acetal group. The choice of 1H-phenanthro[9,10-d]imidazole is based on the consideration that it can function both as fluorescence dye and electron donor in an intramolecular charge transfer (ICT) based system. In addition, upon conjugation with an electron acceptor group, the emission of this dye exhibits pronounced red shift due to the effective ICT process [52]. We envisioned that the acetal group in compound 1 could be deprotected into aldehyde by Fe3+ , and thus compound 1 is converted to 2. Due to the difference in ␲-conjugation, 1 and 2 will exhibit distinct emission profiles. In compound 1, the 1H-phenanthro[9,10-d]imidazole dye is deconjugated with the acetal group, thus the ICT between the two moieties should be turned off, and the compound 1 mainly displays the locally excited (LE) emission. Whereas in compound 2, the 1H-phenanthro[9,10-d]imidazole dye is essentially conjugated with the aldehyde group. Therefore, upon excitation, effective ICT from the 1H-phenanthro[9,10-d]imidazole to aldehyde should proceed, which results in red shift ICT emission. Consequently, upon treated with Fe3+ , compound 1 will show a substantial ratiometric response.

3.2. Synthesis and optical properties Probe 1 was readily prepared in two steps (Scheme 1). Treatment of 9,10-phenanthroquinone with terephthalaldehyde afforded the intermediate compound 2, which was then reacted with trimethyl orthoformate to give compound 1. The products were well characterized by 1 H NMR, 13 C NMR, and ESI-MS spectroscopy. Indeed, as designed, the probe 1 exhibits strong locally excited emission (˚F = 0.636) at 390 nm, whereas compound 2 displays obviously red shift emission (˚F = 0.559) at 522 nm (Fig. S1). The red shift emission is apparently due to the effective ICT process from 1H-phenanthro[9,10-d]imidazole to aldehyde. Thus, it is clear that probe 1 is promising as a ratiometric fluorescent probe for Fe3+ provided that probe 1 could be converted by Fe3+ to compound 2 under appropriate condition.

Fig. 1. (a) Protection of aldehydes as acetals and deprotection by Fe3+ ; (b) design concept of fluorescent ratiometric probe 1 for Fe3+ .

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Scheme 1. The synthetic route to probe 1.

3.3. Optical response of probe 1 to Fe3+ The fluorescence response of probe 1 to Fe3+ was investigated in 20 mM potassium phosphate buffer/acetone (pH 7.0, 1:4 (v/v)) at room temperature. Notably, upon addition of Fe3+ , the emission of 1 at 390 nm gradually decreased and a new emission at 522 nm which quite resembles the emission of compound 2 appeared (Fig. 2a). This implied that the probe 1 was converted to compound 2, in good agreement with the aforementioned design. Importantly, the fluorescence spectra of the probe 1 showed significant red shift (up to 132 nm) before and after addition of Fe3+ . Such a large red shift makes the two emissions resolve very well, which not only enables the accurate measurement of the two emission intensities, but also induces a large ratiometric value changes. In fact, when 30 equivalents of Fe3+ were introduced, an approximately 2362fold enhancement in emission ratio value (I522 /I390 ) is achieved. Moreover, the emission ratio (I522 /I390 ) was linearly proportional to the concentration of Fe3+ in the range of 0–30 ␮M (Fig. S2). The

Fig. 2. Changes in emission spectra (ex = 360 nm) (a) and absorption spectra (b) of probe 1 (5 ␮M) with various amount of Fe3+ (0–150 ␮M). Inset in (a): the emission ratio (I522 /I390 ) of probe 1 to various concentrations of Fe3+ .

probe also showed high sensitivity toward Fe3+ with a detection limit of 0.12 ␮M (S N−1 = 3), which is much lower than the typical concentration of Fe3+ in human blood serum (5–39 ␮M) in normal individuals [56]. In line with red shift of emission spectra, the absorption spectra of probe 1 also showed red shift upon addition of Fe3+ . Probe 1 itself exhibited absorption centered at 362 nm (Fig. 2b), which is ascribed to the ␲–␲* transition of the 1H-phenanthro[9,10d]imidazole dye [57]. However, upon introduced increasing Fe3+ , a new broad band at 373 nm appeared and increased. The red-shift broad band was similar to absorption of compound 2 (Fig. S3) and apparently assigned to the ICT transition. The ratiometric responses of 1 toward other biologically relevant metal ions were explored. Upon introducing 150 ␮M Na+ , K+ , Ca2+ , Mg2+ , Ba2+ , Zn2+ , Cd2+ , Cu2+ , Hg2+ , Co2+ , Mn2+ , Fe2+ , Ni2+ , Cu+ , Ag+ , and Pb2+ , no significant change was observed in the emission ratio (I522 /I390 ) of 1 (Fig. 3). The only prominent response appeared when Fe3+ was added. Furthermore, the visual fluorescence response of probe 1 to various metal ions (Fig. 3, inset) demonstrates that the probe can be used conveniently for Fe3+ detection by simple visual inspection. We further examined the ratiometric response of the probe toward Fe3+ in the presence of other metal ions. Most of other metal ions only displayed minimum

Fig. 3. Ratiometric fluorescence response of probe 1 (5 ␮M) to 150 ␮M of various metal ions. (1) The organic aqueous solution, (2) Na+ , (3) K+ , (4) Ca2+ , (5) Mg2+ , (6) Ba2+ , (7) Zn2+ , (8) Cd2+ , (9) Cu2+ , (10) Hg2+ , (11) Co2+ , (12) Mn2+ , (13) Fe2+ , (14) Ni2+ , (15) Cu+ , (16) Ag+ , (17) Pb2+ , and (18) Fe3+ . Excitation wavelength was 360 nm. Inset: visual fluorescence color changes of probe 1 (5 ␮M) in the presence of various metal ions (150 ␮M), the photo was taken under illumination of a handheld UV lamp. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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interference (Fig. S4). Despite some metal ions such as Ce3+ , In3+ and Er3+ can also be used to deprotection of acetal in mild condition [58–60], however, the content of these ions in living organism is rare. Thus, probe 1 could selectively respond to Fe3+ in the presence of other biologically relevant metal ions. The free probe 1 is stable in the assay conditions (Fig. S5). The time course of 1 toward Fe3+ (150 ␮M) was displayed in Fig. S6. Upon addition of Fe3+ , a dramatic enhancement in the emission ratios was noted, and the ratios essentially reached the maximum in 30 min. In addition, the reaction of probe 1 (5 ␮M) with 1 ␮M Fe3+ was also investigated by fluorescence emission spectra. As shown in Fig. S7, probe 1 could be completely converted to compound 2 within 38 h. The effect of pH value on the ratiometric response of 1 to Fe3+ was also investigated. In the absence of Fe3+ , 1 showed little change in fluorescence spectra (Fig. S8) and emission ratios (Fig. S9) in the pH range of 3.99–9.94. When Fe3+ was added to 1 at various pH values, marked emission ratio enhancement was observed at pH 4.0–8.0 (Fig. S9). These indicated that probe 1 can be employed to detect Fe3+ under the neutral pH condition.

3.4. Reaction products of probe 1 with Fe3+ To further confirm that the ratiometric response is due to the conversion of probe 1 to compound 2, the reaction product of the probe 1 with Fe3+ was isolated and subjected to standard characterization. The 1 H NMR spectrum of the isolated product is essentially identical with that of the standard compound 2 (Fig. 4), confirming the formation of compound 2. This is further verified by mass spectrometry analysis (Fig. S10). Together with evidence of emission spectra and excitation spectra (Fig. S11), we can conclude that the ratiometric response of the probe to Fe3+ is indeed due to the probe 1 was transformed to compound 2.

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Fig. 4. Partial 1 H NMR (400 MHz) spectra in DMSO-d6 of (a) probe 1, (b) the isolated product of probe 1 + Fe3+ , and (c) the standard compound 2.

3.5. Theoretical calculation To get insight into the optical response of probe 1 to Fe3+ , probe 1 and compound 2 were examined by density function theory (DFT) calculations at the B3LYP/6-31+G** level using Gaussian 09 program. The optimized geometries of 1 and 2 are shown in Fig. S12. The probe 1 has almost 90◦ tilted conformation between the 1H-phenanthro[9,10-d]imidazole and acetal group, indicating that ␲-conjugation between the two moieties is interrupted. In contrast, compound 2 adopts a nearly planar conformation, which makes the 1H-phenanthro[9,10-d]imidazole and aldehyde in good ␲-conjugation, consistent with our design. The TD-DFT calculation indicated that the S0 → S1 (HOMO → LUMO) electronic transitions with oscillator strength f = 0.6616 and 0.6601 are identified as the allowable transitions of the probe 1 and compound 2, respectively (Table S1). Therefore, the HOMO–LUMO transitions contribute to the fluorescence for probe 1 and compound 2. The electron distributions in HOMO and LUMO of 1 and 2 are shown in Fig. 5, the

Fig. 5. The HOMO and LUMO energy levels and the orbitals of probe 1 and compound 2.

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quantitative measurement, a standard addition method with Fe(NO3 )3 as the standard was employed to estimate the unknown concentration of Fe3+ in the commercial human blood serum (Fig. S13). The concentration of Fe3+ in the serum was found to be 21.1 ± 0.8 ␮M. The result is in good agreement with the value (23.6 ± 0.4 ␮M) obtained for our sample by employing a reported method [14]. These values are well within the range (5–39 ␮M) of reported Fe3+ concentrations from normal human blood serum [56]. 3.7. Fluorescence imaging of live cells

Fig. 6. Relationship between the emission ratios (I522 /I390 ) of probe 1 (5 ␮M) and the volume of deproteinized serum added.

electron densities in HOMO for both 1 and 2 reside mainly on the 1H-phenanthro[9,10-d]imidazole and phenyl moieties. However, 1 and 2 differ at electron distributions in LUMO. The LUMO of 1 is still located on 1H-phenanthro[9,10-d]imidazole and phenyl moieties, whereas the LUMO of 2 is distributed over the phenyl and aldehyde moieties. Thus, upon excitation, the ICT process will take place from 1H-phenanthro[9,10-d]imidazole to the aldehyde in compound 2. Moreover, the energy gap between the HOMO and LUMO of 2 was smaller than that of 1, in good agreement with the red shift of absorption and emission spectra of compound 2. 3.6. Application of probe 1 to monitoring Fe3+ level in human blood serum

Because of the highly sensitive, selective, and ratiometric fluorescent responses for Fe3+ , probe 1 could be considered as a promising tool for imaging Fe3+ in living cells. Thus, the bioimaging application of probe 1 for Fe3+ in pancreatic cancer cells was investigated. Firstly, to evaluate the cytotoxicity of probe 1, we performed 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays using pancreatic cancer cells at 1 and 5 ␮M of probe 1 for 24 h, respectively. The results clearly showed that probe 1 was nontoxic to the cultured cells under experimental conditions (Fig. S14). Then staining the pancreatic cancer cells with probe 1 (1 ␮M) for 30 min gave intense intracellular fluorescence in the blue channel (Fig. 7b), and almost no fluorescence was observed in green channel (Fig. 7c). These indicated that the probe was cell membrane permeable. When the cells were supplemented with 50 ␮M Fe(NO3 )3 in the growth medium for 30 min and then stained with 1 under the same conditions, giving intense green fluorescence (Fig. 7g) but essentially no blue fluorescence (Fig. 7f). These results establish that the probe is capable of imaging Fe3+ in living cells. 4. Conclusion

With these desirable properties of probe 1 taken into account, it was further employed to detect Fe3+ in human blood serum. The blood serum was firstly treated with trichloroacetic acid to release the Fe3+ from protein according to a reported procedure [53]. And then, aliquots of the deproteinized serum sample were added to a solution of probe 1. As exhibited in Fig. 6, the increase in the amount of deproteinized serum sample elicited a linear enhancement in the emission ratios (I522 /I390 ). These studies revealed that the probe is capable of ratiometric sensing Fe3+ in the serum sample. For

In summary, a novel ratiometric fluorescent probe 1 for Fe3+ has been rationally constructed via the Fe3+ -mediated deprotection of acetal reaction. It is worth noting that this reaction was firstly used for fluorescent Fe3+ probe design. The favorable features of the probe include high sensitivity and selectivity, large emission shift, large emission ratio variation. Importantly, we have demonstrated that the new probe is capable of monitoring Fe3+ level in human blood serum. Thus, probe 1 represents the first ratiometric

Fig. 7. Fluorescence and brightfield images of pancreatic cancer cells. (a) Brightfield image of the cells stained with probe 1 (1 ␮M) for 30 min; (b) fluorescence image of (a) with emission at 445 ± 10 nm; (c) fluorescence image of (a) with emission at 530 ± 10 nm; (d) an overlay image of (a)–(c); (e) brightfield image of cells pretreated with Fe3+ (50 ␮M) for 30 min and then further incubated with probe 1 (1 ␮M) for 30 min; (f) fluorescence image of (e) with emission at 445 ± 10 nm; (g) fluorescence image of (e) with emission at 530 ± 10 nm; (h) an overlay image of (e)–(g). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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fluorescent Fe3+ probe that suitable for detection of Fe3+ level in blood serum. In addition, the probe has been successfully applied for imaging Fe3+ in living cells. We expect that probe 1 will be a useful analytical tool for assessing Fe3+ levels in biological system. Moreover, the Fe3+ -mediated deprotection of acetal reaction will be widely applicable for development of fluorescent Fe3+ probes. Acknowledgments This research was supported by the National Natural Science Foundation of China (21202063, 50925207, 51172100, 21103073), the Ministry of Science and Technology of China (2009DFA50620, 2011DFG52970), the Ministry of Education of China (IRT1064), the Natural Science Foundation of Jiangsu Province (BK2012281), the China Postdoctoral Science Foundation (2012M511200), Jiangsu Innovation Research Team, and the Research Foundation of Jiangsu University (11JDG078). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2013.12.024. References [1] R. Crichton, Inorganic Biochemistry of Iron Metabolism, 2nd ed., Wiley, Chichester, 2001. [2] T. Rouault, Nat. Chem. Biol. 2 (2006) 406–414. [3] I.D. Domenico, D.M. Ward, J. Kaplan, Nat. Rev. Mol. Cell Biol. 9 (2008) 72–81. [4] R.R. Crichton, R.J. Ward, Biochemistry 31 (1992) 11255–11264. [5] S. DuBois, D.J. Kearney, Am. J. Gastroenterol. 100 (2005) 453–459. [6] D.C. Rockey, J.P. Cello, N. Engl. J. Med. 329 (1993) 1691–1695. [7] B. Halliwell, J.M.C. Gutteridge, FEBS Lett. 307 (1992) 108–112. [8] S. Toyokuni, Cancer Sci. 100 (2009) 9–16. [9] K.V. Kowdley, Gastroenterology 127 (2004) S79–S86. [10] R.R. Crichton, D.T. Dexter, R.J. Ward, Coord. Chem. Rev. 252 (2008) 1189–1199. [11] B. Benyamin, M.A.R. Ferreira, G. Willemsen, S. Gordon, R.P.S. Middelberg, B.P. McEvoy, J. Hottenga, A.K. Henders, M.J. Campbell, L. Wallace, I.H. Frazer, A.C. Heath, E.J.C.D. Geus, D.R. Nyholt, P.M. Visscher, B.W. Penninx, D.I. Boomsma, N.G. Martin, G.W. Montgomery, J.B. Whitfield, Nat. Genet. 41 (2009) 1173–1175. [12] K. Punnonen, K. Irjala, A. Rajamäki, Blood 89 (1997) 1052–1057. [13] B. de Valk, M.A. Addicks, I. Gosriwatana, S. Lu, R.C. Hider, J.J.M. Marx, Eur. J. Clin. Invest. 30 (2000) 248–251. [14] F. Jones, Anal. Chem. 21 (1949) 1216–1217. [15] M. Poljak-Blazi, M. Jaganjac, M. Mustapic, N. Pivac, D. Muck-Seler, Immunobiology 214 (2009) 121–128. [16] A. Jafarian-Dehkordi, L. Saghaie, N. Movahedi, DARU J. Pharm. Sci. 16 (2008) 76–82. [17] J.D. Fassett, L.J. Powell, J. Moore, Anal. Chem. 56 (1984) 2228–2233. [18] H. Tanaka, Z. Li, K. Ikuta, L. Addo, H. Akutsu, M. Nakamura, K. Sasaki, T. Ohtake, M. Fujiya, Y. Torimoto, J. Glass, Y. Kohgo, Cancer Sci. 103 (2012) 767–774. [19] M.E.D.C. Busto, M. Montes-Bayón, J. Bettmer, A. Sanz-Medel, Analyst 133 (2008) 379–384. [20] X. Chen, T. Pradhan, F. Wang, J.S. Kim, J. Yoon, Chem. Rev. 112 (2012) 1910–1956.

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A ratiometric fluorescent probe for iron(III) and its application for detection of iron(III) in human blood serum.

A simple and versatile ratiometric fluorescent Fe(3+) detecting system, probe 1, was rationally developed based on the Fe(3+)-mediated deprotection of...
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