Highly Sensitive Ratiometric Fluorescent Detection of Indium(III) Using

Aug 28, 2018 - Job's plot analysis indicated that 1 preferred to form a 2:1 complex with In(III) and the binding affinity for In(III) was measured to ...
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Highly Sensitive Ratiometric Fluorescent Detection of Indium(III) Using Fluorescent Probe Based on Phosphoserine as a Receptor Pramod Kumar Mehta, Gi Won Hwang, Joohee Park, and Keun-Hyeung Lee Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01440 • Publication Date (Web): 28 Aug 2018 Downloaded from http://pubs.acs.org on August 28, 2018

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Analytical Chemistry

Highly Sensitive Ratiometric Fluorescent Detection of Indium(III) Using Fluorescent Probe Based on Phosphoserine as a Receptor Pramod Kumar Mehta, Gi Won Hwang, Joohee Park, and Keun-Hyeung Lee* Center for Design and Applications of Molecular Catalysts, Department of Chemistry and Chemical Engineering, Inha University, Incheon, 402-751, South Korea *E-mail: [email protected]

ABSTRACT: Indium is one of the most widely used scarce metals for manufacturing various electronic devices including notebooks, mobile phones, and PC monitors. Recent studies revealed that indium and its compound could cause several toxicities to human beings and animals. However, there is no report about ratiometric fluorescent detection of In(III) in aqueous solutions. We synthesized a fluorescent probe (1) for In(III) based on a phosphoserine as a receptor with a pyrene fluorophore using solid phase synthesis. 1 showed highly sensitive ratiometric response to In(III) in purely aqueous solutions by increasing excimer emission intensity at 476 nm with a concomitant decrease in monomer emission intensity at 395 nm. 1 showed sensitive ratiometric responses to In(III) over a wide range of pH (2 < pH 8), In(III) precipitated as a solid form of In(OH)3.36,37 Acid leaching was one of most critical process for producing indium(III) from ores as well as secondary materials including the ITO. Furthermore, turn-on response was not ideal because the enhanced emission intensity by analytes could be affected by environmental effects, such as pH, polarity of the media, and temperature. Thus, ratiometric fluorescent probes using two different emission bands were highly recommended in real applications because the ratiometric probes could overcome the limitations of turn on probes by a built-in correction for environmental effects.19,20 In the present study, we reported a fluorescent probe (1) based on phosphoserine as a receptor bearing a pyrene fluorophore for ratiometric detection of In(III) in aqueous solution. Phosphoserine in the protein was reported to interact with trivalent ions and phosphoserine was stable in acidic conditions.38,39 Thus, we chose a phosphoserine as a receptor for the fluorescent probe for In(III). To our delight, 1 exhibited a remarkable sensitive ratiometric response to In(III) in purely aqueous buffered solution. Interestingly, the probe exhibited a selective ratiometric response to In(III) in tested 18 metal ions including Al(III), Ga(III), Zn(II), and Pb(II) in aqueous buffered solution at acidic pHs. To the best of the authors’ knowledge, this is the first example of a fluorescent probe that detects In(III) in aqueous solutions by a ratiometric response.

Scheme 1. Structure of 1 and the binding mode of 1 with In(III) at acidic pHs

EXPERIMENTAL SECTION Reagents. Rink Amide MBHA resin (100–200 mesh, 0.43 mmol/g) and 1-hydroxybenzotriazole (HOBt) were purchased from BeadTech. Fmoc-Ser(PO(OBzl)OH)-OH was purchased from Novabiochem. 2-(1H-Benzotriazole-1yl)-1,1,3,3-tetramethyluronium tetrafluoro-borate (TBTU),

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N,N’-diisopropylcarbodiimide (DIC), anisole and thioanisole were acquired from TCI. N,N-dimethylformamide (DMF), trifluoroacetic acid (TFA), and triisopropylsilane (TIS) were supplied by Acros Organics. The other reagents for the synthesis such as 1-pyreneacetic acid, diethyl ether, N,N-Diisopropylethylamine (DIPEA), 1,2-Ethanedithiol, 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU), and piperidine were purchased from Sigma Aldrich. Solid-Phase Synthesis and Charcterization of 1. 1 was synthesized using solid phase synthesis with 9-fluorenyl methoxycarbonyl (Fmoc) chemistry (Scheme S1). After the coupling of the phosphoserine and 1-pyreneacetic acid was complete in solid phase synthesis, cleavage and deprotection were carried out by adding a cleavage mixture of trifluoroacetic acid/ thioanisole/ 1,2-ethanedithiol/anisole (90:5:3:2, v/v/v/v) at room temperature for 4 hours. Excess trifluoroacetic acid was removed by nitrogen purging and then the precipitated product was obtained by addition of diethyl ether. The precipitate was centrifuged, washed with diethyl ether, and lyophilized using a freeze dryer. The product was purified further by HPLC using a water (0.1% TFA)/acetonitrile (0.1% TFA) gradient. The homogeneity (97 %) of 1 was confirmed by analytical HPLC on a C18 column. 1 was characterized by ESI-Mass spectrometry, 1H NMR, and 13C NMR. 1: White solid, Yield: 60%; 1H NMR (400 MHz, DMSOd6) δ: 8.56 (1H, d, J = 8.4 Hz), 8.40 (1H, d, J = 8.2 Hz), 8.27 (1H, d, J = 8.1 Hz), 8.24-8.06 (7H, m), 7.50 (1H, s), 7.25 (1H, s), 4.49-4.45 (1H, m) 4.37-4.27 (2H, m) 4.05 (2H, s); 13C NMR (400 MHz, DMSO-d6) δ: 170.75, 170.29, 130.88, 130.80,130.36, 129.70, 129.07, 128.71, 127.38, 127.22, 126.79, 126.13, 125.01, 124.87, 124.70, 124.16, 124.04, 123.90, 65.14, 53.09, 38.88; ESI–MS (m/z): [M – H+]- calculated for C21H18N2O6P: 425.0902, observed: 425.0915. General Procedure for UV/Vis, Fluorescent, and Circular Dichroism Spectrophotometric Experiments. The probe was dissolved in CH3CN/H2O (1:1, v/v) to prepare a stock solution at 500 µM. The stock solution was diluted to prepare aqueous buffered solution containing 1% CH3CN and the concentration of the probe was measured by the absorbance at 342 nm for the pyrene fluorophore of the probe. All spectroscopic measurements except CD were carried out in aqueous buffered solutions containing 1% acetonitrile. The UV/Vis absorption spectra of 1 were measured in a 1 cm path length cuvette using a UV/Vis spectrophotometer (Perkin-Elmer, model Lambda 45). The fluorescence emission spectra of 1 were measured in a 1 cm path length cuvette using a fluorescence spectrophotometer (Perkin Elmer, model LS 55) with excitation at 342 nm. The circular dichroism (Jasco, model J-815) spectra of 1 were measured in a 1 cm path length cuvette. All CD spectra were accumulated three times at a scan rate of 50 nm/min. Aqueous buffered solutions at pH ranging from 1.5 to 3.5 were prepared using 10 mM acetate buffering agent (pKa = 4.76) Aqueous buffered solutions at pH ranging from 4.0 to 6.0 were prepared using 10 mM Hexamine

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buffering agent (pKa = 4.89) Aqueous buffered solutions at pH ranging from 6.5 to 7.4 were prepared using 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffering agent (pKa = 7.58). Aqueous buffered solutions at pH ranging from 8.5 to 11.5 were prepared using 10 mM CHES (N-cyclohexyl-2-aminoethanesulfonic acid) buffering agent (pKa = 9.3). Ground water and tap water was diluted 20 times with aqueous buffered solution to prepare sample solutions (10 mM, acetate, pH 3.5) containing 1 % CH3CN. Determination of the Association Constant of 1 for In(III). The association constant for 2:1 complex was determined by a nonlinear least squares fit of the data with the following equation as referenced elsewhere.40 y=

୶ ଶୟୠ(ଵି୶)మ

+

ୠ୶ ଶ

(1)

Where x is I-Io/Imax-Io, y is the concentration of In(III), a is the association constant, and b is the concentration of 1. Determination of Detection Limit of 1 for In(III). To determine the S/N ratio, the fluorescence emission intensity ratio (I476/I395) of 1 (10 µM) in aqueous solutions was measured 10 times, and the standard deviation of the probe without In(III) was measured. The mean intensity ratio of three separate measurements was plotted as a function of the In(III) concentration to determine the slope. The detection limit was calculated using the following equation: Detection limit = 3σ/m

(2)

where σ is the standard deviation of the intensity ratio of 1 in the absence of In(III), and m is the slope of the emission intensity ratio (I476/I395) of 1 as a function of the In(III) concentration.41 Titration of In(III) using EDTA and EBT. Eriochrome black T (EBT) was used as a colorimetric indicator for the titration of In(III) according to the previously reported procedure.15 EBT solution (10.8 mM), EDTA solution (10.0 mM), and Zn(II) solution (1.0 mM) was prepared in de-ionized distilled water, respectively. Excess amount of EDTA was added into the sample solution containing In(III) and then the solution was boiled for 10 min. After the solution was cooled at room temperature, the pH was adjusted to 10 using ammonia solutions as buffering agent and then few drops of EBT as an indicator was added. The uncomplexed EDTA of the solution was titrated using Zn(II) standard solution and the concentration of In(III) in sample solutions was measured by the volume of added Zn(II) standard solution for the titration. Determination of In(III) using 4-(2-pyridylazo) resorcinol. 0.01% 4-(2-pyridylazo) resorcinol (PAR) standard solution were prepared in de-ionized distilled water containing 1% methanol. When In(III) in a given sample solution was added to PAR solution, a pink color complex was

formed at pH 6 (10 mM acetate buffer). The concentration of In(III) was calculated based on the absorbance of In(III)PAR complex at 510 nm using Lambert-Beer's law .16-18

RESULTS AND DISCUSSION Design and Synthesis of Fluorescent Probe (1). In the present study, we searched for the new possible ligands for In(III) and chose a phosphoserine as a key ligand for the fluorescent probe for In(III) because phosphoserine in the protein was reported to interact with trivalent ions and phosphoserine was stable in acidic conditions.38,39 To synthesize a ratiometric fluorescent probe, a pyrene was used as a fluorophore for ratiometric detection of In(III) due to its good photophysical properties, such as a high resistance to photo-bleaching, long-lived fluorescence, insensitivity for pH, and formation of excimer emissions via π− π interactions.42,43 The fluorescent probe (1, PyreneP(Ser)-NH2) was easily synthesized in solid phase synthesis with a high yield (60%). Details on the synthesis and characterization of 1 are described in experimental section. The high purity of 1 (97%) were confirmed by HPLC with ESI mass spectrometer (Figure S1~S4). Fluorescent and UV/Vis Response of 1 to In(III). All photochemical experiments were performed in aqueous buffered solution containing 1 % (v/v) CH3CN. The detection of In(III) was carried out in aqueous solutions at acidic pH because In(III) dissolved well in aqueous solution at acidic pHs and the complex, In(OH)3 was formed at high pHs and precipitated due to the relative insolubility.36,37 Considering the high solubility of In(III) in acidic pH (~3) and the acid leaching methods for producing indium(III) from ores and the recycling process, we investigated the fluorescence response of 1 to In(III) in aqueous buffered solutions at acidic pH (10 mM acetate, pH 3.5). In the absence of the metal ions, significant typical monomer emission bands at 378 and 395 nm were observed, as shown in Fig 1. Upon addition of In(III), a significant increase of the excimer emission at 476 nm and a concomitant decrease of the monomer emissions at 395 nm with an isoemissive point at 443 nm were observed. The emission intensity ratio (I476/I395) at 476 and 395 nm increased significantly as the concentration of In(III) increased. Approximately, 2 equivalent of In(III) was sufficient for the complete of the emission intensity change. The enhancement of the excimer emission at 476 nm indicates that two probes interacted with In(III) and the pyrene fluorophores might dimerize in the presence of In(III).42 1 exhibited a significant ratiometric response to In(III) by the intensity ratio change in aqueous buffered solutions. Interestingly, 1 showed “OFF-On” response to In(III) by the excimer emission change at 480 nm. The fluorescent probe showed a highly sensitive response to In(III) in aqueous buffered solution because approximately, 20 µM (2.0 equiv) of In(III) was required for the complete change of the emission intensity. We measured the quantum yield of 1 in the absence and presence of In(III), respectively. The quantum yield of 1 was 0.177 in the absence of In(III),

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whereas the quantum yield of 1 was 0.241 in the presence of In(III).

Figure 1. (a) Fluorescence emission spectra (λex = 342 nm) and (b) visible emission color images of 1 (10 µM) under UV light (λex = 365 nm) in the presence of In(III) in aqueous buffered solution (10 mM acetate, pH 3.5) containing 1% CH3CN.

We investigated response times of 1 for the detection of In(III). Upon addition of In(III), the enhanced emission intensity at 476 nm was measured as a function of time (Figure S5). Upon addition of relatively high amount (7.5 ~30 µM) of In(III), the emission intensity increased rapidly and changed completely within 5 min. However, upon addition of relatively low concentration (0~5 µM) of In(III), the emission intensity increased and changed completely within 10 min. As the concentration of In(III) decreased, the response time increased. However, the spectrofluorometer was not equipped with a stirrer. Thus, when we mixed the solution vigorously by pipet after addition of the low concentrations of In(III), the emission intensity changed completely within less than 5 mins. Thus, the response time of 1 for the detection of In(III) was quite fast in purely aqueous solution. Fig. 1b shows the visible emission color images of 1 in the presence of In(III) under UV light (365 nm) with a UV lamp. 1 exhibited the purple color in the absence of In(III). As the concentration of In(III) increased, the purple color was changed to light blue and cyan depending on the concentration of In(III). Pyrene was frequently used as a fluorophore for the fluorescent probes because of its good photophysical properties such as high photochemical stability and high resistance to photo bleaching.39,40 Thus, we measured the photochemical stability of 1 in the presence and absence of In(III) under continuous UV light irradiation (Figure S6). 1 exhibited an almost constantly enhanced emission intensity ratio (I476/I395) in the presence of In(III) for 2 hrs under UV light irradiation, which supports that 1 had a high photochemical stability.

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Selectivity Study and pH Effect on the Fluorescence Response. To evaluate the selective detection of In(III), the emission intensity ratio (I476/I395) of 1 were measured in the presence of various metal ions such as Na(I), Mg(II), Al(III), K(I), Ca(II), Cr(III), Mn(II), Fe(II), Fe(III), Co(II), Ni(II), Cu(II), Zn(II), Ga(III), Pb(II), Ag(I), Cd(II), and Hg(II) in aqueous buffered solutions (10 mM acetate, pH 3.5). As shown in Figure 2, 1 showed a highly selective ratiometric response to In(III) among 18 metal ions including Al(III), Zn(II), and Pb(II). Fig 2b showed the emission intensity ratio as a function of pH in the presence of trivalent ions such as In(III), Ga(III), and Al(III), because these metal ions belonging to group 13 in the periodic table have the same charge and relatively similar chemical properties. At acidic and neutral pH, 1 showed significant ratiometric responses to In(III). Interestingly, 1 showed highly selective response to In(III) among Al(III), Ga(III), and In(III) at acidic pHs; as pH was lower than 5.0, 1 exhibited highly selective ratiometric responses to In(III) among the trivalent ions. As pH reached to 2, 1 did not show a response to In(III). Considering the pKa value (pKa1 = 1~2, pKa2 = 6~8) of phosphate group,44,45 the phosphoserine of 1 played a critical role in the binding with In(III). When the pH is increased over 8.0, 1 did not show responses to In(III), Ga(III), and Al(III), respectively. As the pH increased over 8, trivalent metal ions formed the hydroxyl complexes (In(OH)3, Al(OH)3, and Ga(OH)3) with OH− that had poor solubilities in aqueous solutions. The high selectivity of 1 for In(III) at acidic pHs could be explained by two factors. First, the phosphate group of 1 played a pivotal role for the binding with trivalent metal ions and the negative charge of the phosphate group decreased as pH decreased below 7. Thus, the binding affinity for trivalent metal ions might decrease as pH decreased. However, highly charged trivalent metal ions were tightly hydrated in aqueous solution. Thus, when the probe recognized the metal ions, the probe competed with water molecules. As pH decreased below 7, the hydration for the metal ions might be weakened due to the protonation of oxygen of water molecules. As pH decreased below 7, the negative charge of the phosphate group decreased for the interaction with In(III) but the hydration for In(III) among trivalent metal ions was weakened mostly so the probe could have high selectivity for In(III) at acidic pHs. Considering the size of trivalent metal ions, the hydration for In(III) by water molecules was weakened mostly at acidic pHs. The standard hydration enthalpy of Al(III) and Ga(III) was 4659.7 kJ/mol and -4684.8 kJ/mol, whereas the standard hydration enthalpy of In(III) was -4108.7 kJ/mol. The hydration enthalpy of trivalent metal ions also suggests that the interactions between water and In(III) decreased mostly as pH decreased. Thus, even though the negative charge of the phosphate group of 1 decreased at acidic pHs, the probe showed a high selectivity for In(III) at acidic pHs.

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Figure 2. (a) Emission intensity ratio of 1 in the presence of metal ions in aqueous buffered solution (10 mM acetate, pH 3.5) containing 1% CH3CN, (b) emission intensity ratio of 1 with In(III), Ga(III), and Al(III), as a function of pH, and (c) visible emission color images of 1 with In(III) as a function of pH under UV irradiation with UV lamp. [Note: concentration of 1 = 10 µM and metal ion = 2 equiv.]

taminated metal ions in the producing process of In(III) from ores. However, the emission intensity ratio induced by In(III) was affected by Cr(III) and Fe(III), respectively due to the strong quenching effects and/or the absorbance effect of Fe(III).19,46-48 Thus, fluorescent titration experiments of 1 with In(III) were carried out in the presence of Cr(III) and Fe(III), respectively (Figure S7). First, 1 was incubated with Cr(III) in aqueous buffered solution. After addition of increasing concentrations of In(III) into the solution containing 1 and Cr(III), fluorescent spectrum was measured. Upon addition increasing concentration of In(III), the monomer emissions at 395 nm decreased significantly and the excimer emissions at 472 nm increased considerably. Approximately, 2 equiv. of In(III) was enough for the completion of the intensity ratio change (I476/I395). However, upon addition of In(III) in the presence of Fe(III), the monomer emission decreased significantly but the excimer emissions did not increase. Thus, we investigated fluorescence response of 1 to In(III) in presence of Fe(III) and 0.5 mM of ascorbic acid because ascorbic acid was reported to reduce Fe(III) to Fe(II).49 We carried out fluorescence titration experiment of 1 with In(III) in the presence of Fe(III) and 0.5 mM of ascorbic acid (Figure S7). In this case, 1 showed ratiometric responses to In(III) by the decrease of the monomer emission at 395 nm and concomitant increase of the excimer emission at 476 nm because Fe(III) was reduced to Fe(II). Approximately, 2 equiv. of In(III) was enough for the saturation of the intensity ratio change. Overall results indicate that even though the intensity ratio change induced by In(III) decreased in the presence of Cr(III) and Fe(III), 1 could detect In(III) by ratiometric response in the presence of Cr(III) and Fe(III), respectively.

1 1 + In(III) + Other metal ion

2.5

Interestingly, 1 exhibited ratiometric responses to In(III) over a wide range of pH (2 < pH