Dansyl-containing boronate hydrogel film as fluorescent chemosensor of copper ions in water

Abstract

A new type of boronate hydrogel with covalently bound dansyldiethylenetriamine as an indicator has been developed; the gel networks are based on boronate esterification of poly(vinylalcohol) with benzene-1,4-diboronic acid. In this approach, phenylboronic acid-appended N-dansyldiethylenetriamine 1 was newly synthesized to be incorporated into the gel matrix. The resulting gel film showed an absorption band at 336 nm and fluorescence at 511 nm when excited at 340 nm in water. The fluorescence measurements indicated that at neutral conditions using a HEPES buffer, the gel film was selectively quenched after immersion in an aqueous solution of Cu²⁺ for 30 min. The response had minimal interference from other metal ions such as Na⁺, K⁺, Mg²⁺, Ca²⁺, Fe³⁺, Co²⁺, Ni²⁺, Zn²⁺, Cd²⁺, Hg²⁺, Al³⁺, and Pb²⁺, which was noteworthy because dansyldiethylenetriamine alone responds to Hg²⁺ and Ni²⁺ in addition to Cu²⁺ in water. The reversible sensing capability was also evaluated by rinsing the film with an aqueous solution of ethylenediaminetetraacetic acid (EDTA). The gel was found to be a reusable and free-standing film capable of visually detecting Cu²⁺, providing a simple and expedient tool for on-site monitoring of Cu²⁺ in environmental applications such as water analysis.

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Introduction

Chemosensors capable of fluorometrically detecting transition metal ions in water¹ have been recognized as powerful sensing tools for applications in environmental and biological fields. Accordingly, numerous efforts have been devoted to the design and development of selective fluorescent sensors in the disciplinary areas of supramolecular chemistry and analytical chemistry.² Dansyl fluorophore (5-dimethylamino-1-naphthalenesulfonate) is one of the most useful candidates for designing the desired chemosensors, and it is characterized by a charge transfer excited state that exhibits solvatochromism and high emission quantum yields.³ The synthetic availability of the sulfonic acid group, together with its optical properties, led to the development of fluorescent sensors for the detection of not only cations^4–8 but also anions⁹ in water. In particular, Cu²⁺ is a significant metal pollutant due to its widespread use, such as in plating technology.¹⁰ The use of the dansyl moiety has been a feasible approach for highly sensitive Cu²⁺ sensing by monitoring changes in fluorescence.^5–8 However, a major drawback of dansyl-based systems is that they often suffer from cross-sensitivity toward other ions, particularly potential competitors containing Hg²⁺, due to their similar chemical behavior to Cu²⁺.^7,8 Further, solution-based sensors are inconvenient for not only real-time and real-space measurements but also repetitive use. In this context, solid surface-supported fluorescent sensors are of growing interest for Cu²⁺ detection. For instance, Cu²⁺-binding ligands and dansyl reporters were immobilized on silica nanoparticles.¹¹ Dansyl reporters have also been applicable to polymer-based¹² as well as film-based¹³ fluorescent chemosensors for Cu²⁺.

In an alternative approach, the use of boronate hydrogels¹⁴ has attracted our attention for film fabrication. Although it is well-known that the addition of borate as a cross-linking agent leads to the formation of a three-dimensional polyvinyl alcohol (PVA) network,^15,16 to the best of our knowledge, gel indicators based on the PVA–borate network have been as yet unknown, possibly because PVA–borate gels are dissolved in water (vide infra). We realized that the employment of suitable boronic acid-appended cross-linkers and fluorophores would allow the fabrication of PVA-based gel films that provide not only a preferable microenvironment for the detection of the analyte in water but also immobilize the working units. Subsequent systems have potential as portable devices that would be user-friendly detection tools. As a proof-of-concept study, we report here a new type of reusable gel film that is composed of a covalently attached dansyl indicator in a PVA–boronate matrix. Importantly, the film was highly selective toward copper ion-induced fluorescence response in water, with minimal interference from competitive metal ions such as Hg²⁺.

Results and discussion

Synthesis of boronic acid-appended N-dansyldiethylenetriamine

To fabricate fluorescent boronate gels, it is required that the synthetic connection of phenylboronic acid into the dansyl indicator skeleton does not interfere with the metal ion binding and optical properties. With this in mind, we decided to use N-dansyldiethylenetriamine 2^6,17 because of the easy connection of phenylboronic acid at the terminal amine moiety of 2; the reductive amination of 2 with MIDI (N-methyliminodiacetic acid)-protected formylphenylboronic acid in the presence of NaBH₄ afforded 1 after purification, in 18% yield. The assignment of the new compound 1 was carried out using several spectroscopic data (see ESI,† Fig. S1–4). The optical properties came from UV-vis and fluorescence measurements; 1 shows an absorption band at 329 nm (ε = 4.4 × 10³ M⁻¹ cm⁻¹) in 2% DMSO aqueous solution (5.0 mM HEPES, pH = 7.0), which is consistent with that of 2. This suggests that the incorporation of phenylboronic acid into 2 causes no perturbation toward dansyl fluorophore. Indeed, when excited at 340 nm in 2% DMSO aqueous solution (5.0 mM HEPES, pH = 7.0), an orange emission was observed with a fluorescence maximum at 545 nm and a quantum yield of 0.02 against a quinine sulphate (Φ = 0.546 in 0.5 M H₂SO₄ aqueous solution).¹⁸

Scheme 1 Synthesis of boronic acid-appended N-dansyldiethylenetriamine 1.

Formation of boronate–PVA gel

We initially explored suitable cross-linkers for stable boronate gels in water. It is known that PVA–borate systems form homogeneous gels.^15,16 Thus, when the phase diagram reported in the paper¹⁶ is considered, dansyl-containing PVA–borate gel was prepared from PVA (M.W. = 146 000–186 000, 6.4 × 10⁻¹ M¹⁹), borax (9.4 × 10⁻² M), and 1 (1.3 × 10⁻⁴ M) (see ESI,† Fig. S5). The resulting fluorescent gel was found to dissolve in water within 90 min. To prepare stable boronate gels in water, we studied how benzene-1,4-diboronic acid (DBA) with a hydrophobic character would behave as a cross-linking agent in the PVA matrix. For this preparation, DBA was initially allowed to react with PVA in DMSO to provide an organogel because of its low solubility in water. Fig. 1 shows the phase diagram of the boronate–PVA gel in DMSO. 1.5 mL samples were prepared by mixing various amounts of PVA and DBA. For a solution containing a low concentration of PVA (<0.1 M), the sol phase was characterized even at a relatively high concentration of DBA. In contrast, when the PVA concentration became adequate to ensure intermolecular cross-linking through boronate esterification with DBA, gelation occurred. After several trials to obtain suitable gels to this end, we employed the following conditions: PVA = 4.0 × 10⁻¹ M¹⁹ and DBA = 1.2 × 10⁻² M. Under these conditions, excess amounts of free hydroxyl groups of PVA remained to participate in boronate esterification with 1.

Fig. 1 Phase diagram of the PVA–DBA system in DMSO (1.5 mL) at room temperature; (●) sol, (○) gel.

The DMSO gel could be transferred to the corresponding hydrogel by immersing it in water (see experimental section). The obtained hydrogels were stable in water and their characterization was based on the FT-IR analysis. When the xerogel was measured by the KBr method, a characteristic intense peak assigned to the boronate ester formation between PVA and DBA was observed at 664 cm⁻¹, together with a peak corresponding to the B–O stretch at 1304 cm⁻¹ (Fig. 2).²⁰ This result confirmed that the gel formation was driven by benzene diboronate-diol cross-linking in PVA. The gel is stable in water and pH stability tests indicated that the gel never decomposes between pH 1 and 11 (see ESI,† Fig. S6).

Fig. 2 FT-IR spectrum of xerogel produced by PVA and DBA. A characteristic intense peak at 664 cm⁻¹ (*) was assignable to the boronate esters.

Fabrication of boronate gel films with dansyl moieties

Taking the phase diagram of the PVA–DBA system into account (Fig. 1), the target boronate gels with indicators were fabricated as follows. A DMSO solution (0.15 mL) of indicator 1 (6.7 × 10⁻⁴ M) and DBA (6.0 × 10⁻² M) at 25 °C was added to a DMSO solution (0.60 mL) of PVA (5.0 × 10⁻¹ M¹⁹). The resulting solution containing PVA (4.0 × 10⁻¹ M¹⁹), DBA (1.2× 10⁻² M), and 1 (1.3 × 10⁻⁴ M) was cast into a slab at room temperature to immediately afford DMSO gel films with a molar cross-linking ratio of DBA to the PVA monomer unit of 0.12. After aging for 24 h at room temperature, the film was again immersed in DMSO, and then in solvents of increasing water ratio to obtain the desired fluorescent hydrogel. The film was a swollen gel with dimensions of 30 × 10 × 1 mm (Fig. 3a).²¹ The content of the indicator that was immobilized in the PVA matrix was estimated via UV-vis measurement, and the absorbance of the gel film suggested that (3.8 ± 0.3) × 10⁻⁸ mol of the indicator was incorporated into the boronate gel. A plausible structure is shown in Fig. 3b. The morphological analysis of the xerogel was performed by field-emission scanning electron microscopy (FE-SEM), which suggested the formation of a fibrous network structure (Fig. 3c).

Fig. 3 (a) Photograph of the boronate hydrogel film with dansyl moieties; (b) proposed structure of the indicator hydrogel wherein OH-inserted sp³-hybridized tetrahedral boronate esters would be partly produced; (c) FE-SEM image of the xerogel.

Chemosensor capability of boronate hydrogel film

The boronate film exhibited fluorescence at 511 nm when excited at 340 nm in a HEPES aqueous solution; the emission maxima was blue shifted by 34 nm and the fluorescence quantum yield increased significantly (Φ = 0.37) in comparison with dye 1 in 2% DMSO aqueous solution (5.0 mM HEPES, pH = 7.0), possibly due to the hydrophobic microenvironment of the gel matrix.²² These optical properties enabled us to detect green emission from the film upon irradiation with UV light. We thus investigated how Cu²⁺ would induce a change in the fluorescence spectra of the gel film. The boronate film was immersed in a HEPES aqueous solution (5 mM, pH = 7.0) containing various amounts of Cu²⁺. The resulting solution was shaken at 125 rpm for 30 min. As shown in Fig. 4, the fluorescence of the gel was significantly quenched upon addition of incremental amounts of Cu²⁺. A non-linear curve fitting procedure (Fig. 4, inset) was carried out under the assumption of a 1 : 1 stoichiometric complex formation between the dansyldiethylenetriamine moiety and Cu²⁺,⁶ and we reproduced the fluorescence titration data wherein the association constant of the film with Cu²⁺ was estimated to be (3.74 ± 1.02) × 10⁶ M⁻¹. For practical applicability, the detection limit was estimated to be 8.83 × 10⁻⁷ M (883 nM) from the fluorescence profile in Fig. 4.

Fig. 4 Change in fluorescence spectra of gel film (30 × 10 × 1 mm, 1 = 1.25 × 10⁻⁵ M) after immersion in Cu²⁺ in HEPES aqueous solution (5 mM, pH = 7.0, 3.0 mL) for 30 min: λ_ex = 340 nm. Inset: fluorescent intensity changes at 511 nm plotted as a function of incremental amounts of Cu²⁺.

The time dependency for Cu²⁺-induced fluorescence quenching was also examined. The gel film was immersed in a HEPES aqueous solution of Cu²⁺ (1.25 × 10⁻⁵ M), being almost consistent with the amount of dansyl moiety incorporated into the gel. After shaking (125 rpm) the resulting solution, the emission intensity was recorded at different times (Fig. 5a). A good linear relation between ln[(I₀ − I_lim)/(I_lim − I)] versus time was obtained to estimate the first-order rate constant, 8.7 × 10⁻² min⁻¹. This means that the quenching of fluorescence is almost saturated (≈90%) for 30 min (Fig. 5b and 5c).

Fig. 5 (a) Time dependency for the Cu²⁺-induced fluorescent response of the gel film. (Inset: corresponding plot of ln[(I₀ − I_lim)/(I_lim − I)] versus time for fluorescence quenching at 511 nm after immersion in a HEPES aqueous solution of Cu²⁺ (1.25 × 10⁻⁵ M).) Photograph of gel films under UV light (365 nm) after being immersed in HEPES aqueous solution (5 mM, pH = 7.0, 3.0 mL) for 30 min: (b) copper ion-free conditions; (c) in the presence of Cu²⁺ (1.25 × 10⁻⁵ M).

Next, we investigated the selectivity of the gel toward other metal ions in water. Aqueous solutions (3 mL) of metal ions (1.25 × 10⁻⁵ M) such as Na⁺, K⁺, Mg²⁺, Ca²⁺, Fe³⁺, Co²⁺, Ni²⁺, Zn²⁺, Cd²⁺, Hg²⁺, Al³⁺ and Pb²⁺ were prepared in water. The gel film was immersed into each solution. After shaking the resulting solution (125 rpm) for 30 min, the fluorescence was measured. As shown in Fig. 6, the film exhibited a remarkable selectivity toward Cu²⁺. Other metal ions induced no fluorescence quenching of the film. Although it is known that dansyloligoamines respond to not only Cu²⁺ but also Hg²⁺ in water,⁷ dansyl-containing boronate hydrogel films respond to only Cu²⁺ in water.

Fig. 6 Fluorescence intensity changes in film gels after immersion in aqueous solutions of Na⁺, K⁺, Mg²⁺, Ca²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Hg²⁺, Al³⁺ and Pb²⁺, respectively. [Mⁿ⁺] = 1.25 × 10⁻⁵ M, λ_ex = 340 nm, λ_em = 511 nm.

To obtain further evidence regarding selectivity, competitive experiments were carried out by adding Cu²⁺ (1.25 × 10⁻⁵ M) to the buffered solution in the presence of excess amounts of other metal ions (1.25 × 10⁻⁴ M). As shown in Fig. 7, there is almost no fluorescence quenching of the films in the presence of competitive metal ions, with the exception of 10 equiv. Ni²⁺, which causes a 17% decrease in the emission. This can be explained on the basis of energy transfer from dansyl to Ni²⁺, which should be attributed to quenching.⁶ In contrast, the addition of a relatively low amount of Cu²⁺ (1.25 × 10⁻⁵ M) to the solutions containing each metal ion other than Fe^3+ 23 quenched the emission to 30% of its initial value. As a result, although its selectivity toward Cu²⁺ slightly decreased in the case of Ni²⁺ and Fe³⁺, the film is still most responsive to Cu²⁺. Thus, co-existent ions of Hg²⁺ (Fig. 7) have some or a negligible interference effect on Cu²⁺-sensing in the boronate gel film.

Fig. 7 Competitive experiments for fluorescence quenching of film. The red bars represent the emission intensity of the film in the presence of other metal ions (1.25 × 10⁻⁴ M). The blue bars represent the emission intensity of the film that occurs upon addition of Cu²⁺ (1.25 × 10⁻⁵ M) in the presence of other metal ions (1.25 × 10⁻⁴ M).

The fluorescence quenching could be recovered by simple rinsing of the film with an aqueous solution of ethylenediaminetetraacetic acid (EDTA). We investigated the reversible sensing capability by alternatively exposing the film to 3 mL of an aqueous solution (5 mM HEPES buffer, pH = 7.0) containing Cu²⁺ (1.25 × 10⁻⁵ M) and EDTA (1.25 × 10⁻⁵ M). As shown in Fig. 8, when the film was immersed in EDTA solution with shaking at 125 rpm for 30 min at room temperature, approximately 85% of emission could be restored as a result of a competitive binding phenomenon between the film and EDTA toward Cu²⁺. The reversible fluorescence switch was obtained after alternating treatment with Cu²⁺ and EDTA, although the fluorescence intensity of the film decreased gradually over several cycles. Compared to homogeneous solution-based chemosensors, the reversibility of the film has significant merits for practical use as an indicator.

Fig. 8 Fluorescence intensity change in the gel film upon alternating treatment by aqueous solutions of Cu²⁺ (1.25 × 10⁻⁵ M) and EDTA (1.25 × 10⁻⁵ M): λ_ex = 340 nm, λ_em = 511 nm.

Conclusion

We prepared for the first time a PVA–boronate hydrogel via boronate esterification between DBA and PVA. The incorporation of dansyldiethylenetriamine into the gel matrix using 1 allowed us to develop an emissive film through casting into a slab, exhibiting highly selective fluorescence quenching toward Cu²⁺ over other metal ions tested in aqueous HEPES solution (pH = 7.0). This result is worthwhile for developing chemosensors because dansyloligoamines significantly respond to Hg²⁺ as well as Cu²⁺ in a solution. In addition, the reusability of the sensing suggests that the film has potential as a simple and expedient tool for on-site monitoring of Cu²⁺ in environmental applications such as water analysis. We believe that the ready binding of functional phenylboronic acids into the PVA matrix would lead to the preparation of a variety of indicator gels; for instance, two different indicators could be incorporated into the gel matrix through boronate esterification wherein a synergistic effect between the indicators would provide advanced chemosensor systems. Further research in this regard is currently under way in our laboratory.

Experimental section

Materials

Unless otherwise indicated, reagents and solvents used for this study were commercially available and used as supplied (Tokyo Kasei, Kanto Kagaku, Wako, Aldrich, and Merck). N-Dansyldiethylenetriamine was synthesized according to the procedure in a previous paper.¹⁷ As for the buffer solution, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer solution (5 mM, pH = 7, I = 0.01) was used. The pH and ionic strength were adjusted by the addition of aqueous sodium hydroxide (1.0 M) and sodium chloride solution (1.0 M).

Measurements

NMR spectra were recorded on a Bruker AVANCE-500 spectrometer using tetramethylsilane (TMS) as an internal standard (0 ppm) for ¹H and ¹³C NMR analysis and BF₃·OEt₂ as an external standard (0 ppm) for ¹¹B NMR analysis. All spectra were recorded at 298 K. Fast atom bombardment (FAB) mass spectra were obtained on a JEOL JMS-700 spectrometer where 3-nitrobenzyl alcohol was used as a matrix. Field-emission scanning electron microscopy (FE-SEM) was performed using a JEOL JSM-7500F (acceleration voltage of 5 kV). For FE-SEM measurement, xerogel of the boronate ester gel was prepared by a freeze-drying method and was coated with Au on an EIKO IB3 ION COATER. Infrared spectra were recorded on a JASCO FT-IR-5300 spectrometer using a KBr pellet technique. Elemental analysis was performed on a Yanaco CHNcoder MT-5 analyzer. Absorption and fluorescence spectra were measured on a Shimadzu UV-3100PC and a JASCO FP-6300 spectrophotometer, respectively. For UV-vis absorption measurement, the dansyl-functionalized boronate gel was put into a 1 cm × 1 cm cell filled with water. For fluorescence emission measurements, the films were put into a cell filled with the solution at an angle of 45° with respect to the incident light using a quartz glass plate (42 mm × 13 mm). The slit size for excitation and emission was 5 nm. Fluorescence quantum yields of dye 1 and dansyl-functionalized boronate gel were determined using quinine sulphate (Φ = 0.546 in 0.5 M H₂SO₄ aqueous solution) with 340 nm excitation at 25 °C as a reference.¹⁸

Synthesis of 3-[({(N-dansylamino)ethylamino}ethylamino)methyl]phenylboronic acid (1) (Scheme 1). To m-formylphenylboronic acid MIDA ester (0.34 g, 1.3 mmol) in dry ethanol (250 mL) containing molecular sieves 3 Å, was added N-dansyldiethylenetriamine (0.44 g, 1.3 mmol) dissolved in dry ethanol (100 mL) under a N₂ atmosphere at room temperature. The mixture was stirred for 40 h at room temperature. After checking the progress of the reaction by TLC, a dry ethanol solution (130 mL) of NaBH₄ (0.099 g, 2.6 mmol) was added to the solution and the mixture was further stirred at room temperature for 1 h. After filtration, the solution was evaporated in vacuo. The resulting residue was partitioned between CH₂Cl₂ (100 mL) and saturated saline (100 mL), the water phase being extracted with CH₂Cl₂ (100 mL × 2). The organic phase was dried over anhydrous Na₂SO₄ and filtered. The material obtained was chromatographed on silica gel (Silica gel 60 silanized) using a gradient of CH₃OH (0–1.5% v/v)) in CHCl₃ as an eluent. In this way, 111 mg of 1 was obtained in 18% yield. UV-vis: λ_max = 341 nm (ε = 4.5 × 10³ M⁻¹ cm⁻¹) in DMSO and λ_max = 329 nm (ε = 4.4 × 10³ M⁻¹ cm⁻¹) in 5 mM HEPES buffer solution (pH 7.0, I = 0.01); fluorescence: λ_em = 519 nm (Φ = 0.29, λ_ex = 340 nm) in DMSO and λ_em = 545 nm (Φ = 0.02, λ_ex = 340 nm) in 2% DMSO aqueous solution (5.0 mM HEPES, pH = 7.0); ¹H NMR (500 MHz, CD₃OD) δ 8.59 (d, 1H, J = 8.5 Hz), 8.32 (d, 1H, J = 8.7 Hz), 8.22 (dd, 1H, J = 7.3, 1.2 Hz), 7.74 (s, 1H), 7.68 (s, 1H), 7.60 (dd, 1H, J = 8.65, 7.6 Hz), 7.59 (dd, 1H, J = 8.6, 7.35 Hz), 7.53 (d, 1H, J = 7.8 Hz), 7.44 (t, 1H, J = 7.5 Hz), 7.28 (d, 1H, J = 7.6 Hz), 4.17 (s, 2H, CH₂), 3.12 (t, 2H, CH₂, J = 6.1 Hz), 3.04 (t, 2H, CH₂, J = 5.75), 3.01 (t, 2H, CH₂, J = 5.9 Hz), 2.88 (m, 8H, CH₂ and CH₃); ¹³C NMR (125 MHz, CD₃OD) δ 153.42, 136.20, 135.96, 135.46, 133.02, 132.01, 131.60, 131.32, 130.90, 130.54, 129.50, 129.45, 124.39, 120.25, 116.63, 52.78, 46.07, 45.82, 45.50, 41.87; ¹¹B NMR (160 MHz, CD₃OD) δ 28.39; FAB-MS: m/z 741 ([M + 2(3-nitrobenzyl alcohol) − 2H₂O + H]⁺); elemental analysis: anal. calcd for C₂₃H₃₁BN₄O₄S·1.6HCl: C 52.25; H 6.21; N 10.60%, found: C 52.47; H 6.09; N 10.34%.

Fabrication of dansyl-functionalized boronate hydrogel

A DMSO solution (0.6 mL) of PVA (M.W. = 146 000∼186 000, 5.0 × 10⁻¹ M)¹⁹ was added to a DMSO solution (0.15 mL) containing benzene-1,4-diboronic acid (6.0 × 10⁻² M) and 1 (6.7 × 10⁻⁴ M) in a vial. The resulting solution was immediately cast into a slab to afford DMSO gel films (approximately 48 × 16 × 2 mm) and aged for 24 h at room temperature. The film was immersed in DMSO (5 mL) again, and then shaken at 100 rpm for 30 min. Similar procedures were carried out in solvents of increasing water ratio (5 mL, DMSO : water = 50 : 50 , 25 : 75, 12.5 : 87.5 (v/v)) to obtain the desired fluorescent hydrogel. The film was a swollen gel with dimensions of 30 × 10 × 1 mm. The amount of immobilized 1 in the PVA–boronate gel matrix was calculated based on the absorption intensity of the immobilized 1 at 336 nm in the gel matrix and the extinction coefficient of 1 in DMSO (ε = 4.5 × 10³ M⁻¹ cm⁻¹ at 341 nm).

Fluorescence measurements of dansyl-functionalized boronate hydrogel

For the measurements, nitrate salts of metal ions (Na⁺, K⁺, Mg²⁺, Ca²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Hg²⁺, Al³⁺, and Pb²⁺) were used. The dansyl-functionalized boronate gel was immersed in HEPES buffer solutions (3 mL) containing the metal ions and shaken at 125 rpm with a shaking apparatus for 30 min at room temperature. The binding constant (K) of the immobilized dansyltriamine part with Cu²⁺ in a PVA–boronate matrix was determined from the titration curve in the fluorescence spectra upon the addition of incremental amounts of Cu²⁺ (Fig. 4) wherein the change in fluorescence intensity was expressed in the following equations.

A = K × M₀ + K × S₀ + 1

I, I₀, and ΔI_lim are the observed fluorescence intensity, the fluorescence intensity in the absence of a metal ion, and the saturation change in fluorescence intensity, respectively. In the equation, K, S₀, and M₀ are the binding constant, the initial concentration of 1, and the concentration of the metal ion added, respectively. The K and ΔI_lim values were evaluated by a nonlinear least-squares method.

For the determination of the detection limit toward Cu²⁺, the intersection of the regression line obtained from the value of (I − I₀) in the dynamic range of the titration curve allowed us to estimate the value of the detection limit by the following equation.

Y = I_avg − 3 × σ

I _avg and σ are the average value and the standard deviation of fluorescence intensity at 511 nm, respectively, in the absence of a metal ion, measured three times.

Reusability of the film

For the estimation, after being immersed in a Cu²⁺ (1.25 × 10⁻⁵ M in 5 mM HEPES buffer (3 mL) at pH 7.0) aqueous solution for 30 min with shaking at 125 rpm at room temperature, the film was immersed in aqueous EDTA solution (1.25 × 10⁻⁵ M in 5 mM HEPES buffer (3 mL) at pH 7.0) for 30 min with shaking at 125 rpm at room temperature. The fluorescence measurement was then carried out.

Acknowledgements

This research was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (Nos. 21550137, 23750167), the Yamada Science Foundation, and the JGC-S Scholarship Foundation.

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Footnote

† Electronic supplementary information (ESI) available: NMR and FAB mass spectra of compound 1 and experimental details. See DOI: 10.1039/c2ra20516e

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