Visual Simultaneous Detection and Real-Time Monitoring of Cadmium Ions Based on Conjugated Polydiacetylenes.

We prepared the monomer PCDA-HP composed of 5-hydroxy-N 1,N 3-bis(pyridin-2-ylmethyl)isophthalamide (HP) as a cadmium ion tweezer and then polymerized them to form a polydiacetylene (PDA)-based sensor (PDA-HP), which displayed selective and sensitive colorimetric and fluorometric change upon addition of a cadmium ion (Cd2+) at both pH 7.4 and 6.8. The PDA-HP polymer was highly selective for Cd2+ over other metal ions with colorimetric change. In addition, the PDA-HP chemosensor also showed a red fluorescence change in the presence of Cd2+ at both pH 7.4 and 6.8. Naked-eye detection of Cd2+ was accomplished in an aqueous solution through a PDA-based sensor system. Finally, the lowest energy structure of an HP chelator was obtained by the crystal structure and density functional theory (DFT) calculations.

Introduction

p-Conjugated polydiacetylenes (PDAs) have unique characteristics with intense bichromatic properties as a component of sensors.[1] Disruption of the preferred ene–yne conjugated backbone conformation promotes a blue-to-red color transition, which has been applied in the design of colorimetric chemosensors.[2] The blue-colored PDAs, which typically have a maximum absorption at 640 nm, can show a chromatic switch to red in response to external stimuli including temperature,[3] pH,[4] metal ions,[5] anions,[6] surfactants,[7] and other biologically important molecules.[8] Additionally, blue-phase PDAs with no-fluorescence can shift to become fluorescent in response to a stimuli, which facilitate the development of various chemosensors using the polymers.[9]

On the other hand, heavy metals accumulate in the environment and they are not biodegradable.[10] Sensitive and selective detection and quantitative monitoring of toxic heavy metals play an important role in the control of heavy metal-based pollutants. Among these toxic heavy metals, cadmium was ranked as a priority substance in water policy by Directive 2008/105/EC of the European Union Parliament.[11] In this rank, cadmium was identified as very toxic to mammals both during acute and chronic exposures. In this regard, considerable effort has been devoted to developing methods of analysis for toxic heavy metals in both industrial processes and environmental systems. However, the traditional analysis methods for detecting traces of Cd2+ are expensive, such as atomic absorption spectrometry (AAS),[12] inductively coupled plasma mass spectrometry (ICP-MS),[13] and fluorometric[14] and colorimetric sensors.[15] Many fluorescent probes for cadmium have been developed in the past decade. Nevertheless, only a few sensors have rapidly distinguished a cadmium ion in a high selectivity from zinc[16] because of their similarity in chemical properties. Therefore, the design of the sensing interface constitutes the key challenge in a Cd2+-selective sensor, which can recognize Cd2+ over Zn2+ under physiological conditions. The most commonly used approach in the design of metal ion chemosensors is to determine with chelation reactions, and incorporate proper ionophore groups that can exist exclusively in coordination complexes with any specific metal ions. In a previous study, although the PDA-CP polymer, which consists of chelidamic acid and picolylamine, could detect Cd2+, it showed incomplete selectivity and only did so at pH 7.4; and the detection limit of PDA-CP was 1.85 × 10–5 M.[17] Herein, we report the design and synthesis of a new diacetylene monomer (PCDA-HP) composed of 5-hydroxy-N1,N3-bis(pyridin-2-ylmethyl)isophthalamide (HP) and its application for the visual detection method for Cd2+ in an aqueous solution. Due to the high affinity between the Schiff base and heavy metal,[18]PDA-HP can fabricate a cadmium-complexed chelation. The prepared PDA-HP solutions show rapid color changes toward Cd2+ in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer at both pH 7.4 and 6.8. On the other hand, there was no significant color or fluorescence change with other metal solutions.

Results and Discussion

Scheme depicts the synthetic route of monomer PCDA-HP. First, chloride-activated acids reacted with 2-picolylamine to form HP with an yield of 75%. In addition, these moieties reacted with PCDA to form PCDA-HP as a yellow oil with 35% yield. The synthesized monomer PCDA-HPs were characterized using 1H NMR and 13C NMR, and electrospray ionization (ESI) mass spectrometry was used to confirm the target molecule (Figures S1–S6 in the Supporting Information (SI)).

Scheme 1

Synthetic Route of Monomer PCDA-HP

Monomer PCDA-HP and PCDA (PCDA-HP/PCDA = 1:9) were self-assembled in an aqueous solution and photopolymerization was carried out by UV irradiation (254 nm, 1 mW·cm2) for 15 min, resulting in a blue-colored solution of PDAs (PDA-HP) (Scheme ). The attempt to convert to a PDA polymer using only PCDA-HP monomers failed, probably because of the steric hindrance of the bulky head group. The ratio of 1:9 and a time of 15 min were optimized by carrying out a series of experiments.

Scheme 2

(a) Self-Assembly and Photopolymerization of PCDA-HP with PCDA and (b) Conventional Sensing Principle for Ca2+ Using PDAs

The selectivity profiles of PDA-HP were investigated by various metal ions via colorimetric transitions, absorption, and fluorescence spectroscopic changes in HEPES buffer (10 mM) at both pH 7.4 and 6.8. Various metallic ions of Li+, Na+, K+, Cs+, Mg2+, Ca2+, Cr3+, Mn2+, Fe2+, Fe3+, Ni2+, Cu2+, Ag+, Zn2+, Cd2+, and Hg2+ using perchlorate (ClO4–) as the counter anion were used to investigate the colorimetric titrations of PDA-HP (200 μM). The concentrations of metal ions were 2.0 and 1.0 equiv in HEPES buffer at pH 7.4 and 6.8, respectively (Figure a). Among the various potential interferents, only Cd2+ displayed a color change of PDA-HP immediately (∼60 s). Other metal ions exerted a negligible effect on the colorimetric and fluorometric response of PDA-HP. Figure b shows the colorimetric changes of PDA-HP (200 μM) in HEPES buffer at both pH 7.4 and 6.8, and is dependent on the Cd2+ concentration. These results indicate that naked-eye detection of Cd2+ was accomplished using PDA-HP. In addition, the Cd2+-activated blue-to-red (violet) color change of PDA-HP was recorded within 0–90 s (Figure S9). The red phase of PDA-HP clearly appeared after 60 s, treated by 2 equiv of Cd2+, which indicates the high sensitivity of the sensor. We hypothesize that Cd2+ detection of polymers shows the difference at pH 7.4 and 6.8 due to the contribution of not only the nitrogen atoms of the aromatic ring and amide groups but also the stereoscopic effect of an HP unit in the polymers. In a previous study, the PDA solution, which contains only the PCDA monomer, showed negligible color changes with metal ions,[7a] which suggested that the Schiff base moiety of the PCDA-HP monomer may form complexes with Cd2+. Furthermore, the Cd2+-responsive PDA-HP was examined in higher and lower pH conditions (Figure S10). The color change of PDA-HP was observed after adding a Cd2+ cation at pH 8.0 and 5.0. However, the color transition of PDA-HP was remarkable in a neutral or slightly acidic condition.

Figure 1

(a) Selectivity of PDA-HP (200 μM) with potential metal ions (2.0 equiv at pH 7.4, 1.0 equiv at pH 6.8); (b) colorimetric titration with various concentrations of Cd2+ in HEPES buffer at pH 7.4 or 6.8.

The Cd2+-triggered color transition of PDAs was also monitored by UV–vis spectroscopy. The absorption spectra of PDA-HP solutions displayed a clear blue color peak around 640 nm. Upon the addition of Cd2+, the original peak decreased gradually, accompanied by a new blue-shift peak around 540 nm at pH 7.4 (Figure a) and pH 6.8 (Figure c). Furthermore, the presence of Cd2+ was detected by the fluorescence enhancement because of the blue-to-red color change that accompanied the fluorescence emission. PDA-HP was excited at 530 nm in the presence of Cd2+ as a perchlorate anion.

Figure 2

UV–vis spectra of PDA-HP (100 μM) at various concentrations of Cd2+ in HEPES buffer at (a) pH 7.4 and (c) pH 6.8. Fluorescence change of PDA-HP (100 μM) at different concentrations of Cd2+ in HEPES buffer at (b) pH 7.4 and (d) pH 6.8 (excitation at 530 nm, slit: 5 nm/5 nm).

We measured the fluorescence (FL) of PDA-HP (100 μM) immersed at various concentrations of Cd2+ at pH 7.4 (Figure b) and pH 6.8 (Figure d). When the concentration of the added Cd2+ was increased, the fluorescence of PDAs gradually increased (Cd2+ ion concentration from 0 to 400 μM).

Furthermore, the limit of detection (LOD) of PDA-HP for Cd2+ was estimated to be 16.48 μM at pH 7.4 and 8.6 μM at pH 6.8 (3δ/S) (Figure S7). The 1:2 and 1:1 stoichiometries of the sensor PDA-HP with Cd2+ at pH 7.4 and 6.8, respectively, were further confirmed by Job’s plot (continuous-variation plots) analysis (Figure S8). The maxima with a mole fraction at about 0.3 and 0.5 indicate 1:1 and 1:2 stoichiometries, respectively.

The selectivity of PDA-HP could be attributed to the possible coordination with the HP chelator and Cd2+. Cd2+ might form complexes with HP chelator and disturb the conjugated backbone of the PDA polymer, resulting in the release of the strain energy. Finally, the release of the side chain strain might induce partial distortion of the well-aligned p-orbitals, which could be determined by noting the optical changes.

The PCDA-HP monomer and Cd2+ coordination was further studied by 1H NMR titration experiments in acetone-d6 (Figure ). After the addition of Cd2+ in the solution of PCDA-HP, the aromatic proton peaks shifted to downfield, which indicates the interaction between aromatic rings and Cd2+ (Figure ). Additionally, aromatic proton’s broadening was observed, which also demonstrates the hetero atoms playing important roles in chelating Cd2+. Especially, the Hc peak was split into two peaks, and the resulting Cd2+ binding site is one-sided in PCDA-HP, instead of being located in the center, simultaneously chelating with two pyridine moieties. These results suggest that the pyridines and isophthalic acid moieties of PCDA-HP are key structures in coordination with Cd2+.

Figure 3

Partial 1H NMR spectra (acetone-d6) of PCDA-HP (2 mM), with the addition of Cd2+ (Cd(ClO4)2·xH2O) (400 MHz).

To confirm the binding of Cd2+ with PCDA-HP, we performed density functional theory (DFT) calculations starting from various conformations of MeCO-HP with and without Cd2+ using the 6-311+G(2d,p) basis set for C, H, O, and N as well as LanL2DZ for a Cd atom. The diffuse function (+) has an effect of lengthening the bonds involving cadmium. Calculations on transition metal compounds often benefit from including diffuse functions. The second d polarization function is required for an accurate ring slippage angle for these structures.[19] We obtained various structures corresponding to a local minima after optimizations from a large number of starting conformations. The optimized conformation with the lowest Gibbs free energy is considered as the global minimum and others are considered as the local minima. The lowest energy structure of MeCO-HP obtained with DFT (Figures and S12) is quite similar to the crystal structure of HP (Figures and S11), indicating that the basis set used in the DFT calculations is large enough to predict the structure appropriately.

Figure 5

Calculated conformation and lowest Gibbs free energy (global minimum) of MeCO-HP with Cd2+ at 1:1 (a) and 1:2 (b) ratios.

Figure 4

X-ray crystal structure of HP.

Two oxygen molecules of two C=O groups tend to direct up/down or down/up, following the plane of the center benzene ring (Figure ). In the ratios 1:1 and 1:2, Cd2+ tends to be located on one side of the central benzene ring and chelate with a nitrogen atom of a pyridine moiety and an oxygen atom of a C=O group with a distance of about 2.3–2.4 Å (Figures , S13 and S14). Furthermore, the charge distribution of the nitrogen atom of the pyridine moiety and the oxygen atom of the C=O group decreased in the presence of a Cd2+ cation (Table S2), indicating the interaction of the metal cation with these atoms. At the ratio 1:1, the pyridine ring turns to favorably construct to chelate of Cd2+ with nitrogen and oxygen atoms (Figures a and 6b). At the ratio 1:2, two oxygen molecules of the two C=O groups are downward so as to increase the distance as well as decrease the interaction between two Cd2+ cations (Figures b and 6c). Furthermore, ΔG2 ∼ 2 × ΔG1, where ΔG1 and ΔG2 are the difference between the Gibbs free energy of MeCO-HP and MeCO-HP with Cd2+ at 1:1 and 1:2 ratios, respectively, indicating the chelating unification of one or two Cd2+ cations with an MeCO-HP ligand. The binding constants K1 and K2 were calculated to be 4.7 × 10298 and 5.9 × 10291, respectively. In addition, the calculated highest occupied molecular orbital–lowest occupied molecular orbital (HOMO–LUMO) gaps of 5.32, 4.59, and 5.18 eV correspond to 233.05, 270.12, and 239.35 nm, respectively, which are quantitatively consistent with the color change of PDA-HP from blue to red when it interacts with Cd2+ (Figure S15).

Figure 6

Two-dimensional (2D) global minimum conformation of MeCO-HP without (a) and with Cd2+ at 1:1 (b) and 1:2 (c) ratios.

Conclusions

In this study, we have prepared PCDA-HP by the reaction of 10,12-pentacosadiynoic acid with a5-hydroxyisophthalic acid-2-picolylamine (HP) as a chelating moiety. PDA-HP copolymers containing a PCDA-HP monomer and a PCDA monomer, in ratios of 1:9, were polymerized in an aqueous solution. On forming complexes with the Cd2+ ion, the PDA-HP showed a highly selective blue-to-red color change as well as an increase of fluorescence. Specifically, PDA-HP can detect Cd2+ at a low concentration under pH 6.8. The interactions between PCDA-HP and Cd2+ were confirmed by NMR analysis and DFT calculation.

Experimental Section

Materials and Methods

10,12-Pentacosadiynoic acid (PCDA), oxalyl chloride, 5-hydroxyisophthalic acid, and pyridin-2-ylmethanamine were purchased from TCI, South Korea. 1H and 13C NMR spectra were recorded on a Bruker Avance 400 and 600 MHz. HR-ESI MS data were acquired using a maXis-HD (Bruker). UV absorption spectra (V-730 UV–Visible Spectrophotometry, Jasco) and fluorescence emission spectra (F-7000 Fluorescence Spectrophotometry, Hitachi High-Tech) were obtained at room temperature. All concentrations were based on the total monomer (PCDA and PCDA-HP).

Synthesis of 5-Hydroxy-N1,N3-bis(pyridin-2-ylmethyl)isophthalamide (HP)

Oxalyl chloride (2.0 mL, 22.9 mmol) was added dropwise to an acetonitrile (ACN; 30 mL) solution containing 5-hydroxyisophthalic acid (1.0 g, 5.5 mmol) at 0 °C under nitrogen gas. After 1 h of stirring, a drop of dimethylformamide (DMF) was added to the solution and the resulting mixture was stirred for 4 h. After the removal of the solvent, diacyl chloride as a yellow solid was dissolved in ACN (30 mL). This solution was added to 10 mL of ACN containing pyridin-2-ylmethanamine (2 mL, 21.8 mmol). The reaction mixture was stirred overnight at room temperature under nitrogen gas. After evaporating the organic solvent, the crude product was purified by silica gel column chromatography using dichloromethane (DCM) and MeOH (v/v = 90:10) as the eluent. This afforded HP as a white solid (1.5 g, 75%). 1H NMR (400 MHz, DMSO-d6) δ 10.01 (s, 1H), 9.11 (t, J = 6.0 Hz, 2H), 8.51 (ddd, J = 4.9, 1.9, 0.9 Hz, 2H), 7.90 (t, J = 1.5 Hz, 1H), 7.76 (td, J = 7.7, 1.8 Hz, 2H), 7.45 (d, J = 1.4 Hz, 2H), 7.36–7.23 (m, 4H), 4.56 (d, J = 5.9 Hz, 4H). 13C NMR (100 MHz, DMSO-d6) δ 166.60, 159.22, 157.97, 149.37, 137.28, 136.40, 122.64, 121.46, 117.55, 117.43, 46.08, 45.26. ESI HRMS m/z = 363.1452 [M + H]+, calcd. for C20H18N4O3 = 362.38.

Synthesis of 3,5-Bis((pyridin-2-ylmethyl)carbamoyl)phenyl pentacosa-10,12-diynoate (PCDA-HP)

Oxalyl chloride (0.7 mL, 5.51 mmol) was added to a 10,12-pentacosadiynoic acid (PCDA) solution (0.5 g, 1.34 mmol) in 20 mL of DCM at room temperature under nitrogen gas. After 1 h of stirring, a drop of DMF was added to the solution and the resulting mixture was stirred for 4 h. After the removal of the solvent, the obtained PCDA-Cl was dissolved in 30 mL of ACN without purification. This solution was added to a solution of 10 mL ACN 5-hydroxy-N1,N3-bis(pyridin-2-ylmethyl)isophthalamide (HP) (200 mg, 0.55 mmol) and triethylamine (TEA; 0.4 mL, 4 mmol). The reaction mixture was stirred overnight at room temperature under nitrogen gas. After evaporation, the crude product was dissolved in EA, washed with water, and dried over MgSO4. The organic phase was purified by silica gel column chromatography using DCM and MeOH (v/v = 95:5) as eluents, followed by Sephadex LH-20 using chloroform and MeOH (v/v = 1:1) to obtain a yellow oil (138 mg, 35 %). 1H NMR (600 MHz, DMSO-d6) δ 9.29 (t, J = 6.0 Hz, 2H), 8.51 (ddd, J = 4.9, 1.9, 1.0 Hz, 2H), 8.39 (t, J = 1.5 Hz, 1H), 7.83 (d, J = 1.5 Hz, 2H), 7.76 (td, J = 7.7, 1.8 Hz, 2H), 7.34 (dt, J = 7.8, 1.0 Hz, 2H), 7.27 (ddd, J = 7.6, 4.8, 1.1 Hz, 2H), 4.59 (d, J = 5.9 Hz, 4H), 3.17 (d, J = 5.3 Hz, 1H), 2.63 (t, J = 7.4 Hz, 2H), 2.26 (q, J = 7.0 Hz, 4H), 1.66 (p, J = 7.5 Hz, 2H), 1.49–1.19 (m, 34H), 0.84 (t, J = 7.0 Hz, 3H). 13C NMR (150 MHz, DMSO-d6) δ 171.75, 164.96, 158.42, 150.40, 148.88, 136.71, 135.83, 123.79, 123.60, 122.13, 121.05, 77.93, 65.34, 48.58, 44.83, 33.37, 31.28, 28.99, 28.93, 28.85, 28.69, 28.55, 28.37, 28.34, 28.26, 28.15, 27.70, 27.67, 24.25, 22.08, 18.26, 13.92. ESI HRMS m/z = 719.4531 [M + H]+, calcd. for C20H18N4O3 = 718.45.

Computational Details

The DFT calculations of the isolated molecule MeCO-HP complexed with a Cd2+ ion were performed using the Gaussian 09 program package[19] Geometry optimizations of the molecule were performed using the B3LYP hybrid functional.[20] Various conformations were selected as initial structures for optimizations, and the energies of the optimized structures were obtained. We used the 6-311+G(2d,p) basis sets[21] for C, H, N, and O, while LanL2DZ[22] was used for Cd, for which core electrons were represented by LanL2DZ ECPs. Geometry optimizations were performed in an acetone solvent. The solvent was modeled by the polarizable continuum model (PCM) using the integral equation formalism variant (IEFPCM)[23] as implemented in Gaussian 09. After optimizing structures, vibrational frequencies were computed to ensure that there were no imaginary frequencies. The Gibbs free energies were also obtained. Boltzmann’s formula was used to calculate the ratio of populations of two conformations i and jwhere the value of kT is roughly equal to 1 millihartree at room temperature. We estimated the relative abundance of two conformations by simply computing the free-energy difference (in millihartree).[24] The binding constant (K) was calculated via the equationwhere R is the ideal gas constant and T = 298.15 K. NBO was calculated using the 6-311G(2d,p) basis sets for C, H, N, and O, while LanL2DZ was used for Cd (Table S3).

Single Crystals of MeCO-HP

X-ray data were recorded by a Bruker SMART automatic diffractometer facilitated with graphite-monochromated Mo Kα radiation (λ = 0.71073 Å). The effects of absorption were revised using the multiscan method (SADABS)[25] and structures were corrected using the direct method (SHELXS),[26] and refined by full-matrix least-squares techniques (SHELXL 2018/3).[27] The crystal parameters, procedural information, and structure refinement are mentioned in Table S1.