A piezogravimetric sensor platform for sensitive detection of lead (II) ions in water based on calix[4]resorcinarene macrocycles: Synthesis, characterization and detection

2.1.1 Compound A

Compound A (C-dec-9-en-1-ylcalix[4]resorcinarene), was synthesized and characterized using (FTIR, ¹H NMR, ¹³C NMR, TG-DSC-MS, and XRPD), as described in our previous work (Eddaif et al., 2019c).

2.1.2 Novel enantiomer compounds B and C

Compound B (C-dec-9-enylcalix[4]resorcinarene-O-(S-)-α-methylbenzylamine) and compound C (C-dec-9-enylcalix[4]resorcinarene-O-(R+)-α-methylbenzylamine), were prepared according to the following recipe: 0.05 mol. of compound A was dissolved in a THF-methanol mixture (1:1), then added 0.05 mol. of paraformaldehyde. Then added to the mixture: 0.025 mol. of (S-)-α-methylbenzylamine to get Compound B and 0.025 mol. of (R+)-α-methylbenzylamine for compound C, as shown in Fig. 2. All components were stirred under argon atmosphere for 1 h, then refluxed for 12 hrs. After the reaction’s completion, all solvents were removed by rotary evaporator and the residue was crystallized twice from acetonitrile yielding compounds B and C as yellowish-orange solid (Yield: 50%, m.p.: 70–72 °C).

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Fig. 2

Synthetic steps of compounds A, B and C.

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2.2.1 FTIR studies

FTIR measurements were performed on a Varian 2000 FTIR spectrometer (Scimitar Series) (Varian Inc., US), the instrument is equipped with an MCT (Mercury–Cadmium–Telluride) detector, with an ATR single reflection diamond unit (Specac Ltd, UK), and with a resolution of 4 cm⁻¹. For each spectrum, a number of 64 individual scans was averaged, and all spectra were ATR-corrected by the data acquisition software (Varian ResPro 4.0).

2.2.2 NMR measurements

NMR analysis were performed on a Varian VNMR SYSTEM^TM spectrometer in the following conditions (CDCl₃, 400 MHz, 25 °C), the ¹H and ¹³C chemical shifts were referenced to the residual solvent signals (δ_1H = 7.26 ppm, δ_13C = 77 ppm). And, an indirect detection triple resonance ¹H {¹³C, X} Z-gradient probe was used.

2.2.3 Thermal analysis

Thermogravimetric analysis were carried out on a Setaram Labsys Evo thermal analyzer, with a 90 ml min⁻¹ flowrate, and with a 20 °C min⁻¹ heating rate in helium (6.0) atmosphere in the temperature range of 20–500 °C. The procedure consisted on placing 7–8 mg of the sample into 100 μl aluminum crucibles. The gotten output data was evaluated and baseline-corrected with the thermoanalyzer’s software (Calisto Processing, version 1.492).

Simultaneously with the thermogravimetric-differential scanning calorimetric (TGA-DSC) measurements, the products decomposition analysis (EGA: evolved gas analysis) were performed on a Pfeiffer Vacuum OmniStar^TM Gas Analysis System coupled to the TGA-DSC described previously. The spectrometer was functioning in the electron impact mode; the scan speed was around 20 ms amu⁻¹ in the mass range of 10–111 amu. Both transfer lines and gas splitters were thermostated (230 °C), and the analysis were performed in SEM Bar Graph Cycles acquisition mode, in this last, the analog mass spectra, the TIC: Total Ion Current, and the SIC: Separate Ion current (for each scanned mass individually: 101 masses) were recorded.

2.2.4 X-ray powder diffraction

XRPD was used to analyze the crystallinity degree of the subjected samples, which were in grinded form, then appropriate sample quantity was placed in the sample holder under pressure. The diffractograms were recorded by means of a Philips Powder Diffractometer 1810/3710 assisted with a Bragg–Brentano para-focusing geometry, a CuKa monochromatic radiation was used (l = 0.154056 nm), and the data were collected in the reflection mode at room temperature, with an exposure time of 1.00 s at each point, and a step of 0.04° in the (3–77°) 2θ range.

2.2.5 Enantiomeric discrimination by means of polarimetry

The specific rotation [α]_D, is generally calculated using the expression : [α]_D = (100 × α)/(l × c), where ‘c’ stands for the solution’s concentration in (g/100 ml) ‘α’ is the detected rotation, ‘l’ is the optical path length (dm), and ‘D’ is the light wavelength (nm).

The optical rotation activities were performed on a JascoP-2000 Polarimeter. The products were dissolved in chloroform with a 0.1 g/100 ml in a 1 dm polarimeter tube at room temperature. A Sodium ‘D line’ was used (589.3 nm).

2.3.1 Macrocyclic immobilization on QCM resonators

Analytical grade chemicals and Milli-Q purity deionized water (18.2 MΩ cm) were used throughout the experiments. The immobilization of the layers on the Au-surface of the resonator electrode is established through the following protocol: A calixresorcinarene solution was prepared by dissolving 2 mg/ml of the synthesized macrocycle in chloroform, then 10 µl was drop casted on the quartz crystals (formerly cleaned by means of acetone for 10 min, piranha solution (1/3 H₂O₂ + 2/3 H₂SO₄) for 10 min, then rinsed with Milli-Q water, and finally let dry). The coated crystals were dried at room temperature in a desiccator.

2.3.2 QCM-I tests

Heavy metal solutions of various concentrations (5, 25, 250, 500 and 1000 ppm) were prepared by diluting Lead(II) nitrate (Pb(NO₃)₂) in deionized water_. The flow-cell volume is ∼40 µl and the flow rate was 200 µl/min maintained by a peristaltic pump. The resonance frequency and dissipation shifts were recorded for the selected overtones (n = 1, 3, 5, 7 for the frequencies of 5, 15, 25, 35 MHz, respectively). The measurement was driven by the BioSense Software V. 3.1, (developed by MicroVacuum Ltd, Budapest, Hungary), which can depict any two variable combinations vs. time, for any overtone of the following variables (F, ΔF, D, ΔD, Q, FWHM, ΔFWHM, G_max, G_offs, B_offs, Fi, T, Sauerbrey mass (ΔM), Residual and Resonance).

3.1.1 FTIR results

The IR spectroscopy is a powerful tool dedicated to the molecular functional groups determination, furthermore to the molecular structure confirmation. The IR spectra of both enantiomers were collected in the (4000–400 cm⁻¹) wave number range, and are shown in Fig. 3, while Table 1 presents all functional groups, their wave numbers, and the corresponding vibration natures.

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Fig. 3

FT-IR spectra of compounds B and C.

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By analyzing both spectra, an obvious similarity is seen, where both compounds show a large and strong band (3340 cm⁻¹) corresponding to the stretching vibration of the hydroxyl groups (O—H), it may result from the free OH groups in the molecule, or even from the physically absorbed water, the (O—H) bond is confirmed by the existence of the (C—O) stretching (1226 cm⁻¹), and the (O—H) bond’s in-plan deformation (1348 cm⁻¹). The vinyl chain is characterized by the (⚌C—H) stretching (3070, 3027 cm⁻¹), the (C—H) deformation harmonics (1822 cm⁻¹), the (C⚌C) stretching (1640 cm⁻¹), and the (⚌C—H) out-plan deformation (907,880 cm⁻¹). Considering the aromatic cycles, the (⚌C—H) stretching is gotten (3030 cm⁻¹), the (C⚌C) stretching is presented by a quadruplet (1602, 1560, 1540, 1468 cm⁻¹), and the (C—H) aromatic bond is confirmed by the deformation harmonics (1980 cm⁻¹), furthermore the (⚌C—H) out-plan deformation is existing (880, 778, 750 cm⁻¹).

The presence of the cyclic ether is confirmed by the (C—O) stretching (1181 cm⁻¹), and the existence of the tertiary amine is indicated by the (C—N) amine stretching (1145 cm⁻¹). Although, the alkane chain is established by the (CH₃) symmetric stretching (2853 cm⁻¹), the (CH₃) symmetric plane deformation (1468 cm⁻¹), the (CH₂) asymmetric stretching (2925 cm⁻¹), the (CH₂) rocking (700 cm⁻¹), and by the (C—H) stretching (2970 cm⁻¹) as well as the (C—H) out-plane deformation (1346 cm⁻¹).

3.1.2 NMR results

To correctly determine a newly synthesized compound’s molecular structure, the IR studies are not enough, therefore a much powerful technique is required, mostly the Nuclear Magnetic Resonance spectroscopy (NMR) is used. Therefore, the acquired ¹H NMR and ¹³C NMR spectra of both compounds are similar as presented in Figs. 4 and 5(a and b). The results show the typical aliphatic and aromatic carbon and proton peaks which confirms the validity of the proposed structures displayed in Fig. 2.

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Fig. 4

¹H (a) and ¹³C (b) NMR spectra of compound B.

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Fig. 5

¹H (a) and ¹³C (b) NMR spectra of compound C.

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¹H NMR (CDCl₃, 400 MHz, 25 °C): 7.67 (4H, s); 7.18 (8H, d, J = 7.5 Hz); 7.10 (4H, s); 7.05 (8H, m); 6.96 (4H, t, J = 7.5 Hz); 5.82 (4H, m); 5.15 (4H, d, J = 10.4 Hz); 5.04 – 4.90 (12H, m); 4.20 (4H, t, J = 7.8 Hz); 3.96 (4H, d, J = 17.5 Hz); 3.81 (4H, q, J = 6.2 Hz); 3.73 (4H, d, J = 17.5 Hz); 2.18 (8H, m); 2.04 (8H, q, J = 6.0 Hz); 1.50 – 1.20 (60H, m).

¹³C NMR (CDCl₃, 100 MHz, 25 °C): 149.6; 148.7; 144.5; 139.2; 128.2; 127.0; 124.3; 123.4; 121.1; 114.2; 108.9; 80.9; 58.0; 44.5; 33.8; 33.7; 32.7; 29.7; 29.6; 29.2; 29.0; 28.1; 21.4.

3.1.3 Thermal analysis

The coupled thermal investigations and evolved gas analysis (TGA-DSC-MS: EGA), were used to study both the thermal stability and the purity of the novel enantiomeric compounds, furthermore to determine their calorimetric melting points (In order to make a comparison with those determined directly), as well to verify all the released content, meaning to examine if there’s any volatile contaminant. In Fig. 6a and b, the TGA (Thermogravimetric)/ DSC (Heat flow) curves of both compounds are presented versus temperature.

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Fig. 6

Thermogravimetric and heat-flow curves of both enantiomers versus T (°C).

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Seemingly the TG and DSC patterns look similar, however, both enantiomers present high thermal stabilities, since no mass loss occurs in the TG curves up to 180 °C. Beyond this mentioned temperature, the compounds degrade in two steps (bi-step decomposition), furthermore the total mass losses of compounds B and C up to 500 °C are −82.52% and −82.38%, respectively.

In the heat flow curves, between 50 °C and 75 °C temperature, a small broad endotherm is observed, which corresponds to the melting of the two molecules {Compound B: (Onset temp. (T₀): 48.6 °C, pic maximum (T_m): 61 °C}, {Compound C: (Onset temp. (T₀): 49.22 °C, pic maximum (T_m): 60 °C}, the mentioned calorimetric melting points are in good agreement with those determined directly (70–72 °C), and generally such shape of melting endotherms matches with a semi-crystallized compound. Though, the thermal events detected after 180 °C, corresponds to the molecules degradation.

In order to evaluate the purity of both compounds, the evolved gas analysis (EGA) were performed. EGA revealed that the major released component was water (m/z: 18, 17) and the characteristic peaks of residual air were also detected (m/z: 44, 32, 28, 16). Those results confirm the evaporation of physically bounded water molecules from one point and the presence of some entrapped air in the transfer line, and the purity of both molecules from another point.

3.1.4 Powder X-ray diffraction

As a complementary characterization method to those carried out above, the Powder X-ray diffraction analysis (P-XRD) was used to define the crystalline/amorphous character of both novel synthesized enantiomers, thus the P-XRD patterns are presented in Fig. 7. Analyzing the X-Ray diffractograms showed a semi-crystalline behavior, which correlates in a decent manner with the Differential Scanning Calorimetric (DSC) results, as shown previously, when both compounds, on the heat curves, had a small wide endotherm as a melting stage, denoting that both P-XRD investigations and thermal analysis are in good agreement.

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Fig. 7

Powder X-ray diffractograms of compounds B and C.

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3.1.5 Enantiomeric discrimination

One of the extensively used techniques for enantiomeric discrimination purposes, is the so called ‘polarimetry’, it’s based on measuring the polarized light deviation passing through a solution containing chiral compounds (Optically active). While studying enantiomers, their physical and chemical properties are identical, but polarized light is differently effected by them; the rotation amount (Specific rotation) tend to be similar but in opposite directions. The specific optical rotation ([α]_D) values of samples “B” and “C” are the follows: sample B: [α]_D = −185.9°, sample C: [α]_D = +185.0°. The obtained values are almost similar and in opposite sign, evidencing that compounds ‘B’ and ‘C’ are enantiomers.

3.2.1 Frequency and dissipation energy variations in time

The detection ability of the synthesized enantiomeric calix[4]resorcinarene, drop coated onto quartz resonator, will be explored against lead ions in aqueous solutions via In-situ QCM-I measurements with an advanced simultaneous layer characterization parameter (Dissipation Energy). A further aspect of the study is to analyze and compare the configuration effect (‘R’ and ‘S’) of both compounds B and C on lead ions detection, aiming to determine the best coating material in terms of frequency response, sensitivity, selectivity, and detection limits. Figs. 8 and 9 illustrate sequentially the frequency responses of compounds B and C based chemo-sensors against various concentrations of lead ions in static mode (Figs. 8a, 9a), while Figs. 8 and 9(b) are presenting the dissipation energy shifts of compounds B and C based sensing layers, respectively.

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Fig. 8

Frequency (a) and dissipation shifts (b) of compound B based QCM sensor against various lead ions amounts in time.

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Fig. 9

Frequency (a) and dissipation responses (b) of compound C based QCM sensor against various lead ions amounts in time.

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As emphasized beforehand, Figs. 8a and 9a demonstrate the normalized frequency shifts in time during the exposure of the QCM sensors to aqueous samples containing Pb²⁺ concentrations up to 1000 ppm. As expected the frequency responses decreased while increasing the lead concentration in aqueous solutions (concentration dependence). Additionally, it is observed that compound C has reached a higher frequency variation value (ΔF = −23 Hz) at 1000 ppm compared to (ΔF = −10 Hz) for compound B, representing more than two-folds difference. These alterations can be explained due to the fact that both calixresorcinarene derivatives are bearing chiral moieties containing nitrogen atoms (their free electron pairs are accounted to their basic properties). Chelating agents, containing nitrogen (N) atoms, are favorable to form metal-complexes via electron transfer, especially by binding to heavy metals ions, such as Pb²⁺ (Diamond and Nolan, 2001; Flora and Pachauri, 2010; Rashid et al., 2019; Sharma and Cragg, 2011; Sone et al., 1997).

Calixarenes and calixresorcinarenes are considered as the third generation of host-guest macrocyclic networks, this fact ensures the macrocyclic ability of hosting Pb²⁺. From another point of view, the sensing mechanism could be also described by an adsorption effect between the macrocycles and the Pb²⁺. This interaction causes a Pb²⁺ bulk diffusion process, described by the Sauerbrey estimation that links ΔF directly and proportionally to the adsorbed mass. The diffusion process can lead to an effective mass increase (adsorbed Pb²⁺ amount), and reducing the crystal’s resonance frequency as seen in Figs. 8a and 9a, consequently. Both compounds B and C exhibited frequency responses to all applied Pb²⁺ concentrations (Table 2), which supports the functionality of the novel Pb²⁺ detection technique built on utilizing calixresorcinarene coated QCM resonators.

The accumulated data in Table 2, was registered from the end points of the curves shown in Figs. 8 and 9, as a summary of Δf_n/n and ΔD_n values of the deposited layers. Values of the dissipation energy variations ΔD (Energy loss or damping) are indicators for viscoelastic properties (rigidity, softness), as seen in Figs. 8b and 9b, and Table 2. Furthermore, ΔD variation can provide an accurate prediction concerning the appropriateness of the Sauerbrey estimations. As previously mentioned, the upper ΔD limit for treating a layer as rigid is about 2 × 10⁻⁶ (Saftics et al., 2018), however if its higher than that, the deposited film is considered as soft, and alternative viscoelastic model must be considered to better describe both the deposited layer’s characteristics, and the ions-sensor binding mechanism.

According to the presented dissipation variations, compound B is showing a significant viscoelastic behavior (ΔD_n > 2 × 10⁻⁶) for higher Pb²⁺ concentrations (500, 1000 ppm), but still the rigidity character is dominating for other concentrations (ΔD_n ≤ 2 × 10⁻⁶). This could be due to some loosely bound chains network, giving a soft coverage after the sensor’s exposure to higher Pb²⁺ quantities (C_Pb²⁺ > 250 ppm).

On the other hand, in the case of compound C (ΔD_n < 2 × 10⁻⁶), the acquired data indicated the rigidity of the attached macrocyclic network, hence, suggesting the applicability of the Sauerbrey estimation model of the deposited film’s mass.

3.2.2 Calixresorcinarenes immobilization onto the Au surface

The attachment of the ionophores (macrocycles) on the piezoelectric resonator is due to the interactions occurring between the set of heteroatoms (O, N) existing in the ligands and the (Au) surface of the quartz electrodes, therefore constructing a well-defined sensing platform network that can host the target toxic elements. The (Au) surface is a thermodynamically stable phase, in addition, the high activity of both oxygen and nitrogen atoms is making it easy to form bonds such as Au—O and Au—N confirming the immobilization of the calixresorcinarenes, those bonds are formed by charge transfer interactions between the gold atoms and the free electrons of the oxygen and nitrogen atoms, subsequently, the incorporation of oxygen and nitrogen atoms through the gold adsorbent layer must create a higher electronic density between the N, O, and Au atoms. Also there are potentials of interactions between the aromatic cycles electrons and the gold surface as well.

3.2.3 Detection mechanisms of Pb²⁺

The lead ions detection mechanism strongly depends on the interaction described by the host-guest complexation occurring between the Pb²⁺ ions and the molecules which are already attached to the quartz crystal’s gold surface. Since the calixresorcinarenes are possessing electron donor heteroatoms such as N and O, therefore supporting the electron transfer between the sensing platform and the target metal ions. Accordingly, this process is causing an accumulation of the Pb²⁺ ions onto the sensing network at the gold surface (electrode)–solution (electrolyte) interface, the ions accumulation then could be detected piezo-gravimetrically, it’s presumed that due to the Pb²⁺ ions being chelated and complexed with the calixresorcinarenes ionophores, that both macrocycles can form complexes with lead(II) ions according to the reaction models (a) and (b), where the Ligand is the calixresorcinarenes, and the Mⁿ⁺ signifies the lead ions: Model a:  Mⁿ⁺_solution + Ligand_surface → [Mⁿ⁺Ligand]_surface Model b:  Pb²⁺_solution + Calixresorcinarene_surface → [Pb²⁺Calixresorcinarene]_surface

It is also expected that the accumulated lead ions can fit inside the ionophores cavities (Pb²⁺ adsorption), by a cation-π interaction either with the aromatic cycles double bounds and/or with the free electron doublets of the heteroatoms (O, N), this solid interaction is of huge importance in terms of the ion-binding ability between the ionophores and the target cations, since it was of interest to investigate if the lead ions will be attached to the calixresorcinarenes when immobilized to the gold surface, which was the case by the production of a piezo-gravimetic signal translated to a mass increase (Frequency decrease). However, these interactions are strong enough, and can’t be simply destroyed by using distilled water as regenerating solution (RS) for Pb²⁺ desorption (Model c), as it takes long time. Thus, the studies of developing a suitable regeneration process are in progress. Model c:  Pb²⁺(_{solution+ surface}) + RS_solution → [Pb²⁺RS]_solution

3.2.4 Calibration curves, linear ranges, and detection limits

One of the most important aspects of detection is having excellent sensitivity and lower detection limit (LDL). In order to characterize the sensitivity, linear range, and detection limits, for both sensing platforms (B and C), Fig. 10a and c show the calibration curves (Dynamic ranges) obtained as the normalized frequency variations against various Pb²⁺ concentrations, while Fig. 10b and d present the linear ranges. A noble linearity was obtained in both cases (Compound B: R² = 0.996, Compound C: R² = 0.982) for n = 4, and for specific linear regions described in Table 3; the linear regression equations for compounds B and C were: ΔF(Hz) = 0.008_*C(ppm) + 4.800, for compound B, and ΔF(Hz) = 0.020_*C(ppm) + 2.216, for compound C.

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Fig. 10

Calibration curves obtained for the Calix-QCM based sensor in the Pb²⁺ concentration range of 5–1000 ppm for compound B (a) and compound C (c). The linear range based on calibration curves for compound B (b) and compound C (d).

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The sensitivities were presumed to be the slopes of the linear fitted curves, whereas the detection limits were calculated based on the (3σ/S) relationship, where σ presents the standard deviation of the fitted curve and S presents the slopes obtained from the linear correlation curves.

Different characteristics of the studied calix- coated-QCM chemosensors are tabulated in Table 3. The sensitivities were in the order of 0.008 and 0.020 Hz ppm⁻¹, while the limits of detection were 0.45 and 0.30 ppm for compounds B and C, respectively. The obtained results confirm: the superior sensitivity and selectivity of compound C to Pb²⁺ ions over compound B; and the Pb²⁺ detection prospect by means of Calix-coated-QCM based sensors.

The obtained detection limits, shown in Table 3, are slightly higher than the WHO standard allowed in water (0.05 ppm), nevertheless are promising output. Furthermore, a sensitivity comparison of various methods and sensing platforms on the lead ions detection in aqueous solutions is presented in Table 4.