Squaramide-based tripodal ionophores for potentiometric sulfate-selective sensors with high selectivity†

Yueling Liu ^a, Yu Qin *^a and Dechen Jiang *^ab
aState Key Laboratory of Analytical Chemistry for Life science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, China 210093
^bState Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, China. E-mail: qinyu75@nju.edu.cn; dechenjiang@nju.edu.cn; Fax: +86-25-83592562, +86-25-83594846; Tel: +86-25-83592562, +86-25-83594846

First published on 27th May 2015

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Introduction

Anion recognition is more challenging than metal ion measurement because the anions usually have larger size, polyhedral structure, pH dependence, low charge-density and high solvation energy.¹ Generally, the binding of anions with receptors is mainly based on hydrogen bonds,² electrostatic forces,³ ion pairing,⁴ or Lewis acid–base interactions.⁵ Therefore, the emerging anion detection was primarily performed in organic or organic/aqueous mixed solvents to avoid strong solvation of polar solvents. Adjustable changes in absorbance, fluorescence or chemiluminescence could be observed upon anion complexation.⁶ However, the introduction of organic solvents results in the limited application of these systems for biological measurements, which motivates people to investigate new approaches in aqueous solution.

Carrier-based ion-selective electrodes (ISEs) are competent for that drawback,⁷ in which the ionophore is immobilized in the membrane phase on the electrode to selectively recognize target ions in aqueous solution. Moreover, ISEs enjoy many special advantages such as simple fabrication, easy miniaturization, facile portability, inexpensive installations, and no sample destruction.⁸ The detection of anions near the end of Hofmeister series (organic anions > ClO₄⁻ > SCN⁻ > I⁻ ∼ salicylate⁻ > NO₃⁻ > Br⁻ > Cl⁻ > HCO₃⁻ > HPO₄²⁻ ∼ F⁻ ∼ SO₄²⁻) is difficult because they typically have higher hydrophilicity.⁹ The high hydrophilicity decreases the extraction ability of the ISE membrane to the target ions so that the detection sensitivity and selectivity are restricted.¹⁰ Especially, the sulfate ion with an orthogonal tetrahedral coordination structure is at the end of the Hofmeister series. Sulfate is one of the most important macronutrients in cells and is involved in biological processes, such as the biosynthesis and detoxification of many endogenous and exogenous compounds.¹¹ Also, high levels of sulfate in drinking water might be associated with diarrhea.¹² Therefore, sulfate determination is important in many fields; however, the reported selectivity coefficients of solid-state and ionophore-free ion-exchange sulfate electrodes are not satisfactory for application in complex samples. To fulfil these applications, researchers have devoted attention to the synthesis of size- and shape-selective anion carriers or the usage of new membrane matrix materials for the efficient identification of sulfate.^9,13 Considering the significance of the ionophore topology and the unique anion characteristics on the overall ionophore–ion interaction, acyclic ionophores were usually more favorable.¹⁴ Bachas’s group reported the first tripodal receptor molecule as the ionophore for the sulfate ion, in which sulfate complexation was through H-bonds of urea groups in a three dimensional cavity. The three-dimensional rigid molecular architectures mimicked the recognition of sulfate-binding protein with the sulfate anion,¹⁵ and thus, offered high affinity and selectivity.¹⁴

For further improvement of the sulfate-selective ISE performance, a more robust functional group in the three-dimensional ionophore as a hydrogen bonding donor was needed. The theoretical studies showed that squaramide groups were superior to urea or thiourea as a hydrogen bonding donor,¹⁶ which was explained by aromatic features of the squaramide ring upon complexation of the anions.¹⁷ Moreover, the squaramide group bearing two hydrogen bond donor atoms was validated to be ideal ingredients allowing for selective binding of oxoanions in water.¹⁸ Therefore, in this work, squaramide groups were introduced into the tripodal structure as new ionophores for the fabrication of sulfate-selective sensors. Three receptors (Fig. 1) with increasing polarity, namely unsubstituted (I), p-carbon trifluoride (II), and p-nitro (III) phenyl groups present at the end of arms, were used as the ionophores. These receptors had been reported to recognize sulfate ions in DMSO-d₆.^18b The binding constants for sulfate of Ionophores I, II and III obtained using NMR titration were 4.75, 4.87 and 4.95 M⁻¹, respectively.^18b Therefore, it could be proposed that stronger binding of these receptors with sulfate ions in the membrane could offer a higher electrode selectivity. Different plasticizers were used to study the compatibility of Ionophores I–III within the polymeric membrane. Their detection sensitivity and selectivity were evaluated in detail. Finally, the proposed potentiometric sulfate-selective sensor was applied for sulfate determination in cell lysates and drinking water.

Experimental section

Reagents

High molecular weight poly(vinyl chloride) (PVC), tridodecyl methylammonium chloride (TDMACl), tetrahydrofuran (THF), bis(2-ethylhexyl)sebacate (DOS), 2-nitrophenyl octyl ether (NPOE) and bis(thiourea) ionophore were of selectophore grade and were purchased from Fluka (Switzerland). Dioctyl phthalate (DOP) was from Aldrich (USA). Dimethyl phthalate (DMP) and di-n-butyl phthalate (DBP) were obtained from Alfa Aesar (a Johnson Matthey Company). 2-Fluorophenyl 2-nitrophenyl ether (FPNPE) was also of Selectophore grade and was acquired from Heowns (Tianjin Heowns Biochem LLC). All sodium salts of analytical grade including sulfate, phosphate, chloride, bromide, iodide, nitrate, bromide, iodide, thiocyanate, perchlorate and N-(2 hydroxyethyl)piperazine-N-(2-ethanesulfonic acid) (HEPES) were obtained from Sangon (Shanghai, China). All aqueous solutions were prepared in freshly deionized water (resistance 18.25 MΩ cm⁻¹, Millipore, USA). Ionophores I–III were kindly provided by Professor Chen Lin from School of Chemistry and Chemical Engineering at Nanjing University. Details of the preparation of squaramide-based tripodal ionophores could be found in the ESI† of the literature.^18b

Membranes

Gold disk electrodes were pre-processed as described previously.¹⁹ The sensing membrane cocktails (total mass 100 mg) were prepared by dissolving 0.8 wt% of Ionophores I–III, TDMACl (30, 50 or 70 mol%, relative to the ionophore), a plasticizer and PVC (mass ratio 2:1) in 1 mL of THF and sonicated for 30 min. The various compositions of the membranes used in this study were described in the text. Afterwards, 100 μL of the cocktail was drop cast on the gold electrodes evenly and the solvent was evaporated thoroughly at ambient temperature. Finally the fabricated electrodes were conditioned in a 10 mM sodium sulfate solution overnight before the measurements.^13c,d,14

Measurement procedure

The potentiometric responses were measured with a 16-channel interface (Lawson Labs, Inc.) controlled by a PCI-6281 data acquisition board and LabView 8.5 software (National Instruments, Austin, TX) on a PC at room temperature. A double-junction Ag/AgCl/3 M KCl reference electrode containing 1 M CH₃COOLi bridge electrolyte by Metrohm Ion Meter (Switzerland) was used. Activity coefficients were calculated according to the Debye–Huckel approximation and selectivity coefficients were evaluated using the IUPAC Separate Solution Method (SSM).²⁰ All the potential results were the average from at least three electrodes. The phosphate buffer was prepared with 10 mM NaH₂PO₄ and Na₂HPO₄. The pH values of HEPES solutions (10 mM) were adjusted by a calibrated glass electrode (PB-10, Sartorius, Göttingen, Germany) with NaOH.

Cell lysate

HeLa cells were cultured at 37 °C in Dulbecco's modified Eagle's medium (DEME) containing 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin/streptomycin) under a humidified atmosphere with 5% CO₂. The cells were divided into two groups and incubated in requisite concentrations of sulfate solution and HEPES solution (as the blank control), respectively, for 30 min. After the incubation, they were harvested by centrifugation (Centrifuge CT14RD, TECHCOMP) and washed with 10 mM HEPES solution at least three times. Then, the cells were lysed for 10 min in a JY92-II N ultrasonic disintegrator at 4 °C (Ningbo Scientz Biotechnology Company, China). The cell solutions were centrifuged at 14000 rpm for 20 min, and the pellets were discarded. The supernatants were immediately analyzed by Ionophore III and bis(thiourea) ionophore based electrodes, respectively.

Results and discussion

Optimization of electrode performance

Herein, we explored a new family of tripodal squaramide-based receptors as ISE ionophores for all solid-contact sulfate-selective electrodes. Once sulfate came in contact with a tripodal ionophore incorporated membrane, sulfate was expected to be encapsulated in a three-dimensional cage through hydrogen bonds (N–H⋯O) by the squaramide groups. This binding mimicking the sulfate-binding proteins should lead to better electrode performance.¹⁴ For the three Ionophores I–III used in our work, Ionophore I had un-substituted phenyl groups, while Ionophores II and III had p-carbon trifluoride substituted and p-nitro substituted phenyl groups, respectively. More electron withdrawing ability of the N atoms at squaramide could offer stronger hydrogen bonding with sulfate. Considering that the electron withdrawing ability of nitro groups was higher than carbon trifluoride groups, Ionophore III might have the highest hydrogen bond donating ability. In our study, three ionophores were used for the fabrication of sensors, and the performances of these sensors were compared.

A plasticizer as a membrane solvent has a remarkable influence on the physical properties and mobility of their concomitant constituents.^7,21 Since tripodal squaramide ionophores had poor solubility in DOS (ε = 3.9), DBP (ε = 6.4) and DMP (ε = 8.5) with low dielectric constants, the more polar plasticizers NPOE (ε = 24) and FPNPE (ε = 50) were chosen.²² Their response characteristics are shown in Table 1 together with their membrane composition. Ionophore I and Ionophore II in NPOE-plasticized membranes (M1 and M2, Table 1) demonstrated a near Nernstian response of −27.6 and −25.9 mV per decade, respectively, to sulfate concentrations ranging from 1 μM to 100 mM. However, Ionophore III could not be soluble in NPOE, which might be attributed to the largest polarity of p-nitrophenyl groups attached in the structure. Therefore, the most polar plasticizer FPNPE was used to characterize the Ionophore III incorporated membrane. In FPNPE, Ionophore III-based membranes (M5, Table 1) exhibited near Nernstian slopes of −28.6 mV per decade, and Ionophore I and Ionophore II-based membranes (M3 and M4, Table 1) showed the slopes of −25.0 and −25.6 mV per decade, respectively, to sulfate in the linear range from 1 μM to 100 mM. The results suggested that the polarity of the substituted group in the phenyl ring of the tripodal arms was critical to the compatibility of the ionophores in a plasticized polymer matrix and thus offered a better electrode performance.

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With the recognition between ionophores and ions, the extraction of specific anions from the aqueous solution into the membrane could be enhanced by selective complexation.^10a The selectivity coefficients were calculated using the Separate Solution Method (SSM).²⁰ The selectivity patterns of TDMACl-based membranes without any ionophores were taken as the blank control. Given that the phosphate anions were pH dependent, the determination was performed in 10 mM MES of pH 4.5 so that dihydrogen phosphate was predominant. Under these conditions, as compared with control electrodes, the selectivities of Ionophore I, II or III incorporated electrodes for sulfate over phosphate increased at least four orders of magnitude (see Table 1). For other interfering anions, the selectivity was characterized in 10 mM HEPES of pH 7.0. A significant increase in the selectivity was observed in Ionophore I, II or III incorporated electrodes, which supported that these tripodal squaramide-based receptors were better ionophores for the recognition of sulfate ions. More specifically, the observed highest selectivities of Ionophore III in FPNPE-plasticized membranes (M5, Table 1) over H₂PO₄⁻, Cl⁻, Br⁻, NO₃⁻, SCN⁻, I⁻ and ClO₄⁻ were −3.6, −2.1, −1.2, +0.8, +4.2, +5.7 and +6.3, respectively. In comparison with the blank membranes, the selectivity of M5 over others, such as Cl⁻, Br⁻, NO₃⁻ and ClO₄⁻, improved more than six orders of magnitude. As a result, Ionophore III was chosen as the ionophore for the following optimization.

The mole ratio of the ionophore and the ion-exchanger in the membrane is another essential factor to obtain optimized selectivity.^7,23 We studied the response of Ionophore III-based membranes with different concentrations of the ion-exchanger TDMACl. As shown in Fig. 2, the comparison of the selectivities from membranes containing 30, 50 and 70 mol% TDMACl relative to the ionophore showed that 30 mol% TDMACl based membranes gave the best selectivity. The response slope of five optimized Ionophore III-based sensors with 30 mol% TDMACl relative to the ionophore was −30.2 ± 0.7 mV per decade in the linear range from 1 μM to 100 mM. The relative standard deviation of the electrode response reached 2.3% indicating good reproducibility (Fig. 3). The response time at each sulfate concentration was less than 20 seconds. The sulfate selectivity (logK_{SO4²⁻, J}, SSM) was obtained as follows: H₂PO₄⁻, −4.3; Cl⁻, −3.4; Br⁻, −2.5; NO₃⁻, −0.6; SCN⁻, +3.1; I⁻, +3.4; ClO₄⁻, +5.9 (as shown in Fig. 4, row A).

An anionic additive (NaTFPB) or no additive was attempted to fabricate the ionophore based films; however, no response was observed, as shown in Fig. S1 (ESI†). The results suggested that our probe acted as a neutral ionophore, which generated an anionic response in the presence of cationic sites in the membrane.²⁴ The sensitivity and stability of the sensors were maintained with a calibration slope from −30.2 to −27.9 mV per decade in at least 10 days, which was close to the 7 days reported on the urea-based tripodal ionophore based electrodes.¹⁴ The lifetime could be prolonged to more than one month when stored in air. The discrepancy in the lifetime under different storage conditions might be attributed to the slow decomposition of the ionophore in water. In addition, water could form hydrogen bonds with the ionophore. Therefore, after long time conditions, the membranes were saturated with a sulfate solution leading to poor permselectivity.

To show the excellent selectivity of our electrode, the selectivity patterns of some reported sulfate-selective ionophores, including bis(guanidinium), bis(thiourea) and tripodal ionophore with urea groups, were compared with those of Ionophore III based membranes in Fig. 4. Compared with bis(guanidinium) (row C) and bis(thiourea) ionophore (row D),^13c,d an Ionophore III-based membrane (row A) exhibited at least three orders of magnitude increase in the selectivity over the anions. Considering the planar structure of bis(guanidinium) and bis(thiourea), this improvement confirmed the superiority of tripodal topology. For the previously reported tripodal ionophore with urea groups, the matched potential method (MPM) was used in that work and the selectivity coefficients obtained from MPM could not be compared with the values from SSM in our work. Thus, the selectivity coefficient values were estimated from the calibration curves published in the paper and are shown as row B in Fig. 4.¹⁴ A higher selectivity was observed in our Ionophore III-based electrodes, revealing the reduced interference from interfering anions, which verified the advantages of squaramide groups over urea groups as hydrogen donors in polymeric membranes.

Optimization of detection conditions

The effect of pH on tripodal squaramide ionophore-based membranes was characterized in 10 mM HEPES. As shown in Fig. 5, the electrode response was independent of pH in the range of 3–8. Further increasing the pH to the range of 8–11 resulted in a negative slope of −23.7 mV per pH. The negative slope for pH over the range of 8–11 might be attributed to the response of OH⁻, which was negatively charged and resulted in the negative slope. Afterwards, the responses of tripodal squaramide ionophore-based electrodes toward sulfate ions in different buffer solutions were investigated. The electrodes behaved similarly with Nernstian slopes of −30.2 and 29.6 mV per decade in HEPES (pH 7.0) and unbuffered solution, respectively, with the measuring range from 1 μM to 100 mM. However, the detection of sulfate ions in 10 mM phosphate buffer (pH 7.0) with concentration lower than 1 mM was disturbed. Under these conditions, the ratio of monohydrogen phosphate and dihydrogen phosphate was about 3:2. Since the tripodal squaramide ionophores had a good selectivity to sulfate against dihydrogen phosphate, complexation with monohydrogen phosphate was probably the major interference. Only when the sulfate concentration was higher than 1 mM, the electrodes gave a Nernstian response to sulfate, indicating effective recognition of sulfate in the presence of phosphate ions. Therefore, HEPES buffer of pH 7 was employed to ensure a good electrode performance.

Applications of a tripodal squaramide based electrode

All cells require inorganic sulfate for normal functioning. Since the main anion in the cytosol was chloride and the ionophore-based sensors had good selectivity to sulfate against chloride, our sensors should be feasible for the measurement of sulfate in the cell lysates for the biological study. Thus, the potentiometric sulfate-selective sensor was applied for sulfate detection in cell lysates. Due to the unknown concentration of various ions and some other biological substances in the cell lysate, the standard addition method was applied to ensure detection accuracy. Experimentally, the cells were treated in the medium with 1 M sulfate ions so that the intracellular sulfate ion might reach the same concentration through the diffusion from the extracellular medium to the cytosol. Assuming the individual cell's volume of 1 pL,²⁵ 0.33 and 3.6 million cells gave the cellular volume of 0.33 and 3.6 μL,²⁶ and then, the total sulfate in cells was 0.33 and 3.6 μmol. After lysis in 1 mL of buffer, the final concentrations of 0.33 and 3.6 mM in cellular lysates were obtained. The continuous addition of sulfate ions into the lysate established the calibration curve with a slope of −27.9 ± 0.9 mV per decade. The linear detection range was from 10 μM to 100 mM and the concentrations of sulfate ions in cell lysates were determined to be 337.9 μM and 3.51 mM (Table 2), which were close to the previous estimation. The experimental potentials are shown in Table S1 (ESI†). Commercially available sulfate ionophore bis(thiourea) was employed for sulfate determination in the cell lysates as well. The bis(thiourea)-based electrodes had a slope of −25.7 ± 1.4 mV per decade within the same sulfate concentration range. However, the concentration of sulfate ions in the cellular lysate was determined to be higher than 100 mM, which was away from the estimation. This deviation should be attributed to the relatively poor selectivity of a commercial ionophore based electrode so that the interruption of various ions and other biological substances on the measurement was significant.

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Meanwhile, the squaramide tripodal based sensors were applied for the determination of sulfate ions in drinking water samples. 66 and 272 mg sodium sulfate were added to drinking water to generate the concentrations of ∼9 and 40 mM sulfate. Based on the recovery experiments, calibration plots of Ionophore III-based electrodes were conducted in drinking water. A near Nernstian response of −28.9 ± 0.4 mV per decade within the measuring range from 1 μM to 100 mM was obtained. The amounts of sulfate in these two solutions were measured to be 66.6 mg and 266.3 mg, respectively (Table 2). The experimental potentials are shown in Table S1 (ESI†). The satisfactory outcomes in Table 2 proved that the proposed sensor has the accuracy and reliability for sulfate detection in physiological fluids and the environment.

Conclusions

Sulfate ion-selective sensors based on squaramide-based tripodal ionophores in polymeric membranes have been described in this work. The ionophores provided a suitable cavity and flexibility for sulfate by mimicking the sulfate-binding protein mechanism to offer a better sensor performance. The proposed sensors showed a Nernstian response toward sulfate with the so far best selectivity, which led to the success of the analysis of sulfate in the cellular lysate and drinking water. Our future work will focus on the improvement of the film properties for longer lifetimes. Also, sulfate-selective microelectrodes will be fabricated to determine the local efflux of sulfate from single cells for the study of sulfate transport in the cells.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant 21375061) and the open research fund from State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5an00259a

This journal is © The Royal Society of Chemistry 2015