A comparative study on binding ability of three lanthanide ions with centrin using impedance method

Abstract

Centrin, an EF-hand calcium-binding protein, is involved in the formation of the microtubule organizing center (MTOC). The interactions between Gd³⁺, Eu³⁺, La³⁺ and the N-terminal domain of ciliate Euplotes octocarinatus centrin (N-EoCen) were investigated by electrochemical impedance spectroscopy (EIS) using potassium ferricyanide as a redox probe. Results show that an increase in the lanthanide ion (Ln³⁺) concentration resulted in a lower impedance magnitude in the Nyquist plots when the N-EoCen-modified glassy carbon electrode (N-EoCen-GC) was used. The titrations of N-EoCen with Ln³⁺ confirm the complexation reactivity of Ln³⁺ with N-EoCen and suggest that the Ln₂–N-EoCen complex was formed. In contrast, the binding ability of Ca²⁺ to N-EoCen is far less than that of Ln³⁺. The binding constants of the metal ions to N-EoCen are in the order of Gd³⁺ ≈ Eu³⁺ > La³⁺ ≫ Ca²⁺. Compared with the resonance light scattering (RLS) method, the EIS method can not only distinguish the binding ability of different metal ions to N-EoCen, but can also distinguish clearly the different metal ion binding sites on N-EoCen.

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1 Introduction

Centrin is a kind of low-molecular-weight acidic protein (about 20 kD). It belongs to the highly conservative EF-hand calcium-binding protein family. It plays an important role in centrosome orientation, mitosis and formation of the spindle and microtubule.^1–3 One important function of centrin is fiber contraction, which is calcium-dependent and closely related to the aggregation of centrin.^4–6 EoCen consists of two independent domains tethered by a flexible linker, each domain comprising a pair of EF-hand helix-loop-helix motifs that can potentially bind two calcium ions.⁷

Rare earth ions are widely used in rare earth micronutrient fertilizers in agriculture. They are important in the research on combinations between rare earth ions and various types of protein. In the past few years, our group has been studying the interaction between rare earth ions and centrin using methods such as fluorescence, CD spectra and polyacrylamide gel electrophoresis.^8–12 Rare earth ions can be used as a probe for calcium ions, which are spectrally silent, as their coordination chemistry properties are similar to those of calcium and they have a similar ionic radius. Ln³⁺, including La³⁺, Tb³⁺, and Lu³⁺, can compete with Ca²⁺ at calcium-binding sites. The combination of Ln³⁺ with the four binding sites of EoCen is orderly, similar to that with Ca²⁺.¹³ It has been reported that aggregation is closely related to the biological functions of EoCen, such as initiation of flagellar excision and fiber contraction. Ln³⁺ can induce the aggregation of EoCen, and the degree of aggregation induced by Lu³⁺ is about 5 times that with Ca²⁺. In addition, the importance of the N-terminal domain in the process of EoCen self-association has been proved.¹⁰ In the light of our previous work, the order of metal ion-induced conformational change of EoCen is Ca²⁺ < La³⁺ < Tb³⁺ < Lu³⁺.

Sensitized fluorescence is a necessary signal in monitoring the combination of Ln³⁺ and EoCen when using fluorescence spectrometry. However, as the fluorescence of EoCen cannot be greatly sensitized by most Ln³⁺ ions, this causes some limitations in using fluorescence methods. Recently, our group established a new electrochemical method. Using a cyclic voltammetry (CV) method, the interactions of Eu³⁺ with N-EoCen were studied.¹⁴

Compared with the CV technique, EIS can not only give the exact impedance data, but also reflects the nature of the interface directly. Here, we prepared a truncated ciliate EoCen, N-EoCen, including the first (binding site I) and second (binding site II) EF-hand domains. The combinational properties between Ca²⁺, La³⁺, Eu³⁺ and Gd³⁺ with N-EoCen were explored using EIS, CV and RLS. The mechanism by which the response of CV or EIS varies with the Ln³⁺ ion has also been investigated. The effect of ionic strength on the adsorption of N-EoCen was examined.

2 Experimental

2.1 Materials

A truncated ciliate EoCen, N-EoCen, including the first and second EF-hand domains, was expressed and purified as described earlier.¹² N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes) was purchased from Sigma and used without further purification. La₂O₃, Gd₂O₃, and Eu₂O₃ are 99.99% and purchased from Hunan, China. All other chemicals are of analytical grade. The Gd³⁺, Eu³⁺ and La³⁺ solution was prepared by dissolving the appropriate mass of the Gd₂O₃, Eu₂O₃ or La₂O₃ in hydrochloric acid, which was then standardized with EDTA in 0.1 M HAc–NaAc buffer at pH 5.5. Potassium ferricyanide as the probe reagent was purchased from Shanghai xin yu biological technology Co., Ltd.

2.2 Electrochemical study

A CHI 660C electrochemical analyzer (Chen Hua Instrumental Co., Shanghai), in connection with a glassy carbon working electrode (GCE, Ø = 3 mm), a saturated calomel reference electrode (SCE) and a platinum wire auxiliary electrode, was used for the electrochemical measurement. The GCE surface was freshly polished to a mirror by polishing with an alumina–water slurry (high-purity Al₂O₃, particle size 0.3 and 0.05 μm, BDH) and sonicated briefly, followed by thorough rinsing with water. The electrochemical impedance was tested employing a solution of 1.0 mM K₃Fe(CN)₆ containing different concentrations of KCl. The frequency range was from 10 mHz to 100 kHz, the DC potential was the average potential (0.175 V) of the oxidation and reduction peaks, and the amplitude was 10 mV. Impedance data were fitted to the appropriate model using the ZSimpWin software (Ametek). The potential sweep rate used was 50 mV s⁻¹ in CV experiments, using a potential window from 0.5 to −0.2 V.

2.3 Resonance light scattering

Resonance light scattering (RLS) of samples was monitored by fluorescence in quartz cells of 1 cm optical path at 25 °C. The RLS was performed in 0.01 M Hepes at pH 7.4, 0.02 M KCl with a fluorescence spectrometer (F-2500, Hitachi, Japan) using the same excitation and emission wavelengths. Samples were prepared by gradually adding Ln³⁺ into solutions of proteins. An equilibrium time of 5 min was used between each titration.

3 Results and discussion

3.1 Effect of ionic strength on the adsorption of N-EoCen

The adsorption of N-EoCen has been found to follow a Langmuir adsorption isotherm.¹⁴ Many electrochemical studies have been performed to evaluate the adsorption behavior of biomolecules. It has been shown that adsorption behavior is significantly affected by the ionic strength.^15–17 However, it is unclear what the effect of ionic strength is on the adsorption of N-EoCen.

The impedance under different ionic strengths was monitored versus the concentration of N-EoCen in 10 mM Hepes solutions (pH 7.4). Fig. 1A–D shows the Nyquist plots at a GC electrode using 1.0 mM potassium ferricyanide as probe ions in Hepes solutions containing 0.02, 0.04, 0.07 and 0.1 M KCl, respectively. The impedance results of Fig. 1 were fitted to the equivalent circuit (see Fig. 3), which is characteristic of an insulating layer over a conducting surface^18–22 and yielded the best fit among several other circuits (solid line in Fig. 1).

Fig. 1 The influence of ionic strength on the electrochemical impedance spectra of GC electrode in the presence of different concentrations of N-EoCen (from a to d): 3.9, 7.7, 11.4, 14.9 μM. In all experiments, the probe solution contains 0.01 M Hepes, pH 7.4 buffer solution in the presence of 1.0 mM Fe(CN)₆³⁻, (A): 0.02 M, (B): 0.04 M, (C): 0.07 M, (D): 0.1 M KCl.

Adsorption of N-EoCen onto the electrode surface can be described by the Langmuir isotherm equation.^23–26

The Langmuir isotherm equation can be rewritten as follows:

where C (mol cm⁻³) is the equilibrium concentration of the adsorbate in the bulk solution, Γ (mol cm⁻²) is the amount of protein adsorbed, i.e., surface concentration, Γ_max (mol cm⁻²) is the maximum value of Γ (saturated surface concentration) and the parameter B_ADS (cm³ mol⁻¹) reflects the affinity of the adsorbate molecules towards adsorption sites. Since R_ct is directly proportional to Γ, substitution of R_ct for Γ, and rearrangement of eqn (2) gives:

If the Langmuir isotherm is valid for an observed system, a plot of C/R_ct versus concentration C should yield a straight line with the parameters R_max and B_ADS derived from the slope and intercept, respectively.

The dependence of C/R_ct versus C in Hepes buffer containing different concentrations of KCl is shown in Fig. 2A. It shows that a linear dependence exists in the range 0.02–0.1 M KCl. B_ADS is obtained. Then the plot of B_ADS versus concentration of KCl is shown in Fig. 2B. It can be seen that the affinity of the N-EoCen towards the GC electrode surface increases with decreasing concentration of KCl. The low ionic strength is beneficial for the adsorption of N-EoCen.

Fig. 2 (A): Langmuir adsorption isotherm presented in a linearized form according to eqn (3) for N-EoCen adsorbed onto the GC electrode in 0.01 M Hepes buffer solution (pH 7.4) in the presence of 1.0 mM Fe(CN)₆³⁻, containing different concentrations of KCl. (B): Dependence of B_ADS and the Gibbs energy (ΔG_ADS) of adsorption on the concentration of KCl for N-EoCen adsorbed onto the GC electrode.

B_ADS can be presented by Gomma and Wahdan.²⁷

where R (J mol⁻¹ K⁻¹) is the gas constant, T (K) the temperature, ΔG_ADS (J mol⁻¹) the Gibbs energy of adsorption and 55.5 is the molar concentration of the water (mol dm⁻³), which is used as a solvent. Using this equation, the Gibbs energy of adsorption of N-EoCen onto the GC electrode surface in the Hepes buffer solution was calculated at 23 °C. The plot of ΔG_ADS versus concentration of KCl is also shown in Fig. 2B. It indicates that the Gibbs energy of adsorption decreases with decreasing concentration of KCl. It shows that N-EoCens are more easily adsorbed spontaneously at the glassy carbon electrode surface in conditions of low ionic strength. It can be concluded that N-EoCen adsorption decreases with increasing ionic strength. It indicates that electrostatic interaction plays the key role in the adsorption of N-EoCen, because electrostatic affinity between sorbent and protein will decrease in the presence of high concentrations of salt.^28–30 In order to obtain the best adsorption, the N-EoCen-GC electrodes are all prepared using 20 mM KCl solutions.

3.2 The interaction between La³⁺, Eu³⁺, Gd³⁺ and N-EoCen

3.2.1 AC impedance spectroscopy study of the interaction with different concentrations of Gd³⁺, Eu³⁺ and La³⁺. Compared with the CV technique, EIS is a more effective method to probe the features of surface-modified electrodes and for theoretical analysis of the impedance properties of the electrode. It enables one to understand chemical transformations associated with the conductive supports.³¹

EIS was used to study the interaction of the N-EoCen with Gd³⁺, Eu³⁺ and La³⁺. The N-EoCen was immobilized on the surface of the GC electrode according to the method described earlier.¹⁴ To obtain data about the impedance of modified electrodes, which allows tracking of the changes occurring at the interface, the Randles equivalent circuit shown in Fig. 3 was selected to fit the measured results. The changes in the charge transfer resistance (R_ct) indicate the binding process of Ln³⁺ with N-EoCen. Examples of impedance spectra in relation to the concentration of Gd³⁺, La³⁺ and Eu³⁺ are shown in Fig. 4A–C, respectively. The measurements were performed in the electrolyte solution containing 0.02 M KCl, 0.01 M Hepes buffer (pH 7.4), and 1.0 mM [Fe(CN)₆]³⁻ at room temperature. It can be seen that the diameter of the Nyquist circles decreases with increasing concentration of Ln³⁺. It indicates that an increase in Ln³⁺ concentration resulted in a lower impedance magnitude in the Nyquist plots in the low-frequency section. Fig. 4D shows the dependence of R_ct versus [Ln³⁺]/[N-EoCen].

Fig. 3 Equivalent electrical circuit (EEC) used to model the EIS data. The total impedance depends on several parameters such as: R_s, the electrolyte resistance, R_f, an adsorbed protein resistance, CPE₁, an adsorbed protein capacitance as a constant phase element, CPE₂, a double-layer capacitance as a constant phase element, R_ct, charge transfer resistance, and Z_w, Warburg impedance. A constant phase element, CPE, was introduced instead of pure capacitors in the fitting procedure to obtain good agreement between the simulated and experimental data.

Fig. 4 Impedance spectra obtained at the N-EoCen-coated electrode in 0.01 M Hepes, 0.02 M KCl solutions (pH 7.4) in the presence of 1.0 mM Fe(CN)₆³⁻ containing different concentration ratios of Ln³⁺ to N-EoCen, from 1 to 9 for Gd³⁺ (A): 0.37, 0.56, 0.74, 0.93, 1.11, 1.58, 1.95, 2.23, 2.51. From 1 to 10 for La³⁺ (B): 0.45, 0.75, 1.05, 1.20, 1.35, 1.50, 1.80, 2.03, 2.25, 2.48. From 1 to 10 for Eu³⁺ (C): 0.45, 0.63, 0.89, 1.07, 1.43, 1.61, 1.79, 1.97, 2.50, 2.78. (D): Changes of charge-transfer resistance (R_ct) of probe ions with different [Ln³⁺]/[N-EoCen].

It can be seen from Fig. 4D that R_ct decreased slowly below 1.0 equiv. of Ln³⁺. However, R_ct decreased rapidly with an additional 1.0 equiv. of Ln³⁺. It changed slowly above 2.0 equiv. of added Ln³⁺. The titration curve shows that the electron-transfer resistance varies with the concentration of added Ln³⁺, due to the combination of Ln³⁺ with N-EoCen. The first equivalent of added Ln³⁺ appears to be bound quantitatively with minimal response. A stronger electrochemical response was induced with the addition of another equivalent of Ln³⁺, which reached a final plateau at a [Ln³⁺]/[N-EoCen] ratio of up to 2 : 1. The titration curves reveal two breaks at [Ln³⁺]/[N-EoCen] = 1.0 and 2.0, confirming that a 2 : 1 stoichiometric ratio of the Ln₂–N-EoCen complex was formed. It can be seen that there are two inequivalent changes in R_ct with increasing concentration of Ln³⁺, corresponding to the two different binding sites in N-EoCen.

In order to compare the results measured in the same conditions, the binding constants were calculated using a Langmuir isotherm. This approach has been successfully applied for the determination of binding constants between cations and crown ethers.^32–34 The Langmuir isotherm assumes an equal binding energy for all binding sites.

In the experiment presented, the changes in R_ct are related to the binding of Ln³⁺ to N-EoCen. As to the combination with the first binding site of N-EoCen, the relation between the occupied binding sites Θ and the change in R_ct is as follows:

For the second binding site of N-EoCen, the relation is as follows:

R_ct(i), and R_ct(0) mean the charge transfer resistances of the N-EoCen-GC electrode with and without the particular concentration of Ln³⁺ studied, respectively. R_ct(1) and R_ct(∞) are the charge transfer resistances when the first binding site is occupied and when the concentration of Ln³⁺ is infinitely large.

In the case of the Langmuir isotherm, Θ can be related to the binding constant K_a according to the equation:³⁵

where K_a denotes the binding constant and c is the equilibrium concentration of Ln³⁺ in the solution. The linearization of the Langmuir isotherm gives:

Combination of eqn (5), (6) and (8) gives:

Eqn (9) and (10) have been applied for the calculation of the binding constants for Ln³⁺–N-EoCen complexes associated with the first and second binding sites.

The ratio [R_ct(0) − R_ct(i)]/[R_ct(i) − R_ct(1)] varies linearly with the concentration of Gd³⁺ in the range [Gd³⁺]/[N-EoCen] from 0 to 1.0 (Fig. 5A), and [R_ct(1) − R_ct(i)]/[R_ct(i) − R_ct(∞)] also varies linearly in the range from 1.0 to 2.0 (Fig. 5B). Therefore, the binding constants could be calculated from the slope. Similarly, the linear plots for La³⁺ (Fig. S1C and D†) and Eu³⁺ (Fig. S1A and B†) were further explored, and the binding constants were obtained.

Fig. 5 The linear relationship of [R_ct(0) − R_ct(i)]/[R_ct(i) − R_ct(1)] vs. [Gd³⁺] (A). The linear relationship of [R_ct(1) − R_ct(i)]/[R_ct(i) − R_ct(∞)] vs. [Gd³⁺] (B).

The affinities of Gd³⁺, Eu³⁺ and La³⁺ towards binding site I of N-EoCen are (8.42 ± 0.27) × 10⁶, (8.25 ± 0.97) × 10⁶ and (5.21 ± 0.44) × 10⁶ M⁻¹, respectively, while the affinities of Gd³⁺, Eu³⁺ and La³⁺ towards binding site II of N-EoCen are (8.06 ± 0.78) × 10⁶, (7.45 ± 0.55) × 10⁶ and (2.75 ± 0.34) × 10⁶ M⁻¹. The ionic potentials for Ln³⁺ and the binding constants calculated are summarized in Table 1. On the whole, the affinity order of Ln³⁺ binding to N-EoCen is Gd³⁺ ≈ Eu³⁺ > La³⁺, which is consistent with the order of the Ln³⁺ ionic potentials (see Table 1).

Table 1 Ionic potentials and binding constants of metal ions to N-EoCen

Metal ion	Gd^3+	Eu^3+	La^3+	Ca^2+
Ionic potential e/r	0.0320	0.0316	0.0283	0.0202
Binding constant KI/M^−1	(8.42 ± 0.27) × 10^6	(8.25 ± 0.97) × 10^6	(5.21 ± 0.44) × 10^6	(7.35 ± 0.50) × 10^3
Binding constant KII/M^−1	(8.06 ± 0.78) × 10^6	(7.45 ± 0.55) × 10^6	(2.75 ± 0.34) × 10^6	(7.35 ± 0.50) × 10^3

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We speculate that the R_ct change with adding Ln³⁺ may be due to N-EoCen aggregation induced by Ln³⁺. This is because the ferricyanide probe molecules can more easily approach the vacant GC electrode surface resulting from the aggregation of N-EoCen.

EIS measurement can provide a reliable method to detect the interaction between the metal ions and the protein, and can further distinguish the binding abilities of different metal ions to N-EoCen. This measuring system is relatively simple in comparison to other spectroscopic methods. The spectroscopic method often needs to introduce a strong chromophore into N-EoCen, benefiting from the energy transfer between Ln³⁺ and chromophore residues.

3.2.2 Cyclic voltammetry measurement. In order to further confirm the conclusions obtained from EIS experiments, the CV response of Fe(CN)₆³⁻/⁴⁻ to Gd³⁺, Eu³⁺ and La³⁺ binding to N-EoCen film was investigated. The CV method has proved useful for detecting the binding ability of Eu³⁺ to N-EoCen.¹⁴ Fig. 6A shows the CV of Fe(CN)₆³⁻/⁴⁻ in 10 mM Hepes buffer (20 mM KCl) using a N-EoCen-GC electrode containing different concentrations of Gd³⁺. After adding increasing concentrations of Gd³⁺ the redox peaks were increased markedly. This implies that the N-EoCen immobilized on GC electrode can react with Gd³⁺. Following the same procedure, the CV profiles of La³⁺ and Eu³⁺ were also explored and show a similar increasing tendency of the redox peaks (Fig. S2A and B†).

Fig. 6 Cyclic voltammograms of 1.0 mM Fe(CN)₆³⁻ at a N-EoCen-coated GC electrode in 0.01 M Hepes, 0.02 M KCl solutions (pH 7.4) containing different concentrations of Gd³⁺ (A) (from a to l): 0, 2.16, 2.87, 3.58, 4.28, 5.32, 6.01, 6.69, 7.37, 8.37, 9.37, 10.4 μM. (B) The peak separations (ΔE_p) of the redox reaction as a function of [Ln³⁺]/[N-EoCen].

The peak separations (ΔE_p) of the redox reaction as a function of [Ln³⁺]/[N-EoCen] are presented in Fig. 6B. With an increasing concentration of Ln³⁺, the peak separation decreased. It can be seen from Fig. 6B that the peak separation decreased slightly below 1.0 equiv. of Ln³⁺, while with an additional 1.0 equiv. of Ln³⁺ the peak separation decreased rapidly, and then it changed slowly above 2.0 equiv. of Ln³⁺.

The titration curves (Fig. 6B) in the CV experiment show a similar shape to the curves from EIS (Fig. 4D), indicating that the results obtained from the CV and EIS experiments match each other well.

3.2.3 Characterization of resonance light scattering in Gd³⁺–N-EoCen, Eu³⁺–N-EoCen and La³⁺–N-EoCen complexes. RLS is a valuable technique for detecting and characterizing extended aggregates of chromophores,¹⁰ since the aggregations lead to the formation of large fractal structures that exhibit strong RLS signals.³⁶ Here, fluorescent RLS measurements were used to study the protein conformation changes and aggregation properties.

A series of fluorescence RLS measurements was conducted at different wavelengths between 250 and 650 nm. The aggregation of N-EoCen in the presence of Gd³⁺ was monitored (Fig. 7A). A titration curve of RLS versus [Gd³⁺]/[N-EoCen] was plotted from Fig. 7A, as shown in Fig. 7B. When the first 1.0 equiv. Gd³⁺ was added to the solution of N-EoCen, RLS increased clearly. When the additional Gd³⁺ was added, RLS was enhanced significantly and finally reached a larger amplitude at 2.0 equiv. Gd³⁺. RLS for the addition of La³⁺ and Eu³⁺ was also measured in our experiment and this shows a similar increasing tendency of RLS (Fig. S3A and B†). The titration curves for La³⁺ and Eu³⁺ are also shown in Fig. 7B. These RLS experiments show similar titration curves to those obtained from electrochemistry experiments (Fig. 4D and 6B), which implies that aggregation of N-EoCen induced by Ln³⁺ plays a critical role in electrochemical titration experiments. The aggregation degree of N-EoCen induced by Ln³⁺ also conforms to the order Gd³⁺ ≈ Eu³⁺ > La³⁺. This result is consistent with that obtained from EIS measurement.

Fig. 7 (A): Resonance light scattering spectra for the addition of Gd³⁺ to the solution of N-EoCen in 10 mM Hepes at pH 7.4, 0.02 M KCl at [Gd³⁺]/[N-EoCen] = 0 (a); 0.28 (b); 0.55 (c); 0.83 (d); 1.38 (e); 1.66 (f); 2.07 (g); 2.43 (h); 2.71 (i); 2.98 (j). (B): The titration curve of N-EoCen with the addition of Gd³⁺ (), Eu³⁺ () and La³⁺ (), using the resonance light scattering value at 370 nm in 10 mM Hepes at pH 7.4, 0.02 M KCl.

In combination with the high similarity of the three types of experiment and the nature of aggregation reflected by RLS, this can provide valid evidence that the titration curve in electrochemical experiments reflects the aggregation property of N-EoCen to a certain extent.

In RLS titration curves, there is a break at [Ln³⁺]/[N-EoCen] = 1.0, which can be faintly observed. However, there is a clear break at [Ln³⁺]/[N-EoCen] = 1.0 in the EIS (Fig. 4D) and CV titration curves (Fig. 6B), suggesting that electrochemical methods can distinguish these two binding sites more easily than RLS experiments. Moreover, the RLS titration curves of La³⁺, Eu³⁺ and Gd³⁺ are very close. It is difficult to distinguish the aggregation degrees for these three Ln³⁺ clearly. However, due to the sensitivity of probe ions to protein–Ln³⁺ interaction at N-EoCen-GC, EIS and CV can distinguish the different binding abilities of Ln³⁺ to N-EoCen easily.

3.3 The interaction between Ca²⁺ and N-EoCen

Centrin is a calcium-binding protein. The combination of centrin and Ca²⁺ can cause protein aggregation and conformational change in centrin. Based on this characterization of Ca²⁺ binding, its natural biological function can be evaluated. Here, the interaction between Ca²⁺ and N-EoCen has also been investigated by CV. Fig. 8A shows the CVs of Fe(CN)₆³⁻/⁴⁻ in 10 mM Hepes buffer (20 mM KCl) using a N-EoCen-GC electrode containing different concentrations of Ca²⁺. With an increasing concentration of Ca²⁺ the redox peak currents increase slowly. This implies that the N-EoCen immobilized on the GC electrode can react weakly with Ca²⁺.

Fig. 8 Cyclic voltammograms of 1.0 mM Fe(CN)₆³⁻ at a N-EoCen-coated GC electrode in 0.01 M Hepes, 0.02 M KCl solutions (pH 7.4) containing different concentrations of Ca²⁺ (A) (from a to k): 0, 74.5, 149, 222, 295, 367, 721, 1063, 1392, 1711, 2019 μM. (B): The peak separations (ΔE_p) of the redox reaction as a function of [Ca²⁺]/[N-EoCen].

The peak separations (ΔE_p) of the redox reaction as a function of [Ca²⁺]/[N-EoCen] were plotted (Fig. 8B). The titration curve shows that there is little change in CV when the concentration ratio of Ca²⁺ to N-EoCen is 1–10. Peak currents increase sharply and peak separations decrease clearly above [Ca²⁺]/[N-EoCen] ≧ 20. They change slowly for [Ca²⁺]/[N-EoCen] ratios up to 300 : 1. This indicates that Ca²⁺ combines with N-EoCen when excess Ca²⁺ is added. Compared with Fig. 6B, Ca²⁺ exhibits much lower binding ability to N-EoCen. This result is consistent with the conclusions reported.^7,37 Due to the weak affinity of Ca²⁺ for N-EoCen, we can substitute the free concentration of Ca²⁺ with the total concentration of Ca²⁺ and further evaluate the average binding constant of Ca²⁺ to N-EoCen. K_Ca–N-EoCen = (7.35 ± 0.50) × 10³ is obtained and also listed in Table 1. Then, the affinity order of metal ions to N-EoCen is Gd³⁺ ≈ Eu³⁺ > La³⁺ ≫ Ca²⁺, which is consistent with the ionic potential order (Table 1).

4 Conclusion

In this study, it is revealed that the adsorption of N-EoCen at a GC surface depends on ionic strength. With increasing ionic strength, the adsorbed amount decreased. This shows that electrostatic forces play an important role in the adsorption of N-EoCen. Currently, Ln³⁺ ion/N-EoCen interactions are investigated at the same ionic strength by EIS. Results show that the binding of Ln³⁺ to N-EoCen resulted in a lower impedance magnitude in the Nyquist plots. The Ln³⁺–N-EoCen complex obeys a 2 : 1 stoichiometry, and the two binding sites of N-EoCen are not equivalent, from the titration curves. The affinity order is Gd³⁺ ≈ Eu³⁺ > La³⁺ ≫ Ca²⁺. EIS is a more sensitive method than RLS for differentiating the two binding sites of N-EoCen and the binding abilities of different Ln³⁺.

Acknowledgements

This work was supported by the national natural science foundation of the People's Republic of China (Grant nos 20771068 and 20901048) and the PhD Programs Foundation of the Ministry of Education of China (Grant no. 20131401110011).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra08099h

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