Biomimetic membranes based on molecularly imprinted conducting polymers as a sensing element for determination of taurine

Accepted Manuscript Title: Biomimetic membranes based on molecularly imprinted conducting polymers as a sensing element for determination of taurine Author: Justyna Kupis-Rozmysłowicz Michał Wagner Johan Bobacka Andrzej Lewenstam Jan Migdalski PII: DOI: Reference:

S0013-4686(15)30931-2 http://dx.doi.org/doi:10.1016/j.electacta.2015.12.007 EA 26156

To appear in:

Electrochimica Acta

Received date: Revised date: Accepted date:

20-5-2015 29-11-2015 1-12-2015

Please cite this article as: Justyna Kupis-Rozmyslowicz, Michal Wagner, Johan Bobacka, Andrzej Lewenstam, Jan Migdalski, Biomimetic membranes based on molecularly imprinted conducting polymers as a sensing element for determination of taurine, Electrochimica Acta http://dx.doi.org/10.1016/j.electacta.2015.12.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Biomimetic membranes based on molecularly imprinted conducting polymers as a sensing element for determination of taurine

Justyna Kupis-Rozmysłowicza, Michał Wagnerb, Johan Bobackac, Andrzej Lewenstama,c, Jan Migdalskia

a Faculty of Material Science and Ceramics, AGH – University of Science and Technology, PL 30-059 Krakow, Poland b Biomolecular and Organic Electronics, IFM, Linköping University, S-581 83, Linköping, Sweden c Process Chemistry Centre, c/o Centre for Process Analytical Chemistry and Sensor Technology (ProSens), Åbo Akademi University, FI 20500 Turku, Finland *Corresponding Author: [email protected]

Abstract Molecularly Imprinted Conducting Polymer based films (MICP films) devoted for the taurine determination is described. MICP films were electrodeposited from an aqueous solution containing 3,4-ethylenedioxythiophene, acetic acid thiophene, flavin mononucleotide, and taurine. The presence of taurine inside freshly deposited MICP films as well as absence of taurine in the outer layer of the MICP film after extraction process was confirmed by the XPS spectra. It was found that after taurine extraction, MICP films can be used as a potentiometric sensor giving close to Nernstian response towards taurine equal to 53.8 ± 2.6 mV/p[taurine] in the concentration range 10−2 to 10−4 mol·dm-3.

Keywords: Molecular Imprinting, Biosensor, Taurine, Conducting Polymer

1. Introduction Conducting polymers (CPs) have been used in a range of chemical sensing applications, wherein molecular recognition properties are engineered through receptor layers forged through molecular imprinting. Molecular imprinting (Figure 1) is a process where monomeric receptors are embedded into a polymeric mold to impart the mold with molecular selectivity. A prepolymerized complex is formed between a template molecule (e.g. the target analyte of the sensor) and one or more functional monomers by either covalent [1, 2] or non-covalent [3, 4] interactions. An excess of cross-linking agent is added to the pre-polymerized complex to further polymerize the complex into a molecular imprinting polymer (MIP) [5]. Template removal from the MIP matrix is done through solvent washing or through a relatively mild chemical treatment [6]. Removal of the template leaves taurine-selective recognition sites, or characteristic cavities, that are complementary to the template molecule in terms of size, shape, and functional group orientation [7]. These empty cavities are able to rebind analyte in a selective and reversible manner, allowing these cavities to be used as embedded receptors for detecting molecules such as taurine. Taurine (2-aminoethane sulphonic acid) is a naturally occurring organic acid present in most living organisms, with the highest concentrations in the heart, brain, retina, and skeleton muscles [8]. Taurine plays an important role in numerous physiological [9] and pharmacological processes [10] such as membrane stabilization [11], intracellular Ca2+ regulation a known neuromediator and neuromodulator [12,13]. Deviations from normal taurine concentrations is often correlated with several pathological conditions such as diabetes, epilepsy [14], sepsis [15], retinitis pigmentosa [16], and different kinds of cancer [17], as well as different forms of psychosis including trauma [18], depression [19], and schizophrenia [20]. Several methods for evaluating taurine concentrations in bio-relevant samples (e.g. in plasma,

energy drinks, milk, egg white and yolk samples, and urine) have been reported in the literature. For example, the taurine content in plasma has been determined by gas chromatography [21], gas chromatography coupled with mass spectrometry [22], high-performance liquid chromatography [23-27], high-performance anion-exchange chromatography coupled with integrated pulsed amperometric detection [21], capillary electrophoresis [28], and spectrophotometry [29]. High-performance anion-exchange chromatography coupled with integrated pulsed amperometric has also been used to measure taurine concentrations in egg white and yolk samples as well as human serum and urine extracts [30]. Provided the ease and versatility with which potentiometric sensors can reliably and selectively detect a variety of chemicals [31], this work focuses on the development of a potentiometric taurine sensor based on molecularly imprinted conducting polymer (MICP) films. MICP film preparation

involves

the

electrochemical

polymerization

of

a

3,4-ethylenedioxy-

thiophene/acetic acid thiophene (EDOT/AAT) copolymer film doped with flavin mononucleotide (FMN). Each component serves a distinct function: the EDOT is used as the cross-linker, AAT is the monomer, FMN is the dopant, and taurine is the template, or target, molecule. Both the structure of the chemicals used and the hypothetical structure of the resulting MICP film are shown in Figure 2. The molecular imprinting of the PEDOT/AAT-based copolymer with taurine is hypothetically enabled by hydrogen bonding between taurine and the AAT molecules. It is hypothesized that the FMN, which is used as a dopant anion, enhances the imprinting process by introducing additional hydrogen binding sites that immobilize the taurine.

2. Experimental Part 2.1 Chemicals

2-aminoethanesulfonic

acid

(taurine),

3,4-ethylenedioxythiophene

(EDOT),

flavin

mononucleotide (riboflavin-5′-phosphate, FMN) and acetic acid thiophene (3-Thiophene acetic acid, AAT) were obtained from Sigma-Aldrich. The 2-hydroxy-5-sulfobenzoic acid (SSA) was obtained from Fluka. All other chemicals were of analytical grade and were used as received. Aqueous solutions were prepared with freshly deionized water (resistivity = 18.2 MΩ cm) from the ELGA PURELAB® Ultra water system.

2.2 Electrodeposition of the EDOT/AAT-based copolymer films Electrochemical deposition of the EDOT/AAT film was performed using the Autolab PGSTAT100 or PGSTAT20 analyzer (both from Eco Chemie, B.V., Netherlands) equipped with a general-purpose electrochemical system software. The electrodeposition was carried out in a single compartment, three-electrode electrochemical cell. Glassy carbon disc electrodes (GC) with diameter of an area around 0.07 cm2 or Pt-sheet electrodes with an area of about 0.16 cm2 were used as working electrodes that were to be covered with the copolymer film. The auxiliary electrode was a Pt wire with an area of about 0.5 cm2. The reference electrode was a double-junction silver chloride (AgCl) electrode (Metrohm or Mineral), and the bridge was filled with a 0.1 mol·dm-3 SSA solution. Prior to CP deposition, the GC disc electrodes were mechanically polished with 0.3 μm alumina oxide powder. Next, the electrodes were placed in 1 mol·dm-3 nitric acid (HNO3), sonicated in an ultrasonic bath for 5 minutes, rinsed with water, sonicated again in a 0.1 mol·dm-3 potassium hydroxide (KOH)/ethanol solution for 2 minutes, rinsed with water, and finally sonicated for an additional 10 minutes in methanol. This procedure was performed to clean and degrease the disc electrode surface. The Pt-sheet electrodes were cleaned by boiling them in concentrated

nitric acid for 15 minutes. Afterwards, the electrodes were rinsed with water and immediately immersed in the solution used for the electrochemical polymerization of the CP films. The MICP films were deposited from a solution containing 0.01 mol·dm-3 EDOT, 0.1 mol·dm3

AAT, 0.1 mol·dm-3 FMN, and 0.5 mol·dm-3 taurine. For comparison, non-imprinted

conducting polymer (NICP) films were deposited from the same solution, except without taurine. Both MICP and NICP films were deposited by potential cycling from 0 mV to 1100 mV with a scan rate of 50 mVs−1. Approximately 10 – 20 full anodic/cathodic potential scans were applied. The resulting films were rinsed with deionized water and allowed to dry. Prior to additional characterization, the quality of the dried films (adhesion, continuity, lack of cracks, and presence of uncovered GC surface) was inspected with a scanning electron microscope.

2.3 Preparation of MICP film-based taurine sensor In order to engineer MICP films that are sensitive to taurine, the taurine template molecules need to be extracted from the outer layer of the MICP film. The extraction process was performed using several mixtures: acetic acid and methanol, methanol and water, 0.1 mol·dm3

KOH solutions, and 0.1 mol·dm-3 SSA solution. The best results were obtained from a mixture

of methanol and water with volume ratio of 2:5.

2.4 Methods applied for MICP and NICP films investigation Scanning electron microscopy (SEM) images and X-ray photoelectron spectroscopy (XPS) spectra were recorded using a LEO 1530 (LEO Electron Microscopy Ltd.) and a Phi Quantum 2000 XPS (instrument with a base pressure of 1.33 x 10-5 Pa and monochromatized Mg Kα Xrays with photon energy hv = 1253.6 eV), respectively. Raman spectra were taken using a HORIBA LabRAM HR spectrometer with 532 nm excitation.

Electrochemical impedance spectra and cyclic voltammograms were recorded in aqueous solutions using an Autolab analyzer AUT20 equipped with a General-Purpose Electrochemical System and a Frequency Response Analyzer. Impedance spectra were recorded in the frequency range of 100 kHz to 0.01 Hz under open-circuit potential conditions with 10 mV sinusoidal excitation signals. Potentiometric measurements were taken using a Lawson EMF16 interface potentiometer (Lawson Labs, Inc.). The potential changes of the films were measured against a Ag/AgCl/sat. KCl single-junction reference electrode (Metrohm) without correction for the liquid-junction potential. All electrochemical measurements were made at room temperature (22 ± 2 °C) under ambient air.

3. Results and discussions

3.1 Electrochemical synthesis of PEDOT/AAT-based copolymers Cyclic voltammograms recorded during the electrodeposition of the MICP and NICP films are shown in Figure 3. With each subsequent potential scan, the oxidation current continued to decrease, indicating that the resistance associated with mass transfer across the growing films was increasing. Similar polymerization kinetics for electrochemical systems exhibiting relatively low conductivities have been observed in previous studies [34]. Additionally, the use of AAT which can be in a slightly deprotonated form, can also lead to the minor “self” doping of copolymer backbones within the closest chains. Therefore, diffusion through both NICP and MICP films can be limited by the slightly decreased copolymer chain distance. Control measurements (dash line Figure 3) indicated that the PEDOT/AAT-based copolymer films cannot be deposited from a solution containing only EDOT and AAT monomers in the absence of FMN. Under these conditions, very low oxidation currents were observed during potential cycles between 0 mV and 1100 mV (see Figure 3 – Insert: broken line). The current densities

recorded during the electrodeposition of the MICP films were higher than those recorded for the NICP films. This observed difference can be explained by the possible parallel doping effects of the taurine ligands in the early stages of the polymerization process.

3.2 Surface analysis of the NICP and MICP films SEM images were recorded for freshly deposited NICP (1) and MICP (2) films, as well as for MICP (3) films after a week-long extraction in a (2 : 5 v/v) methanol/water solution. The images show that the surface morphologies of the NICP (1), MICP (2), and MICP (3) films differ significantly. The NICP (1) film surface (Figure 4 b-d) is smoother than the MICP (2) surface (Figure 4 e-f). Figures 4 (b-d) and 4 (e-f) also show that the NICP (1) and MICP (2) films uniformly cover the glassy carbon surface. The MICP (3) film after the extraction process conveys a “cauliflower-like” and porous structure (Figure 4 (g-h)). Moreover, the MICP (3) film after extraction appears to have a more open and looser structure compared to the structure of freshly deposited MICP (2) films.

3.3 Raman spectroscopy measurements The Raman spectrum of the MICP (2) film (Figure 5) shows vibrational modes and spectral patterns that are characteristic of poly(3,4-ethylenedioxythiophene) (PEDOT)-based materials [33] . The vibrational modes observed in the spectral range of 1500-1400 cm−1 are ascribed to the stretching motions of the C=C bonds. The major peaks in this range, ~1573, 1498, 1433 and 1368 cm−1, are typical for PEDOT films. Peaks at ~1498 and 1433 cm−1 are attributed to C=C asymmetric and symmetric vibrations in the thiophene ring [34]. The intensity ratios of the symmetric and asymmetric modes (I1433/I1498) can be used for estimating the conjugation length. Although the intensity of the peak at ~1498 cm−1 is lower than the peak intensity at

~1433 cm−1, as expected for polymerized films, the peak at 1498 cm−1 is still considerably higher than expected. This indicates that the MICP film consists of rather short oligomers. As seen from the insert in Figure 5, the imprinting of the taurine molecule into the EDOT/AATbased copolymer resulted in a slight red-shifting of the peak at ~1433 cm−1 and a narrowing of the major vibrational modes surrounding this peak. Similar observations were made for PEDOT:PSS films modified with different kinds of zwitterions, suggesting an extension of the quinoid form of the thiophene rings [33].

3.4 X-ray photoelectron spectroscopy measurements

The chemical composition of the MICP films was investigated by XPS analysis. Measurements were performed for a freshly deposited MICP (2) film and for a MICP (3) film after taurine extraction. The analysis revealed several peaks: O (1s) at 531.0 eV, N (1s) at 398.1 eV, C (1s) at 284.5 eV, P (2p) at 130.0 eV, S (2s) at 228.0 eV and S (2p) at 164.0 eV. High-resolution deconvolution of the C (1s) peak shows different carbon groups inside the MICP (2) film: C-C (~284.3 eV), C-C (sp2, ~284.8 eV), C-N amine (285.6 eV), C=N (~286.4 eV) and C-S (~287.1 eV), C-O (~288.0 eV), C-C (290.0 eV), O=C-OH (~289.2 eV) (Figure 6(1)). Similarly, highresolution deconvolution of the S peak (2p) shows two different sulphur groups inside the MICP (2) film: a thiophene group (~164 eV) characteristic of EDOT and AAT monomers and an Saliphatic group characteristic of taurine (168.5 eV) [35] (Figure 6(3)). The presence of C-N amine carbon groups and S-aliphatic groups confirm that taurine was introduced into the MICP (2) film. The XPS binding energy profiles for the MICP (3) film are shown in Figure 6(2) and 6(4). The absence of signals from the C-N amine carbon group and S-aliphatic sulphur groups, both characteristic of taurine, is a strong indication that the template taurine molecules have been removed from the surface layer of the MICP (3) films.

3.6. Electrochemical properties of the NICP and MICP films

3.6.1 CV and EIS measurements Cyclic voltammograms recorded in extracted taurine solution for the NICP (1) film, freshly deposited MICP (2) film, and MICP (3) film are shown in Figure 7(1). Figure 7(2) shows cyclic voltammograms recorded for the MICP (3) film in 0.1 mol·dm-3 taurine solution and, for comparison, in 0.1 mol·dm-3 KCl solution. Cyclic voltammograms with relatively low current values were recorded in taurine solution for NICP (1) and freshly deposited MICP (2) films. On the other hand, much higher current values were recorded under the same conditions for the MICP (3) film (Figure 7-1). Significantly different voltammograms were recorded for MICP (3) films in taurine and KCl solutions (Figure 7-2). These results show that the extraction process highly influences the MICP film electroactivity and that the MICP (3) film is less electroactive in KCl solution than in taurine solution. Electrochemical impedence spectroscopy (EIS) spectra were recorded under open-circuit potential conditions in 0.1 mol·dm-3 taurine solution. EIS spectra recorded for the NICP (1) and MICP (2) films were similar, except for the higher Z’ values observed for the NICP (1) film (Fig. 8(1)). These films were insensitive to taurine during the potentiometric measurements. The extraction process and extraction time considerably affected the shape and value of the Z' and Z” peaks of the EIS spectra (Figure 8(2)). As shown in Figure 8(2), after extraction, the second semicircle starts to disappear and the Z values decrease, even after short extraction times; taurine sensitivity is concomitantly introduced. As shown in the EIS spectra, relatively high values of charge transfer resistance were observed for NICP (1) and MICP (2) films, but after adequately long extraction times, the charge-transfer resistance of the MICP (3) film was considerably lowered.

3.6.2 Potentiometric measurements Calibration of the NICP and MICP films was performed in taurine solutions with concentrations ranging from 10-5 to 10-1 mol·dm-3 and was preceded by taurine extraction from the outer layer of the MICP films. The slope values for the MICP (3) films were dependent on extraction time. As shown in Figure 9, an almost theoretical cationic slope value was observed after one week of extraction. (A cationic response was expected since the pKa value of taurine is equal to 9.06 and the pHs of the taurine solutions used were below 7). At the same time, a linear dependence of the film potential on the logarithm of taurine concentration was observed in the concentration range of 10-4 to 10-2 mol·dm-3. As shown in Figure 9, the NICP (1) film was not sensitive towards taurine under the same conditions. It should be noted that a decrease in the slope values was observed after repeated calibration measurements with taurine. These results offer promising prospects for chemical sensing applications. For example, taurine concentrations in energy drinks exceed 10-2 mol·dm-3, which lies within the sensitivity limits of this MCIP potentiometric sensor [36]. However, additional improvements in the detection limit of the MCIP sensor must be done for extended biological applications. For example, typical taurine concentrations in human plasma are about 40 μmol·dm-3, below the detection limit of the sensor [37-42]. Since the pKa value of taurine is equal to 9.06, the existence of a zwitterionic form of taurine in the pH range of 8.4 - 9.6, as well as the presence of protonated or deprotonated forms of taurine outside this pH range, is expected. For this reason, the influence of pH of the taurine solution on the potentiometric response of the MICP (3) films was also studied. Figure 10 shows the influence of pH on MICP (3) film potentials. Measurements were performed with constant taurine concentrations of 0.01 mol·dm-3. The potential values were invariant in the pH range of 1.5 - 7.6, where the cationic form of taurine is expected. Moreover, the potential differences recorded for 0.001 mol·dm-3 and 0.01 mol·dm-3 taurine solutions with different pH

values were almost constant and equal to 60 mV within a pH range of 1.5 – 7.1, as shown in Figure 10 (see Insert). These observations indicate that the MICP (3) electrode is insensitive towards hydrogen ions. On the other hand, an anionic response of the MICP (3) film was observed in taurine solutions with pHs above 9.6, where an anionic form of taurine is expected. The selectivity of MICP (3) films towards alkali and alkali earth cations (Na+, K+, Ca2+, and Mg2+ in the form of chloride salts, which are common in physiological solutions) was studied using the fixed-interference method [43]. After a week-long extraction, the sensitivity towards taurine was verified prior to studying selectivity towards the specified cations. The potential changes recorded after the addition of alkali and alkali earth cations to the taurine solutions, with final cation concentrations ranging from 10-3 mol·dm-3 to 10-4 mol·dm-3, are listed in Table 1. These results show that the MICP (3) electrodes are largely insensitive toward these cations.

4. Conclusion Taurine-sensitive and -selective MICP films were obtained by electrochemical deposition of a solution containing 0.01 mol·dm-3 EDOT, 0.1 mol·dm-3 AAT, 0.1 mol·dm-3 FMN, and 0.5 mol·dm-3 taurine. The presence of taurine inside freshly deposited MICP films was confirmed with the presence of Saliphatic and C-NR2 peaks in the XPS spectra. These peaks were not observed after extraction, verifying that the taurine was removed from the surface layer of the MICP film. After taurine extraction, the MICP film was used as a receptor layer in a potentiometric taurine sensor. A linear cationic response towards the protonated form of taurine, with a slope value of 53.8 ± 2.6 mV/p[taurine], was observed for taurine concentrations ranging from 10-4 to 10-2 mol·dm-3. Moreover, these sensors are largely insensitive toward inorganic cations like calcium, magnesium, sodium, and potassium.

Acknowledgements This work is supported by the Polish Ministry of Science and Higher Education, grant N 507 234340. CIMO Fellowship (Finland) is also gratefully acknowledged for financial support.

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Figure legends

Fig. 1.Principles of molecular imprinting process.

Fig. 2. Hypothetical structure of MICP film based on EDOT as a cross linking polymer doped with FMN and functionalized with AAT to bind the taurine molecule. Note: the dashed lines represent hypothetical hydrogen bond interactions.

Fig. 3.Cyclic voltammograms recorded during electrochemical deposition of an MICP film from an aqueous solution containing 0.01 mol dm-3 EDOT, 0.1 mol dm-3 AAT, 0.1 mol dm-3 FMN and 0.5 mol dm-3 taurine. Insert: cyclic voltammograms recorded during electrochemical deposition of an NICP film from an aqueous solution containing 0.01 mol dm-3 EDOT, 0.1 mol dm-3 AAT and 0.1 mol dm-3 FMN (solid line) and cyclic voltammograms recorded in solution containing 0.01 mol dm-3 EDOT, and 0.1 mol dcm-3 AAT only. Scan rate: 50 mV s−1.

Fig. 4.SEM images of the surface of the bare GC electrode (a) and the GC surface covered with NICP (1) film (b-d). Images were taken with different magnifications: (a, b) 5000, (c) 10 000, and (d) 25 000. SEM images of the GC surface covered with MICP film prior to (e-f) and after (g-h) taurine extraction, taken with 25 000 magnification. Conditions of NICP and MICP film deposition: potential cycling range = 0 – 1100 mV, scan rate = 50 mV/s, number of scans =10).

Fig. 5.Raman spectra of the MICP (2) film recorded with 532 nm excitation. Insert: Comparison of the MICP (2) and NICP (1) spectra recorded in extended spectral region around the peaks associated with the major stretching modes of the thiophene ring.

Fig. 6 High-resolution XPS spectra of the MICP film recorded prior to (see Fig. 6(1) and 6(3)) and after (see Fig. 6(2) and 6(4)) taurine extraction. The lack of Saliphatic and C-NR2 peaks (see

Fig. 6(2) and 6(4)) confirm that taurine was removed from the outer layer of the MICP film during extraction.

Fig. 7.(1)- Comparison of the cyclic voltammograms recorded in 0.1 mol·dm-3 taurine solution for (a) NICP (1), (b) MICP (2) and (c) MICP (3) films, (2)- Comparison of the cyclic voltammograms recorded for MICP (3) film (a) in 0.1 mol·dm-3 taurine and (b) 0.1 mol·dm-3 KCl solutions. Range of potential cycling = -0.5 V to 0.5 V, scan rate = 50 mV s-1. Prior to measurements, taurine was removed from the MICP (3) film by a week-long extraction in a (2:5 v/v) methanol/water solution.

Fig. 8(1)- EIS spectra recorded for the NICP (1) film (○), and the MICP (2) film (●) under open circuit potential conditions in 0.1 mol·dm-3 taurine solution. (2)- EIS spectra recorded under open circuit potential conditions in 0.1 mol·dm-3 taurine solution for the MICP (3) film after different extraction times in a mixed methanol/water solution. Extraction time: MICP (3)-A 6 hours, MICP (3)-B 1 day. Insert: High-frequency part of the EIS spectra of MICP (3)-B.

Fig. 9. Potential changes of the MICP (3) film recorded during calibration in taurine solutions with a constant pH of 2.5. Prior to calibration, the film was soaked in a mixture of methanol and water with a volume ratio of 2 : 5 for 6 hours (circles), 3 days (asterixes) and 1 week (rombus) to remove the target taurine from the outer layer of the MICP (3) film. Potential changes of the NICP (1) film electrode are shown for comparison.

Fig. 10. Dependence of MICP (3) electrode potential on pH of taurine solution. Measurements were performed in 0.01 mol·dm-3 taurine solution with different pH values established by HCl or KOH addition. Insert: dependence of MICP (3) electrode potential on pH values and taurine concentrations. Measurements were performed in 0.001 mol·dm-3 (●) and 0.01 mol·dm-3 (○) taurine solutions with different pH values established by HCl addition. Prior to measurements, taurine was removed from the MICP film by a week-long extraction in a methanol/water solution with a volume ratio of 2 : 5.

Table 1. Potential changes of the MICP (3) film caused by addition of the selected cations (in the form of chloride salts, X = Mg2+, Ca2+, Na+, K+, final concentration = 10-3 mol·dm-3) to 104

mol·dm-3 taurine solution. Prior to measurements, taurine was removed from the MICP film

by a week-long extraction in a methanol/water solution with a volume ratio of 2 : 5.

Added cation (X) p[taurine] = 4, p[X] = 3

Mg 2.9

Potential change / mV Ca K 2.7 4.4

Na 4.9