Article Electrochemical Detection of Using a -State -Selective of Single-Piece Type Membrane

Li-Da Chen 1, Wei-Jhen Wang 1 and Gou-Jen Wang 1,2,3,*

1 Department of Mechanical Engineering, National Chung-Hsing University, Taichung 40227, Taiwan; [email protected] (L.-D.C.); [email protected] (W.-J.W.) 2 Graduate Institute of Biomedical Engineering, National Chung-Hsing University, Taichung 40227, Taiwan 3 Regenerative Medicine and Therapy Research Center, Kaohsiung Medical University, Kaohsiung 80708, Taiwan * Correspondence: [email protected]

Abstract: This study aimed to develop simple electrochemical for the fast detection of , and in human serum. A flat thin-film electrode was used as the detection electrode for chloride ions; a single-piece type membrane based solid-state ion-selective electrode (ISE), which was formed by covering a flat thin-film gold electrode with a mixture of 7,7,8,8- tetracyanoquinodimethane (TCNQ) and ion-selective membrane (ISM), was developed for sodium and potassium ions detection. Through cyclic voltammetry (CV) and square-wave voltammetry (SWV), the detection data can be obtained within two minutes. The linear detection ranges in the standard samples of chloride, sodium, and potassium ions were 25–200 mM, 50–200 mM, and 2–10 mM, with the average relative standard deviation (RSD) of 0.79%, 1.65%, and 0.47% and the average recovery rates of 101%, 100% and 96%, respectively. Interference experiments with Na+,K+, Cl−, Ca2+, and Mg2+ ions demonstrated that the proposed detection electrodes have good selectivity.  Moreover, the proposed detection electrodes have characteristics such as the ability to be prepared  under relatively simple process conditions, excellent detection sensitivity, and low RSD, and the − + + Citation: Chen, L.-D.; Wang, W.-J.; detection linear range is suitable for the Cl , Na and K concentrations in human serum. Wang, G.-J. Electrochemical Detection of Electrolytes Using a Solid-State Keywords: solid-state ion-selective electrode; electrochemical detection of electrolytes; cyclic voltam- Ion-Selective Electrode of metry; square-wave voltammetry Single-Piece Type Membrane. Biosensors 2021, 11, 109. https:// doi.org/10.3390/bios11040109 1. Introduction Received: 1 March 2021 The chloride, sodium and potassium ions in the can maintain the phys- Accepted: 4 April 2021 Published: 7 April 2021 iological functions of water metabolism, the neuromuscular system, -base balance and osmotic pressure in the body. Therefore, the metabolic functions of the human body

Publisher’s Note: MDPI stays neutral can be evaluated based on the measurements of the chloride, sodium and potassium ions with regard to jurisdictional claims in concentrations in the body [1–4]. published maps and institutional affil- The concentrations of chloride, sodium, and potassium ions in normal human serum iations. are 98–106 mM [2], 135–145 mM [3] and 3.5–5 mM [3], respectively. Too-high concentra- tions in the human body will cause , hypernatremia, and , while the occurrence of concentrations lower than the normal concentration will cause hypochloremia, hyponatremia, and hypoxemia potassium. Another common chloride- related disease is cystic fibrosis, which is an autosomal recessive genetic disease that affects Copyright: © 2021 by the authors. mucus-producing cells in the body and causes mucus accumulation in the lungs, trachea, Licensee MDPI, Basel, Switzerland. This article is an open access article and digestive tract, leading to the degradation of breathing functions [5]. The general distributed under the terms and clinical diagnosis of this disease is based on measuring the chloride ion content in sweat. conditions of the Creative Commons People with the disease usually have a chloride ion content of over 60 mM in their sweat [2], Attribution (CC BY) license (https:// while the chloride ion content in normal human sweat is below 40 mM [5]. creativecommons.org/licenses/by/ The laboratory autoanalyzer is a general -detecting instrument that can 4.0/). provide doctors with reliable results. However, the specimen must be delivered to a

Biosensors 2021, 11, 109. https://doi.org/10.3390/bios11040109 https://www.mdpi.com/journal/biosensors Biosensors 2021, 11, 109 2 of 12

specific laboratory and diluted before indirect ion-selective electrodes (ISEs) are used for detection. For patients with a severe or life-threatening degree of the illness, the analyzer (BGA) is usually used clinically to achieve rapid detection. The BGA uses direct ISE for direct analysis without the need to first dilute the sample, and the results can be obtained within 1 to 5 min [6–10]. The BGA can be divided into two types: benchtop and portable. The benchtop BGA detection cartridge has a service life of about 28–30 days; hence, it is suitable for detection applications. However, the instrument needs regular calibration and maintenance. The portable BGA usually uses a disposable detection cassette, which can reduce the frequency of instrument calibration and maintenance; however, since the cassette cannot be reused, the portable BGA is only suitable for small- volume detection applications [11]. In recent years, as the utilization rate of point-of-care (POC) testing has significantly increased, the economic value of POC-related products has also increased [12]. Therefore, there are many portable and small BGAs and electrolyte analyzer-related products in the market, such as Abbott i-STAT [13], Siemens epoc® blood analysis system [12], and Arkray Spotchem EL SE-1520 [14]. At present, the electrochemical potentiometric method [13–18] is the main method for electrolyte ions quantification. Other detection methods include the conductivity method [19,20], cyclic voltammetry (CV) [21–26], and coulometry [27,28]. The potentio- metric method uses liquid-state ISEs that need to be regularly replaced. The operating is limited, and the electrodes cannot be effectively miniaturized [29–31]. More- over, the practical application of the conductance method is not yet mature [21]. Therefore, in the current study, a novel detection method combining high-sensitivity voltammetry and solid-state ISE is developed. The solid-state ISE does not need to be filled with a . The ion-selective membrane directly contacts the electrode, and the solid contact material of the intermediate layer is added to enhance the effect of ion and conversion. Therefore, the can be more easily miniaturized, and its durability and operating temperature range are improved. Commonly used solid contact materials include high-molecular-weight conductive polymers such as polypyrroles [32], poly(3-octylthiophene) [33], and poly(3,4-ethylenediox- ythiophene) (PEDOT) [34]. Nanomaterials used as solid contact materials include nanotubes [17], graphene [35], platinum nanoparticles [36], and noble nanostruc- tures [37]. Electroactive substances are also used, such as tetrathiafulvalene [38] and 7,7,8,8-tetracyanoquinodimethane (TCNQ) [39]. Conductive polymers such as PEDOT need to be synthesized via electrochemical [34], while nanomaterials need to be produced via electrochemical deposition [37]. The manufacturing processes of both are relatively cumbersome. Therefore, this study uses the solid-state contact conduction mechanism of high capacitors [29] and adopts the electroactive material TCNQ as the solid-state contact material. TCNQ is a powerful organic electron acceptor with excellent electrical properties and high electron affinity (2.88 eV) [40]; thus, it can easily donate electron acceptors to react with transition or organic compounds and form numerous TCNQ anion–based complexes, such as CuTCNQ [40], AgTCNQ [40], KTCNQ [39], and NaTCNQ [39]. In addition, the use of the TCNQ involves a simple process; the TCNQ only needs to be dropped on the electrode substrate through drop-casting [39]. In this study, electrodes for the effective and fast detection of electrolytes in human serum were developed. A flat thin-film gold electrode was used as the detection electrode for chloride ions and a solid-state ISE, which was formed by covering a flat thin-film gold electrode with a mixture of TCNQ and ion-selective membrane (ISM), was developed for sodium and potassium ions detection, respectively.

2. Materials and Methods 2.1. Materials and Reagents TCNQ, tetrahydrofuran (THF), , , chloride, chloride, human serum, potassium I, poly(vinyl chloride) and Biosensors 2021, 11, x FOR PEER REVIEW 3 of 12

2. Materials and Methods Biosensors 2021, 11, 1092.1. Materials and Reagents 3 of 12 TCNQ, tetrahydrofuran (THF), potassium chloride, sodium chloride, calcium chlo- ride, , human serum, potassium ionophore I, poly(vinyl chloride) and potassium phosphatepotassium phosphatemonobasic monobasic were purcha weresed purchased from Sigma-Aldrich from Sigma-Aldrich (St. Louis, (St. Louis,MO, MO, USA). USA). PotassiumPotassium ferricyanide ferricyanide and potassium and potassium ferrocyanide were were purchased purchased from from SHOWA SHOWA (Tokyo, (Tokyo, Japan).Japan). Potassium Potassium tetrakis tetrakis (4-chlorophenyl)borate, (4-chlorophenyl)borate, 2-nitrophenyl 2-nitrophenyl octyl octyl ether, ether, and and 4-tert- 4-tert-butylcalix[4]arenetetraaceticbutylcalix[4]arenetetraacetic acid acidtetr tetraethylaethyl ester ester were were purchased purchased from from ThermoThermo (Darmstadt, (Darmstadt,Germany). Germany). Sodium Sodium phosphate dibasic dibasic was was purchased purchased from from J. J. T. T. Baker Baker (USA). (USA). Acetone was Acetone waspurchased purchased from from ECHO ECHO Chemical Chemical (Miaoli,(Miaoli, Taiwan). Distilled deionized deionized water water (>18 MΩ) (>18 MΩ) waswas obtained obtained from from ELGA ELGA LabWater LabWater (London, (London, UK). UK).

2.2. Electrode2.2. Fabrication Electrode Fabrication Scheme 1 illustratesScheme1 theillustrates fabrication the fabricationprocess of processthe chloride, of the potassium, chloride, potassium, and sodium and sodium 2 ions detectionions electrodes. detection electrodes.First, a thin First, gold alayer thin goldwas sputtered layer was onto sputtered a 3 × onto5 mm a2 3polyeth-× 5 mm polyethy- ylene terephthalatelene terephthalate (PET) membrane (PET) membrane (Scheme (Scheme1A). Then,1A). the Then, gold the thin-film–sputtered gold thin-film–sputtered PET PET membranemembrane was packaged was packaged to form tothe form detection the detection chip (Scheme chip 1B). (Scheme For the1B). potassium For the potassium µ and sodiumand ions sodium detection ions electrodes, detection 3 electrodes, μL of a TCNQ 3 L ofand a TCNQISM mixture and ISM solution mixture was solution was dripped ontodripped the electrode onto the (Scheme electrode 1C). (Scheme Table1 1C). presents Table1 presentsthe compositions the compositions and contents and contents of of potassiumpotassium and sodium and ISMs. sodium ISMs.

Scheme 1. FabricationScheme process1. Fabrication of the chloride,process of potassium, the chloride, and potassium, sodium ions and detection sodium electrodes: ions detection (A) sputteringelectrodes: of Au thin- film on a polyethylene(A) sputtering terephthalate of Au thin-film (PET) membrane; on a polyethylene (B) packaging; terephthalate (C) dropping (PET) membrane; of 7,7,8,8-tetracyanoquinodimethane (B) packaging; (TCNQ) + ion-selective(C) dropping membrane of 7,7,8,8-tetracyanoquinodimethane (ISM) mixture solution (potassium (TCNQ) and sodium+ ion-selective ions detection membrane electrodes). (ISM) mix- ture solution (potassium and sodium ions detection electrodes). Table 1. Compositions and contents of potassium and sodium-ion-selective membrane. Table 1. Compositions and contents of potassium and sodium-ion-selective membrane. Target Ion Reagents Target Ion K+ Na+ Reagents + + K Na Potassium ionophore I 14 mg 0 Potassium ionophore I 14 mg 0 4-tert- 4-tert-Butylcalix[4]arenetetraacetic 0 9.9 mg Butylcalix[4]arenetetraacetacid tetraethyl ester 0 9.9 mg ic acid tetraethylPotassium ester tetrakis 3 mg 2.5 mg Potassium tetrakis(4-chlorophenyl)borate (4- 3 mg 2.5 mg chlorophenyl)boratePolyvinyl chloride 328 mg 329 mg Polyvinyl chloride(high-molecular-weight) (high- 328 mg 329 mg molecular-weight)2-Nitrophenyl octyl ether 0.63 mL 0.63 mL 2-Nitrophenyl octyl ether 0.63 mL 0.63 mL 2.3. Apparatus 2.3. Apparatus Cyclic voltammetry and square-wave voltammetry (SWV) were conducted with an Cyclic SP-150voltammetry potentiostat and square-wave (BioLogic, voltammetry Seyssinet-Pariset, (SWV) France) were conducted connected with to a an computer. A SP-150 potentiostatthree-electrode (BioLogic, system, Seyssinet-Pa consistingriset, of a France) working connected electrode, to a platinuma computer. counter A electrode, three-electrodeand system, an Ag/AgCl consisting reference of a workin electrode,g electrode, was applied. a platinum Field-emission counter electrode, scanning electron microscopy (FE-SEM, Zeiss UltraPlus, Potsdam, Germany) was used for the electrode surface characterization. The analysis system and the electrodes are shown in Figure1. Biosensors 2021, 11, x FOR PEER REVIEW 4 of 12

Biosensors 2021, 11, 109 and an Ag/AgCl reference electrode, was applied. Field-emission scanning electron4of mi- 12 croscopy (FE-SEM, Zeiss UltraPlus, Potsdam, Germany) was used for the electrode sur- face characterization. The analysis system and the electrodes are shown in Figure 1.

Figure 1. The analysis system: (A) experimental setup; (B) electrodes for the experiment. Figure 1. The analysis system: (A) experimental setup; (B) electrodes for the experiment.

3.3. Results and Discussion 3.1.3.1. Optimization of Detection ElectrodesElectrodes TwoTwo parametersparameters ofof thethe detectiondetection electrodeelectrode needneed toto bebe optimized—theoptimized—the organicorganic solventsolvent toto dissolvedissolve TCNQTCNQ andand thethe volume volume ratio ratio of of TCNQ TCNQ to to ISM. ISM. Regarding Regarding the the organic organic , solvent, a suitablea suitable solvent solvent for for TCNQ TCNQ was was selected selected from from two two organic organic : solvents: acetone acetone and and THF. THF. The + + TCNQThe TCNQ solution solution was mixedwas mixed with thewith corresponding the corresponding ISM (Na ISM and(Na+ K and) with K+) awith volume a volume ratio ofratio 1:1 of to 1:1 determine to determine which which was the was more the suitable more suitable solvent. solvent. After the After suitable the suitable TCNQ solvent TCNQ wassolvent selected, was selected, TCNQ/ISM TCNQ/ISM mixed solutionsmixed solution with differents with different volume volume ratios (1:1, ratios 1:2, (1:1, 2:1, 3:1)1:2, were2:1, 3:1) prepared were prepared to optimize to optimize the volume the ratio.volume ratio. + + FigureFigure2 2A,BA,B show show the the cyclic cyclic voltammograms voltammograms for for Na Na+ (140(140 mMmM NaCl)NaCl) andand KK+ (5(5 mMmM KCl) detections, respectively. The acetone-dissolved TCNQ resulted in peak oxidation KCl) detections, respectively. The+ acetone-+ dissolved TCNQ resulted in peak oxidation currentscurrents atat 0.460.46 andand 0.500.50 VV forfor Na Na+ andand KK+ detections,detections, respectively.respectively. TheThe peakpeak oxidationoxidation currents were much higher than those obtained from the THF-dissolved TCNQ. Therefore, currents were much higher than those obtained from the THF-dissolved TCNQ. There- acetone was selected as the TCNQ solvent. The cyclic voltammograms for Na+ and K+ fore, acetone was selected as the TCNQ solvent. The cyclic voltammograms for Na+ and detections with different TCNQ/ISM volume ratios are shown in Figure2C,D, respectively. K+ detections with different TCNQ/ISM volume ratios are shown in Figure 2C,D, respec- The results show that for the Na+ detection, the TCNQ/ISM volume ratio of 2:1 resulted in tively. The results show that for the Na+ detection, the TCNQ/ISM volume ratio of 2:1 the maximum peak oxidation current at 0.45 V, while for the K+ detection, the ratio of 3:1 resulted in the maximum peak oxidation current at 0.45 V, while for the K+ detection, the resulted in the maximum peak oxidation current at 0.43 V. In Figure2C,D, as the TCNQ ratio of 3:1 resulted in the maximum peak oxidation current at 0.43 V. In Figure 2C,D, as content increases, the oxidation potentials of Na+ and K+ shift to the left. Therefore, using the TCNQ content increases, the oxidation potentials of Na+ and K+ shift to the left. There- a lower potential can drive the oxidation reaction and reduce the scanning range fore, using a lower potential can drive the oxidation reaction and reduce the scanning during CV. voltage range during CV. 3.2. Characterization of Fabricated Electrodes Figure3 displays the scanning electron microscopy (SEM) images of the fabricated electrodes. The top view shows many typical TCNQ black blocks and a few white flake structures. The white flake structures are TCNQ erected under the influence of gravity [41]. The cross-sectional view (Figure3B) shows that the thickness of the deposited Na + ISM and K+ ISM layers: about 24.4 and 14.6 µm, respectively. Biosensors 2021, 11, x FOR PEER REVIEW 5 of 12

(A) (B) 2.0 2.0 Biosensors 2021, 11, 109x FOR1.5 PEER REVIEW 5 of 12 1.5 1.0 1.0 0.5 0.5 0.0 0.0 (A)- 0.5 (B) 2.0 - 0.5 - 1.0 Current (µA) Current Current (µA) Current + 2.0 + 1.5 TCNQ/ISM (Na ) = 1/1 - 1.0 TCNQ/ISM (K ) = 1/1 - 1.5 1.5 TCNQ in Acetone - 1.5 TCNQ in Acetone 1.0 TCNQ in THF TCNQ in THF - 2.0 1.0 0.5 - 2.0 - 2.5 0.5 0.0- 0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 - 0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.0 - 0.5 Ewe/V vs. Ag/AgCl/NaCl (3M) Ewe/V vs. Ag/AgCl/NaCl (3M) (C) (D)- 0.5 - 1.0 Current (µA) Current Current (µA) Current 3.0 TCNQ/ISM (Na+) = 1/1 -3.0 1.0 TCNQ/ISM (K+) = 1/1 - 1.5 2.0 TCNQ in Acetone TCNQ in Acetone -2.0 1.5 - 2.0 TCNQ in THF TCNQ in THF 1.0 - 2.0 - 2.5 1.0 0.0- 0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 - 0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.0 Ewe/V vs. Ag/AgCl/NaCl (3M) Ewe/V vs. Ag/AgCl/NaCl (3M) - 1.0 (C) (D)- 1.0 3.0 + + - 2.0 TCNQ/ISM (Na ) 3.0 TCNQ/ISM (K ) Current (µA) Current 1:1 - 2.0 1:1 2.0 Current (µA) - 3.0 1:2 2.0 1:2 2:1 - 3.0 2:1 1.0 3:1 3:1 - 4.0 1.0 - 4.0 0.0 - 0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.0- 0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 - 1.0 Ewe/V vs. Ag/AgCl/NaCl (3M) Ewe/V vs. Ag/AgCl/NaCl (3M) - 1.0 + + - 2.0 TCNQ/ISM (Na ) TCNQ/ISM (K ) Current (µA) Current 1:1 - 2.0 1:1 Figure 2. Cyclic voltammograms for TCNQ solvent selection and Current (µA) TCNQ/ISM volume ratio optimization: (A,B) cyclic volt- - 3.0 + + 1:2 1:2 + ammograms for Na (140 mM NaCl) and K (5 mM 2:1 KCl) detections,- 3.0 respectively; (C,D) cyclic voltammograms 2:1 for Na and + 3:1 3:1 K detections with- 4.0 different TCNQ/ISM volume ratios, respectively. - 4.0 - 0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 - 0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Ewe/V3.2. Characterizationvs. Ag/AgCl/NaCl of(3M) Fabricated Electrodes Ewe/V vs. Ag/AgCl/NaCl (3M)

Figure 3 displays the scanning electron microscopy (SEM) images of the fabricated Figure 2 2.. CyclicCyclic voltammograms voltammogramselectrodes. for for TCNQ TCNQ The solvent top solvent view selectio selection showsn and andmany TCNQ/ISM TCNQ/ISM typica volumel TCNQ volume ratio black ratio optimization: blocks optimization: and (A ,aB )few ( Acyclic,B )white cyclic volt- flake + + + ammogramsvoltammograms for Na for Na(140+ (140mMstructures. mMNaCl) NaCl) and The andK (5 Kwhite mM+ (5 KCl) mM flake KCl)detections, structures detections, respectively; are respectively; TCNQ (C ,Derected ()C cyclic,D) cyclic undervoltammograms voltammograms the influence for Na for of and Na gravity+ K+ detections with different TCNQ/ISM volume ratios, respectively. and K+ detections with different[41]. TCNQ/ISMThe cross-sectional volume ratios, view respectively. (Figure 3B) shows that the thickness of the deposited Na+ ISM and K+ ISM layers: about 24.4 and 14.6 μm, respectively. 3.2. Characterization of Fabricated Electrodes Figure 3 displays the scanning electron microscopy (SEM) images of the fabricated electrodes. The top view shows many typical TCNQ black blocks and a few white flake structures. The white flake structures are TCNQ erected under the influence of gravity [41]. The cross-sectional view (Figure 3B) shows that the thickness of the deposited Na+ ISM and K+ ISM layers: about 24.4 and 14.6 μm, respectively.

Figure 3. Scanning electron microscopy (SEM) images of electrode surface: (A) TCNQ/ion-selective membrane (ISM) de- Figure 3. Scanning electron microscopy (SEM) images of electrode surface: (A) TCNQ/ion-selective membrane (ISM) posited electrode (top view); (B) cross-sectional view. deposited electrode (top view); (B) cross-sectional view.

3.3. The Electrochemical Behavior of Cl−, Na+, and K+ Electrodes The electron diffusion rate can be used to study the characteristics of electrochemical Figure 3. Scanning electron electrodes.microscopy (SEM The oxidation) images of andelectrode reduction surface: potentials, (A) TCNQ/ion-selective as well as the membrane reaction current(ISM) de- of the posited electrode (top view);analyte, (B) cross-sectional can be obtained view. based on a CV analysis of the reaction between the analyte and the electrode. The results can be used to determine whether the reaction is diffusion-controlled.

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3.3. The Electrochemical Behavior of Cl−, Na+, and K+ Electrodes The electron diffusion rate can be used to study the characteristics of electrochemical Biosensors 2021, 11, 109 electrodes. The oxidation and reduction potentials, as well as the reaction current of6 ofthe 12 analyte, can be obtained based on a CV analysis of the reaction between the analyte and the electrode. The results can be used to determine whether the reaction is diffusion-con- trolled. FigureFigure 4 showsshows the the CV CV results results of ofthe the proposed proposed different different electrodes electrodes at scan at scanrates ratesrang- ingranging from from 100 to 100 500 to 500mV/s mV/s in 50 in mM 50 mM KCl, KCl, 140 140 mM mM NaCl, NaCl, and and 1 1mM mM KCl KCl . solutions. The The cycliccyclic voltammograms voltammograms displayed displayed in Figure4 4(A1,B1,C1)(A1–C1) show show that that the the absolute absolute value value of the of theoxidation oxidation and and reduction reduction peak peak currents currents increased increased with with the increasethe increase in scan in scan rate. rate. Generally, Gen- erally,the Randles–Sevcik the Randles–Sevcik equation equation (Equation (Equation (1) is used(1) is toused evaluate to evaluate the diffusion-controlled the diffusion-con- trolledcharacteristic characteristic of a sensing of a sensing electrode. electrode. i = 2.69=××××××× 105 ×5n3/2 3/2× A × C × D1/2 1/2× v 1/2 (1) p inACDvp 2.69 10 (1)

wherewhere ip isis the peakpeak current,current,n nis is the the number number of of electrons appearing appearing in ain half-reaction a half-reaction for for the 2 3 theredox redox couple, couple,A is A the is electrodethe electrode area area (cm (cm), C2is), C the is analytethe analyte concentration concentration (/cm (mole/cm), D3is), 2 Dthe is analytethe analyte diffusivity diffusivity (cm /s),(cm2 and/s), andv is thev is scanthe scan rate ofrate potential of potential (V/s). (V/s). For stationaryFor stationaryn, A, nC, ,A and, C,D and, ip isD a, i linearp is a linear function function of the squareof the square root of theroot scanning of the scanning rate. The rate. corresponding The corre- spondinglinear relationship linear relationship between between the peak the current peak andcurrent the and square the rootsquare of theroot scanning of the scanning rate of ratethe proposedof the proposed electrodes electrodes for the cyclicfor the voltammograms cyclic voltammograms shown in shown Figure in4A1–C1 Figure are (4A1,B1, given 2 andin Figure C1) are4A2–C2, given in respectively. Figure 4(A2,B2, The andR values C2), respectively. were calculated The R to2 values be 0.9986, were 0.9981, calculated and to0.9989, be 0.9986, respectively. 0.9981, Theand results0.9989, demonstraterespectively. that The the results proposed demonstrate electrodes that possess the proposed a typical electrodesdiffusion-controlled possess a typical electrochemical diffusion-controlle characteristic;d electrochemical hence, the electrodes characteristic; are suitable hence, the for electrodespractical quantitative are suitable analyses.for practical quantitative analyses.

(A1) (A2)2.0 2.0

2 1.5 1.8 R =0.9986 100 mV/s 1/2 200 mV/s Y=0.0655v+0.4029 1.0 300 mV/s 1.6 400 mV/s 500 mV/s 0.5 1.4 0.0 Current (mA) Current Current Current (mA) 1.2 - 0.5 1.0 - 1.0 0.0 0.3 0.6 0.9 1.2 1.5 1.8 10 12 14 16 18 20 22 24 1/2 Ewe/V vs. Ag/AgCl/NaCl(3M) Square root of scan rate (mV/s) (B1) (B2) 4 3.4 3.2 2 R =0.9981 2 3.0 1/2 Y=0.1127v+0.6473 2.8 0 2.6 2.4 100 mV/s - 2 200 mV/s 2.2 Current (µA) Current 300 mV/s (µA) Current 400 mV/s 2.0 500 mV/s - 4 1.8 1.6 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 10 12 14 16 18 20 22 24 1/2 Ewe/V vs. Ag/AgCl/NaCl(3M) Square root of scan rate (mV/s)

Figure 4. Cont. BiosensorsBiosensors 20212021,, 1111,, x 109 FOR PEER REVIEW 77 of of 12 12

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(C1) (C2) (C1) 4 (C2)3.8

4 3.8 2 3.6 R =0.9989 1/2 2 2 3.43.6 Y=0.1082v+1.2476R =0.9989 2 1/2 3.4 Y=0.1082v+1.2476 0 3.2 0 3.03.2 - 2 100 mV/s 2.83.0 200 mV/s - 2 100 mV/s Current Current (µA) 300 mV/s Current (µA) 2.8 200 mV/s 2.6 - 4 400 mV/s Current Current (µA) 300 mV/s Current (µA) 500 mV/s 2.6 - 4 400 mV/s 2.4 500 mV/s 2.4 - 6 2.2 - 6 2.2 - 0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 10 12 14 16 18 20 22 24 1/2 - 0.1 0.0Ewe/V 0.1 0.2vs. Ag/AgCl/NaCl(3M) 0.3 0.4 0.5 0.6 0.7 0.8 10Square 12 root 14 of 16 scan 18 rate 20(mV/s) 22 24 1/2 Ewe/V vs. Ag/AgCl/NaCl(3M) Square root of scan rate (mV/s)

FigureFigure 4. 4. ElectrochemicalElectrochemical characteristics characteristics of of the the proposed proposed electrode electrode for for electrolyte electrolyte detection: detection: ( (A1A1,B1,B1,C1,C1)) cyclic cyclic voltammo- voltammo- grams of the proposed different electrodes at scan rates ranging from 100 to 500 mV/s in 50 mM KCl, 140 mM NaCl, and Figuregrams of4. theElectrochemical proposed different characteristics electrodes of atthe scan proposed rates ranging electrode from for 100electrolyte to 500 mV/s detection: in 50 ( mMA1,B1 KCl,,C1 140) cyclic mM voltammo- NaCl, and 1grams mM KClof the solutions, proposed respectively; different electrodes (A2,B2,C2 at) scanthe corresponding rates ranging fromRandles–Sevcik 100 to 500 mV/s curves in of50 the mM cyclic KCl, voltammograms140 mM NaCl, and in 1 mM KCl solutions, respectively; (A2,B2,C2) the corresponding Randles–Sevcik curves of the cyclic voltammograms in (1A1 mM,B1 ,KClC1), solutions,respectively. respectively; (A2,B2,C2) the corresponding Randles–Sevcik curves of the cyclic voltammograms in (A1,B1,C1), respectively. (A1,B1,C1), respectively. 3.4. Detection of Cl−, Na+, and K+ − + + 3.4. Detection of Cl− , Na+ , and+ K 3.4. Detection of Cl , Na , and K − Cyclic voltammetry was used for the detection of Cl− (KCl) because it can be easily oxidizedCyclicCyclic by voltammetry gold with a was wassuitable used oxidationfor the detection potential. of ClClSquare-wave− (KCl)(KCl) because because voltammetry it it can can be be easily was adoptedoxidizedoxidized for by the gold detections with aa suitablesuitableof Na+ (NaCl) oxidationoxidation and potential.Kpotential.+ (KCl) because Square-wave the oxidation voltammetry process was in- + + volvedadoptedadopted the for for ISM. the the detections detections of of Na Na+ (NaCl)(NaCl) and and K+ K (KCl)(KCl) because because the theoxidation oxidation process process in- involved the ISM. (1)volved Cl− detectionthe ISM. − (1) Cl− detection (1) ClFigure detection 5 shows the detection results of Cl− with various concentrations (25, 50, 100, − 150, andFigureFigure 200 5 5mM). shows shows Clear the the detection oxidationdetection results andresults reduction of Clof Clwith− with current various various peaks concentrations concentrations can be observed (25, (25, 50, in 100,50, Figure 100, 150, 5A.150,and The and 200 absolute mM).200 mM). Clear value Clear oxidation of oxidation the oxidation and and reduction reduction and reduction current current peaks peak peaks can currents can be observedbe increasedobserved in Figure inwith Figure 5theA . increase5A.The The absolute inabsolute Cl value− concentration. value of the of oxidation the Figure oxidation and 5B reduction displays and reduction peakthe corresponding currents peak currents increased calibration increased with the curve with increase forthe − − Clincreasein− Cldetection,concentration. in Cl −with concentration. the normal Figure Figure5concentrationB displays 5B displays the range corresponding the of correspondingCl− in human calibration blood calibration identified curve curve for Clby for a − barCldetection,− detection,icon. The with linearwith the the detection normal normal concentration range, concentration sensitivity, range range correlation of of Cl Cl−in in humancoefficient,human bloodblood relative identifiedidentified standard by a deviationbarbar icon. The The(RSD), linear linear and detection recovery range, range,rate of sensitivity, sensitivity,the electrode correlation were calculated coefficient, coefficient, to be relative 5–200 mM, standard 0.27 mAdeviationdeviation mM−1 (RSD),cm (RSD),−2, 0.9988, and and recovery 0.79%, recovery and rate rate 101%, of ofthe therespectively. electrode electrode were were calculated calculated to be to 5–200 be 5–200 mM, mM,0.27 −1 −2 mA0.27 mM mA− mM1 cm−2, 0.9988,cm , 0.9988,0.79%, 0.79%,and 101%, and respectively. 101%, respectively. (A) (B) (A)4.0 (B)4.0 3.54.0 2 25 mM 3.54.0 R =0.9988 3.03.5 50 mM Y=0.0191X+0.01962 25 mM 3.5 R =0.9988 2.5 100 mM 3.0 3.0 150 50 mMmM Y=0.0191X+0.0196 2.02.5 200100 mMmM 2.53.0 150 mM 1.5 2.0 200 mM 2.02.5 1.01.5 1.52.0 0.51.0 Current (mA) Current Current (mA) Current 1.5 0.00.5 1.0 Current (mA) Current Current (mA) Current - 0.50.0 0.51.0 -- 1.00.5 0.00.5 - 1.0 0.0 0.3 0.6 0.9 1.2 1.5 1.8 0.0 20 40 60 80 100 120 140 160 180 200 220 Ewe/V vs. Ag/AgCl/NaCl(3M) Concentration (mM) 0.0 0.3 0.6 0.9 1.2 1.5 1.8 20 40 60 80 100 120 140 160 180 200 220 Ewe/V vs. Ag/AgCl/NaCl(3M) Concentration (mM) Figure 5. Cl− detection using cyclic voltammetry (CV): (A) cyclic voltammograms (100 mV/s) for various Cl− concentrations (25–200Figure 5. mM); Cl− detection detection(B) linear using usingcalibration cyclic cyclic voltammetry voltammetrycurve representing (CV): ( Athe) cyclic relation voltammograms between the (100stable(100 mV/s)mV/s) oxidization for variousvarious peak ClCl current−− concentrationsconcentrations and con- centration;(25–200 mM); mM); scanning ( (BB)) linear linear range: calibration calibration 0–1.6 V, curveN curve = 3. representing representing the the relation relation between between the thestable stable oxidization oxidization peak peak current current and con- and centration;concentration; scanning scanning range: range: 0–1.6 0–1.6 V, N V, = N 3. = 3.

Biosensors 2021, 11, 109 8 of 12 BiosensorsBiosensors 20212021,, 1111,, xx FORFOR PEERPEER REVIEWREVIEW 88 ofof 1212

+ (2)(2)(2) NaNaNa++ detectiondetectiondetection FigureFigureFigure 66 showsshows thethe SWVSWV detectiondetection resultsresults ofof NaNa++ withwithwith variousvarious various concentrationsconcentrations concentrations (50,(50, (50, 100,100, 100, 150,150, andand 200200 mM).mM). FigureFigure 66A6AA displaysdisplays clearclear oxidationoxidation currentcurrent peaks,peaks,peaks, whichwhich increaseincreaseincrease withwithwith thethethe increaseincrease inin the the NaNa++ concentrations.concentrations. FigureFigure Figure 66B6BB showsshowsshows thethethe correspondingcorrespondingcorresponding calibrationcalibrationcalibration curvecurvecurve forfor for NaNa Na++ detection,detection,detection, withwith with thethe the normalnormal normal NaNa Na+++ concentrationconcentrationconcentration rangerange range inin in humanhuman human bloodblood blood markedmarked marked bybyby aa a barbar bar icon.icon. icon. TheThe The linearlinear linear detectiondetection detection range,range, range, sensitivity,sensitivity, sensitivity, correlationcorrelation correlation coefficient,coefficient, coefficient, RSD,RSD, RSD, andand and re-re- −1 −2 coverycoveryrecovery raterate rate ofof thethe of the electrodeelectrode electrode werewere were calculatedcalculated calculated toto bebe to 50–20050–200 be 50–200 mM,mM, mM,0.1250.125 0.125μμA·mMA·mMµA−−1·1cmmMcm−−22,, 0.9786,0.9786,cm , 1.65%1.65%0.9786, andand 1.65% 100%,100%, and respectively.respectively. 100%, respectively.

(A)(A) (B)(B) 99 8.48.4 8.2 8 8.2 8 5050 mMmM 22 100100 mMmM 8.0 RR=0.9786=0.9786 8.0 Y=0.0088X+6.3948 77 150150 mMmM Y=0.0088X+6.3948 200200 mMmM 7.87.8 6 6 7.67.6 7.4 55 7.4 I delta/µA I delta/µA 7.27.2 Current (µA) Current 44 (µA) Current 7.07.0 33 6.86.8 22 6.66.6 0.00.0 0.1 0.1 0.2 0.2 0.3 0.3 0.4 0.4 0.5 0.5 4040 60 60 80 80 100 100 120 120 140 140 160 160 180 180 200 200 220 220 EE step/Vstep/V ConcentrationConcentration (mM)(mM)

+ + FigureFigure 6. 6. NaNa+ detectiondetection usingusing square-wave square-wave voltammetry voltammetry (SWV): (SWV): ( (AA)) cycliccyclic voltammogramsvoltammograms (100(100 mV/s)mV/s)mV/s) forfor various various Na Na+ concentrationsconcentrations (50–200(50–200 (50–200 mM);mM); mM); (( BB))) linearlinear calibrationcalibration curvecurve curve representingrepresenting thethe the relationrelation betweenbetween thethe stablestable oxidizationoxidization peakpeak current and concentration; scanning range: 0–0.5 V, pulse height 100 mV, pulse width 200 ms, step height 20 mV, N = 3. currentcurrent and and concentration; concentration; scanning scanning rang range:e: 0–0.5 0–0.5 V, V, pulse height 100 mV, pulsepulse widthwidth 200200 ms,ms, stepstep heightheight 2020 mV,mV, NN = = 3.3.

(3)(3) KK++ detectiondetection (3) K+ detection TheThe SWVSWV detectiondetection resultsresults ofof KK++ withwith variousvarious concentrationsconcentrations (2,(2, 3.5,3.5, 5,5, 7.5,7.5, andand 1010 mM)mM) Theareare shown SWVshown detection inin FigureFigure results 7.7. InIn ofFigureFigure K+ with 7A,7A, various thethe oxidationoxidation concentrations currentcurrent (2, peakspeaks 3.5, 5,increaseincrease 7.5, and withwith 10 mM) in-in- creasingcreasingare shown KK++ in concentrations.concentrations. Figure7. In Figure FigureFigure7A, the7B7B oxidationshowsshows ththe currente correspondingcorresponding peaks increase quadraticquadratic with increasingcalibrationcalibration + + curvecurveK concentrations. forfor KK++ detection,detection, Figure whichwhich7Bshows isis moremore the appropriateappropriate corresponding forfor fittingfitting quadratic thethe measured calibrationmeasured data.data. curve TheThe for de-de- K tectiontectiondetection, range,range, which sensitivity,sensitivity, is more appropriatecorrelationcorrelation coefficicoeffici for fittingent,ent, theRSD,RSD, measured andand recoveryrecovery data. rate Therate detectionofof thethe electrodeelectrode range, wereweresensitivity, measuredmeasured correlation toto bebe 2–102–10 coefficient, mM,mM, 0.56–7.980.56–7.98 RSD, and μμA·mMA·mM recovery−−11 cmcm rate−−22,, 0.9778,0.9778, of the electrode 0.47%,0.47%, andand were 96%,96%, measured respec-respec- −1 −2 tively.tively.to be 2–10 mM, 0.56–7.98 µA·mM cm , 0.9778, 0.47%, and 96%, respectively.

(A)(A) (B)(B) 1313 12.512.5 22 12 RR== 0.97780.9778 12 2 mM 22 2 mM Y= -0.0328X+0.6953X+8.6420 3.5 mM Y= -0.0328X+0.6953X+8.6420 11 3.5 mM 12.012.0 11 55 mMmM 7.5 mM 10 7.5 mM 11.511.5 10 1010 mMmM 9 9 11.011.0 88

I delta/µA 10.5 I delta/µA 10.5 Current (µA) Current 77 (µA) Current 10.010.0 66 9.59.5 55 0.20.2 0.3 0.3 0.4 0.4 0.5 0.5 12345678910111234567891011 EE step/Vstep/V ConcentrationConcentration (mM)(mM)

FigureFigure 7. 7. SWVSWV detection detection results results ofof KK++ withwith various various concentrations: concentrations: ( (AA)) cyclic cyclic voltammogramsvoltammograms (100(100 mV/s)mV/s)mV/s) forfor various various K K++ concentrationsconcentrations (2–10(2–10 (2–10 mM);mM); (( BB))) quadraticquadratic calibrationcalibration curvecurve curve representingrepresenting representing thethe the relationrelation betweenbetween thethe stablestable oxidization oxidization peakpeak currentcurrent andand concentration; concentration; scanning scanning rang range:range:e: 0.2–0.60.2–0.6 V,V, pulsepulse heightheight 100100 mV,mV, pulse pulsepulse width widthwidth 200 200200 ms, ms,ms, step stepstep height heightheight 20 2020 mV, mV,mV, N NN = == 3. 3.3.

Biosensors 2021, 11, x FOR PEER REVIEW 9 of 12

(4) Interference detection We used normal concentrations of Na+, K+, Cl−, Ca2+, and Mg2+ in human blood to examine the influence of interference substances on the oxidation current response of dif- ferent electrodes. The experimental results are depicted in Figure 8. The results of the Cl− detection interference experiments (Figure 8) showed that the oxidation currents of four (KCl, NaCl, CaCl2, and MgCl2) having the same con- centration (100 mM) were between 1.84 and 1.94 mA, with an average current of 1.89 mA. The RSD was calculated to be 2.35%. The experimental results indicate that the Cl− detec- tion electrode was not interfered by cations. The results of the Na+ detection interference experiments (Figure 8) showed that the current response of the Na+ detection electrode in the NaCl solution was 5.55 μA. After KCl, CaCl2, and MgCl2 were added to the KCl solution, the current interferences were measured to be 1.08%, 7.31%, and 1.68%, respectively. Among the introduced compounds, CaCl2 had the greatest impact on the current response. However, there was no statistically significant difference between the current response of the NaCl solution containing any of the three interference substances and that of the pure NaCl solution. The experimental Biosensors 2021, 11, 109 9 of 12 results show that the proposed Na+ detection electrode has good selectivity. As shown in Figure 8, the current response of the K+ detection electrode in pure KCl solution was measured to be 8.92 μA. The current interferences after the addition of NaCl, (4)CaClInterference2, and MgCl2 detection were measured to be 5.23%, 1.01%, and 4.21%, respectively. Similar to the results of the interference experiment of the Na+ detection electrode, there was no sta- We used normal concentrations of Na+,K+, Cl−, Ca2+, and Mg2+ in human blood tistically significant difference between the current response of the KCl solution contain- to examine the influence of interference substances on the oxidation current response of ing any of the three interference substances and that of the pure KCl solution. The exper- different electrodes. The experimental results are depicted in Figure8. imental results show that the proposed K+ detection electrode possesses good selectivity.

+ 10 K

8

Na+ 6

4 - Current (µA) Current Cl 2

0

− FigureFigure 8.8. InterferencesInterferences detection. Cl Cl− detection:detection: 100 100 mM mM KCl, KCl, 100 100 mM mM NaCl, NaCl, 50 mM 50 mM CaCl CaCl2, and, and 50 + 2 mM MgCl2; Na detection:+ 140 mM NaCl separately mixed with 4 mM KCl, 2.5 mM CaCl2, and 1 50 mM MgCl2;+ Na detection: 140 mM NaCl separately mixed with 4 mM KCl, 2.5 mM CaCl2, and mM MgCl2; K detection:+ 4 mM KCl separately mixed with 140 mM NaCl, 2.5 mM CaCl2, and 1 1 mM MgCl2;K detection: 4 mM KCl separately mixed with 140 mM NaCl, 2.5 mM CaCl2, and mM MgCl2. 1 mM MgCl2. 3.5. Performance Comparisons The results of the Cl− detection interference experiments (Figure8) showed that the oxidationIn recent currents years, the of electrochemical four chlorides (KCl, techniques NaCl, CaClfor measuring2, and MgCl electrolyte2) having concentra- the same concentrationtion have been (100 rapidly mM) increasing. were between The 1.84materials and 1.94for Cl mA,− detection with an electrode average currentare mostly of 1.89nano- mA. and The micro-structures RSD was calculated of carbon, to be 2.35%., Theand experimentalplatinum. Sodium-ion results indicate detection that elec- the Cltrode− detection materials electrode are mainly was manganese not interfered –mo by cations.dified carbon and –modified glassThe carbon. results Potassium of the Na ion+ detection electrode interference materials include experiments manganese (Figure oxide8) showed nanorod–mod- that the currentified carbon response electrode of the as Na well+ detection as carrier- electrode and DNA-modified in the NaCl solutiongold electrode. was 5.55 TableµA. 2 After com- KCl,pares CaCl the 2detection, and MgCl performances2 were added of tothe the prop KClosed solution, detection the currentelectrodes interferences and the existing were measured to be 1.08%, 7.31%, and 1.68%, respectively. Among the introduced compounds, CaCl2 had the greatest impact on the current response. However, there was no statistically significant difference between the current response of the NaCl solution containing any of the three interference substances and that of the pure NaCl solution. The experimental results show that the proposed Na+ detection electrode has good selectivity. As shown in Figure8, the current response of the K + detection electrode in pure KCl solution was measured to be 8.92 µA. The current interferences after the addition of NaCl, CaCl2, and MgCl2 were measured to be 5.23%, 1.01%, and 4.21%, respectively. Similar to the results of the interference experiment of the Na+ detection electrode, there was no sta- tistically significant difference between the current response of the KCl solution containing any of the three interference substances and that of the pure KCl solution. The experimental results show that the proposed K+ detection electrode possesses good selectivity.

3.5. Performance Comparisons In recent years, the electrochemical techniques for measuring electrolyte concentration have been rapidly increasing. The materials for Cl− detection electrode are mostly nano- and micro-structures of carbon, silver, and platinum. Sodium-ion detection electrode materials are mainly manganese oxide–modified carbon and iron sulfate–modified carbon. Potassium ion electrode materials include manganese oxide nanorod–modified carbon electrode as well as carrier- and DNA-modified gold electrode. Table2 compares the Biosensors 2021, 11, 109 10 of 12

detection performances of the proposed detection electrodes and the existing technologies. The proposed detection electrode has characteristics such as the ability to be prepared under relatively simple process conditions, excellent detection sensitivity, and low RSD, and the detection linear range is suitable for the chloride, sodium, and potassium ions concentrations in human serum.

Table 2. Performance comparison of the proposed ion detection electrode and existing technologies.

Sensitivity Linear Range RSD Ions Electrodes Reference (µA·mM−1) (mM) (%) Ag/SPE 1.98 10–200 <10 [2] AgGNP/SPE 0.0000193 0.0005–0.09 2.22 [22] Cl− SPPtE −24.147 0.76–150 5.8 [23] 3D-GN/CPE 0.044 0.5–1000 - [25] This study 19.1 25–200 0.79

Mn3O4/CPE 19.5 0.02–0.2 - [26] + Na FePO4/GCE 0.015 25–250 - [42] This study 0.126 50–200 1.65

MnO2 nanorods/GCE 0.0004 0.002–0.09 - [21] 4-aminobenzo-18-Crown-6 - 0.5–7 - [43] K+ ether/11-Mercaptoundecanoic acid/Au DNA/Au 113 10−7–100 <5 [44] This study 0.56–7.98 2–10 0.47 Note: Normal Cl−, Na+,K+ concentrations in human blood: 98–106 mM, 135–145 mM, and 3.5–5 mM, respectively.

4. Conclusions In this study, Cl−, Na+, and K+ detection electrodes were successfully developed through a simple process to effectively improve the electrolyte detection speed and obtain an appropriate linear detection range, good sensitivity, RSD and recovery rate. Through CV and SWV, the detection data can be obtained in two minutes. The linear detection ranges in the standard samples of Cl−, Na+, and K+ were measured to be 25–200 mM, 50–200 mM, and 2–10 mM, with the average RSDs of 0.79%, 1.65%, and 0.47%, respectively, and the average recovery rates of 101%, 100% and 96%, respectively. The proposed detection electrode has characteristics such the ability to be prepared under relatively simple process conditions, excellent detection sensitivity, and low RSD; moreover, the detection linear range is suitable for the Cl−, Na+, and K+ concentrations in human serum.

Author Contributions: Conceptualization, G.-J.W. and L.-D.C.; methodology, G.-J.W. and L.-D.C.; validation, G.-J.W. and L.-D.C.; formal analysis, G.-J.W. and W.-J.W.; data curation, L.-D.C.; writing— original draft preparation, G.-J.W.; writing—review and editing, G.-J.W. and W.-J.W.; supervision, G.-J.W.; funding acquisition, G.-J.W. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Ministry of Science and Technology of Taiwan, grant number MOST-109y-002. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data that support the findings of this study are available from the corresponding author upon reasonable request. Conflicts of Interest: The authors declare no conflict of interest.

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