Coated Copper Wire Calcium Selective Microelectrode for Applications in Dental Medicine

EUGENIA EFTIMIE TOTU1, IBRAHIM ISILDAK2, OZLEM TAVUKCUOGLU2, ISMAIL AGIR3, RIDVAN YILDIRIM2, MUSTAFA NIGDE2, AURELIA CRISTINA NECHIFOR1, CORINA MARILENA CRISTACHE4* 1University Politehnica of Bucharest, Faculty of Applied Chemistry and Material Science, 1-5 Polizu Str., 11061 Bucharest, Romania. 2Department of Bioengineering, Faculty of Chemical and Metallurgical Engineering, Yildiz Technical University, 34210 EsenlerIstanbul, Turkey 3Bioengineering Department, Istanbul Medeniyet University, Goztepe, 34700 Istanbul, Turkey 4 University of Medicine and Pharmacy Carol Davila, Faculty of Midwifery and Medical Assisting (FMAM), Department of Dental Techniques, 8, Eroilor Sanitari Blvd., 050474,Bucharest, Romania.

During last years, important progress has been obtained in the area of biological and clinical analysis using ion selective electrodes with membranes based on macrocyclic transporters [1][2][3]. In potentiometric methodology, a significant development was represented by the ion selective electrodes. These selective devices allowed the application of the potentiometric method for complex samples analysis, including for clinical environment. One of the well-known application of the microelectrodes with membranes based on ionophores is the usage of flux injection analysis systems for determining Na + and K + ions in blood [4]. A successful class of selective microelectrodes is the microelectrodes realized by covering conducting wires with selective membranes, being simple and without requiring internal reference electrode. Although in such system a thermodynamic equilibrium between selective membrane and its solid support is not established, the covered wire microelectrodes are easy to be prepared and the function is satisfactory [5], with a Nernstian behavior and a good selectivity. Lot of efforts have been directed towards the development of sensitive microdevices for applications in pharmaceutical formulations analysis. Depending on their dimension, form and configuration, the microsensors could be also used as portable tools for medical diagnosis.
In the context of our keen interest to develop and introduce new sensitive microdevices able to determine various inorganic species into saliva and gingival crevicular fluid, we developed a coated wire calcium selective microelectrode as an alternative to an ISFET structure based sensor [6]. Because both saliva and the gingival crevicular fluid (GCF) are considered as possible markers for periodontal disease evolution [7][8][9][10] we consider that the development of various types of microsensors able to assess the level of different ions in GCF could be of great importance for early diagnosis and evaluation of periodontal disease.
The aim of the present paper is to introduce a coated wire calcium selective microsensor for applications in dental medicine.

Experimental part
In the performed experiments, the following chemicals have been used: tetrahydrofuran (THF) (Fluka) as solvent, high molecular weight polyvinylchloride (PVC) (Fluka) for the polymeric matrix, o-nitro phenyl octyl ether (NPOE) (Fluka) as plasticizer, [12, (4-ethylphenyl) dodecyl]) (Fluka), potassium tetrakis (p-chloro) phenyl borate (KTpClPB) (Fluka) as lipophilic agent and graphite (Fluka). In addition, there has been used epoxy resin (Ultrapure SU 2227, Victor, Italy), hardener (Desmodur RFE) (Bayer AG). As electroactive agent it was the calcium ionophore IV (N, N-Dicyclohexyl-N ', N'-dioctadecyl-3-oxapentanamide (Merck). The electrolyte solutions have been prepared using highly pure salts provided by Merck and ultrapure water (18.2 MΩ, ELGA System). Standard solution of calcium have been obtained from calcium chloride (Merck) and the interfering solutions from the corresponding chloride salt for: magnesium, sodium, lithium, potassium, barium, strontium and ammonium (Merck). The analyte solutions have been prepared by subsequent dilution from an initial stock solution, 10 -1 mol/L CaCl 2 . The studies dedicated to the pH influence on the potentiometric answer of the calcium microelectrode have been conducted in phosphate buffered solutions (Merck).
For microelectrode manufacturing, copper wires of approximately 0.4-0.5 mm diameter and 5-10 cm length have been used. Electrochemical measurements have been performed on a multichannel potentiometer with associated software (ISEMS-4, Medisen). As well known, in all electrochemical measurements, the indicating electrode, the calcium selective electrode to be developed, and the reference electrode need to be present simultaneously. Therefore, in order to comply with the specificity of the indicating electrode, a homemade microsized solid-state Ag/AgCl reference electrode [11] was used.

Calcium selective membrane
The calcium selective membrane was prepared using a PVC cocktail [12]. In 5 mL of THF solvent were thoroughly mixed: PVC 29%, plasticizer NPOE 68%, calcium ionophore IV 2% and KpTClPB 1%, all added in mass percentage (w/ w). This membrane was further used to obtain the microelectrode.

Solid-state calcium selective microelectrode
The solid-state contact mixture containing: graphite, 50% (wt), epoxy resin, 35% (wt) and hardener, 15% (wt) was dissolved in THF solvent. While mixing, the appropriate viscosity was reached and the copper wires were dipped in the mixture in order to be covered. This procedure was repeated 6 to 8 times to assure a correct and uniform coverage of the copper wires. The wires were left overnight in opened air, at room temperature.
The previously prepared calcium selective membrane was used as dipping media for the conditioned copper wires. As consequence, the membrane matrix was coextruded being deposited over the solid-state contact material. The calcium selective electrode obtained was conditioned for 24h in 10 -2 mol/L CaCl 2 solution before its usage. In figure 1 some of the obtained calcium selective microelectrodes are presented. calcium (mV), E 0' -standard electrochemical potential of the calcium-selective microelectrode (mV), R -the universal gases constant, 8.314 J K "1 mol "1 , T -absolute temperature, (K), F-the Faraday constant (coulombs/ mole of electrons), 96,485.34 C/mol, and a Ca -chemical activity of calcium.
If, by contrary, the solution would contain only the interfering ion, i, calcium -selective microelectrode's potentiometric answer becomes: (2) where: E Ca,i -electrochemical potential of the calcium selective microelectrode in solution containing only interfering ion, i (mV), a i -chemical activity of interfering ion, i, and stands for selectivity coefficient of calcium against interfering ion, i.
When working at equal activities for calcium and interfering ion, i, then from the previous relationships one obtains: We could also approach this method using the potentiometric determinations of the two solutions (calcium and interfering ion) when the activities a Ca and a i are varied and the potentials defined by equations (1) and (2) are graphically represented. Such diagram should contain two linear graphs from where it would be determined the activities values, a Ca and a i , for which the two electrochemical potentials would become equal, E Ca,i = E Ca . In this situation, the selectivity coefficient is calculated according to: (5) Saliva content in calcium ions is 1.2x10 -3 -2.80x10 -3 mol/ L, which is similar to the calcium content in plasma. Calcium is one of the inorganic ions, which has been intensely studied as a potential indicator for periodontal disease in saliva. Saliva with an increased calcium concentration proved to be characteristic for patients with periodontitis [7]. The gingival crevicular fluid could contain 10 -2 mol/L calcium in healthy patients and up to 1.59 x 10 -2 mol/L calcium for in moderate periodontitis [10]. Following our intentions to develop a solid-state contact calcium-selective microsensor for assessing the calcium level in the gingival crevicular fluid (GCL) and saliva, the calcium micro-device should be tailored in such way to be highly sensitive for calcium within the specific concentration range 10 -2 mol/L -1.2x10 -3 mol/L.

Results and discussions
The Nernstian behavior of the obtained Ca 2+ -selective microelectrode was tested for Ca +2 solutions with concentration ranging between 10 -6 and 10 -1 mol/L. The specific behavior could be followed in figure 2 where the potentiometric response of two identical Ca 2+ -selective microelectrodes is presented. The specific potentiometric performance of the prepared Ca 2+ -selective microelectrode was studied against the following cations: Mg 2+ , Ca 2+ , Li + , Na + , K + , NH 4 + , Sr 2+ , Ba 2+ with concentrations ranging between 10 -6 and 10 -1 mol/L. It was noticed that the prepared microelectrode exhibited fast, selective and Electrochemical characterization The calcium microelectrodes were electrochemically characterized. The specific potentiometric behavior has been evaluated, as well as the selectivity against main interfering ions. These interfering ions were chosen taking into account the final application of the microelectrodesfor the clinical area. In addition, the response time, detection limit and working pH range of the prepared Ca 2+selective microelectrode have been investigated.
The separate solution method was applied for the determination of selectivity coefficients. Considering that our newly prepared calcium selective microelectrode is answering to a solution containing only the ion Ca 2+ , then the electrochemical potential varies according to: (1) where: E Ca -electrochemical potential of the calcium selective microelectrode in solution containing only reproducible response against Ca 2+ ion in the presence of interfering ions.
One of the most important dynamic characteristics is the detection limit of ion selective electrodes. For the obtained calcium-selective microelectrode the detection limit was calculated using the calibration curve, which is presented in figure 3. For the Ca 2+ -selective microelectrode the determined detection limit was 3.26-10 -6 mol/L, which is satisfactory for the performances that we are seeking for.
In order to establish the selectivity constants, potentiometric measurements of the calcium solution and interfering ion solutions with the PVC matrix based Ca 2+selective microelectrode were done. The potentiometric behavior of Ca 2+ -selective microelectrode against different interfering ions is presented in figure 4.
During this study, the selectivity coefficients have been calculated applying the separate solution method [13,14] using the electrochemical potentials equalization. Therefore, the concentrations of the calcium ion solution that gives equal potential measured in the 10 -2 mol/L solution of the interfering ion have been determined. The calculated values are presented in table 1.
The experimental results recorded showed that the prepared Ca 2+ -selective microelectrode is highly selective against the various interfering ions as presented in figure 4.
The calcium microelectrode described in the present paper has been developed for further clinical applications. For such applications, the response time is very important, as we are seeking to obtain a quick and accurate potentiometric answer. In figure, 5 the variation of the response time for the calcium microelectrode could be followed. The microelectrode has been immersed in standard calcium chloride solutions applying two subsequent concentration sequences: from 10 -1 to 10 -6 mol/ L and form 10 -6 to 10 -1 mol/L, respectively. In the transition from 10 -4 mol/L Ca 2+ to 10 -3 mol/L Ca 2+ solution, the time corresponding to 95% of the equilibrium time of the microelectrode potential has been calculated according to IUPAC [13]. The response time (t95) of the PVC-matrix Ca 2+ -selective microelectrode was lower than 6 s. We could conclude that the obtained selective device presents a very short and satisfactory response time.
Another important dynamic characteristic of the calcium microelectrode is the repeatability. It is known that for getting reliable results for repeatability, we must perform the same electrochemical procedure multiple times. In order to avoid supplementary error, during this experiment, the following working conditions were secured: the same researcher applied the same procedure using the same materials and equipment keeping unchanged the environmental conditions and all determinations were done  in a short period of time. For determining the repeatability parameter, the microelectrode was immersed in different calcium chloride solutions, namely 10 -4 , 10 -3 , and 10 -2 mol/ L, respectively. The immersion procedure was performed/ repeated 25 times in a row, when approximately the same potential values were recorded after each measurement. This could be considered as an indication regarding a very good repeatability of the calcium microelectrode. The specific behavior during the applied procedure to determine the repeatability is presented in figure 6. The test run for 10 -2 mol/L Ca +2 solution gave for the standard deviation of the mean 0.20 compared with that ones for 10 -3 mol/L Ca +2 solution which was 0.09 and for 10 -4 mol/L Ca +2 solution when 0.08 was recorded. In table 2 the statistical parameters based on the measurements shown in figure  5 are presented. The lower value of the standard deviation of the mean (table 2) indicates a higher reliability of the results.
The environment of the clinical samples to be analyzed using the obtained calcium microelectrode is dependent upon the pH variation. As consequence, it is important to establish the pH working range for the microelectrode.
After potentiometric calibration of the microelectrode for standard calcium solutions with concentrations ranging between 10 -1 mol/L to 10 -6 mol/L, measurements to follow the influence of pH on the electrochemical answer of the microelectrode were performed. Phosphate buffer solutions (5-10 -3 mol/L) were used to obtain a variation of pH frm 4 to 9. In these buffered solutions, the calcium ion concentration was maintained constant to 10 -3 mol/L or to 10 -4 mol/L. In figure 7 the microelectrode electrochemical behavior in solutions with varied pH could be observed.
The prepared Ca 2+ -selective microelectrode does not show a significant potential change between pH = 4 and pH = 9. Therefore, we could conclude that the microelectrode is functioning without being significantly affected by the pH of the medium ranging between 4 and 9. It is known that, in oral cavity, saliva's pH is maintained within the neutral range between 6.7 to 7.3 and the resting pH is not lower than 6.3 [15]. It has to be noted that for the physiological pH range 6.3 -7.3, the recorded electrochemical potentials do not vary with more than 5 mV in answer.

Conclusions
Due to its characteristics and miniaturization, the obtained calcium microelectrode could be easily used in biomedical environment, clinical studies and particularly in dental medicine for gingival crevicular fluid assessment in periodontal disease. Our previous presented CHEMFET [6] could be considered as a natural extension of the actual presented coated-wire calcium microsensor. The obtained solid-state contact coated wire calcium microsensor proved high selectivity for calcium against main interfering ions present in GCF or saliva. The electrochemical dynamic characteristics of the calcium microsensor: nernstian response, 6s for response time, detection limit of 3.26 -10 -6 mol/L allow its usage for assessing the calcium level from complex biological matrices. Much more, the stability of its electrochemical answer within the physiological pH range (6.3-7.3) transforms the developed solid-state calcium microelectrode into a useful tool for investigations over the evolution of the calcium level in GCF or saliva.