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Coated Copper Wire Calciu m Selective Microelectrode for Applications in
Dental Medicine
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REV.CHIM.(Bucharest) ♦69♦No.11♦2018 http://www.revistadechimie.ro 4113Coated 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 Esenler-
Istanbul, 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.
A coated wire calcium selective microelectrode for biological use was developed, comprising a PVC selective
matrix containing calcium ionophore IV coated on copper wire, previously covered with a solid-state contact
mixture. The obtained calcium microsensor presented a Nernstian answer in a concentration range of 10-1 to
10-6
mol/L. The selectivity coefficients over the main interfering ions of biological interest proved that the
calcium microelectrode is highly selective. Also, the response time (6s) and repeatability have been
determined. The pH variation did not significantly modify the calcium microelectrode answer, being stable
over the pH range (6.7-7.3) of interest. The obtained calcium microelectrode is simple, inexpensive and able
to give reliable electrochemical response, recommending itself as a solution for assessing the level of
inorganic ions of the gingival crevicular fluid and saliva.
Keywords : calcium microsensor, coated wire microsensor, solid-state contact mixture, PVC selective mixture,
gingival crevicular fluid
*email: [anonimizat] last years, important progress has been obtained
in the area of biological and clinical analysis using ionselective electrodes with membranes based on
macrocyclic transporters [1-3]. In potentiometric
methodology, a significant development was representedby the ion selective electrodes. These selective devices
allowed the application of the potentiometric method for
complex samples analysis, including for clinicalenvironment. One of the well-known application of the
microelectrodes with membranes based on ionophores is
the usage of flux injection analysis systems for determiningNa
+ and K+ ions in blood [4]. A successful class of selective
microelectrodes is the microelectrodes realized by covering
conducting wires with selective membranes, being simpleand 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 beendirected towards the development of sensitive
microdevices for applications in pharmaceutical
formulations analysis. Depending on their dimension, formand 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 selectivemicroelectrode as an alternative to an ISFET structure based
sensor [6]. Because both saliva and the gingival crevicular
fluid (GCF) are considered as possible markers forperiodontal disease evolution [7-10] we consider that the
development of various types of microsensors able toassess 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 saltfor: magnesium, sodium, lithium, potassium, barium,
strontium and ammonium (Merck). The analyte solutions
have been prepared by subsequent dilution from an initialstock solution, 10
-1mol/L CaCl2. The studies dedicated to
the pH influence on the potentiometric answer of the
calcium microelectrode have been conducted inphosphate buffered solutions (Merck).
For microelectrode manufacturing, copper wires of
approximately 0.4-0.5 mm diameter and 5-10 cm length

http://www.revistadechimie.ro REV.CHIM.(Bucharest) ♦69♦No.11♦2018 4114have 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 presentsimultaneously. Therefore, in order to comply with the
specificity of the indicating electrode, a homemade micro-
sized solid-state Ag/AgCl reference electrode [11] wasused.
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 ionophoreIV 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 wireswere 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 wereleft overnight in opened air, at room temperature.
The previously prepared calcium selective membrane
was used as dipping media for the conditioned copperwires. As consequence, the membrane matrix was
coextruded being deposited over the solid-state contact
material. The calcium selective electrode obtained wasconditioned for 24h in 10
-2
mol/L CaCl2 solution before its
usage. In figure 1 some of the obtained calcium selective
microelectrodes are presented.calcium (mV), E0’ – 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 aCa – 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: ECa,i – electrochemical potential of the calcium
selective microelectrode in solution containing only
interfering ion, i (mV), ai – 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 aiare varied and the potentials defined by equations (1) and(2) are graphically represented. Such diagram shouldcontain two linear graphs from where it would be
determined the activities values, a
Ca and ai, for which the
two electrochemical potentials would become equal, ECa,i= ECa. In this situation, the selectivity coefficient is
calculated according to:

(5)
Saliva content in calcium ions is 1.2×10-3 – 2.80×10-3 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 calciumconcentration 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 calciumlevel 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 specificconcentration range 10
-2 mol/L – 1.2×10-3 mol/L.
Results and discussions
The Nernstian behavior of the obtained Ca2+-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 Ca2+-selective
microelectrodes is presented. The specific potentiometricperformance of the prepared Ca
2+-selective microelectrode
was studied against the following cations: Mg2+, Ca2+, Li+,
Na+, K+, NH4+, Sr2+, Ba2+ with concentrations ranging
between 10-6 and 10-1 mol/L. It was noticed that the
prepared microelectrode exhibited fast, selective and
Fig. 1. Calcium selective
microelectrode
Electrochemical characterization
The calcium microelectrodes were electrochemically
characterized. The specific potentiometric behavior hasbeen evaluated, as well as the selectivity against main
interfering ions. These interfering ions were chosen taking
into account the final application of the microelectrodes -for the clinical area. In addition, the response time,
detection limit and working
pH range of the prepared Ca2+-
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 isanswering to a solution containing only the ion Ca
2+, then
the electrochemical potential varies according to:
(1)
where: ECa – electrochemical potential of the calcium
selective microelectrode in solution containing only(3)
(4)

REV.CHIM.(Bucharest) ♦69♦No.11♦2018 http://www.revistadechimie.ro 4115reproducible response against Ca2+ 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 ispresented 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 seekingfor.
In order to establish the selectivity constants,
potentiometric measurements of the calcium solution andinterfering ion solutions with the PVC matrix based Ca
2+ –
selective microelectrode were done. The potentiometric
behavior of Ca2+ – 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 Ca2+-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 theresponse 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 Ca2+ to 10-3 mol/L Ca2+ solution, the time
corresponding to 95% of the equilibrium time of the
microelectrode potential has been calculated accordingto 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 presentsa very short and satisfactory response time.
Another important dynamic characteristic of the calcium
microelectrode is the repeatability. It is known that forgetting 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 theenvironmental conditions and all determinations were done
Fig.2 Potential-time plots for two identical PVC matrix based Ca2+-
selective microelectrodes
Fig.3 Calibration curve for Ca2+ – selective microelectrodeFig. 4 Potentiometric behavior of PVC-matrix based Ca2+
selective microelectrode against different interfering ions
Table 1
SELECTIVITY CONSTANTS CALCULATED FOR PVC-MATRIX CA2+ –
SELECTIVE MICROELECTRODE
Fig. 5 Response time of the Ca2+-selective microelectrode

http://www.revistadechimie.ro REV.CHIM.(Bucharest) ♦69♦No.11♦2018 4116
Table 2
STATISTICAL PARAMETERS
Fig.6. Repeatability of the obtained Ca2+-selective microelectrode
Fig.7 pH working range of the PVC-matrix based
Ca2+-selective microelectrode
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 verygood 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 10-1mol/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 Ca2+-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 recordedelectrochemical 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 assessmentin periodontal disease. Our previous presented CHEMFET
[6] could be considered as a natural extension of the actual
presented coated-wire calcium microsensor. The obtainedsolid-state contact coated wire calcium microsensor
proved high selectivity for calcium against main interfering
ions present in GCF or saliva. The electrochemical dynamiccharacteristics 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-statecalcium microelectrode into a useful tool for investigations
over the evolution of the calcium level in GCF or saliva.
Acknowledgements: This work was supported by a grant of the
Romanian National Authority for Scientific Research and Innovation,
CCCDI – UEFISCDI, project number 39/2018 COFUND-MANUNET III-
HAMELDENT, within PNCDI III and by the Scientific and T echnological
Research Council of Turkey (TÜBITAK), Grant no: BIYOTEG-9170032.
Also, the authors acknowledge the support of the grant of the Romanian
National Authority for Scientific Research and Innovation, CCDI-
UEFISCDI, project number 30/2016, MANUNET II – PRIDENTPRO within
PNCDI III.
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Manuscript received:21.02.2018
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