- Email: [email protected]
RETRACTED: Development of electrode carbon paste modified by molecularly imprinted polymer as sensor for analysis of creatinine by potentiometric
Results in Physics 7 (2017) 1808–1817
Contents lists available at ScienceDirect
Results in Physics journal homepage: www.journals.elsevier.com/results-in-physics
Development of electrode carbon paste modified by molecularly imprinted polymer as sensor for analysis of creatinine by potentiometric Handoko Darmokoesoemo a,⇑, Miratul Khasanah a, Nunung Mareta Sari a, Yassine Kadmi b,c,d,e, Hicham Elmsellem f, Heri Septya Kusuma a,⇑ a
Department of Chemistry, Faculty of Science and Technology, Airlangga University, 60115, Indonesia Université d’Artois, EA 7394, Institut Charles Viollette, Lens F-62300, France ISA Lille, EA 7394, Institut Charles Viollette, Lille F-59000, France d Ulco, EA 7394, Institut Charles Viollette, Boulogne sur Mer F-62200, France e Université de Lille, EA 7394, Institut Charles Viollette, Lille F-59000, France f Laboratoire de chimie analytique appliquée, matériaux et environnement (LC2AME), Faculté des Sciences, B.P. 717, 60000 Oujda, Morocco b c
a r t i c l e
i n f o
Article history: Received 15 April 2017 Accepted 8 May 2017 Available online 12 May 2017 Keywords: Creatinine Molecularly imprinted polymer Carbon paste Potentiometric
a b s t r a c t This research aims to develop an electrode carbon paste/molecularly imprinted polymer (MIP) as a sensor for the analysis of creatinine by potentiometric. The preparation of a non-imprinted polymer (NIP) is done by mixing aniline, creatinine, and potassium peroxodisulfate with mole ratio of 2:0.1:1. Creatinine is extracted from the polymer using hot water to form mold creatinine, this so-called MIP. In this study, the optimum electrode has a composition made of activated carbon, paraffin, and MIP of 40:35:25 (mass ratio), pH of working solution 6, and response time of 91–192 s. The Nernst factor obtained from this study was 23.2 mV/decade, the measuring range of 106–103 M, lower detection limit of 5.49 106 M, and the upper detection limit of 1.07 103 M. The accuracy of methods for concentration of 106–103 M was 76.40–165.80%, and the precision expressed by the coefficient of variation for the concentration is 0.05–0.32%. The electrodes have up to 83 times of usage. Urea does not interfere with the performance of the electrode carbon paste MIP on the analysis of creatinine. Ó 2017 The Author. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction Creatinine is a metabolic waste product of the breakdown of muscle creatine. The creatinine levels in the blood serum indicate an equilibrium between production and excretion by the kidney and is an indicator of kidney function [1]. Normal concentrations of creatinine in the blood serum for adult males is 0.62 to 1.10 mg/dL and 0.45 to 0.75 mg/dL for adult women [2]. Increased serum creatinine levels are comparable with the decline in the quality of the kidneys; thus, analysis of creatinine levels in the blood is important for understanding the quality of work in the kidneys. The Jaffe method is generally used for creatinine analysis in the medical field. Analysis of creatinine using the Jaffe method has a principle, namely, by forming a colored complex solution, which can be analyzed by a UV–Vis spectrophotometer. The advantages of this method is an easy and simple analytical process, while ⇑ Corresponding authors. E-mail addresses: [email protected] (H. Darmokoesoemo), [email protected] (H.S. Kusuma).
the drawback is the low selectivity. Other common methods used for the analysis of creatinine is the enzymatic method. Analysis of creatinine with this method is hardly bothered by another matrix, but it takes a long time for the analysis, and the cost is quite expensive [3]. Another method developed for the analysis of creatinine is voltammetry. Lakshmi et al. (2006) [4] conducted analysis of creatinine in bloodserumby differential pulse cathodic stripping voltammetry with electrode hanging mercury drop (HMD) modified molecularly imprinted polymer (MIP) made from melamine as monomer and chloranyl. The analysis showed that the modified electrode has high selectivity toward creatinine because it is not disturbed by the presence of NaCl, creatine, urea, glucose, phenylalanine, tyrosine, histidine, and cytosine. Rizki (2015) [5] has modified carbon paste electrodes with MIP using the same monomer for analysis of creatinine by stripping voltammetry. Analysis of creatinine using this modified electrode has low selectivity in urea matrix when creatinine levels are low. The potentiometric method also has been used for the analysis of creatinine. This method uses two electrodes: the working electrode and reference electrode. The function of the working elec-
http://dx.doi.org/10.1016/j.rinp.2017.05.012 2211-3797/Ó 2017 The Author. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
H. Darmokoesoemo et al. / Results in Physics 7 (2017) 1808–1817
trode is to censor the analyte in the solution being analyzed, so that the working electrode must be selective and sensitive to analyte. Electrodes used for potentiometric analysis can be modified with the aim to increase the selectivity and sensitivity to analyte. Palupi (2014) [6] modified carbon paste electrodes using MIP with methacrylic acid (MAA) as a monomer for creatinine potentiometric analysis. The results of the analysis using the modified electrode has high selectivity toward creatinine in samples that also contain urea. The presence of urea up to 60 mg/dL do not interfere with the analysis of creatinine. Chandra (2014) [7] have modified carbon electrodes with imprinted zeolite for creatinine potentiometric analysis. The analysis of creatinine by potentiometric using the electrode has high accuracy. In this research, analysis of creatinine by potentiometric using carbon paste electrodes are modified with MIP made from aniline as the monomer. The parameters studied were the optimum composition of the MIP, carbon nanoporous, and paraffin in the preparation of carbon paste electrodes. Additionally, we studied the optimum pH solution in order to obtain good analytical results. Performance of modified electrodes is studied through parameter response time, life time, measurement range, Nernst factors, detection limit, selectivity, sensitivity, accuracy, and precision.
Materials and methods Materials and chemicals The chemicals used in this study were creatinine (C4H7N3O), aniline (C6H5NH2), potassium peroxodisulfate (K2S2O8), hydrochloric acid (HCl), acetic acid (CH3COOH), sodium acetate (CH3COONa), sodium hydrogenphosphate (Na2HPO4, 2H2O), sodium dihydrogenphosphate (NaH2PO4, 2H2O), urea (CO(NH2)2), activated carbon, solid paraffin, and Ag wire. Chemicals used have a degree of purity of pro analysis. Distilled water is also used.
Preparation of polyaniline Polyaniline was made with a 0.3 mL pipette of aniline and then mixed with 7.5 mL of HCl 1 M. The solution was stirred with a magnetic stirrer and heated for 30 min at 50 °C. After 30 min, a potassium peroxodisulfate solution made of 0.5000 g potassium peroxodisulfate and dissolved in 2.5 mL of distilled water was added. The addition of potassium peroxodisulfate was done slowly, with slow stirring. Then the solution stood at room temperature for 12 h. The solids that formed were washed using HCl 1 M. The solids were then dried and characterized using FTIR and XRD.
Preparation of molecularly imprinted polymer (MIP) Half of the NIP is used to make a molecularly imprinted polymer (MIP). MIP is made by extracting creatinine from synthesis, as a result of NIP using hot water (50 °C) through the centrifugation technique for 20 min. Extraction was performed 10 times. The solids obtained were dried and characterized using FTIR. Preparation of carbon Preparation of carbon is done by chemical and physical activation. The chemical activation is done by soaking carbon in a solution of HCl 4 N for 24 h and then soaked in n-hexane. Physical activation is done by heating the carbon at a temperature of 500 °C in a furnace. Preparation of electrode carbon paste MIP The electrode carbon paste MIP was made with a set Ag wire in a 1 mL micropipette tip. The function of the Ag wire is to act as a liaison between the electrodes with a potentiometer. Then, the micropipette tip was filled with solid paraffin up to threequarters. The remaining parts are still empty; the micropipette tip is filled with a mixture of activated carbon, paraffin, and MIP. The mixture of activated carbon, paraffin, and MIP was added with a certain ratio. Before placing the micropipette tip, solid paraffin is heated in a watch glass at 60 °C using a hotplate. Filling the mixture into micropipette tip is done by way of emphasis. The surface of electrode carbon paste MIP was scrubbed using the HVS to be smooth. Construction MIP carbon paste electrodes are shown in Fig. 1. Optimization of electrode composition Optimization of electrode composition is performed to determine comparison of electrode constituent material, which gives optimum performance of the electrode. The mixture composition of activated carbon, paraffin, and MIP is shown in Table 1 [8]. The optimum composition of electrodes was determined by measuring potential of creatinine standard solutions using electrodes based on the composition as shown in Table 1. The electrodes that generate a great Nernst factor and wide range of measurement are the electrodes showing good performance. The optimum composition of the electrodes is used as the basis for preparation electrode carbon paste modified NIP and electrode carbon paste modified polyaniline. The three electrodes were used to measure the potential of creatinine standard solution to determine its performance.
Preparation of nonmolecularly imprinted polymer (NIP) The preparation of nonmolecularly imprinted polymer (NIP) was done by mixing the monomer, initiator, and templates (analyte) with a mole ratio of 2:1:0.1. Monomers used in this study are made of aniline; the initiator is potassium peroxodisulfate, along with astemplateis creatinine. A 0.3 mL aniline pipette, mixed with 7.5 mL of HCl 1 M, was added as much as 0.0183 g of creatinine. The mixture was stirred with a magnetic stirrer and heated at 50 °C for 30 min. Then, we added a potassium peroxodisulfate solution (made of 0.5000 g of potassium peroxodisulfate in 2.5 mL of distilled water) done dropwise, with stirring slowed. The solution was allowed to stand at room temperature for 12 h and washed using HCl 1 M. The solid was dried and characterized using FTIR.
1809
Fig. 1. Construction-MIP carbon paste electrode.
1810
H. Darmokoesoemo et al. / Results in Physics 7 (2017) 1808–1817
Table 1 Composition of carbon, paraffin, and MIP in the preparation of electrodes. Electrode
E1 E2 E3 E4 E5 E6 E7
Mass (g)
Comparison of the mass of activated carbon, paraffin and MIP
Activated carbon
Paraffin
MIP
0.195 0.180 0.174 0.165 0.150 0.135 0.120
0.105 0.105 0.105 0.105 0.105 0.105 0.105
0 0.015 0.021 0.030 0.045 0.060 0.075
65:35:0 60:35:5 58:35:7 55:35:10 50:35:15 45:35:20 40:35:25
Optimization of pH creatinine solution
Determination of detection limit
A solution used for the optimization of pH is a creatinine solution with concentrations of 108–103 M. Each creatinine solution has a certain concentration, as much as 5.0 mL put in 50 mL volumetric flask, then added with 2 mL buffer solution pH 3, 4, 5, 6, 7, and 8 and diluted with water up to the mark. The solution was shaken until homogeneous and analyzed using working electrode carbon paste MIP. The optimum pH is pH when the solution has potential value is relatively constant.
The detection limit is the smallest or the largest concentration of analyte that can still be measured by electrodes. The detection limit is determined using the graph between the potential (E) against log concentration, which determines the cut point between the linear curve (measuring range) with a nonlinear curve potential of measurement results on the measurement of creatinine standard solutions. If at the cut point is drawn as the line toward the x axis, the obtained log is the concentration of detection limit. The antilog value of log concentration (x) of the detection limit is the detection limit value.
Measurement of creatinine standard solution The creatinine solution with a concentration of 102 M–108 M, which has been made with optimum pH, was measured for potential using electrode carbon paste MIP. Then we graphed potential (E) against log concentration of creatinine.
Determination of selectivity
The determination of response time of the working electrode carbon paste MIP to the analyte is done by measuring the potential of creatinine standard solutions with a concentration of 106–103 M at pH optimum. We measured the potential and the observed time for each creatinine standard solution to show a constant potential.
In this study, the selectivity of the electrode is expressed by the coefficient of selectivity. Selectivity coefficient values obtained by measuring the potential of the creatinine solution in accordance with normal levels in the serum is a concentration of 104 M. Measurements were performed by preparing the four pieces of a 50 mL volumetric flask, each filled with 5 mL of creatinine with a concentration of 103 M. Then the second, third, and fourth flasks are added to 5 mL of urea solution with a concentration of 102 M, 103 M, and 104 M, respectively. We subsequently added 2 mL of buffer solution pH optimum diluted with water up to the mark limit. The solution mixture is a measured potential using electrodes that have been optimized. The potential measurement results of a solution containing creatinine and urea solution is substituted into Eq. (2):
Determination of measurement range
K ij ¼
Determination of electrode performance Determination of response time
The measurement range was studied using electrodes from the optimization results to determine the potential of creatinine standard solution 102–108 M. Then we graphed the potential (E) against log concentration and determined the linear regression equation. The measurement range, indicated by the range of concentrations, is a straight line graph, which still meets the Nernst equation. Determination of Nernst factors and linearity The Nernst factor is determined by plotting between measurement potential data (E) of creatinine standard solution against log concentration, which is obtained by Eq. (1). The Nernst factor value is shown by the slope of the curve (b):
y ¼ bx þ a
ð1Þ
where y is the potential of the solution, x the value of the log concentration of the analyte, b is the slope of the curve, and a is the intercept. The linearity of the curve is shown by the correlation coefficient (r), which has a range of 1 r 1.
ai ð10ðE2 E1 =SÞ 1Þ ajn=x
ð2Þ
where ai is the activity of measured analytes, aj is the activity of the compounds that interfere, E1 is the potential before the addition of the compounds that interfere, E2 is the potential after the addition of the compounds that interfere, S is the slope of the calibration curve, n is the charge of main ions, and x is the charge of ions that interfere. If kij 1, the electrode is more selective to creatinine solution rather than the urea solution. This shows that urea does not interfere with the performance of the electrode carbon paste MIP in the analysis of creatinine. Determination of accuracy Accuracy is the degree of closeness between the levels obtained from the analysis with actual levels. A creatinine solution with a concentration in the measurement range measures potential by using electrode carbon paste MIP. The potential value of the measurement results is an analogy as y and substituted in the obtained linear regression equation, so that we can determine the creatinine concentration. Accuracy was determined by using Eq. (3):
H. Darmokoesoemo et al. / Results in Physics 7 (2017) 1808–1817
xt R ¼ 100% xi
ð3Þ
where R is the accuracy value, xt is the concentration from the measurement result, and xi is the actual concentration. Determination of precision Precision is the closeness between the value of the analysis results on the same analyte, the same conditions, and in a short time interval. Precision values are determined by measuring the potential of the creatinine solution on the measuring range. Potential measurements are performed at each concentration by three times replication using electrode carbon paste MIP. Precision is determined by calculating the standard deviation (SD) and coefficient of variation (CV) using Eqs. (4) and (5):
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi PN 2 i¼1 ðx xÞ SD ¼ N1 s CV ¼ 100% x
1811
line has become conductive emeraldine salt [11]. Solids that have been washed are then dried to obtain polyaniline powder. Synthesis result of nonimprinted polymer (NIP) The preparation of NIP is done by mixing the aniline monomer, initiator potassium peroxodisulfate, and creatinine as a template with a ratio of 2:1:0.1 [12]. Aniline 0.3 mL in 7.5 mL of HCl is added with 0.0183 g of creatinine. Then it is stirred using a magnetic stirrer for 30 min at 50 °C and added to the potassium peroxodisulfate solution dropwise. The solution is initially colorless after a few drops of potassium peroxodisulfate, then it turned into dark green, which thickens. The solution was allowed to stand for 12 h at room temperature to form into solids. The obtained solids were washed using HCl 1 M. Washing using HCl aims at removing residual potassium peroxodisulfate and residual unreacted aniline. NIP solids obtained a dark green color.
ð4Þ Synthesis result of molecularly imprinted polymer (MIP)
ð5Þ
with x being the value of each measurement, xis the average value of all the observations, and N the number of measurements. Determination of electrode life time The life time of the electrodes is the measured time span because the used electrode still shows good performance up to the time the electrodes had significant reduction in performance. The life time of the electrode is determined by measuring the electrode potential for the analysis of creatinine standard solution every week in order to obtain the electrode Nernst factor. Measurements were made to show the Nernst factors that deviate from the permissible value. Results and discussion Result of polymer synthesis Synthesis result of polymer control (polyaniline) In this study, analysis of creatinine by a potentiometric using electrodecarbon paste modified molecularly imprinted polymer (MIP) with aniline as the monomer. In the initial phase, synthesis of polyaniline needs to be done. Aniline is used as monomer because, in that structure, there are functional groups ANH, which are expected to interact with the functional groups of C@O contained in creatinine through hydrogen bonds. Nanostructures that are owned by polyaniline generates the conductive nanowire system, which allows direct electrical connection between the electrode and the MIP [9]. Polyaniline is also a conductive polymer that adds stability to heat, air, and moisture [10]. The preparation of polyaniline was made by mixing 0.3 mL of aniline and 7.5 mL 1 M HCl, then stirred with a magnetic stirrer for 30 min and heated at 50 °C. We also added a potassium peroxodisulfate solution, which is made of 0.5000 g of potassium peroxodisulfate crystal dissolved in 2.5 mL of distilled water. The addition of potassium peroxodisulfate as the initiator is done slowly, with slow stirring. Initially, a colorless solution turned into a dark green color, which thickens after the potassium peroxodisulfate solution is added. The green solution was allowed to stand for 12 h at room temperature. This process is intended so long as the polymerization reaction of aniline is running perfectly. Then the obtained solids are washed using HCl 1 M. Washing using HCl aims to eliminate residual unreacted potassium peroxodisulfate and ensures all formed polyani-
MIP is made by extracting creatinine of NIP powders using hot water with a temperature of 50 °C. Extraction is done many times by centrifugation to obtain neutral pH. Each extraction is done for 20 min. The purpose of extraction is to remove creatinine from the polymer; thus, we obtained mold creatinine. In this study, the MIP obtained is dark green. Characterization of control polymer, NIP, and MIP using FTIR Polymer control, NIP, and MIP from the synthesis result are characterized using FTIR. Characterization is performed to determine the functional groups contained in aniline, polyaniline, NIP, and MIP. FTIR spectra of aniline and polyaniline are shown in Fig. 2. Fig. 2 shows that the peak spectra between aniline and polyaniline appearing in wavenumbers are almost the same. That is because they are the same constituent. The same spectra between polyaniline and aniline are stretching C-Hsp2, which appears at wavenumber 3228 cm1 for aniline and 3215 cm1 for polyaniline, spectra of aromatic CAC bonds appears at wavenumber 1494 cm1 for aniline and 1483 cm1 for polyaniline. The difference spectra between aniline and polyaniline are contained in stretching CAN; the polyaniline spectra contained at wavenumber 1246 cm1 is steeper than the spectra contained in aniline at wavenumber 1273 cm1. The difference spectra between aniline and polyaniline are also contained in stretching ANH. In aniline stretching, ANH appeared in two peaks, at the wavenumbers 3431 and 3365 cm1, whereas polyaniline appears only at one peak at wavenumber 3421 cm1. That is because the aniline has structure primary ANH, and polyaniline has structure secondary ANH. The similarities and differences contained in aniline and polyaniline spectra showed that the polyaniline has been formed. FTIR data of aniline and polyaniline are shown in Table 2. The FTIR spectra of polyaniline, NIP, and MIP are shown in Fig. 3. The wavenumber data of polyaniline, NIP, and MIP are shown in Table 3. The spectra of NIP and MIP should also emerge as a peak, stating the OAH bond contained in the wavenumber around 3600 cm1. The hydrogen bond indicates the bond between creatinine with polyaniline, as shown in Fig. 4. The peak spectra hould also emerge as a typical spectral of creatinine that indicates C@O, which should appear at a wavenumber around 1700 cm1. The typical spectra of creatinine that does not appear is possible because the mole creatinine was too small. In this study, analysis using FTIR was not carried out quantitatively, so that the intensity cannot be used as a benchmark in the success of the synthesis of NIP and MIP.
1812
H. Darmokoesoemo et al. / Results in Physics 7 (2017) 1808–1817
Fig. 2. The FTIR spectra of aniline and polyaniline. Table 2 FTIR data of aniline and polyaniline. Wave number (cm1)
Description
Aniline
Polyaniline
3431 and 3365 3228 1494 1273
3421 3215 1483 1246
Stretching ANH Stretching CAH sp2 Aromatic CAC bond Stretching CAN
Characterization of polymer control using XRD The characterization using X-ray diffraction (XRD) was conducted to determine crystallinity of polyaniline. A polyaniline diffraction pattern can be seen in Fig. 5, which shows that the emerged sharp peak diffraction pattern at 2h = 25.5°. The diffraction patterns emerge due to periodicity perpendicular to the benzoid ring and quinoid of polyaniline polymer chain. Preparation of carbon In this study, we conducted carbon reactivation of activated carbon as the preparation phase. Carbon reactivation is to obtain car-
bon with a larger surface area and to improve the conductivity of carbon in order to produce great potential when used as an electrode. The carbon reactivation process is done in two steps, that is, carbon immersion using HCl 4 N and soaked with an n-hexane solution. The first step is carbon soaked in a solution of HCl 4 N, which functions as an activator. The HCl solution was chosen because it has properties of a dehydrating agent, which can improve the structure of porous carbon, whereas the concentration used 4 N because, at these concentrations, pores open to easily extract soluble impurities [13]. After soaking with HCl 4 N for ±24 h, the carbon is washed using distilled water until neutral. The neutral carbon is then dried using an oven, so that water remnants in the pores can evaporate. The second stage is soaking carbon in an n-hexane solution for ±24 h. Soaking carbon in acid solution only removes hydrogen on the outer surface of carbon, so that the required organic solvents remove hydrocarbons [14]. The organic solvent used is n-hexane. The loss of hydrogen and hydrocarbons can make the surface of the carbon become increasingly widespread. Carbon that has been soaked with n-hexane is then heated in a furnace at temperature of 500 °C. Carbon that
Fig. 3. FTIR spectra of polyaniline, NIP and MIP.
1813
H. Darmokoesoemo et al. / Results in Physics 7 (2017) 1808–1817 Table 3 FTIR data of polyaniline, NIP and MIP. Wave number (cm1)
Description
Polyaniline
NIP
MIP
3421 3228 1483 1246
– – 1494 1244
3464 3211 1491 1238
Stretching NAH Stretching CAH sp2 Aromatic CAC bond Stretching CAN
has been reactivated is tested at its surface area and pore size using the BET (Brunaeur-Emmet-Teller) and BJH (Barrett-JoynerHallenda) tests. In this study, the surface area value of porous carbon is 877.463 m2/g. The surface area value of the carbon shows that the size of the used carbon is mesoporous, while the carbon pore diameter value in this study is 3.835 nm. Performance of carbon would be good if it has a large surface area with small pore size.
Optimization results of electrode carbon paste MIP composition The optimization of electrode composition is performed to find the electrode that has maximum performance. In this study, the working electrode is made from a mixture of actived carbon, solid paraffin, and MIP. The addition of MIP as polymers with specific binding sites to the analyte aims to improve the sensitivity and selectivity of the electrode. Activated carbon is used as the electrode, as it has a large surface area that is expected to extend the binding of the analyte. Activated carbon is conductive material, so it can deliver the response to the potentiometer through the silver wire (Ag). The addition of paraffin serves as an adhesive between the activated carbon and MIP in order not to be separated when it is inserted into the micropipette tip. In this study, we created seven electrodes with composition of activated carbon and MIP, which varies with fixed paraffin composition, as shown in Table 1. The electrodes were then soaked in creatinine solution 103 M before being used for conditioning. Further electrodes were used to measure the potential of creatinine solution 108–101 M, which has been added with KCl solution as the supporting electrolyte. The measurements results of Nernst factor values, measurement range, and linearity (r) using electrodes carbon paste MIP with certain variations are shown in Table 4. The performance of electrodes in potentiometric is good if it generates good Nernst factor value and linearity and has a wide measurement range. The Nernst factor is said to be good if it satis-
fies ð59;1 ð1 2ÞÞ mV, where n is the valence of the analyte. Anan lytes measured in this study are creatinine (monovalent molecule because it releases one electron), so that it has Nernst factor of 59.1 mV. In this study, E2 and E7 have the best Nernst factor values, which are 25.17 mV and 24.54 mV, respectively. This optimization result shows that E2 has a better Nernst factor, but the linearity is less favorable than E7. Thus, pH needs tobe optimized to determine the composition of the electrode carbon paste MIP, which has maximum performance. Optimization results of pH creatinine solution The optimization of pH is performed to determine the pH range, which produces stable potential measurement. This optimization is done in a creatinine solution with concentrations of 108–103 M with a pH range of 4–8. Electrodes used were E2 and E7 as well as E1 for comparison. The Nernst factor value and measurement range at various pH using E1, E2, and E7 are shown in Table 5. The results in Table 5 show that the best Nernst factor for electrode carbon paste MIP was obtained at pH 6 using E7. But the best Nernst factor with the same measurements range was obtained at pH 8 using E1. E1 is a reference electrode, which, in its composition, does not contain MIP. The Nernst factor produced from measurements using E1 at pH 8 was also good. This is because carbon has good activity. The creatinine theoretically has a pH of 7–9 at a temperature of 25 °C. In this study, the mother solution of creatinine had a pH of 6.57. The creatinine has two dissociation constant values: pKa1 = 4.8 and pKa2 = 9.2. The creatinine in acidic condition (below pH 4.8) is in the form of cations, whereas in alkaline condition (around pH 9.2) it is in the form of anion. In this study, creatinine will produce a good Nernst factor when it is in the shape of a molecule, so that it has optimum pH 6. Standard curves of creatinine The standard curves of creatinine were created by measuring the potential of the creatinine solution with a concentration of 108–102 M at pH 6 using E7, which is the optimum pH and composition of the electrode. The potential measurement results of creatinine solution at pH 6 using E7 are shown in Table 6. The curve also shows the relationship between log concentration of creatinine solution with the measured potential of electrode. The formed curve shows a selected concentration range via straight line approaching a Nernst factor of 59.1 mV/decade and linearity approaching 1. The curve, which shows a straight line, is then used as a standard curve of creatinine. The curve showing
HN
CH3 N
HN
N
H N O
HN
NH
N H
Fig. 4. The hydrogen bond between creatinine with polyaniline.
NH
1814
H. Darmokoesoemo et al. / Results in Physics 7 (2017) 1808–1817
using E7. The measurement results of potential graph between potential (E) with log concentrations are shown in Fig. 8. Fig. 8 shows the creatinine concentration measurement range is 106–103 M with a Nernst factor value of 23.2 mV/decade and linearity 0.986. Nernst factor and linearity can be seen in the standard curve of creatinine (Fig. 8). The Nernst factor is the slope values of the standard curve of creatinine, while linearity is the correlation coefficient (r) of a standard curve. In this research, the linear regression equation y = 23.2x + 1460, and the correlation coefficient of 0.986.
Determination results of the detection limit
Fig. 5. The diffraction pattern of polyaniline.
the relationship between log concentration of creatinine with potential can be seen in Fig. 6, whereas the standard curve of creatinine is shown in Fig. 7. Performance test result of electrode and validity of method Determination results of electrode response time The electrode response time is the length of time required by the electrodes in response to the analyte in the sample solution. A faster electrode response time to the analyte indicates that the performance of the electrode is better. The electrode response time shows electrode sensitivity toward the analyte. In this study, the determination of the electrode response time is done in a creatinine solution with concentrations of 106–103 M at pH 6 using E7. The results of determination of the electrode response time can be seen in Table 7. Data in Table 7 show that the higher concentration of creatinine can make the electrode response time also faster. This is because, with the higher concentration, it can cause the amount of analyte in the solution to increase, so that the displacement of the analyte from the solution to the electrode becomes faster. Determination results of the measurement range, Nernst factor, and the linearity The measurement range is the range of concentrations that shows the linear line on the graph between the potential (E) against the log concentration and still meets the Nernst equation. The measurement method is good if it has wide measurement range. In the potentiometric, determination of measurement range also must consider the good linearity and Nernst factor. The measurement range is determined by measuring the potential standard solution of creatinine with concentrations of 108–102 M at pH 6
The detection limit is the lowest or higher concentration of analyte that can still be measured by electrodes. The detection limit can be known by determining the cut point between the linear and nonlinear curve from the curve with a relationship between log concentration of creatinine and potential, as shown in Fig. 9. In this research, the lower detection limit is 5.49 106 M, and the upper detection limit is 1.07 103 M. These results indicate that the electrode carbon paste MIP is quite good because it has fairly low detection limits. The upper detection limit of a method for the measurement of creatinine is required. It aims to facilitate the measurement of creatinine levels for patients with renal impairment who have high creatinine levels.
Determination result of selectivity The determination of selectivity is important because it can be used to determine the ability of the method to measure, without any interference, that matrices are together with the sample. The selectivity test in this study is conducted with the addition of urea solution because urea is in blood serum along with creatinine and other compounds. The selectivity test was conducted by adding urea solution with a concentration of 103 M, 104 M, and 105 M in creatinine solution of 104 M. The normal concentration of creatinine solution is 104 M, whereas the concentration of urea solution selected in this study is below, equal, and above the normal concentration of creatinine. The potential of solution is measured using E1 and E7 at pH 6 and calculated using the selectivity coefficient (kij) value. The electrode used for the measurement is E7 because this electrode has optimum composition, while E1 is used as the comparison. The result of calculation kij for the creatinine solution 104 M with a matrix of urea solution is shown in Table 8. In the calculation of kij, the value of kij < 1 indicates that these methods are more selective to creatinine and urea matrix, which were added and do not interfere with the analysis, although with concentrations below, equal, and above the normal level of creatinine in the blood serum. Data in Table 8 show that the obtained results of selectivity coefficient using electrode 7 is better than electrode 1, so that the electrode carbon paste MIP is more selec-
Table 4 The measurements results of Nernst factor values, measurement range and linearity (r) using electrodes carbon paste-MIP with certain variations. Electrode
Comparation of the mass of activated carbon, paraffin, and MIP
Nernst factor
Measurement range
Linearity (r)
E1 E2 E3 E4 E5 E6 E7
65:35:0 60:35:5 58:35:7 55:35:10 50:35:15 45:35:20 40:35:25
10.37 25.17 22.28 15.77 16.71 18.14 24.54
108–103 108–103 108–103 108–103 108–103 108–103 108–103
0.697 0.933 0.910 0.935 0.977 0.921 0.967
1815
H. Darmokoesoemo et al. / Results in Physics 7 (2017) 1808–1817 Table 5 Nernst factor value and measurement range at various pH using E1, E2, and E7. pH
4 5 6 7 8
Electrode 1
Electrode 2
Electrode 7
Measurement range
Factor Nernst
Measurement range
Nernst factor
Measurement range
Nernst factor
108–103 108–103 108–103 108–103 108–103
21.74 15 54 1.28 20.37 24.65
108–103 108–103 108–103 108–103 108–103
6.17 3.65 3.60 13.65 10.11
108–103 108–103 108–103 108–103 108–103
11.94 11.94 20.94 7.88 13.77
Table 6 The potential measurement results of creatinine solution at pH 6 using E7. Concentration (M)
Potential (mV)
108 107 106 105 104 103 102
1 335 1329 1319 1350 1366 1391 1380
Fig. 8. The curve with relationship between potential (E) with log concentration.
Fig. 6. The curve with relationship between log concentration of creatinine with potential.
Fig. 9. The curve with relationship between log concentration and potential (linear and non-linear).
Table 8 The calculation data of kij for the creatinine solution 104 M with matrix of urea solution. Concentration of urea solution
105 M 104 M 103 M
kij value Electrode 1
Electrode 7
9.40 0.85 0.04
9.99 0.99 0.09
Fig. 7. The standard curve of creatinine.
Table 7 The electrode response time on the potential measurement of various concentration of creatinine. Concentration (M) 6
10 105 104 103
Response time (sec)
Potential (mV)
192 158 142 91
997 1018 1014 1025
Table 9 The result of the accuracy calculation of the potentiometric method using E7 to measure the creatinine solution of 106–103 M. Concentration [actual] (M)
Concentration [analysis result] (M)
Accuracy (%)
106 105 104 103
0.76 106 1.65 105 0.81 104 0.97 103
76.40 165.80 81.20 97.10
1816
H. Darmokoesoemo et al. / Results in Physics 7 (2017) 1808–1817
Table 10 The calculation of standard deviation (SD) and coefficient of variation (CV) for the measurement of creatinine concentrations of 106–103 M. Concentration (M)
Measurement replication
106 105 104 103
1
2
3
1132 1159 1180 1205
1139 1160 1181 1205
1134 1161 1179 1204
Table 11 The data of Nernst factor and measurement range on the determination result of electrode life time. Total usage
Nernst factor
Measurement range
16 76 83 118
28.40 18.30 23.20 12.20
106–103 M 106–103 M 106–103 M 106–103 M
SD
CV
3.60 1.00 1.00 0.57
0.32 0.09 0.08 0.05
The accuracy value is good if close to 100%, so there is no difference between the concentration of the analysis results with actual concentrations. The accuracy values, which are statistically acceptable for concentration 106 and 105 M is 80–110%, 104 M is 90– 107%, and 103 M is 95–105% [15]. Based on these data, in this study only the solution with a concentration of 103 M has good accuracy. Determination result of precision Precision is the closeness between the value of the results of analysis on the same analyte, the same conditions, and in a short time interval. The creatinine solution with concentrations of 106–103 M at pH 6 was measured potential using E7, with each concentration done three times for repeatability of measurements. At first calculated precision is a determination of the standard deviation (SD) value, then we calculated the coefficient of variation (CV). The calculation result of standard deviation (SD) and coefficient of variation (CV) for the measurement of creatinine concentration solution of 106–103 M is shown in Table 10. The coefficient of variation values, which is statistically acceptable for concentration of 106 M is 22.60%, 105 M is 16%, 104 M is 11.30%, and 103 M is 8% [15]. Based on these data, this method has good precision.
Fig. 10. The curve with relationship between log concentration against potential of electrode carbon paste-polyaniline, electrode carbon paste-NIP, and electrode carbon paste-MIP.
tive to the creatinine solution than the urea solution when compared with electrode 1, which does not contain MIP. Determination results of accuracy Accuracy is the degree of closeness between the levels obtained from the analysis with actual levels. The determination of accuracy is done by measuring the potential of the creatinine solution with concentrations of 106–103 M at pH 6 using E7. The obtained potential value is substituted into the regression equation of the standard curve to obtain the concentration of creatinine. Accuracy value can be calculated using Eq. (3). The result of the accuracy calculation of the potentiometric method using E7 to measure the creatinine solution of 106–103 M can be seen in Table 9.
Determination results of electrode life time The electrode life time has a limit but it is still fit for use. In this study, the life time of the electrode is determined by counting the number of times the electrode can be used to measure the potential of the creatinine solution and show good performance. Every dip of the electrode into creatinine solution is considered one usage. The electrode used to determine the life time is E7 because this electrode has optimum composition. This electrode is used to measure creatinine solution with concentrations of 106–103 M; then we determined the Nernst factor and measurement range. The determination result of electrode life time is shown in Table 11. Table 11 shows usage up to 83 times, and the obtained Nernst factor value is still quite good. In the usage of 118 times, the obtained Nernst factor is further reduced. It shows that, with the longer time of electrode usage, the electrode performance will also
Table 12 The comparison of analysis methods of creatinine. Methods
HPLC [17]
Voltammetry [4]
Potentiometric (this study)
Detection limit Measurement range Response time Accuracy Selectivity
0.0083 103 M
1.30 109 M
5.49 106 M
4.40 106– 3.52 104 M 252 s 94–104% –
2.20 108–7.40 104 M
106–103 M
Life time
–
– 99–105% Selective in the creatinine matrix rather than NaCl, creatine, urea, glucose, phenylalanine, tyrosine, histidine, and cytosine –
91–192 s 76.40–165.80% Selective in the creatinine matrix rather than urea 83 times
H. Darmokoesoemo et al. / Results in Physics 7 (2017) 1808–1817
decrease. The decline in the performance of the electrode can be caused by dissolution of the electrode material into the solution, so that the electrode surface becomes uneven. The usage of electrodes too often also can allow changes in the mold of the active electrode to the analyte. The life time of an electrode can be affected by several things, including the flexibility of the electrode membrane, the resistance of membrane against organic compounds, pH, oxidizing agents, and the solubility level of electrode membrane [16].
1817
factor is 23.2 mV/decade, the lower detection limit of 5.49 106 M, and the upper detection limit of 1.07 103 M. Accuracy of methods for concentration of 106–103 M is 76.40–165.80%, and the obtained coefficient of variation is 0.05–0.32%. The measurement of creatinine with concentrations of 104 M that contain a urea solution of 103, 104, and 105 M produces a selectivity coefficient of 0.09, 0.99, and 9.99. The electrode carbon paste modified with MIP can be used up to 83 times. Competing interests
Comparison of the performance electrode carbon paste polyaniline, electrode carbon paste NIP, and electrode carbon paste MIP This study compared the performance electrode carbon paste polyaniline, electrode carbon paste NIP, and electrode carbon paste MIP. These electrodes are made with the same composition and are used to measure the potential of the creatinine solution with concentrations of 106–103 M at pH 6. The measurement results show a curve with a relationship potential against the log concentration. The curve with a relationship between log concentration against the potential of electrode carbon paste polyaniline, electrode carbon paste NIP, and electrode carbon paste MIP is shown in Fig. 10. Fig. 10 shows that the measurement result using electrode carbon paste MIP has better Nernst factors and linearity compared with the others. That is because the MIP has an active specific site to the analyte, so that performance is more optimal than the electrode carbon paste-polyaniline and electrode carbon paste NIP. Comparison of potentiometric methods using electrode carbon paste MIP with previous method for analysis of creatinine In this study, we conducted a comparison analysis of creatinine using the potentiometric method with previous methods, such as HPLC [17] and stripping voltammetry [4]. The comparison results of these methods showed that each method has advantages and disadvantages. The potentiometric method has wider measuring range and better response time compared with the HPLC method. But the voltammetry method has lower detection limits and better accuracy than the potentiometric method. The comparison of analysis methods of creatinine is shown in Table 12. Conclusion The research concludes that the optimum composition of the constituent material of electrodecarbon paste modified with MIP for analysis of creatinine by potentiometric is activated carbon, paraffin, and MIP with a mass ratio of 40:35:25. The electrode carbon paste modified with MIP made from aniline as the monomer has the optimum performance for the analysis of creatinine by potentiometric at pH 6. The electrode carbon paste modified with MIP made from aniline as a monomer leads to the analysis of creatinine by potentiometric response times for 91–192 s. The electrode measurement range is 106–103 M, the obtained Nernst
The authors declare no competing financial interests. References [1] Horne MM, Swearingen PL. Fluids, electrolytes and acid-base balance. 2nd ed. St Louis: Mosby; 1993. [2] Skurup A, Kristensen T, Wennecke G. New creatinine sensor for point-of-care testing of creatinine meets the National Kidney Disease Education Program guidelines. Clin Chem Lab Med 2008;46(1):3–8. [3] Guo MD, Guo HX. Voltammetric behaviour study of creatinine at phosphomolybdic-polypyrrole film modified electrode. J Electroanal Chem 2005;585:28–34. [4] Lakshmi D, Prasad BB, Sharma PS. Creatinine sensor based on a molecularly imprinted polymer-modified hanging mercury drop electrode. Talanta 2006;70(2):272–80. [5] Rizki DA. Analysis creatinine by voltammetry using electrodes carbon paste modified molecularly imprinted polymer (in Bahasa Indonesia), Thesis, Faculty of Science and Technology. Airlangga University; 2015. [6] Palupi WA. The development of electrodes based carbon nanoporous/molecularly imprinted polymer as potentiometric sensor of creatinine (in Bahasa Indonesia), Thesis, Faculty of Science and Technology. Airlangga University; 2014. [7] Chandra PAN. The development of electrodes carbon nanoporous/imprinted zeolite for analysis of creatinine by potentiometric (in Bahasa Indonesia), Thesis, Faculty of Science and Technology. Airlangga University; 2014. [8] Herdiningtyas ATM. Electrode based carbon nanoporous/molecularly imprinted polymer as potentiometric sensor of fructose in honey (in Bahasa Indonesia), Thesis, Faculty of Science and Technology. Airlangga University; 2012. [9] Berti F, Todros S, Lakshmi D, Whitcombe MJ, Chianella I, Ferroni M, et al. Quasimonodimensional polyaniline nanostructures for enhanced molecularly imprinted polymer-based sensing. Biosens Bioelectron 2010;26(2):497–503. [10] Chandrakanthi N, Careem MA. Thermal stability of polyaniline. Polym Bull 2000;44(1):101–8. [11] Maddu A, Wahyudi ST, Kurniati M. Synthesis and characterization of nanofiber polyaniline (in Bahasa Indonesia). J Nanosains dan Nanoteknologi 2008;1 (2):74–8. [12] Sreenivasan K. Synthesis and evaluation of molecularly imprinted polymers for nucleic acid bases using aniline as a monomer. React Funct Polym 2007;67 (10):859–64. [13] Safi’i FF, Mitarlis M. Utilization of solid waste of furfural synthesis process from rice husk as carbon active (in Bahasa Indonesia). UNESA J Chem 2013;2 (2):1–9. [14] Widhianti WD. The preparation of active charcoal from kapok seeds (Ceiba pentandra L.) as dye adsorbant of Rhodamine B(in Bahasa Indonesia), Thesis, Faculty of Science and Technology. Airlangga University; 2010. [15] Taverniers I, Loose MD, Bockstaele EV. Trends in quality in the analytical laboratory. II. Analytical method validation and quality assurance. TrAC Trends Anal Chem 2004;23(8):535–52. [16] Kembaren A. Preparation of the ion selective electrode for Pb2+ using membrane from mixture of PbS, PVC, and DBP (in Bahasa Indonesia), Thesis, Faculty of Mathematics and Natural Sciences. State University of Medan; 2013. [17] George SK, Dipu MT, Mehra UR, Singh P, Verma AK, Ramgaokar JS. Improved HPLC method for the simultaneous determination of allantoin, uric acid and creatinine in cattle urine. J Chromatogr B 2006;832(1):134–7.
Contents lists available at ScienceDirect
Results in Physics journal homepage: www.journals.elsevier.com/results-in-physics
Development of electrode carbon paste modified by molecularly imprinted polymer as sensor for analysis of creatinine by potentiometric Handoko Darmokoesoemo a,⇑, Miratul Khasanah a, Nunung Mareta Sari a, Yassine Kadmi b,c,d,e, Hicham Elmsellem f, Heri Septya Kusuma a,⇑ a
Department of Chemistry, Faculty of Science and Technology, Airlangga University, 60115, Indonesia Université d’Artois, EA 7394, Institut Charles Viollette, Lens F-62300, France ISA Lille, EA 7394, Institut Charles Viollette, Lille F-59000, France d Ulco, EA 7394, Institut Charles Viollette, Boulogne sur Mer F-62200, France e Université de Lille, EA 7394, Institut Charles Viollette, Lille F-59000, France f Laboratoire de chimie analytique appliquée, matériaux et environnement (LC2AME), Faculté des Sciences, B.P. 717, 60000 Oujda, Morocco b c
a r t i c l e
i n f o
Article history: Received 15 April 2017 Accepted 8 May 2017 Available online 12 May 2017 Keywords: Creatinine Molecularly imprinted polymer Carbon paste Potentiometric
a b s t r a c t This research aims to develop an electrode carbon paste/molecularly imprinted polymer (MIP) as a sensor for the analysis of creatinine by potentiometric. The preparation of a non-imprinted polymer (NIP) is done by mixing aniline, creatinine, and potassium peroxodisulfate with mole ratio of 2:0.1:1. Creatinine is extracted from the polymer using hot water to form mold creatinine, this so-called MIP. In this study, the optimum electrode has a composition made of activated carbon, paraffin, and MIP of 40:35:25 (mass ratio), pH of working solution 6, and response time of 91–192 s. The Nernst factor obtained from this study was 23.2 mV/decade, the measuring range of 106–103 M, lower detection limit of 5.49 106 M, and the upper detection limit of 1.07 103 M. The accuracy of methods for concentration of 106–103 M was 76.40–165.80%, and the precision expressed by the coefficient of variation for the concentration is 0.05–0.32%. The electrodes have up to 83 times of usage. Urea does not interfere with the performance of the electrode carbon paste MIP on the analysis of creatinine. Ó 2017 The Author. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction Creatinine is a metabolic waste product of the breakdown of muscle creatine. The creatinine levels in the blood serum indicate an equilibrium between production and excretion by the kidney and is an indicator of kidney function [1]. Normal concentrations of creatinine in the blood serum for adult males is 0.62 to 1.10 mg/dL and 0.45 to 0.75 mg/dL for adult women [2]. Increased serum creatinine levels are comparable with the decline in the quality of the kidneys; thus, analysis of creatinine levels in the blood is important for understanding the quality of work in the kidneys. The Jaffe method is generally used for creatinine analysis in the medical field. Analysis of creatinine using the Jaffe method has a principle, namely, by forming a colored complex solution, which can be analyzed by a UV–Vis spectrophotometer. The advantages of this method is an easy and simple analytical process, while ⇑ Corresponding authors. E-mail addresses: [email protected] (H. Darmokoesoemo), [email protected] (H.S. Kusuma).
the drawback is the low selectivity. Other common methods used for the analysis of creatinine is the enzymatic method. Analysis of creatinine with this method is hardly bothered by another matrix, but it takes a long time for the analysis, and the cost is quite expensive [3]. Another method developed for the analysis of creatinine is voltammetry. Lakshmi et al. (2006) [4] conducted analysis of creatinine in bloodserumby differential pulse cathodic stripping voltammetry with electrode hanging mercury drop (HMD) modified molecularly imprinted polymer (MIP) made from melamine as monomer and chloranyl. The analysis showed that the modified electrode has high selectivity toward creatinine because it is not disturbed by the presence of NaCl, creatine, urea, glucose, phenylalanine, tyrosine, histidine, and cytosine. Rizki (2015) [5] has modified carbon paste electrodes with MIP using the same monomer for analysis of creatinine by stripping voltammetry. Analysis of creatinine using this modified electrode has low selectivity in urea matrix when creatinine levels are low. The potentiometric method also has been used for the analysis of creatinine. This method uses two electrodes: the working electrode and reference electrode. The function of the working elec-
http://dx.doi.org/10.1016/j.rinp.2017.05.012 2211-3797/Ó 2017 The Author. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
H. Darmokoesoemo et al. / Results in Physics 7 (2017) 1808–1817
trode is to censor the analyte in the solution being analyzed, so that the working electrode must be selective and sensitive to analyte. Electrodes used for potentiometric analysis can be modified with the aim to increase the selectivity and sensitivity to analyte. Palupi (2014) [6] modified carbon paste electrodes using MIP with methacrylic acid (MAA) as a monomer for creatinine potentiometric analysis. The results of the analysis using the modified electrode has high selectivity toward creatinine in samples that also contain urea. The presence of urea up to 60 mg/dL do not interfere with the analysis of creatinine. Chandra (2014) [7] have modified carbon electrodes with imprinted zeolite for creatinine potentiometric analysis. The analysis of creatinine by potentiometric using the electrode has high accuracy. In this research, analysis of creatinine by potentiometric using carbon paste electrodes are modified with MIP made from aniline as the monomer. The parameters studied were the optimum composition of the MIP, carbon nanoporous, and paraffin in the preparation of carbon paste electrodes. Additionally, we studied the optimum pH solution in order to obtain good analytical results. Performance of modified electrodes is studied through parameter response time, life time, measurement range, Nernst factors, detection limit, selectivity, sensitivity, accuracy, and precision.
Materials and methods Materials and chemicals The chemicals used in this study were creatinine (C4H7N3O), aniline (C6H5NH2), potassium peroxodisulfate (K2S2O8), hydrochloric acid (HCl), acetic acid (CH3COOH), sodium acetate (CH3COONa), sodium hydrogenphosphate (Na2HPO4, 2H2O), sodium dihydrogenphosphate (NaH2PO4, 2H2O), urea (CO(NH2)2), activated carbon, solid paraffin, and Ag wire. Chemicals used have a degree of purity of pro analysis. Distilled water is also used.
Preparation of polyaniline Polyaniline was made with a 0.3 mL pipette of aniline and then mixed with 7.5 mL of HCl 1 M. The solution was stirred with a magnetic stirrer and heated for 30 min at 50 °C. After 30 min, a potassium peroxodisulfate solution made of 0.5000 g potassium peroxodisulfate and dissolved in 2.5 mL of distilled water was added. The addition of potassium peroxodisulfate was done slowly, with slow stirring. Then the solution stood at room temperature for 12 h. The solids that formed were washed using HCl 1 M. The solids were then dried and characterized using FTIR and XRD.
Preparation of molecularly imprinted polymer (MIP) Half of the NIP is used to make a molecularly imprinted polymer (MIP). MIP is made by extracting creatinine from synthesis, as a result of NIP using hot water (50 °C) through the centrifugation technique for 20 min. Extraction was performed 10 times. The solids obtained were dried and characterized using FTIR. Preparation of carbon Preparation of carbon is done by chemical and physical activation. The chemical activation is done by soaking carbon in a solution of HCl 4 N for 24 h and then soaked in n-hexane. Physical activation is done by heating the carbon at a temperature of 500 °C in a furnace. Preparation of electrode carbon paste MIP The electrode carbon paste MIP was made with a set Ag wire in a 1 mL micropipette tip. The function of the Ag wire is to act as a liaison between the electrodes with a potentiometer. Then, the micropipette tip was filled with solid paraffin up to threequarters. The remaining parts are still empty; the micropipette tip is filled with a mixture of activated carbon, paraffin, and MIP. The mixture of activated carbon, paraffin, and MIP was added with a certain ratio. Before placing the micropipette tip, solid paraffin is heated in a watch glass at 60 °C using a hotplate. Filling the mixture into micropipette tip is done by way of emphasis. The surface of electrode carbon paste MIP was scrubbed using the HVS to be smooth. Construction MIP carbon paste electrodes are shown in Fig. 1. Optimization of electrode composition Optimization of electrode composition is performed to determine comparison of electrode constituent material, which gives optimum performance of the electrode. The mixture composition of activated carbon, paraffin, and MIP is shown in Table 1 [8]. The optimum composition of electrodes was determined by measuring potential of creatinine standard solutions using electrodes based on the composition as shown in Table 1. The electrodes that generate a great Nernst factor and wide range of measurement are the electrodes showing good performance. The optimum composition of the electrodes is used as the basis for preparation electrode carbon paste modified NIP and electrode carbon paste modified polyaniline. The three electrodes were used to measure the potential of creatinine standard solution to determine its performance.
Preparation of nonmolecularly imprinted polymer (NIP) The preparation of nonmolecularly imprinted polymer (NIP) was done by mixing the monomer, initiator, and templates (analyte) with a mole ratio of 2:1:0.1. Monomers used in this study are made of aniline; the initiator is potassium peroxodisulfate, along with astemplateis creatinine. A 0.3 mL aniline pipette, mixed with 7.5 mL of HCl 1 M, was added as much as 0.0183 g of creatinine. The mixture was stirred with a magnetic stirrer and heated at 50 °C for 30 min. Then, we added a potassium peroxodisulfate solution (made of 0.5000 g of potassium peroxodisulfate in 2.5 mL of distilled water) done dropwise, with stirring slowed. The solution was allowed to stand at room temperature for 12 h and washed using HCl 1 M. The solid was dried and characterized using FTIR.
1809
Fig. 1. Construction-MIP carbon paste electrode.
1810
H. Darmokoesoemo et al. / Results in Physics 7 (2017) 1808–1817
Table 1 Composition of carbon, paraffin, and MIP in the preparation of electrodes. Electrode
E1 E2 E3 E4 E5 E6 E7
Mass (g)
Comparison of the mass of activated carbon, paraffin and MIP
Activated carbon
Paraffin
MIP
0.195 0.180 0.174 0.165 0.150 0.135 0.120
0.105 0.105 0.105 0.105 0.105 0.105 0.105
0 0.015 0.021 0.030 0.045 0.060 0.075
65:35:0 60:35:5 58:35:7 55:35:10 50:35:15 45:35:20 40:35:25
Optimization of pH creatinine solution
Determination of detection limit
A solution used for the optimization of pH is a creatinine solution with concentrations of 108–103 M. Each creatinine solution has a certain concentration, as much as 5.0 mL put in 50 mL volumetric flask, then added with 2 mL buffer solution pH 3, 4, 5, 6, 7, and 8 and diluted with water up to the mark. The solution was shaken until homogeneous and analyzed using working electrode carbon paste MIP. The optimum pH is pH when the solution has potential value is relatively constant.
The detection limit is the smallest or the largest concentration of analyte that can still be measured by electrodes. The detection limit is determined using the graph between the potential (E) against log concentration, which determines the cut point between the linear curve (measuring range) with a nonlinear curve potential of measurement results on the measurement of creatinine standard solutions. If at the cut point is drawn as the line toward the x axis, the obtained log is the concentration of detection limit. The antilog value of log concentration (x) of the detection limit is the detection limit value.
Measurement of creatinine standard solution The creatinine solution with a concentration of 102 M–108 M, which has been made with optimum pH, was measured for potential using electrode carbon paste MIP. Then we graphed potential (E) against log concentration of creatinine.
Determination of selectivity
The determination of response time of the working electrode carbon paste MIP to the analyte is done by measuring the potential of creatinine standard solutions with a concentration of 106–103 M at pH optimum. We measured the potential and the observed time for each creatinine standard solution to show a constant potential.
In this study, the selectivity of the electrode is expressed by the coefficient of selectivity. Selectivity coefficient values obtained by measuring the potential of the creatinine solution in accordance with normal levels in the serum is a concentration of 104 M. Measurements were performed by preparing the four pieces of a 50 mL volumetric flask, each filled with 5 mL of creatinine with a concentration of 103 M. Then the second, third, and fourth flasks are added to 5 mL of urea solution with a concentration of 102 M, 103 M, and 104 M, respectively. We subsequently added 2 mL of buffer solution pH optimum diluted with water up to the mark limit. The solution mixture is a measured potential using electrodes that have been optimized. The potential measurement results of a solution containing creatinine and urea solution is substituted into Eq. (2):
Determination of measurement range
K ij ¼
Determination of electrode performance Determination of response time
The measurement range was studied using electrodes from the optimization results to determine the potential of creatinine standard solution 102–108 M. Then we graphed the potential (E) against log concentration and determined the linear regression equation. The measurement range, indicated by the range of concentrations, is a straight line graph, which still meets the Nernst equation. Determination of Nernst factors and linearity The Nernst factor is determined by plotting between measurement potential data (E) of creatinine standard solution against log concentration, which is obtained by Eq. (1). The Nernst factor value is shown by the slope of the curve (b):
y ¼ bx þ a
ð1Þ
where y is the potential of the solution, x the value of the log concentration of the analyte, b is the slope of the curve, and a is the intercept. The linearity of the curve is shown by the correlation coefficient (r), which has a range of 1 r 1.
ai ð10ðE2 E1 =SÞ 1Þ ajn=x
ð2Þ
where ai is the activity of measured analytes, aj is the activity of the compounds that interfere, E1 is the potential before the addition of the compounds that interfere, E2 is the potential after the addition of the compounds that interfere, S is the slope of the calibration curve, n is the charge of main ions, and x is the charge of ions that interfere. If kij 1, the electrode is more selective to creatinine solution rather than the urea solution. This shows that urea does not interfere with the performance of the electrode carbon paste MIP in the analysis of creatinine. Determination of accuracy Accuracy is the degree of closeness between the levels obtained from the analysis with actual levels. A creatinine solution with a concentration in the measurement range measures potential by using electrode carbon paste MIP. The potential value of the measurement results is an analogy as y and substituted in the obtained linear regression equation, so that we can determine the creatinine concentration. Accuracy was determined by using Eq. (3):
H. Darmokoesoemo et al. / Results in Physics 7 (2017) 1808–1817
xt R ¼ 100% xi
ð3Þ
where R is the accuracy value, xt is the concentration from the measurement result, and xi is the actual concentration. Determination of precision Precision is the closeness between the value of the analysis results on the same analyte, the same conditions, and in a short time interval. Precision values are determined by measuring the potential of the creatinine solution on the measuring range. Potential measurements are performed at each concentration by three times replication using electrode carbon paste MIP. Precision is determined by calculating the standard deviation (SD) and coefficient of variation (CV) using Eqs. (4) and (5):
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi PN 2 i¼1 ðx xÞ SD ¼ N1 s CV ¼ 100% x
1811
line has become conductive emeraldine salt [11]. Solids that have been washed are then dried to obtain polyaniline powder. Synthesis result of nonimprinted polymer (NIP) The preparation of NIP is done by mixing the aniline monomer, initiator potassium peroxodisulfate, and creatinine as a template with a ratio of 2:1:0.1 [12]. Aniline 0.3 mL in 7.5 mL of HCl is added with 0.0183 g of creatinine. Then it is stirred using a magnetic stirrer for 30 min at 50 °C and added to the potassium peroxodisulfate solution dropwise. The solution is initially colorless after a few drops of potassium peroxodisulfate, then it turned into dark green, which thickens. The solution was allowed to stand for 12 h at room temperature to form into solids. The obtained solids were washed using HCl 1 M. Washing using HCl aims at removing residual potassium peroxodisulfate and residual unreacted aniline. NIP solids obtained a dark green color.
ð4Þ Synthesis result of molecularly imprinted polymer (MIP)
ð5Þ
with x being the value of each measurement, xis the average value of all the observations, and N the number of measurements. Determination of electrode life time The life time of the electrodes is the measured time span because the used electrode still shows good performance up to the time the electrodes had significant reduction in performance. The life time of the electrode is determined by measuring the electrode potential for the analysis of creatinine standard solution every week in order to obtain the electrode Nernst factor. Measurements were made to show the Nernst factors that deviate from the permissible value. Results and discussion Result of polymer synthesis Synthesis result of polymer control (polyaniline) In this study, analysis of creatinine by a potentiometric using electrodecarbon paste modified molecularly imprinted polymer (MIP) with aniline as the monomer. In the initial phase, synthesis of polyaniline needs to be done. Aniline is used as monomer because, in that structure, there are functional groups ANH, which are expected to interact with the functional groups of C@O contained in creatinine through hydrogen bonds. Nanostructures that are owned by polyaniline generates the conductive nanowire system, which allows direct electrical connection between the electrode and the MIP [9]. Polyaniline is also a conductive polymer that adds stability to heat, air, and moisture [10]. The preparation of polyaniline was made by mixing 0.3 mL of aniline and 7.5 mL 1 M HCl, then stirred with a magnetic stirrer for 30 min and heated at 50 °C. We also added a potassium peroxodisulfate solution, which is made of 0.5000 g of potassium peroxodisulfate crystal dissolved in 2.5 mL of distilled water. The addition of potassium peroxodisulfate as the initiator is done slowly, with slow stirring. Initially, a colorless solution turned into a dark green color, which thickens after the potassium peroxodisulfate solution is added. The green solution was allowed to stand for 12 h at room temperature. This process is intended so long as the polymerization reaction of aniline is running perfectly. Then the obtained solids are washed using HCl 1 M. Washing using HCl aims to eliminate residual unreacted potassium peroxodisulfate and ensures all formed polyani-
MIP is made by extracting creatinine of NIP powders using hot water with a temperature of 50 °C. Extraction is done many times by centrifugation to obtain neutral pH. Each extraction is done for 20 min. The purpose of extraction is to remove creatinine from the polymer; thus, we obtained mold creatinine. In this study, the MIP obtained is dark green. Characterization of control polymer, NIP, and MIP using FTIR Polymer control, NIP, and MIP from the synthesis result are characterized using FTIR. Characterization is performed to determine the functional groups contained in aniline, polyaniline, NIP, and MIP. FTIR spectra of aniline and polyaniline are shown in Fig. 2. Fig. 2 shows that the peak spectra between aniline and polyaniline appearing in wavenumbers are almost the same. That is because they are the same constituent. The same spectra between polyaniline and aniline are stretching C-Hsp2, which appears at wavenumber 3228 cm1 for aniline and 3215 cm1 for polyaniline, spectra of aromatic CAC bonds appears at wavenumber 1494 cm1 for aniline and 1483 cm1 for polyaniline. The difference spectra between aniline and polyaniline are contained in stretching CAN; the polyaniline spectra contained at wavenumber 1246 cm1 is steeper than the spectra contained in aniline at wavenumber 1273 cm1. The difference spectra between aniline and polyaniline are also contained in stretching ANH. In aniline stretching, ANH appeared in two peaks, at the wavenumbers 3431 and 3365 cm1, whereas polyaniline appears only at one peak at wavenumber 3421 cm1. That is because the aniline has structure primary ANH, and polyaniline has structure secondary ANH. The similarities and differences contained in aniline and polyaniline spectra showed that the polyaniline has been formed. FTIR data of aniline and polyaniline are shown in Table 2. The FTIR spectra of polyaniline, NIP, and MIP are shown in Fig. 3. The wavenumber data of polyaniline, NIP, and MIP are shown in Table 3. The spectra of NIP and MIP should also emerge as a peak, stating the OAH bond contained in the wavenumber around 3600 cm1. The hydrogen bond indicates the bond between creatinine with polyaniline, as shown in Fig. 4. The peak spectra hould also emerge as a typical spectral of creatinine that indicates C@O, which should appear at a wavenumber around 1700 cm1. The typical spectra of creatinine that does not appear is possible because the mole creatinine was too small. In this study, analysis using FTIR was not carried out quantitatively, so that the intensity cannot be used as a benchmark in the success of the synthesis of NIP and MIP.
1812
H. Darmokoesoemo et al. / Results in Physics 7 (2017) 1808–1817
Fig. 2. The FTIR spectra of aniline and polyaniline. Table 2 FTIR data of aniline and polyaniline. Wave number (cm1)
Description
Aniline
Polyaniline
3431 and 3365 3228 1494 1273
3421 3215 1483 1246
Stretching ANH Stretching CAH sp2 Aromatic CAC bond Stretching CAN
Characterization of polymer control using XRD The characterization using X-ray diffraction (XRD) was conducted to determine crystallinity of polyaniline. A polyaniline diffraction pattern can be seen in Fig. 5, which shows that the emerged sharp peak diffraction pattern at 2h = 25.5°. The diffraction patterns emerge due to periodicity perpendicular to the benzoid ring and quinoid of polyaniline polymer chain. Preparation of carbon In this study, we conducted carbon reactivation of activated carbon as the preparation phase. Carbon reactivation is to obtain car-
bon with a larger surface area and to improve the conductivity of carbon in order to produce great potential when used as an electrode. The carbon reactivation process is done in two steps, that is, carbon immersion using HCl 4 N and soaked with an n-hexane solution. The first step is carbon soaked in a solution of HCl 4 N, which functions as an activator. The HCl solution was chosen because it has properties of a dehydrating agent, which can improve the structure of porous carbon, whereas the concentration used 4 N because, at these concentrations, pores open to easily extract soluble impurities [13]. After soaking with HCl 4 N for ±24 h, the carbon is washed using distilled water until neutral. The neutral carbon is then dried using an oven, so that water remnants in the pores can evaporate. The second stage is soaking carbon in an n-hexane solution for ±24 h. Soaking carbon in acid solution only removes hydrogen on the outer surface of carbon, so that the required organic solvents remove hydrocarbons [14]. The organic solvent used is n-hexane. The loss of hydrogen and hydrocarbons can make the surface of the carbon become increasingly widespread. Carbon that has been soaked with n-hexane is then heated in a furnace at temperature of 500 °C. Carbon that
Fig. 3. FTIR spectra of polyaniline, NIP and MIP.
1813
H. Darmokoesoemo et al. / Results in Physics 7 (2017) 1808–1817 Table 3 FTIR data of polyaniline, NIP and MIP. Wave number (cm1)
Description
Polyaniline
NIP
MIP
3421 3228 1483 1246
– – 1494 1244
3464 3211 1491 1238
Stretching NAH Stretching CAH sp2 Aromatic CAC bond Stretching CAN
has been reactivated is tested at its surface area and pore size using the BET (Brunaeur-Emmet-Teller) and BJH (Barrett-JoynerHallenda) tests. In this study, the surface area value of porous carbon is 877.463 m2/g. The surface area value of the carbon shows that the size of the used carbon is mesoporous, while the carbon pore diameter value in this study is 3.835 nm. Performance of carbon would be good if it has a large surface area with small pore size.
Optimization results of electrode carbon paste MIP composition The optimization of electrode composition is performed to find the electrode that has maximum performance. In this study, the working electrode is made from a mixture of actived carbon, solid paraffin, and MIP. The addition of MIP as polymers with specific binding sites to the analyte aims to improve the sensitivity and selectivity of the electrode. Activated carbon is used as the electrode, as it has a large surface area that is expected to extend the binding of the analyte. Activated carbon is conductive material, so it can deliver the response to the potentiometer through the silver wire (Ag). The addition of paraffin serves as an adhesive between the activated carbon and MIP in order not to be separated when it is inserted into the micropipette tip. In this study, we created seven electrodes with composition of activated carbon and MIP, which varies with fixed paraffin composition, as shown in Table 1. The electrodes were then soaked in creatinine solution 103 M before being used for conditioning. Further electrodes were used to measure the potential of creatinine solution 108–101 M, which has been added with KCl solution as the supporting electrolyte. The measurements results of Nernst factor values, measurement range, and linearity (r) using electrodes carbon paste MIP with certain variations are shown in Table 4. The performance of electrodes in potentiometric is good if it generates good Nernst factor value and linearity and has a wide measurement range. The Nernst factor is said to be good if it satis-
fies ð59;1 ð1 2ÞÞ mV, where n is the valence of the analyte. Anan lytes measured in this study are creatinine (monovalent molecule because it releases one electron), so that it has Nernst factor of 59.1 mV. In this study, E2 and E7 have the best Nernst factor values, which are 25.17 mV and 24.54 mV, respectively. This optimization result shows that E2 has a better Nernst factor, but the linearity is less favorable than E7. Thus, pH needs tobe optimized to determine the composition of the electrode carbon paste MIP, which has maximum performance. Optimization results of pH creatinine solution The optimization of pH is performed to determine the pH range, which produces stable potential measurement. This optimization is done in a creatinine solution with concentrations of 108–103 M with a pH range of 4–8. Electrodes used were E2 and E7 as well as E1 for comparison. The Nernst factor value and measurement range at various pH using E1, E2, and E7 are shown in Table 5. The results in Table 5 show that the best Nernst factor for electrode carbon paste MIP was obtained at pH 6 using E7. But the best Nernst factor with the same measurements range was obtained at pH 8 using E1. E1 is a reference electrode, which, in its composition, does not contain MIP. The Nernst factor produced from measurements using E1 at pH 8 was also good. This is because carbon has good activity. The creatinine theoretically has a pH of 7–9 at a temperature of 25 °C. In this study, the mother solution of creatinine had a pH of 6.57. The creatinine has two dissociation constant values: pKa1 = 4.8 and pKa2 = 9.2. The creatinine in acidic condition (below pH 4.8) is in the form of cations, whereas in alkaline condition (around pH 9.2) it is in the form of anion. In this study, creatinine will produce a good Nernst factor when it is in the shape of a molecule, so that it has optimum pH 6. Standard curves of creatinine The standard curves of creatinine were created by measuring the potential of the creatinine solution with a concentration of 108–102 M at pH 6 using E7, which is the optimum pH and composition of the electrode. The potential measurement results of creatinine solution at pH 6 using E7 are shown in Table 6. The curve also shows the relationship between log concentration of creatinine solution with the measured potential of electrode. The formed curve shows a selected concentration range via straight line approaching a Nernst factor of 59.1 mV/decade and linearity approaching 1. The curve, which shows a straight line, is then used as a standard curve of creatinine. The curve showing
HN
CH3 N
HN
N
H N O
HN
NH
N H
Fig. 4. The hydrogen bond between creatinine with polyaniline.
NH
1814
H. Darmokoesoemo et al. / Results in Physics 7 (2017) 1808–1817
using E7. The measurement results of potential graph between potential (E) with log concentrations are shown in Fig. 8. Fig. 8 shows the creatinine concentration measurement range is 106–103 M with a Nernst factor value of 23.2 mV/decade and linearity 0.986. Nernst factor and linearity can be seen in the standard curve of creatinine (Fig. 8). The Nernst factor is the slope values of the standard curve of creatinine, while linearity is the correlation coefficient (r) of a standard curve. In this research, the linear regression equation y = 23.2x + 1460, and the correlation coefficient of 0.986.
Determination results of the detection limit
Fig. 5. The diffraction pattern of polyaniline.
the relationship between log concentration of creatinine with potential can be seen in Fig. 6, whereas the standard curve of creatinine is shown in Fig. 7. Performance test result of electrode and validity of method Determination results of electrode response time The electrode response time is the length of time required by the electrodes in response to the analyte in the sample solution. A faster electrode response time to the analyte indicates that the performance of the electrode is better. The electrode response time shows electrode sensitivity toward the analyte. In this study, the determination of the electrode response time is done in a creatinine solution with concentrations of 106–103 M at pH 6 using E7. The results of determination of the electrode response time can be seen in Table 7. Data in Table 7 show that the higher concentration of creatinine can make the electrode response time also faster. This is because, with the higher concentration, it can cause the amount of analyte in the solution to increase, so that the displacement of the analyte from the solution to the electrode becomes faster. Determination results of the measurement range, Nernst factor, and the linearity The measurement range is the range of concentrations that shows the linear line on the graph between the potential (E) against the log concentration and still meets the Nernst equation. The measurement method is good if it has wide measurement range. In the potentiometric, determination of measurement range also must consider the good linearity and Nernst factor. The measurement range is determined by measuring the potential standard solution of creatinine with concentrations of 108–102 M at pH 6
The detection limit is the lowest or higher concentration of analyte that can still be measured by electrodes. The detection limit can be known by determining the cut point between the linear and nonlinear curve from the curve with a relationship between log concentration of creatinine and potential, as shown in Fig. 9. In this research, the lower detection limit is 5.49 106 M, and the upper detection limit is 1.07 103 M. These results indicate that the electrode carbon paste MIP is quite good because it has fairly low detection limits. The upper detection limit of a method for the measurement of creatinine is required. It aims to facilitate the measurement of creatinine levels for patients with renal impairment who have high creatinine levels.
Determination result of selectivity The determination of selectivity is important because it can be used to determine the ability of the method to measure, without any interference, that matrices are together with the sample. The selectivity test in this study is conducted with the addition of urea solution because urea is in blood serum along with creatinine and other compounds. The selectivity test was conducted by adding urea solution with a concentration of 103 M, 104 M, and 105 M in creatinine solution of 104 M. The normal concentration of creatinine solution is 104 M, whereas the concentration of urea solution selected in this study is below, equal, and above the normal concentration of creatinine. The potential of solution is measured using E1 and E7 at pH 6 and calculated using the selectivity coefficient (kij) value. The electrode used for the measurement is E7 because this electrode has optimum composition, while E1 is used as the comparison. The result of calculation kij for the creatinine solution 104 M with a matrix of urea solution is shown in Table 8. In the calculation of kij, the value of kij < 1 indicates that these methods are more selective to creatinine and urea matrix, which were added and do not interfere with the analysis, although with concentrations below, equal, and above the normal level of creatinine in the blood serum. Data in Table 8 show that the obtained results of selectivity coefficient using electrode 7 is better than electrode 1, so that the electrode carbon paste MIP is more selec-
Table 4 The measurements results of Nernst factor values, measurement range and linearity (r) using electrodes carbon paste-MIP with certain variations. Electrode
Comparation of the mass of activated carbon, paraffin, and MIP
Nernst factor
Measurement range
Linearity (r)
E1 E2 E3 E4 E5 E6 E7
65:35:0 60:35:5 58:35:7 55:35:10 50:35:15 45:35:20 40:35:25
10.37 25.17 22.28 15.77 16.71 18.14 24.54
108–103 108–103 108–103 108–103 108–103 108–103 108–103
0.697 0.933 0.910 0.935 0.977 0.921 0.967
1815
H. Darmokoesoemo et al. / Results in Physics 7 (2017) 1808–1817 Table 5 Nernst factor value and measurement range at various pH using E1, E2, and E7. pH
4 5 6 7 8
Electrode 1
Electrode 2
Electrode 7
Measurement range
Factor Nernst
Measurement range
Nernst factor
Measurement range
Nernst factor
108–103 108–103 108–103 108–103 108–103
21.74 15 54 1.28 20.37 24.65
108–103 108–103 108–103 108–103 108–103
6.17 3.65 3.60 13.65 10.11
108–103 108–103 108–103 108–103 108–103
11.94 11.94 20.94 7.88 13.77
Table 6 The potential measurement results of creatinine solution at pH 6 using E7. Concentration (M)
Potential (mV)
108 107 106 105 104 103 102
1 335 1329 1319 1350 1366 1391 1380
Fig. 8. The curve with relationship between potential (E) with log concentration.
Fig. 6. The curve with relationship between log concentration of creatinine with potential.
Fig. 9. The curve with relationship between log concentration and potential (linear and non-linear).
Table 8 The calculation data of kij for the creatinine solution 104 M with matrix of urea solution. Concentration of urea solution
105 M 104 M 103 M
kij value Electrode 1
Electrode 7
9.40 0.85 0.04
9.99 0.99 0.09
Fig. 7. The standard curve of creatinine.
Table 7 The electrode response time on the potential measurement of various concentration of creatinine. Concentration (M) 6
10 105 104 103
Response time (sec)
Potential (mV)
192 158 142 91
997 1018 1014 1025
Table 9 The result of the accuracy calculation of the potentiometric method using E7 to measure the creatinine solution of 106–103 M. Concentration [actual] (M)
Concentration [analysis result] (M)
Accuracy (%)
106 105 104 103
0.76 106 1.65 105 0.81 104 0.97 103
76.40 165.80 81.20 97.10
1816
H. Darmokoesoemo et al. / Results in Physics 7 (2017) 1808–1817
Table 10 The calculation of standard deviation (SD) and coefficient of variation (CV) for the measurement of creatinine concentrations of 106–103 M. Concentration (M)
Measurement replication
106 105 104 103
1
2
3
1132 1159 1180 1205
1139 1160 1181 1205
1134 1161 1179 1204
Table 11 The data of Nernst factor and measurement range on the determination result of electrode life time. Total usage
Nernst factor
Measurement range
16 76 83 118
28.40 18.30 23.20 12.20
106–103 M 106–103 M 106–103 M 106–103 M
SD
CV
3.60 1.00 1.00 0.57
0.32 0.09 0.08 0.05
The accuracy value is good if close to 100%, so there is no difference between the concentration of the analysis results with actual concentrations. The accuracy values, which are statistically acceptable for concentration 106 and 105 M is 80–110%, 104 M is 90– 107%, and 103 M is 95–105% [15]. Based on these data, in this study only the solution with a concentration of 103 M has good accuracy. Determination result of precision Precision is the closeness between the value of the results of analysis on the same analyte, the same conditions, and in a short time interval. The creatinine solution with concentrations of 106–103 M at pH 6 was measured potential using E7, with each concentration done three times for repeatability of measurements. At first calculated precision is a determination of the standard deviation (SD) value, then we calculated the coefficient of variation (CV). The calculation result of standard deviation (SD) and coefficient of variation (CV) for the measurement of creatinine concentration solution of 106–103 M is shown in Table 10. The coefficient of variation values, which is statistically acceptable for concentration of 106 M is 22.60%, 105 M is 16%, 104 M is 11.30%, and 103 M is 8% [15]. Based on these data, this method has good precision.
Fig. 10. The curve with relationship between log concentration against potential of electrode carbon paste-polyaniline, electrode carbon paste-NIP, and electrode carbon paste-MIP.
tive to the creatinine solution than the urea solution when compared with electrode 1, which does not contain MIP. Determination results of accuracy Accuracy is the degree of closeness between the levels obtained from the analysis with actual levels. The determination of accuracy is done by measuring the potential of the creatinine solution with concentrations of 106–103 M at pH 6 using E7. The obtained potential value is substituted into the regression equation of the standard curve to obtain the concentration of creatinine. Accuracy value can be calculated using Eq. (3). The result of the accuracy calculation of the potentiometric method using E7 to measure the creatinine solution of 106–103 M can be seen in Table 9.
Determination results of electrode life time The electrode life time has a limit but it is still fit for use. In this study, the life time of the electrode is determined by counting the number of times the electrode can be used to measure the potential of the creatinine solution and show good performance. Every dip of the electrode into creatinine solution is considered one usage. The electrode used to determine the life time is E7 because this electrode has optimum composition. This electrode is used to measure creatinine solution with concentrations of 106–103 M; then we determined the Nernst factor and measurement range. The determination result of electrode life time is shown in Table 11. Table 11 shows usage up to 83 times, and the obtained Nernst factor value is still quite good. In the usage of 118 times, the obtained Nernst factor is further reduced. It shows that, with the longer time of electrode usage, the electrode performance will also
Table 12 The comparison of analysis methods of creatinine. Methods
HPLC [17]
Voltammetry [4]
Potentiometric (this study)
Detection limit Measurement range Response time Accuracy Selectivity
0.0083 103 M
1.30 109 M
5.49 106 M
4.40 106– 3.52 104 M 252 s 94–104% –
2.20 108–7.40 104 M
106–103 M
Life time
–
– 99–105% Selective in the creatinine matrix rather than NaCl, creatine, urea, glucose, phenylalanine, tyrosine, histidine, and cytosine –
91–192 s 76.40–165.80% Selective in the creatinine matrix rather than urea 83 times
H. Darmokoesoemo et al. / Results in Physics 7 (2017) 1808–1817
decrease. The decline in the performance of the electrode can be caused by dissolution of the electrode material into the solution, so that the electrode surface becomes uneven. The usage of electrodes too often also can allow changes in the mold of the active electrode to the analyte. The life time of an electrode can be affected by several things, including the flexibility of the electrode membrane, the resistance of membrane against organic compounds, pH, oxidizing agents, and the solubility level of electrode membrane [16].
1817
factor is 23.2 mV/decade, the lower detection limit of 5.49 106 M, and the upper detection limit of 1.07 103 M. Accuracy of methods for concentration of 106–103 M is 76.40–165.80%, and the obtained coefficient of variation is 0.05–0.32%. The measurement of creatinine with concentrations of 104 M that contain a urea solution of 103, 104, and 105 M produces a selectivity coefficient of 0.09, 0.99, and 9.99. The electrode carbon paste modified with MIP can be used up to 83 times. Competing interests
Comparison of the performance electrode carbon paste polyaniline, electrode carbon paste NIP, and electrode carbon paste MIP This study compared the performance electrode carbon paste polyaniline, electrode carbon paste NIP, and electrode carbon paste MIP. These electrodes are made with the same composition and are used to measure the potential of the creatinine solution with concentrations of 106–103 M at pH 6. The measurement results show a curve with a relationship potential against the log concentration. The curve with a relationship between log concentration against the potential of electrode carbon paste polyaniline, electrode carbon paste NIP, and electrode carbon paste MIP is shown in Fig. 10. Fig. 10 shows that the measurement result using electrode carbon paste MIP has better Nernst factors and linearity compared with the others. That is because the MIP has an active specific site to the analyte, so that performance is more optimal than the electrode carbon paste-polyaniline and electrode carbon paste NIP. Comparison of potentiometric methods using electrode carbon paste MIP with previous method for analysis of creatinine In this study, we conducted a comparison analysis of creatinine using the potentiometric method with previous methods, such as HPLC [17] and stripping voltammetry [4]. The comparison results of these methods showed that each method has advantages and disadvantages. The potentiometric method has wider measuring range and better response time compared with the HPLC method. But the voltammetry method has lower detection limits and better accuracy than the potentiometric method. The comparison of analysis methods of creatinine is shown in Table 12. Conclusion The research concludes that the optimum composition of the constituent material of electrodecarbon paste modified with MIP for analysis of creatinine by potentiometric is activated carbon, paraffin, and MIP with a mass ratio of 40:35:25. The electrode carbon paste modified with MIP made from aniline as the monomer has the optimum performance for the analysis of creatinine by potentiometric at pH 6. The electrode carbon paste modified with MIP made from aniline as a monomer leads to the analysis of creatinine by potentiometric response times for 91–192 s. The electrode measurement range is 106–103 M, the obtained Nernst
The authors declare no competing financial interests. References [1] Horne MM, Swearingen PL. Fluids, electrolytes and acid-base balance. 2nd ed. St Louis: Mosby; 1993. [2] Skurup A, Kristensen T, Wennecke G. New creatinine sensor for point-of-care testing of creatinine meets the National Kidney Disease Education Program guidelines. Clin Chem Lab Med 2008;46(1):3–8. [3] Guo MD, Guo HX. Voltammetric behaviour study of creatinine at phosphomolybdic-polypyrrole film modified electrode. J Electroanal Chem 2005;585:28–34. [4] Lakshmi D, Prasad BB, Sharma PS. Creatinine sensor based on a molecularly imprinted polymer-modified hanging mercury drop electrode. Talanta 2006;70(2):272–80. [5] Rizki DA. Analysis creatinine by voltammetry using electrodes carbon paste modified molecularly imprinted polymer (in Bahasa Indonesia), Thesis, Faculty of Science and Technology. Airlangga University; 2015. [6] Palupi WA. The development of electrodes based carbon nanoporous/molecularly imprinted polymer as potentiometric sensor of creatinine (in Bahasa Indonesia), Thesis, Faculty of Science and Technology. Airlangga University; 2014. [7] Chandra PAN. The development of electrodes carbon nanoporous/imprinted zeolite for analysis of creatinine by potentiometric (in Bahasa Indonesia), Thesis, Faculty of Science and Technology. Airlangga University; 2014. [8] Herdiningtyas ATM. Electrode based carbon nanoporous/molecularly imprinted polymer as potentiometric sensor of fructose in honey (in Bahasa Indonesia), Thesis, Faculty of Science and Technology. Airlangga University; 2012. [9] Berti F, Todros S, Lakshmi D, Whitcombe MJ, Chianella I, Ferroni M, et al. Quasimonodimensional polyaniline nanostructures for enhanced molecularly imprinted polymer-based sensing. Biosens Bioelectron 2010;26(2):497–503. [10] Chandrakanthi N, Careem MA. Thermal stability of polyaniline. Polym Bull 2000;44(1):101–8. [11] Maddu A, Wahyudi ST, Kurniati M. Synthesis and characterization of nanofiber polyaniline (in Bahasa Indonesia). J Nanosains dan Nanoteknologi 2008;1 (2):74–8. [12] Sreenivasan K. Synthesis and evaluation of molecularly imprinted polymers for nucleic acid bases using aniline as a monomer. React Funct Polym 2007;67 (10):859–64. [13] Safi’i FF, Mitarlis M. Utilization of solid waste of furfural synthesis process from rice husk as carbon active (in Bahasa Indonesia). UNESA J Chem 2013;2 (2):1–9. [14] Widhianti WD. The preparation of active charcoal from kapok seeds (Ceiba pentandra L.) as dye adsorbant of Rhodamine B(in Bahasa Indonesia), Thesis, Faculty of Science and Technology. Airlangga University; 2010. [15] Taverniers I, Loose MD, Bockstaele EV. Trends in quality in the analytical laboratory. II. Analytical method validation and quality assurance. TrAC Trends Anal Chem 2004;23(8):535–52. [16] Kembaren A. Preparation of the ion selective electrode for Pb2+ using membrane from mixture of PbS, PVC, and DBP (in Bahasa Indonesia), Thesis, Faculty of Mathematics and Natural Sciences. State University of Medan; 2013. [17] George SK, Dipu MT, Mehra UR, Singh P, Verma AK, Ramgaokar JS. Improved HPLC method for the simultaneous determination of allantoin, uric acid and creatinine in cattle urine. J Chromatogr B 2006;832(1):134–7.