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molecularly imprinted polymer (MIP) with methacrylic acid as monomer to analyze glucose by potentiometry
Accepted Manuscript Development of Electrode Carbon Paste/Molecularly Imprinted Polymer (MIP) with Methacrylic Acid as Monomer to Analyze Glucose by Potentiometry Handoko Darmokoesoemo, Lendhy Kustyarini, Yassine Kadmi, Miratul Khasanah, Heri Septya Kusuma, Hicham Elmsellem PII: DOI: Reference:
S2211-3797(17)30634-4 http://dx.doi.org/10.1016/j.rinp.2017.05.015 RINP 700
To appear in:
Results in Physics
Received Date: Accepted Date:
14 April 2017 9 May 2017
Please cite this article as: Darmokoesoemo, H., Kustyarini, L., Kadmi, Y., Khasanah, M., Kusuma, H.S., Elmsellem, H., Development of Electrode Carbon Paste/Molecularly Imprinted Polymer (MIP) with Methacrylic Acid as Monomer to Analyze Glucose by Potentiometry, Results in Physics (2017), doi: http://dx.doi.org/10.1016/j.rinp. 2017.05.015
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1 Development of Electrode Carbon Paste/Molecularly Imprinted Polymer (MIP) with Methacrylic Acid as Monomer to Analyze Glucose by Potentiometry Handoko Darmokoesoemo 1,*, Lendhy Kustyarini1, Yassine Kadmi2,3,4,5, Miratul Khasanah1, Heri Septya Kusuma1,* and Hicham Elmsellem6 1
Department of Chemistry, Faculty of Science and Technology, Airlangga University, 60115, Indonesia 2
Université d'Artois, EA 7394, Institut Charles Viollette, Lens, F-62300, France 3
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5
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ISA Lille, EA 7394, Institut Charles Viollette, Lille, F-59000, France
Ulco, EA 7394, Institut Charles Viollette, Boulogne sur Mer, F-62200, France
Université de Lille, EA 7394, Institut Charles Viollette, Lille, F-59000, France
Laboratoire de chimie analytique appliquée, matériaux et environnement (LC2AME), Faculté des Sciences, B.P. 717, 60000 Oujda, Morocco.
Email: [email protected]; [email protected]
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2 Abstract Electrodes with carbon paste and molecularly imprinted polymer (MIP) have been developed to analyze glucose by potentiometry. The MIP was made by mixing glucose as template, methacrylic acid as monomer and ethylene glycol dimethacrylate as cross-linker with a mole ratio of 1:4:12. The electrode made by mixing activated carbon, MIP and paraffin with a ratio of 50:35:15 (% by weight) was observed to perform optimally. The results obtained show that analysis of glucose gives optimum results at a pH of 5 (without pH adjustment). The analysis of glucose using the carbon paste/MIP electrode gives a Nernst factor of 28.80 mV/decade; the measurement range is 10 -2-10 -5 M, and the limit of detection is 5.87.10 -5 M. In this study, it can be shown that urea did not interfere in the analysis of glucose using this method. The accuracy of this electrode is 70.7-129%, and the coefficient of variation is 0.06-0.18% for the concentration range 10-5-10 -2 M. This electrode showed a response time of less than 2 minutes and could be used 145 times. Keyword: glucose, molecularly imprinted polymer, potentiometry, electrode carbon paste
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3 Introduction Diabetes mellitus is a disease in which the body cannot control blood sugar levels automatically. In a healthy body, the pancreas will release the hormone insulin, which functions to transport sugar through the blood to the muscles and the tissues of the body to supply energy [1]. The impact of excess sugar, or hyperglycemia, namely glikosuria, can lead to changes in fat metabolism. On the other hand, a shortage in blood sugar levels can cause health problems such as hypoglycemia (excess insulin) [2]. Glucose levels can be analyzed using several methods, including spectrophotometry [3], high performance liquid chromatography (HPLC) [4] and liquid chromatography-mass spectroscopy (LC-MS) [5]. Analysis of glucose as sugar levels are reduced by the NelsonSomogyi method is conducted by spectrophotometry. This method has the disadvantage of being less selective because the reagent can give a positive response to reductions in compounds other than glucose, such as fructose and galactose [3]. Ratnayani et al. (2008) [4], in their study, analyzed glucose and fructose in samples of cottonwood honey and long an honey with the HPLC method. This method shows more specific results than other methods in determining glucose and fructose in the sample. The LC-MS method for the analysis of glucose shows high selectivity, but operational costs are also quite high [5]. In this study, an alternative method was developed for the analysis of glucose that is simpler and cheaper but still sensitive. It uses potentiometric techniques through the development of the working electrode. Potentiometric analysis technique is based on the measurement of electrochemical cell potential at zero current. The most important part of the potentiometric method is the electrode. One type of working electrode that is used, namely the carbon paste electrode, is made by mixing carbon powder and paraffin. The performance of the electrode can be improved through modification. The advantage of the modified carbon paste electrode is the manufacturing process, which is relatively easy. The electrodes are
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4 cheap and can be regenerated easily [6]. One of the materials used to modify the electrode is molecularly imprinted polymer (MIP). MIP is manufactured by a technique that is specific to a particular compound [7]. The advantages of MIP are high selectivity for capturing the target analytes and good stability in organic solvents, pH and extreme temperatures [8]. The advantages of MIP make it considered an ideal material in the manufacture of electrochemical sensors. In this research, MIP glucose was synthesized from methacrylic acid as monomer, ethylene glycol dimethacrylate as cross-linker and glucose as template. The research on the development of potentiometric sensors through modified electrodes with MIP has been done previously. Noryani (2015) [9], using carbon paste electrodes modified with MIP, made from melamine as monomer and kloranil as potentiometric sensor, analyzed glucose in the blood. Safitri (2011) [10] developed carbon paste/MIP electrodes with methacrylic acid monometer as a potentiometric sensor for melamine. The parameters studied in this research are the composition of activated carbon, paraffin and MIP in the manufacture of carbon paste/MIP electrodes and the optimum pH conditions of glucose solution. The performance of the electrode and the validity of the analytical methods studied include the electrode response time, detection limit, Nernst factor, measurement range, accuracy, precision, selectivity and lifetime of the electrode. The selectivity was studied by adding urea solution to the glucose solution analyzed. Urea was selected as the interference matrix because it is contained in blood serum and has the same binding site as glucose. From the results of this study, we expect to obtain electrodes based on carbon paste/MIP that are sensitive to and selective for glucose, thus offering an alternative for the measurement of glucose in the blood potentiometrically with accurate, fast, easy and inexpensive results.
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5 Materials and methods Materials and chemicals The materials used in this study are glucose, urea, methacrylic acid, chloroform, ethylene glycol dimethacrylate (EGDMA), benzoyl peroxide (BPO), methanol, glacial acetic acid, sodium acetate trihydrate, disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, carbon, solid paraffin and Ag wire. All chemicals used have a purity degree of proanalysis. The water used was distilled.
Research Procedure Preparation of control polymer (polymethacrylic acid) First, 0.0688 g methacrylic acid was weighed and dissolved with 5 mL of chloroform in a glass beaker. In another glass beaker, 0.4752 g ethylene glycol dimethacrylate (EGDMA) was weighed and 0.2422 g benzoyl peroxide was added; these were dissolved in 1 mL of chloroform. The two solutions were mixed and heated at a temperature of 60 oC without stirring until they formed a solid. Solids were then dried in open air. The dried solids were washed with ethanol three times and then dried in an oven to obtain polymer powder. The polymer was characterized by Fourier transform infrared spectroscopy (FTIR) and viscosity tested using an Ostwald viscometer to determine its molecular weight. A polymer control was weighed to 0.0375 g and dissolved with 10 mL of methanol and then diluted with methanol in a 25 mL volumetric flask up to the mark. This solution was labeled concentration C. From this solution, other solutions with varying concentrations were prepared through dilution. The glucose solution made had concentrations of 0.1 C, 0.3 C, 0.5 C and 0.7 C. The viscosity was measured by inserting as much as 5 mL of solution with varying concentrations and determining the flow time (t) using the Ostwald viscometer. Methanol flow time was also counted as t0. From the obtained data, we then can determine specific viscosity and reduction
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6 viscosity and graph it, where x is the concentration (C) and y is the reduction viscosity. The obtained intercept values are then put in the Mark-Houwink-Sakurada equation.
Preparation of non-imprinted polymer (NIP) First, methacrylic acid was weighed to 0.0688 g and dissolved with 5 mL of chloroform, and glucose as template was weighed to 0.0360 g and diluted with distilled water in a glass beaker. Furthermore, the solution is mixed. In a different glass beaker 0.4752 g of ethylene glycol dimethacrylate (EGDMA) was weighed, to which was added 0.2422 g of benzoyl peroxide; these were dissolved in 1 mL of chloroform. The mixture of methacrylic acid and glucose were added to the mixture of EGDMA and benzoyl peroxide and then heated at a temperature of 60 oC without stirring. The solids that formed then were dried in the open air. The solid was subsequently crushed, washed using a mixture of acetic acid and methanol in a ratio of 2:8 and dried in open air. The obtained NIP was then characterized by FTIR.
Preparation of molecularly imprinted polymer (MIP) Half of the NIP obtained from the previous procedure was extracted with 10 mL of hot water three times. The filtrate was separated from the solids by centrifuge. The resulting solids then were dried in an oven. The obtained MIP was characterized by FTIR.
Preparation of carbon Carbon was chemically activated by immersion in H3PO4 10 -1 N for 24 hours. The carbon was then filtered using a Buchner funnel and washed using distilled water until the filtrate reached a neutral pH. After filtration, the carbon was heated in an oven at a temperature of about 60 oC to obtain a dry carbon powder. The obtained carbon was then characterized by Brunauer-Emmett-Teller (BET).
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Preparation of working electrode of carbon paste/MIP The working electrode of carbon paste/MIP was prepared by mixing paraffin, activated carbon and MIP of varying mass ratios in 1 mL micropipette tip. The mixture of paraffin, activated carbon and MIP was heated to form a paste. Further into the micropipette tip, we installed Ag wire to serve as the liaison between electrode and potentiometer. Paraffin was inserted into the micropipette tip to fill three-quarters of the micropipette tip section, and the remaining space was filled with the paste mixture of paraffin, activated carbon and MIP. The material was pressed to completely fill the micropipette tip. The electrode surface was rubbed with HVS paper until smooth. The construction of electrodes of carbon paste/MIP can be seen in Figure 1. In this research, the preparation of the electrode was carried out with different composition ratios of paraffin, activated carbon and MIP, as shown in Table 1. The total mass of the mixture was 0.3 g. To discover which electrode has optimum performance, we measured the potential of glucose solution with a concentration of 10-1-10 -8 M using the electrodes made with composition as shown in Table 1. After determining the optimum electrode composition, this composition was used as the basis for an electrode modified by NIP and polymer control. The carbon paste/NIP electrodesand the carbon paste/polymer control electrodes were used as a reference to determine the performance of the carbon paste/MIP electrodes.
Optimization of pH glucose solution Optimization of pH glucose solution was performed on standard glucose solution with a concentration of 10 -8-10-2 M at pH 4, 5, 6, 7 and 8. In this step, we prepared five 10 mL volumetric flasks. Each flask was filled respectively with 5 mL of glucose 10-2 M and 2 mL
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8 of buffer solution at pH 4, 5, 6, 7 and 8 and then diluted with distilled water up to the mark and shaken until homogeneous. The same procedure was done for glucose solution with a concentration of 10 -3-10-8 M. Next, the solution was inserted into the sample container and analyzed potentiometrically using a working electrode of carbon paste/MIP and a reference electrode of Ag/AgCl. The optimum pH is the pH of glucose solution that produces regression equations with Nernst factor (slope) approaching the theoretical value (
.
± 1-
2) mV.
Preparation of glucose standard curve To make the standard curve of glucose, potential measurements were conducted on the working solution of glucose with a concentration of 10 -2-10-8 M at optimal pH using the working carbon paste/MIP electrode and the reference Ag/AgCl electrode. From the obtained potential data, we can make the curve with the relationship between potential and log concentration of glucose. The curve that gives a straight line is the standard curve or calibration curve of glucose.
Determination of electrode performance and validity of analysis methods The response time of electrodes The response time of the carbon paste/MIP electrode was obtained by measuring the potential of the standard solution of glucose with a concentration on the linear range using a working carbon paste/MIP electrode and a reference Ag/AgCl electrode. The electrode response time is the time required by the electrodes to respond to the analyte and give a constant potential.
The range of measurement 8
9 The measurement range was obtained by measuring the potential of the standard glucose solution with a concentration of 10 -2-10 -8 M using a working carbon paste/MIP electrode and reference Ag/AgCl electrode. From the results of the measurements, we can make a standard curve with the relationship between potential and log concentration of glucose. The measurement range can be obtained from the curve in the concentration range that still gives a linear line.
Nernst factor The Nernst factor is obtained by measuring the potential of the standard solution of glucose in the measurement range using the working carbon paste/MIP electrode and reference Ag/AgCl electrode. From the obtained potential results, we can make a standard curve with the relationship between potential and log concentration of glucose to obtain the linear regression equation as follows: y = bx + a
(1)
where b is the slope of the curve, and a is the intercept. From these equations, the slope of the curve is the Nernst factor.
The detection limit The determination of the detection limit is done by making the cutoff point between the linear and non-linear of regression line on the curve with the relationship between potential and log concentration of glucose. If the cutoff point of the two lines is extrapolated to the xaxis, we can obtain the log concentration of the detection limit.
Selectivity
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10 The selectivity of the electrode is determined by measuring the potential of the glucose solution with a concentration of 10-3 M, then measuring the potential of the glucose solution with a concentration 10 -3 M that also contains urea solution of various concentrations. The 10-3 M glucose solution that contains a urea solution of 5×10-3 M is made by taking 10 mL of 10-2 M glucose and placing it in the 100 mL volumetric flask, then adding 5.00 mL of 10-1 M urea solution and 2 mL of buffer solution of optimum pH and diluting with distilled water up to the mark. The solution is shaken until homogeneous. The glucose solution of 10-3 M concentration that contains 6×10-3 M urea solution is made by taking 10 mL of 10 -2 M glucose and inserting it in the 100 mL volumetric flask, then adding 6 mL of 10-1 M urea solution and 2 mL of buffer solution of optimum pH and diluting with distilled water up to the mark. The solution is shaken until homogeneous. The glucose solution with a concentration of 10 -3 M that contains 7×10 -3 M urea solution is made by taking 10 mL of 10 -2 M glucose and inserting it in the 100 mL volumetric flask, then adding 7 mL of 10-1 M urea solution and 2 mL of buffer solution of pH optimum and diluting with distilled water up to the mark. The solution is shaken until homogeneous. The glucose solution with a concentration of 10 -3 M that contains 10 -2 M urea solution is made by taking 10 mL of 10 -2 M glucose and inserting it in the 100 mL volumetric flask, then adding 10 mL of 10 -1 M urea solution and 2 mL of buffer solution of optimum pH and diluting with distilled water up to the mark. The solution is shaken until homogeneous. The potential value obtained from the measurement of glucose solution and glucose solution containing urea is given in Equation 2: =
⁄
(2)
⁄
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 10
11 charge of main ions and x is the charge of ions that interfere. If the value of kij>1, the electrode responds more selectively to interference solution than to glucose, and if the value of kij <1, the electrode more selectively responds to glucose [11].
Accuracy The accuracy is obtained by measuring the potential of the working solution of 10 -2-10 5
M glucose using the working carbon paste/MIP electrode and reference Ag/AgCl
electrode. The results of potential measurement (as the value of Y) are substituted into the linear regression equation from the calibration curve and yield values of log concentration so that the glucose concentration can be calculated. The concentration of 10 -2-10-5 M glucose is regarded as the actual concentration so that the recovery value can be determined by Equation 3:
= × 100%
(3)
where R is recovery or accuracy value, xt is the value from the measurement result and xi is the actual value.
Precision The precision is obtained by measuring the potential of the standard solution of glucose in the linear range using the working carbon paste/MIP electrode and reference Ag/AgCl electrode. The measurements for each concentration were repeated three times. From the measurement results, we can determine standard deviation (s) and coefficient of variation (CV) according to Equations 4 and 5: $ = &
∑) * ̅
(4)
.
(5)
+
,- = ̅ × 100% 11
12 where x is the value of each measurement, /̅ is the average value of all the observations and N is the number of measurements.
The lifetime of electrodes The lifetime of electrodes is obtained by measuring the potential of the standard solution of glucose with a concentration of 10 -2-10-5 M using the working carbon paste/MIP electrode and reference Ag/AgCl electrode. The lifetime of the electrode is determined at the start time of the electrode used to measure the potential of the solution until the good performance of the electrode is decreased, as shown by the deviation value of the Nernst factor from its .
allowed values: 0
± 1 − 24 mV.
Results and discussion Preparation results of polymer control, NIP and MIP A polymer is a macro molecule that is composed of a series of small molecules called monomers. In this study, polymethacrylic, referred to as polymer control, is made from monomers without the addition of templates. NIP is made by adding templates on monomers, and MIP is made by adding templates on monomers, with further templates extracted to produce a mold with the shape and size of the template. In this study, the monomers used are methacrylic acid and ethylene glycol dimethacrylate, and the template is glucose. Methacrylic acid was chosen as the monomer because it has carboxyl groups that easily bind non-covalently, particularly via hydrogen bonding. In this study, the cross-linker serves as the backbone to stabilize the structure of the polymer chains.
Preparation results of control polymer 12
13 The polymer control was made as a material for modifying the electrode carbon paste. This polymer control has no specific identified side to glucose. The polymer control was made by reacting methacrylic acid, ethylene glycol dimethacrylate and benzoyl peroxide with a solvent, (porogen) chloroform. The mixture of three materials was then heated at a temperature of 60 oC until it produced white precipitate. The precipitate was then washed with distilled water, and washing continued with the use of ethanol three times. This washing serves to remove residual, unreacted reactants. The next step was drying of solids in an oven at a temperature of 60±2oC. The dried polymer control was then pulverized and weighed. Some of the polymers were characterized using FTIR and viscosity test to determine their molecular weight. The determination of molecular weight was made using the viscometry method because at a constant temperature the viscosity of a macromolecule solution is affected by molecular weight. Based on Figure 2, we obtained the linear regression equation where the intrinsic viscosity value of the polymer is equal to the intercept value. Furthermore, we conducted the calculation of molecular weight using the Mark-Houwink-Sakurada Equation of [η] = K[Mv]a with the intrinsic viscosity value of control polymer as 0.111; K and a values of this system are 242×10 -6 and 0.51, respectively. From these calculations, the molecular weight of the control polymer is 158.05 kg/mole.
Preparation results of NIP NIP preparation followed the same step sused in control polymer preparation, but in NIP preparation we added glucose as templates, so that the mixture consisted of methacrylic acid, ethylene glycol dimethacrylate and glucose with a mole ratio of 4:1:12 [12]. In this ratio, the mole content of monomer is greater than the template because it is expected that the monomer will produce maximum active side by surrounding template. Thus, glucose can
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14 be trapped within the mold. The bond formed between glucose and methacrylic acid can be seen in Figure 3. The bond that occurs is estimated to the hydrogen bonding between O and H. Cross-linker and initiators were added to the mixture of glucose and methacrylic acid. The NIP that had been formed was then crushed and washed with acetic acid and methanol with a ratio of 2:8 (v/v) to remove residual unreacted reactants. The powder then dried in open air. The final result of NIP is yellowish-white powder. Some of the powder was subsequently weighed and then characterized by FTIR. The estimationof NIP formation can be seen in Figure 4. In this study, the polymerization that occurs is addition polymerization because the final result is not accompanied by the formation of small molecules such as H2O, which would disrupt the bond between monomers and template. The initiator is used to assist the polymerization reaction. In this study, benzoyl peroxide was used to initiate methacrylic acid to form radicals. Further radicals that formed experience propagation (chain elongation) and termination (the cessation of the polymerization process) [13].
Preparation results ofMIP In the preparation of MIP is advanced step of NIP preparation. After obtaining the yellow ish-white powder, we then extracted the glucose trapped in the polymer chain to form the glucose mold in the polymers [12]. Hydrogen bonds formed between monomers and glucose is easily brokenso that glucose is easily extracted from the polymer chain [14]. The estimation of formed mold on the MIP can be seen in Figure 5.
Characterization using FTIR
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15 Characterization using FTIR aims to compare the functional groups contained in the template, control polymer, NIP and MIP. The wavenumber data of the glucose and NIP spectrum can be seen in Table 2. From the wave number data shown in Table 2, it can be seen that for glucose and NIP the functional group –C-O spectrum does not shift significantly. Functional groups –C-C on glucose are indicated by wave number 1377.22 cm-1, while NIP is indicated by wave number 1224.84 cm-1, which gives a sharp peak. The possibility of a shift in the wave numbers of NIP is due to the formation of new bonds between glucose and other constituents of NIP [15]. The functional group O-H is characterized by a very wide spectrum in the wave number, in the range of 3200-3600 cm-1. The functional group O-H on glucose is indicated by wave number 3271.38 cm-1, while the functional group O-H on NIP is indicated by wavenumber 3446.91 cm-1. The FTIR results of glucose and NIP can be seen in Figure 6. The wavenumber data of control polymer, NIP and MIP can be seen in Table 3. Based on Table 3, it can be seen that the polymerisation process on the control polymers, NIP and MIP is characterized by the functional group -C=O that conjugated -C=C. The functional group -C=O that conjugated -C=C is indicated by wavenumber 1728.28cm-1 for control polymer, 1759.14 cm-1 for NIP and 1732.13 cm-1 for MIP. The presence of hydrogen bonding to the template and monomers in NIP and MIP is indicated by the presence of functional group O-H, which widened at the wave number 3446.42 cm-1 for NIP and 3483.56 cm-1 for MIP. Control polymers that have not been added to the template cause no formation of hydrogen bonding. Therefore, in the polymer control, the functional group O-H was free and sharp at wavenumber 3527.92 cm-1. The FTIR results of the control polymer, NIP and MIP can be seen in Figure 7.
Preparation results of carbon
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16 Preparation of carbon in this study was conducted through the reactivation of the carbon, which aims to produce carbon with a larger surface area and to improve the conductivity of carbon. Carbon reactivation starts with soaking the carbon in a solution of H3PO4 10-1 N. Phosphoric acid was selected as the activator because it has strong dehydrating agent properties and can refine the pores in the carbon structure. The soaking of carbon serves to open the pores of the carbon to increase surface area. Carbon was soaked for 24 hours, and after the immersion process was filtered using a Buchner funnel and washed with distilled water. The washing using distilled water was performed repeatedly until the filtrate had a neutral pH. The carbon then was dried using an oven at a temperature of ±60oC. Drying aims to separate carbon from residual water left from the washing process. The carbon produced is then tested by BET to determine its surface area. BET test showed that the carbon has a surface area of 877.463 m2/g, so it can be said that carbon has good quality. In addition, the carbon has a pore diameter of 3.835 nm, so it can be said that carbon resulting from reactivation is nanoporous. The range for the size of nanoporous carbon is 2.1 to 6.5 nm [16].
Optimization results of composition in the preparation of carbon/MIP electrode and pH solution The optimization of electrode composition and pH is done to maximize the performance of the electrode to obtain optimum measurement results. Optimization results of composition in the preparation of carbon/MIP electrode The electrodes in this study were made from a mixture of activated carbon, solid paraffin and MIP. The activated carbon acts as a conductive material that forwards the response from the silver wire (Ag) to the potentiometer so that the potential can be read by tools. The addition of solid paraffin serves as an adhesive between MIP and activated carbon so that it
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17 can form a paste that cannot easily be separated when the mixture is inserted into the micropipette tip; this makes the electrode material less soluble when used to measure a solution. The addition of MIP in the mixture serves to increase the sensitivity and selectivity of the electrodes to the analyte because MIP is a polymer that has a specific side to the analyte. In this research, the preparation of five electrodes was carried out with different composition ratios ofparaffin, activated carbon and MIP, as shown in Table 1. The five electrodes that were made were soaked in 10-5 M glucose solution for conditioning. The electrode was used to measure the potential of 10 -8-10-1 M glucose solution. The measurement results show that each electrode has a different performance, as can be seen from the calculation results of the Nernst factor, measurement range and linearity (r), which can be seen in Table 4. The performance of the working electrode can be said to be good if it has a good Nernst factor and linearity and a wide measurement range. The Nernst factor of the electrode can be .
said to be good if it satisfies 0
± 1 − 24 mV/decade, where n is the charge of the analyte
ions. The analyte measured in this research is glucose (divalent molecule). Glucose undergoes an oxidation process that releases two electrons to form gluconic acid so that the value of the Nernst factor is 29.5 mV/decade [11]. The measurement results of each electrode showed that E4 has a Nernst factor approaching 29.5mV/decade (28.8 mV/decade). Linearity is expressed by the correlation coefficient (r) of the calibration curve. The linearity can be said to be good if the correlation coefficient (r) approaches 1. The correlation coefficient (r) can show the sensitivity of the method or device used. From the measurement results, E4 has a good correlation coefficient (r) of 0.9917. The measurement range is indicated by the curve that still gives a straight line at a measurement range with a certain concentration [17]. From the measurement results, it can be seen that each electrode has the same measurement range at a concentration of 17
18 10-5-10-2 M. The curve with the relationship between log glucose and potential of measurement results can be seen in Figure 8. Based on Figure 8, it can be seen that the electrode that gives optimum measurement results is E4 with a mass ratio of activated carbon:paraffin:MIP of 50%:35%:15%. In this study, the addition of more than 15% MIP gives a less optimum response because the resulting electrode is possibly rigid, so it gives a weak response [12]. After obtaining the optimum electrode composition, this composition was used to make electrodes modified by control polymerand NIP. This electrode was used as the reference to determine the effect of glucose mold on MIP. The performance produced by an electrode modified by control polymer, MIP and NIP can be seen in Table 5, including the Nernst factor, measurement range and linearity (r) from the measurements of glucose standard solution. Based on Table 5, it can be seen that the Nernst factor, measurement range and linearity (r) of E4 is better than ENIP and EPOL, because E4 containing MIP has a specific identified side to glucose molecules. The measurement range can be seen in Figure 9.
Optimization results of pH In this study, the optimization of pH is conducted with glucose concentrations of 10-8-10-2 M using E4 with a pH range of 4-8. This range was selected to evaluate the response produced by the electrodes when the glucose solution is measured at acidic, neutral and alkaline pH. The calculation results of Nernst factor, measurement range and linearity (r) of the 10 -8-10 -2 M glucose solution using E4 on the pH range 4-8 can be seen in Table 6. Based on Table 6, we can see that the potential measurement results of glucose by adjusting pH with the addition of pH 5 buffer solution show the obtained Nernst factor is quite good (22.50 mV/decade). However, this study did not conduct pH adjustment because the glucose solution before adding buffer solution already has a pH of 5. Analysis of standard
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19 glucose solution without pH adjustment produces a Nernst factor of 28.8 mV/decade. This is probably because the electrode used was saturated with glucose solution and so produced the same potential.
Preparation results of glucose standard curves The preparation of glucose standard curves is done by measuring the working solution of glucose with a concentration of 10-8-10 -2 using the optimum electrode E4. From the measurements results, we made a curve with the relationship between log glucose and measured potential. From the curve we selected a range that still shows a straight line with the Nernst factor approaching 29.5 mV/decade and linearity approaching 1. From the selected range of concentrations, we then determined the linear regression equation and used it as the glucose standard curve. The data on the potential measurement from glucose solution with a concentration of 10-8-10-2 M using E4 can be seen in Table 7. The curve of the relationship between log glucose and the measured potential can be seen in Figure 10, and the glucose standard curve can be seen in Figure 11.
Performance test results of electrodes and validity of analysis method The determination results of electrode response time The electrode response time is the average time that is taken by the electrodes to respond to analytes at a constant potential [18]. In this study, the response time was measured using E4 with the 10 -5-10-2 M glucose solution. The response time measurement aims to determine the sensitivity of the electrode. With a lower electrode response time to the analyte, the electrode is more sensitive. The data on electrode response time measurement using E4 can be seen in Table 8.
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20 From Table 8, we can see that the response time of the electrodes becomes faster with higher glucose concentration. This occurs because the higher concentration of glucose solution causes the number of molecules contained in the solution to increase and quickly attach to the electrodes’ surface due to frequent collisions between molecules.
The determination results of measurement range The measurement range is the concentration range of the curve with the relationship between potential (E) against log concentration (log C), which results in a straight line and still meets the Nernst equation [17]. The potentiometric method can be said to be good if it produces a wide measurement range, has a Nernst factor approaching 0.0592 mV/decade and has linearity (r) close to 1 [11]. In this study, we conducted a comparison of the measurement range for E3, E4 and E5 while considering the Nernst factor and linearity (r). The data on the measurement range for E3, E4 and E5 can be seen in Table 9.
The determination results of Nernst factor Nernst factor is the slope of the glucose standard curve. In this research, we obtained the standard curve of glucose with the following linear regression equation: y = 28.80x + 380.8. Thus, in this study, the Nernst factor is 28.80 mV/decade.
The determination results of detection limit The limit of detection is the minimum or maximum amount of analyte that can be detected by an analytical method [19]. The limit of detection can be determined by marking the intersection between the linear line and nonlinear line of the standard curve. In this research, the lower detection limit is 5.87×10-5 M. Based on the calculation of the limit of
20
21 detection, it can be seen that the carbon paste/MIP electrode produced has quite a low detection limit so that it can be applied to the analysis of glucose in the blood.
The determination results of selectivity The determination of the selectivity of a method aims to determine the ability of the method to measure a specific species (analyte) carefully in a sample without interference from other species that may exist together in the analyte. In this study, the electrode selectivity is determined by adding urea solution to the glucose solution because the developed method is intended to analyze blood samples in which glucose molecules are found along with urea molecules. The selectivity of electrodes for glucose in a urea matrix was determined by adding urea solution to 10-3 M glucose solution to a final urea concentration of 5×10-3 M, 6×10-3 M, 7×10 -3 M and 10 -2 M. The 10-3 M glucose solution was selected because generally glucose is found in the blood at these concentrations, whereas the urea concentrations selected are below normal, normal and above normal. Potential measurements were conducted using E4, and then Kij was calculated. The calculation result of Kij for glucose concentrations 10-3 M with the addition of urea matrix can be seen in Table 10. According to Table 10, the addition of urea solution to the glucose solution gives a value of Kij <1. Thus, it can be concluded that the method used in this study is more selective for glucose and is not interfered with by urea.
The determination results of accuracy Accuracy is the conformity between the value of the measurement results and the real value and can be expressed as the recovery value [20]. Recovery is expressed by the ratio between the value of the standard solution concentrations that are obtained and the actual
21
22 concentration of standard solution. In determining accuracy, the working solution of glucose used a concentration of 10 -5-10-2 M because this concentration range is the measurement range of the electrode. From the concentration range, we obtained the linear regression equation from the calibration curve; the potential value of each concentration then was inserted into the equation to obtain the concentration of the glucose solution. The concentration of glucose solution from the calculation result was then used to calculate the accuracy; results for the 10-5-10-2 M glucose solution using E4 can be seen in Table 11. Based on Table 11, it can be seen that the accuracy obtained for the 10-5-10-2 M glucose solution is 70.7-129%, which indicates that the accuracy range is far from the real range [21]. This may be due to the electrode used being saturated with glucose solution so that the resulting potential is varied. It shows that with the greater of the accuracy value so the used method is more accurate to apply on the measurement of glucose in the blood.
The determination results of precision Precision is repeatability, which represents the similarity of measurement results between two or more repeated measurements made using the same method and in the same sample [19]. The amount of precision is expressed by the coefficient of variation (CV). In this study, precision was determined on the 10-5-10-2 M glucose solution using E4; the measurements were performed repeatedly on the same sample. The calculation results of the precision can be seen in Table 12. According to Taylor (1994) [22], precision is good if the value of CV is ≤3%. The smaller the precision value, the more conscientious the method used. Based on Table 12, it can be seen that the potentiometric method using carbon paste/MIP electrodes gives a small value of CV, and thus this method has good precision (accuracy).
22
23
The determination results of electrode lifetime The lifetime of the electrode is the time range during which the electrode has good performance [18]. The determination of the lifetime of the electrode can be done by measuring the potential of the electrode for the measurement of standard solutions, which then can be used to determine the Nernst factor. If the measurement range and the value of the Nernst factor stray away from the characteristics value of the electrode, the electrode is not eligible for use [23]. The calculation of lifetime was conducted in this study by counting the number of times the electrodes were used while still giving good performance for the analysis of glucose solution; one measurement is counted as one usage. In certain application ranges, the electrodes were used to measure the potential of the working solution of 10 -5-10-2 M glucose, then used to determine the Nernst factor and measurement range. The measurement range and Nernst factor used in determining the lifetime of electrodes can be seen in Table 13. Based on Table 13, it can be seen that with 145 usages, the electrode still gives good values for Nernst factor and measurement range. However, at 149 usages, there is a decrease in the Nernst factor, away from the theoretical value, so it can be said that the performance of the electrode has decreased. Use of the electrode affects the performance of the electrode itself; when the electrodes are used often it allows a change of the mold (binding site) on the electrode material. The decrease in the electrode’s performance can also be caused by long usage time, which causes the electrode material to dissolve in the solution so that the surface becomes uneven. The lifetime of the electrode depends on the durability of the electrode material against organic compounds, oxidizing agent, pH and solubility and the flexibility of the electrode material [23].
23
24
The performance comparison of carbon paste/MIP electrodes and validity of potentiometric method The results of this study include the validity of the method and the performance of carbon paste/MIP electrode by potentiometric measurement compared with the results of previous studies, such as analysis of glucose using carbon paste/zeolite electrodes [24] and electrodes with carbon paste/MIP with melamine chloranil as monomer [9]. The results of the method’s validity and the performance of the electrode can be seen in Table 14. From Table 14, it can be seen that the potentiometric method with different components of MIP has some advantages, such as wide measurement range, precision and lifetime. The measurement range of carbon paste/polymethacrylic acid electrodes is wider (10-5-10-2 M) than that of the carbon paste/zeolite electrodes, precision is quite good and the lifetime is quite long (145 times of usage). Based on the development of electrodes of carbon paste/MIP with methacrylic acid as monomer, the optimum electrode is the fourth electrode (E4) with a Nernst factor of 28.80 and a measurement range of 10 -5-10-2 M. In this study, the value of Kij>1, so it can be said that urea does not interfere in the analysis of glucose.
Conclusion The optimum composition of activated carbon, paraffin and MIP in producing the carbon paste/MIP electrodes is 50:35:15 (%, by weight). The measurement of glucose solution is conducted potentiometrically using carbon paste/MIP electrodes gives optimum results at pH 5 (without pH adjustment). The analysis of glucose using carbon paste/MIP electrodes gives a Nernst factor of 28.80 mV/decade with a linearity (r) of 0.9917. The resulting measurement range is 10-5-10 -2 M, while the glucose detection limit is 5.87.10-5 M.
24
25 The selectivity is stated with Kij and gives a value for 10-2 M urea solution of 0.086. From the value of Kij, it can be concluded that the electrode is selective toward glucose. The resulting accuracy of the 10 -5-10-2 M glucose solution is 70.7-129%. The precision stated by the coefficient of variation for the 10-5-10 -2M glucose solution is 0.06-0.18%. This electrode has an average response time of less than two minutes, and the lifetime of the electrode is up to 145 usages.
References 1.
Sustrani, L., 2006, Hypertension (in Bahasa Indonesia), PT Gramedia Pustaka Utama, Jakarta
2.
Murray, R.K., Granner, D.K., Mayes, P.A., Rodwell, V.W., 1996, Harpers Biochemistry, 24th edition, Appleton and Lange, Stamford, Connecticut, United States
3.
Sudarmadji, S., Haryono B., Suhardi, 1997, Analysis procedure for foodstuff and agriculture (in Bahasa Indonesia), Liberty, Yogyakarta
4.
Ratnayani, K., Adhi, S.N.M.A.D., Gitadewi, I.G.A.M.A.S., 2008, Determination of glucose and fructose on the randu honey and kelengkeng honey with high performance liquid chromatography method (in Bahasa Indonesia), Journal of Chemistry, 2(2), 77-86
5.
Kamal, M.A., Klein, P., 2011, Determination of sugar in honey by liquid chromatography, Saudi Journal of Biological Sciences, 18(1), 17-21
6.
Gholivand, M.B., Malekzadeh, G., Torkashvand, M., 2013, Determination of lamotrigine by using molecularly imprinted polymer-carbon paste electrode, Journal of Electroanalytical Chemistry, 692, 9-16
7.
Yan, H., Row, K.H., 2006, Characteristic and synthetic approach of molecularly imprinted polymer, International Journal of Molecular Sciences, 7(5), 155-178
25
26 8.
He, L., Su, Y., Zheng, Y., Huang, X., Wu, L., Lui, Y., Zeng, Z., Chen, Z., 2009, Novel cyromazine imprinted polymer applied to the solid-phase extraction of melamine from feed and milk samples, Journal of Chromatography A, 1216(34), 6196-6203
9.
Noryani, I., 2015, Development of electrodes carbon paste/molecularly imprinted polymer (MIP) with melamine and chloranil as monomer for potentiometry sensor of glucose (in Bahasa Indonesia), Thesis, Faculty of Science and Technology, Airlangga University
10. Safitri, B.A., 2011, Electrodecarbon paste/molecularly imprinted polymer (MIP) withmethacrylate acidas monomer for potentiometry sensor of melamine (in Bahasa Indonesia),Thesis, Faculty of Science and Technology, Airlangga University 11. Cattrall, R.W., 1997, Chemical Sensors, Vol 1, Oxford University Press, New York 12. Liang, R., Zhang, R., Qin, W., 2009, Potentiometric sensor based on molecularly imprinted polymer for determination of melamine in milk, Sensor and Actuators B: Chemical, 141(2), 544-550 13. Odian, G., 2004, Principles of polymerization, Fourth Edition, John Willey and Sons, Inc., New York 14. Prasad, B.B., Lakshmi, D., 2004, Barbituric acid sensor based on molecularly imprinted polymer-modified hanging mercury drop electrode, Electroanalysis, 17(14), 1260-1268 15. Nakamoto, K., 2008, Infrared and Raman Spectra of Inorganic and Coordination Compounds: Part B: Applications in Coordination, Organometallic, and Bioinorganic Chemistry, 7th Edition, John Willey and Sons, Inc., New York 16. Ariyanto, T., Prasetyo, I., Rochmadi, 2012, Effect of Pore Structure on the capacitance of supercapacitor electrodes made fromnanoporous carbon (in Bahasa Indonesia), Reactor, 14(1), 25-32
26
27 17. Bakker, E., Buhlmann, P., Pretsch, E., 1997, Carrier-based ion-selective electrode and bulk optodes. 1. General characteristics, Chemical Reviews, 97(8), 3083-3132 18. Javanbakht, M., Fard, S.E., Mohammadi, A., Abdouss, M., Ganjali, M.R., Norouzi, P., Safaraliee, L., 2008, Molecularly imprinted polymer based potentiometric sensor for the determination of hydroxyzine in tablets and biological fluids, Analytica Chimica Acta,612(1), 65-74 19. Skoog, D.A., Leary, J.J., 1992, Principles of instrumental analysis, 4th Edition, Saunders College Publishing, Philadelphia 20. Skoog, D.A., West, D., Holler, F.J., 1990, Analytical chemistry: An introduction, Fifth Edition, Saunders College Publishing, Philadelphia 21. Taverniers, I., Loosee, M.D., Bockstaele, E.V., 2004, Trends in quality in the analytical laboratory. II. Analytical method validation and quality assurance, TrAC Trends in Analytical Chemistry, 23(8), 535-552 22. Taylor, L.R., Papp, R.B., Pollard, B.D., 1994, Instrumental methods for determining elements, Weinheim/VCH Publishers, New York 23. Kembaren, A., 2013, 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 24. Ilmiyah, B., 2015, Modification of electrodes carbon paste-imprinted zeolite as potentiometry sensor of blood glucose (in Bahasa Indonesia),Thesis, Faculty of Science and Technology, Airlangga University.
Competing interests The authors declare no competing financial interests.
27
28
Table 1. The composition of paraffin, activated carbon and MIP in the preparation of electrodes carbon paste/MIP Comparison of composition (%, w/w) Electrode MIP
Activated carbon Paraffin
EI
0
65
35
E2
5
60
35
E3
10
55
35
E4
15
50
35
E5
20
45
35
Table 2. The wave number data from spectrum of glucose and NIP Wave number (cm-1) Functional groups Glucose
NIP
-C-O (1025-1200 cm-1)
1155.40
1178.55
-C-C (1400-1200 cm-1)
1377.22
1224.84
O-H (3200-3600 cm-1)
3271.38
3483.56
Table 3. The wave number data from spectrum of control polymer, NIP and MIP Wave number (cm-1) Functional groups Control polymer
NIP
MIP
-C=O
1728.28
1759.14
1732.13
-C=C
1637.62
1751.42
1639.55
-O-H
-
3446.42
3483.56
28
29
Table 4. The values of Nernst factor, measurement range and linearity (r) from the measurement of electrode carbon/MIP that made with variation of compositions Mass (%, by weight) Electrode Activated carbon
Measurement Paraffin MIP range (M)
Nernst factor (mV/decade)
Linearity (r)
E1
65
35
0
10-5 - 10 -2
10.90
0.9570
E2
60
35
5
10-5 - 10 -2
14.60
0.8956
E3
55
35
10
10-5 - 10 -2
17.10
0.9296
E4
50
35
15
10-5 - 10 -2
28.80
0.9917
E5
45
35
20
10-5 - 10 -2
11.40
0.9713
Table 5. The values of Nernst factor, measurement range and linearity (r) of glucose solution that measured by electrode modified by MIP, NIP and control polymer Mass (%, by weight) Electrode Activated carbon
Measurement Paraffin MIP range (M)
Nernst factor (mV/decade)
Linearity (r)
E4
50
35
15
10-5 - 10-2
28.80
0.9917
ENIP
50
35
15
10-5 - 10-2
22.90
0.9980
EPOL
50
35
15
10-5 - 10-2
18.40
0.9750
29
30
Table 6. The calculation results of Nernst factor, measurement range, linearity (r) of the glucose solution 10-8 - 10 -2 M using E4 with pH range 4-8 Nernst factors
pH
Measurement range (M)
4
10-5 - 10 -2
17.70
0.9730
5
10-5 - 10 -2
22.50
0.9830
6
10-5 - 10 -2
20.80
0.9840
7
10-5 - 10 -2
18.62
0.9720
8
10-5 - 10 -2
16,90
0.9610
(mV/decade)
Linearity (r)
Table 7. The data of potential measurement from glucose solution with concentration 10-8 10-2 M using E4 The concentration of glucose (M)
Potential (mV)
10-8
225
10-7
239
10-6
241
10-5
236
10-4
269
10-3
290
10-2
325
Table 8. The data of electrode response time measurement using E4 on the glucose solution with concentration 10 -5 - 10-2 M
30
31 The concentration of glucose (M)
Time (sec)
10 -5
62
10 -4
58
10 -3
51
10 -2
45
Table 9. The data of measurement range for E3, E4 and E5 Electrode E3
Measurement
The regression Nernst factor Linearity
range (M)
equation
10 -5 - 10-2
y
=
(mV/decade)
(r)
17.1x
+ 17.1
0.9296
28.8x
+ 28.8
0.9917
11.4x
+ 11.4
0.9713
321.1 10 -5 - 10-2
E4
y
=
380.8 10 -5 - 10-2
E5
y
=
338.4
Table 10. The calculation result of Kij for glucose concentrations with the addition of urea solution The concentration The concentration of Selectivity of glucose (M)
10
-3
urea (M)
(Kij)
5x10-3
-0.168
6x10-3
-0.040
7x10-3
0
10x10 -3
0.086
coefficient
Table 11. The calculation result of accuracy for the glucose solution 10 -5 - 10-2 M using E4 The actual concentration The
calculation
of glucose (M)
concentration of glucose (M)
10 -5
1.20 x 10 -5
Accuracy (%) 120
31
32 10 -4
1.29 x 10 -4
129
10 -3
7.07 x 10 -4
70.7
10 -2
1.17 x 10 -2
117
Table 12. The calculation results of precision for glucose solution with concentration 10-5 10-2 M using E4 The concentration Potential (mV)
Standard
CV
of glucose (M)
Replication 1
Replication 2
Replication 3
Deviation (s)
(%)
10 -5
831
834
833
1.52
0.18
10 -4
863
864
862
1.00
0.11
10 -3
893
891
892
1.00
0.11
10 -2
923
922
923
0.57
0.06
Table 13. The measurement range and Nernst factor in determining the life time of electrodes Usage
Measurement range Nernst factors (M)
(mV/decade)
29
10-5 - 10-2
28.8
125
10-5 - 10-2
22.5
129
10-5 - 10-2
30.6
133
10-5 - 10-2
29.1
137
10-5 - 10-2
30.0
141
10-5 - 10-2
27.8
145
10-5 - 10-2
27.0
149
10-5 - 10-2
18.8
152
10-5 - 10-2
17.1
32
33 10-5 - 10-2
156
11.1
Table 14. The comparison of the validity of some methods and the performance of the electrodes on the analysis of glucose by potentiometric Electrode
carbon Electrode
Electrode carbon paste/poly
Parameter
paste/zeolite [24]
carbon
melamine chloranil paste/poly [9]
methacrylic acid
Limit of detection
5.62x10-5 M
8.75x10-5 M
5.87x10-5 M
Measurement range
10-4 M - 10-2 M
10 -4 M - 10-1 M
10 -5 M - 10-2 M
Response time (sec)
-
85-17
62-45
Accuracy (%)
88.64 - 94.63
51.8 - 141
70.7 - 129
Precision (%)
0.42 - 2.20
0.52 - 0.86
0.06 - 0.18
More selective for glucose
Selectivity
than
ascorbic acid and uric acid
Life
time
amount of usage)
(the
> 63
33
More selective for More
selective
glucose than ascorbic for glucose from acid and uric acid
the urea
96
145
34
Figure Captions Figure 1. The construction of electrodes carbon paste/MIP Figure 2. The reduction viscosity of control polymer at various concentrations Figure 3. The estimation bond between glucose with methacrylic acid Figure 4. The estimation of NIP formation Figure 5. The estimation of formed mold on the MIP [12] Figure 6. The FTIR spectra of glucose and NIP Figure 7. The FTIR spectra of control polymer, NIP and MIP Figure 8. The curve with relationship between log glucose and potential at various of electrode composition Figure 9. The standard curve of glucose from the measurement results using E4, ENIP and EPOL Figure 10. The curve with relationship between log glucose and the measured potential from glucose solution with concentration 10-8 - 10 -2 M Figure 11. The glucose standard curve
34
35
Figure 1. The construction of electrodes carbon paste/MIP
0.12 0.1
y = -0.113x + 0.111 R² = 0.801
ηred
0.08 0.06 0.04 0.02 0 -0.02 0
0.2
0.4
0.6
0.8
1
1.2
C (g/L)
Figure 2. The reduction viscosity of control polymer at various concentrations
35
36
Figure 3. The estimation bond between glucose with methacrylic acid
Figure 4. The estimation of NIP formation
36
37
Figure 5. The estimation of formed mold on the MIP [12]
Figure 6. The FTIR spectra of glucose and NIP
37
38
Figure 7. The FTIR spectra of control polymer, NIP and MIP
E1
350
E2
E4
Potential (mV)
325
E3
300
E5 275 250 225 200 -6
-5
-4
-3
-2
-1
0
Log [glucose] Figure 8. The curve with relationship between log glucose and potential at various of electrode composition
38
39 y = 22.9x + 1361 R² = 0.998
1400
ENIP
1200
EPOL
1000
y = 18.4x + 1087 R² = 0.975
Potential (mV)
E4
800 600 400
y = 28.8x + 380.8 200 R² = 0.991 0 -6
-5
-4
-3
-2
-1
0
Log [glucose] Figure 9. The standard curve of glucose from the measurement results using E4, ENIP and EPOL
350
300
Po te nti al (m V)
y = 28.80x + 380.8 R2 = 0.9917
250
200
y = 11.5x2 + 136.5x + 631 R2 = 1
150
100 -8
-7
-6
-5
-4
-3
-2
-1
0
Log [glucose] Figure 10. The curve with relationship between log glucose and the measured potential from glucose solution with concentration 10 -8 - 10-2 M
39
40 350
y = 28.8x + 380.8 R² = 0.991
300
Potential (mV)
250 200 150 100 50 0 -6
-5
-4
-3
-2
-1
Log [glucose]
Figure 11. The glucose standard curve
40
0
S2211-3797(17)30634-4 http://dx.doi.org/10.1016/j.rinp.2017.05.015 RINP 700
To appear in:
Results in Physics
Received Date: Accepted Date:
14 April 2017 9 May 2017
Please cite this article as: Darmokoesoemo, H., Kustyarini, L., Kadmi, Y., Khasanah, M., Kusuma, H.S., Elmsellem, H., Development of Electrode Carbon Paste/Molecularly Imprinted Polymer (MIP) with Methacrylic Acid as Monomer to Analyze Glucose by Potentiometry, Results in Physics (2017), doi: http://dx.doi.org/10.1016/j.rinp. 2017.05.015
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1 Development of Electrode Carbon Paste/Molecularly Imprinted Polymer (MIP) with Methacrylic Acid as Monomer to Analyze Glucose by Potentiometry Handoko Darmokoesoemo 1,*, Lendhy Kustyarini1, Yassine Kadmi2,3,4,5, Miratul Khasanah1, Heri Septya Kusuma1,* and Hicham Elmsellem6 1
Department of Chemistry, Faculty of Science and Technology, Airlangga University, 60115, Indonesia 2
Université d'Artois, EA 7394, Institut Charles Viollette, Lens, F-62300, France 3
4
5
6
ISA Lille, EA 7394, Institut Charles Viollette, Lille, F-59000, France
Ulco, EA 7394, Institut Charles Viollette, Boulogne sur Mer, F-62200, France
Université de Lille, EA 7394, Institut Charles Viollette, Lille, F-59000, France
Laboratoire de chimie analytique appliquée, matériaux et environnement (LC2AME), Faculté des Sciences, B.P. 717, 60000 Oujda, Morocco.
Email: [email protected]; [email protected]
1
2 Abstract Electrodes with carbon paste and molecularly imprinted polymer (MIP) have been developed to analyze glucose by potentiometry. The MIP was made by mixing glucose as template, methacrylic acid as monomer and ethylene glycol dimethacrylate as cross-linker with a mole ratio of 1:4:12. The electrode made by mixing activated carbon, MIP and paraffin with a ratio of 50:35:15 (% by weight) was observed to perform optimally. The results obtained show that analysis of glucose gives optimum results at a pH of 5 (without pH adjustment). The analysis of glucose using the carbon paste/MIP electrode gives a Nernst factor of 28.80 mV/decade; the measurement range is 10 -2-10 -5 M, and the limit of detection is 5.87.10 -5 M. In this study, it can be shown that urea did not interfere in the analysis of glucose using this method. The accuracy of this electrode is 70.7-129%, and the coefficient of variation is 0.06-0.18% for the concentration range 10-5-10 -2 M. This electrode showed a response time of less than 2 minutes and could be used 145 times. Keyword: glucose, molecularly imprinted polymer, potentiometry, electrode carbon paste
2
3 Introduction Diabetes mellitus is a disease in which the body cannot control blood sugar levels automatically. In a healthy body, the pancreas will release the hormone insulin, which functions to transport sugar through the blood to the muscles and the tissues of the body to supply energy [1]. The impact of excess sugar, or hyperglycemia, namely glikosuria, can lead to changes in fat metabolism. On the other hand, a shortage in blood sugar levels can cause health problems such as hypoglycemia (excess insulin) [2]. Glucose levels can be analyzed using several methods, including spectrophotometry [3], high performance liquid chromatography (HPLC) [4] and liquid chromatography-mass spectroscopy (LC-MS) [5]. Analysis of glucose as sugar levels are reduced by the NelsonSomogyi method is conducted by spectrophotometry. This method has the disadvantage of being less selective because the reagent can give a positive response to reductions in compounds other than glucose, such as fructose and galactose [3]. Ratnayani et al. (2008) [4], in their study, analyzed glucose and fructose in samples of cottonwood honey and long an honey with the HPLC method. This method shows more specific results than other methods in determining glucose and fructose in the sample. The LC-MS method for the analysis of glucose shows high selectivity, but operational costs are also quite high [5]. In this study, an alternative method was developed for the analysis of glucose that is simpler and cheaper but still sensitive. It uses potentiometric techniques through the development of the working electrode. Potentiometric analysis technique is based on the measurement of electrochemical cell potential at zero current. The most important part of the potentiometric method is the electrode. One type of working electrode that is used, namely the carbon paste electrode, is made by mixing carbon powder and paraffin. The performance of the electrode can be improved through modification. The advantage of the modified carbon paste electrode is the manufacturing process, which is relatively easy. The electrodes are
3
4 cheap and can be regenerated easily [6]. One of the materials used to modify the electrode is molecularly imprinted polymer (MIP). MIP is manufactured by a technique that is specific to a particular compound [7]. The advantages of MIP are high selectivity for capturing the target analytes and good stability in organic solvents, pH and extreme temperatures [8]. The advantages of MIP make it considered an ideal material in the manufacture of electrochemical sensors. In this research, MIP glucose was synthesized from methacrylic acid as monomer, ethylene glycol dimethacrylate as cross-linker and glucose as template. The research on the development of potentiometric sensors through modified electrodes with MIP has been done previously. Noryani (2015) [9], using carbon paste electrodes modified with MIP, made from melamine as monomer and kloranil as potentiometric sensor, analyzed glucose in the blood. Safitri (2011) [10] developed carbon paste/MIP electrodes with methacrylic acid monometer as a potentiometric sensor for melamine. The parameters studied in this research are the composition of activated carbon, paraffin and MIP in the manufacture of carbon paste/MIP electrodes and the optimum pH conditions of glucose solution. The performance of the electrode and the validity of the analytical methods studied include the electrode response time, detection limit, Nernst factor, measurement range, accuracy, precision, selectivity and lifetime of the electrode. The selectivity was studied by adding urea solution to the glucose solution analyzed. Urea was selected as the interference matrix because it is contained in blood serum and has the same binding site as glucose. From the results of this study, we expect to obtain electrodes based on carbon paste/MIP that are sensitive to and selective for glucose, thus offering an alternative for the measurement of glucose in the blood potentiometrically with accurate, fast, easy and inexpensive results.
4
5 Materials and methods Materials and chemicals The materials used in this study are glucose, urea, methacrylic acid, chloroform, ethylene glycol dimethacrylate (EGDMA), benzoyl peroxide (BPO), methanol, glacial acetic acid, sodium acetate trihydrate, disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, carbon, solid paraffin and Ag wire. All chemicals used have a purity degree of proanalysis. The water used was distilled.
Research Procedure Preparation of control polymer (polymethacrylic acid) First, 0.0688 g methacrylic acid was weighed and dissolved with 5 mL of chloroform in a glass beaker. In another glass beaker, 0.4752 g ethylene glycol dimethacrylate (EGDMA) was weighed and 0.2422 g benzoyl peroxide was added; these were dissolved in 1 mL of chloroform. The two solutions were mixed and heated at a temperature of 60 oC without stirring until they formed a solid. Solids were then dried in open air. The dried solids were washed with ethanol three times and then dried in an oven to obtain polymer powder. The polymer was characterized by Fourier transform infrared spectroscopy (FTIR) and viscosity tested using an Ostwald viscometer to determine its molecular weight. A polymer control was weighed to 0.0375 g and dissolved with 10 mL of methanol and then diluted with methanol in a 25 mL volumetric flask up to the mark. This solution was labeled concentration C. From this solution, other solutions with varying concentrations were prepared through dilution. The glucose solution made had concentrations of 0.1 C, 0.3 C, 0.5 C and 0.7 C. The viscosity was measured by inserting as much as 5 mL of solution with varying concentrations and determining the flow time (t) using the Ostwald viscometer. Methanol flow time was also counted as t0. From the obtained data, we then can determine specific viscosity and reduction
5
6 viscosity and graph it, where x is the concentration (C) and y is the reduction viscosity. The obtained intercept values are then put in the Mark-Houwink-Sakurada equation.
Preparation of non-imprinted polymer (NIP) First, methacrylic acid was weighed to 0.0688 g and dissolved with 5 mL of chloroform, and glucose as template was weighed to 0.0360 g and diluted with distilled water in a glass beaker. Furthermore, the solution is mixed. In a different glass beaker 0.4752 g of ethylene glycol dimethacrylate (EGDMA) was weighed, to which was added 0.2422 g of benzoyl peroxide; these were dissolved in 1 mL of chloroform. The mixture of methacrylic acid and glucose were added to the mixture of EGDMA and benzoyl peroxide and then heated at a temperature of 60 oC without stirring. The solids that formed then were dried in the open air. The solid was subsequently crushed, washed using a mixture of acetic acid and methanol in a ratio of 2:8 and dried in open air. The obtained NIP was then characterized by FTIR.
Preparation of molecularly imprinted polymer (MIP) Half of the NIP obtained from the previous procedure was extracted with 10 mL of hot water three times. The filtrate was separated from the solids by centrifuge. The resulting solids then were dried in an oven. The obtained MIP was characterized by FTIR.
Preparation of carbon Carbon was chemically activated by immersion in H3PO4 10 -1 N for 24 hours. The carbon was then filtered using a Buchner funnel and washed using distilled water until the filtrate reached a neutral pH. After filtration, the carbon was heated in an oven at a temperature of about 60 oC to obtain a dry carbon powder. The obtained carbon was then characterized by Brunauer-Emmett-Teller (BET).
6
7
Preparation of working electrode of carbon paste/MIP The working electrode of carbon paste/MIP was prepared by mixing paraffin, activated carbon and MIP of varying mass ratios in 1 mL micropipette tip. The mixture of paraffin, activated carbon and MIP was heated to form a paste. Further into the micropipette tip, we installed Ag wire to serve as the liaison between electrode and potentiometer. Paraffin was inserted into the micropipette tip to fill three-quarters of the micropipette tip section, and the remaining space was filled with the paste mixture of paraffin, activated carbon and MIP. The material was pressed to completely fill the micropipette tip. The electrode surface was rubbed with HVS paper until smooth. The construction of electrodes of carbon paste/MIP can be seen in Figure 1. In this research, the preparation of the electrode was carried out with different composition ratios of paraffin, activated carbon and MIP, as shown in Table 1. The total mass of the mixture was 0.3 g. To discover which electrode has optimum performance, we measured the potential of glucose solution with a concentration of 10-1-10 -8 M using the electrodes made with composition as shown in Table 1. After determining the optimum electrode composition, this composition was used as the basis for an electrode modified by NIP and polymer control. The carbon paste/NIP electrodesand the carbon paste/polymer control electrodes were used as a reference to determine the performance of the carbon paste/MIP electrodes.
Optimization of pH glucose solution Optimization of pH glucose solution was performed on standard glucose solution with a concentration of 10 -8-10-2 M at pH 4, 5, 6, 7 and 8. In this step, we prepared five 10 mL volumetric flasks. Each flask was filled respectively with 5 mL of glucose 10-2 M and 2 mL
7
8 of buffer solution at pH 4, 5, 6, 7 and 8 and then diluted with distilled water up to the mark and shaken until homogeneous. The same procedure was done for glucose solution with a concentration of 10 -3-10-8 M. Next, the solution was inserted into the sample container and analyzed potentiometrically using a working electrode of carbon paste/MIP and a reference electrode of Ag/AgCl. The optimum pH is the pH of glucose solution that produces regression equations with Nernst factor (slope) approaching the theoretical value (
.
± 1-
2) mV.
Preparation of glucose standard curve To make the standard curve of glucose, potential measurements were conducted on the working solution of glucose with a concentration of 10 -2-10-8 M at optimal pH using the working carbon paste/MIP electrode and the reference Ag/AgCl electrode. From the obtained potential data, we can make the curve with the relationship between potential and log concentration of glucose. The curve that gives a straight line is the standard curve or calibration curve of glucose.
Determination of electrode performance and validity of analysis methods The response time of electrodes The response time of the carbon paste/MIP electrode was obtained by measuring the potential of the standard solution of glucose with a concentration on the linear range using a working carbon paste/MIP electrode and a reference Ag/AgCl electrode. The electrode response time is the time required by the electrodes to respond to the analyte and give a constant potential.
The range of measurement 8
9 The measurement range was obtained by measuring the potential of the standard glucose solution with a concentration of 10 -2-10 -8 M using a working carbon paste/MIP electrode and reference Ag/AgCl electrode. From the results of the measurements, we can make a standard curve with the relationship between potential and log concentration of glucose. The measurement range can be obtained from the curve in the concentration range that still gives a linear line.
Nernst factor The Nernst factor is obtained by measuring the potential of the standard solution of glucose in the measurement range using the working carbon paste/MIP electrode and reference Ag/AgCl electrode. From the obtained potential results, we can make a standard curve with the relationship between potential and log concentration of glucose to obtain the linear regression equation as follows: y = bx + a
(1)
where b is the slope of the curve, and a is the intercept. From these equations, the slope of the curve is the Nernst factor.
The detection limit The determination of the detection limit is done by making the cutoff point between the linear and non-linear of regression line on the curve with the relationship between potential and log concentration of glucose. If the cutoff point of the two lines is extrapolated to the xaxis, we can obtain the log concentration of the detection limit.
Selectivity
9
10 The selectivity of the electrode is determined by measuring the potential of the glucose solution with a concentration of 10-3 M, then measuring the potential of the glucose solution with a concentration 10 -3 M that also contains urea solution of various concentrations. The 10-3 M glucose solution that contains a urea solution of 5×10-3 M is made by taking 10 mL of 10-2 M glucose and placing it in the 100 mL volumetric flask, then adding 5.00 mL of 10-1 M urea solution and 2 mL of buffer solution of optimum pH and diluting with distilled water up to the mark. The solution is shaken until homogeneous. The glucose solution of 10-3 M concentration that contains 6×10-3 M urea solution is made by taking 10 mL of 10 -2 M glucose and inserting it in the 100 mL volumetric flask, then adding 6 mL of 10-1 M urea solution and 2 mL of buffer solution of optimum pH and diluting with distilled water up to the mark. The solution is shaken until homogeneous. The glucose solution with a concentration of 10 -3 M that contains 7×10 -3 M urea solution is made by taking 10 mL of 10 -2 M glucose and inserting it in the 100 mL volumetric flask, then adding 7 mL of 10-1 M urea solution and 2 mL of buffer solution of pH optimum and diluting with distilled water up to the mark. The solution is shaken until homogeneous. The glucose solution with a concentration of 10 -3 M that contains 10 -2 M urea solution is made by taking 10 mL of 10 -2 M glucose and inserting it in the 100 mL volumetric flask, then adding 10 mL of 10 -1 M urea solution and 2 mL of buffer solution of optimum pH and diluting with distilled water up to the mark. The solution is shaken until homogeneous. The potential value obtained from the measurement of glucose solution and glucose solution containing urea is given in Equation 2: =
⁄
(2)
⁄
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 10
11 charge of main ions and x is the charge of ions that interfere. If the value of kij>1, the electrode responds more selectively to interference solution than to glucose, and if the value of kij <1, the electrode more selectively responds to glucose [11].
Accuracy The accuracy is obtained by measuring the potential of the working solution of 10 -2-10 5
M glucose using the working carbon paste/MIP electrode and reference Ag/AgCl
electrode. The results of potential measurement (as the value of Y) are substituted into the linear regression equation from the calibration curve and yield values of log concentration so that the glucose concentration can be calculated. The concentration of 10 -2-10-5 M glucose is regarded as the actual concentration so that the recovery value can be determined by Equation 3:
= × 100%
(3)
where R is recovery or accuracy value, xt is the value from the measurement result and xi is the actual value.
Precision The precision is obtained by measuring the potential of the standard solution of glucose in the linear range using the working carbon paste/MIP electrode and reference Ag/AgCl electrode. The measurements for each concentration were repeated three times. From the measurement results, we can determine standard deviation (s) and coefficient of variation (CV) according to Equations 4 and 5: $ = &
∑) * ̅
(4)
.
(5)
+
,- = ̅ × 100% 11
12 where x is the value of each measurement, /̅ is the average value of all the observations and N is the number of measurements.
The lifetime of electrodes The lifetime of electrodes is obtained by measuring the potential of the standard solution of glucose with a concentration of 10 -2-10-5 M using the working carbon paste/MIP electrode and reference Ag/AgCl electrode. The lifetime of the electrode is determined at the start time of the electrode used to measure the potential of the solution until the good performance of the electrode is decreased, as shown by the deviation value of the Nernst factor from its .
allowed values: 0
± 1 − 24 mV.
Results and discussion Preparation results of polymer control, NIP and MIP A polymer is a macro molecule that is composed of a series of small molecules called monomers. In this study, polymethacrylic, referred to as polymer control, is made from monomers without the addition of templates. NIP is made by adding templates on monomers, and MIP is made by adding templates on monomers, with further templates extracted to produce a mold with the shape and size of the template. In this study, the monomers used are methacrylic acid and ethylene glycol dimethacrylate, and the template is glucose. Methacrylic acid was chosen as the monomer because it has carboxyl groups that easily bind non-covalently, particularly via hydrogen bonding. In this study, the cross-linker serves as the backbone to stabilize the structure of the polymer chains.
Preparation results of control polymer 12
13 The polymer control was made as a material for modifying the electrode carbon paste. This polymer control has no specific identified side to glucose. The polymer control was made by reacting methacrylic acid, ethylene glycol dimethacrylate and benzoyl peroxide with a solvent, (porogen) chloroform. The mixture of three materials was then heated at a temperature of 60 oC until it produced white precipitate. The precipitate was then washed with distilled water, and washing continued with the use of ethanol three times. This washing serves to remove residual, unreacted reactants. The next step was drying of solids in an oven at a temperature of 60±2oC. The dried polymer control was then pulverized and weighed. Some of the polymers were characterized using FTIR and viscosity test to determine their molecular weight. The determination of molecular weight was made using the viscometry method because at a constant temperature the viscosity of a macromolecule solution is affected by molecular weight. Based on Figure 2, we obtained the linear regression equation where the intrinsic viscosity value of the polymer is equal to the intercept value. Furthermore, we conducted the calculation of molecular weight using the Mark-Houwink-Sakurada Equation of [η] = K[Mv]a with the intrinsic viscosity value of control polymer as 0.111; K and a values of this system are 242×10 -6 and 0.51, respectively. From these calculations, the molecular weight of the control polymer is 158.05 kg/mole.
Preparation results of NIP NIP preparation followed the same step sused in control polymer preparation, but in NIP preparation we added glucose as templates, so that the mixture consisted of methacrylic acid, ethylene glycol dimethacrylate and glucose with a mole ratio of 4:1:12 [12]. In this ratio, the mole content of monomer is greater than the template because it is expected that the monomer will produce maximum active side by surrounding template. Thus, glucose can
13
14 be trapped within the mold. The bond formed between glucose and methacrylic acid can be seen in Figure 3. The bond that occurs is estimated to the hydrogen bonding between O and H. Cross-linker and initiators were added to the mixture of glucose and methacrylic acid. The NIP that had been formed was then crushed and washed with acetic acid and methanol with a ratio of 2:8 (v/v) to remove residual unreacted reactants. The powder then dried in open air. The final result of NIP is yellowish-white powder. Some of the powder was subsequently weighed and then characterized by FTIR. The estimationof NIP formation can be seen in Figure 4. In this study, the polymerization that occurs is addition polymerization because the final result is not accompanied by the formation of small molecules such as H2O, which would disrupt the bond between monomers and template. The initiator is used to assist the polymerization reaction. In this study, benzoyl peroxide was used to initiate methacrylic acid to form radicals. Further radicals that formed experience propagation (chain elongation) and termination (the cessation of the polymerization process) [13].
Preparation results ofMIP In the preparation of MIP is advanced step of NIP preparation. After obtaining the yellow ish-white powder, we then extracted the glucose trapped in the polymer chain to form the glucose mold in the polymers [12]. Hydrogen bonds formed between monomers and glucose is easily brokenso that glucose is easily extracted from the polymer chain [14]. The estimation of formed mold on the MIP can be seen in Figure 5.
Characterization using FTIR
14
15 Characterization using FTIR aims to compare the functional groups contained in the template, control polymer, NIP and MIP. The wavenumber data of the glucose and NIP spectrum can be seen in Table 2. From the wave number data shown in Table 2, it can be seen that for glucose and NIP the functional group –C-O spectrum does not shift significantly. Functional groups –C-C on glucose are indicated by wave number 1377.22 cm-1, while NIP is indicated by wave number 1224.84 cm-1, which gives a sharp peak. The possibility of a shift in the wave numbers of NIP is due to the formation of new bonds between glucose and other constituents of NIP [15]. The functional group O-H is characterized by a very wide spectrum in the wave number, in the range of 3200-3600 cm-1. The functional group O-H on glucose is indicated by wave number 3271.38 cm-1, while the functional group O-H on NIP is indicated by wavenumber 3446.91 cm-1. The FTIR results of glucose and NIP can be seen in Figure 6. The wavenumber data of control polymer, NIP and MIP can be seen in Table 3. Based on Table 3, it can be seen that the polymerisation process on the control polymers, NIP and MIP is characterized by the functional group -C=O that conjugated -C=C. The functional group -C=O that conjugated -C=C is indicated by wavenumber 1728.28cm-1 for control polymer, 1759.14 cm-1 for NIP and 1732.13 cm-1 for MIP. The presence of hydrogen bonding to the template and monomers in NIP and MIP is indicated by the presence of functional group O-H, which widened at the wave number 3446.42 cm-1 for NIP and 3483.56 cm-1 for MIP. Control polymers that have not been added to the template cause no formation of hydrogen bonding. Therefore, in the polymer control, the functional group O-H was free and sharp at wavenumber 3527.92 cm-1. The FTIR results of the control polymer, NIP and MIP can be seen in Figure 7.
Preparation results of carbon
15
16 Preparation of carbon in this study was conducted through the reactivation of the carbon, which aims to produce carbon with a larger surface area and to improve the conductivity of carbon. Carbon reactivation starts with soaking the carbon in a solution of H3PO4 10-1 N. Phosphoric acid was selected as the activator because it has strong dehydrating agent properties and can refine the pores in the carbon structure. The soaking of carbon serves to open the pores of the carbon to increase surface area. Carbon was soaked for 24 hours, and after the immersion process was filtered using a Buchner funnel and washed with distilled water. The washing using distilled water was performed repeatedly until the filtrate had a neutral pH. The carbon then was dried using an oven at a temperature of ±60oC. Drying aims to separate carbon from residual water left from the washing process. The carbon produced is then tested by BET to determine its surface area. BET test showed that the carbon has a surface area of 877.463 m2/g, so it can be said that carbon has good quality. In addition, the carbon has a pore diameter of 3.835 nm, so it can be said that carbon resulting from reactivation is nanoporous. The range for the size of nanoporous carbon is 2.1 to 6.5 nm [16].
Optimization results of composition in the preparation of carbon/MIP electrode and pH solution The optimization of electrode composition and pH is done to maximize the performance of the electrode to obtain optimum measurement results. Optimization results of composition in the preparation of carbon/MIP electrode The electrodes in this study were made from a mixture of activated carbon, solid paraffin and MIP. The activated carbon acts as a conductive material that forwards the response from the silver wire (Ag) to the potentiometer so that the potential can be read by tools. The addition of solid paraffin serves as an adhesive between MIP and activated carbon so that it
16
17 can form a paste that cannot easily be separated when the mixture is inserted into the micropipette tip; this makes the electrode material less soluble when used to measure a solution. The addition of MIP in the mixture serves to increase the sensitivity and selectivity of the electrodes to the analyte because MIP is a polymer that has a specific side to the analyte. In this research, the preparation of five electrodes was carried out with different composition ratios ofparaffin, activated carbon and MIP, as shown in Table 1. The five electrodes that were made were soaked in 10-5 M glucose solution for conditioning. The electrode was used to measure the potential of 10 -8-10-1 M glucose solution. The measurement results show that each electrode has a different performance, as can be seen from the calculation results of the Nernst factor, measurement range and linearity (r), which can be seen in Table 4. The performance of the working electrode can be said to be good if it has a good Nernst factor and linearity and a wide measurement range. The Nernst factor of the electrode can be .
said to be good if it satisfies 0
± 1 − 24 mV/decade, where n is the charge of the analyte
ions. The analyte measured in this research is glucose (divalent molecule). Glucose undergoes an oxidation process that releases two electrons to form gluconic acid so that the value of the Nernst factor is 29.5 mV/decade [11]. The measurement results of each electrode showed that E4 has a Nernst factor approaching 29.5mV/decade (28.8 mV/decade). Linearity is expressed by the correlation coefficient (r) of the calibration curve. The linearity can be said to be good if the correlation coefficient (r) approaches 1. The correlation coefficient (r) can show the sensitivity of the method or device used. From the measurement results, E4 has a good correlation coefficient (r) of 0.9917. The measurement range is indicated by the curve that still gives a straight line at a measurement range with a certain concentration [17]. From the measurement results, it can be seen that each electrode has the same measurement range at a concentration of 17
18 10-5-10-2 M. The curve with the relationship between log glucose and potential of measurement results can be seen in Figure 8. Based on Figure 8, it can be seen that the electrode that gives optimum measurement results is E4 with a mass ratio of activated carbon:paraffin:MIP of 50%:35%:15%. In this study, the addition of more than 15% MIP gives a less optimum response because the resulting electrode is possibly rigid, so it gives a weak response [12]. After obtaining the optimum electrode composition, this composition was used to make electrodes modified by control polymerand NIP. This electrode was used as the reference to determine the effect of glucose mold on MIP. The performance produced by an electrode modified by control polymer, MIP and NIP can be seen in Table 5, including the Nernst factor, measurement range and linearity (r) from the measurements of glucose standard solution. Based on Table 5, it can be seen that the Nernst factor, measurement range and linearity (r) of E4 is better than ENIP and EPOL, because E4 containing MIP has a specific identified side to glucose molecules. The measurement range can be seen in Figure 9.
Optimization results of pH In this study, the optimization of pH is conducted with glucose concentrations of 10-8-10-2 M using E4 with a pH range of 4-8. This range was selected to evaluate the response produced by the electrodes when the glucose solution is measured at acidic, neutral and alkaline pH. The calculation results of Nernst factor, measurement range and linearity (r) of the 10 -8-10 -2 M glucose solution using E4 on the pH range 4-8 can be seen in Table 6. Based on Table 6, we can see that the potential measurement results of glucose by adjusting pH with the addition of pH 5 buffer solution show the obtained Nernst factor is quite good (22.50 mV/decade). However, this study did not conduct pH adjustment because the glucose solution before adding buffer solution already has a pH of 5. Analysis of standard
18
19 glucose solution without pH adjustment produces a Nernst factor of 28.8 mV/decade. This is probably because the electrode used was saturated with glucose solution and so produced the same potential.
Preparation results of glucose standard curves The preparation of glucose standard curves is done by measuring the working solution of glucose with a concentration of 10-8-10 -2 using the optimum electrode E4. From the measurements results, we made a curve with the relationship between log glucose and measured potential. From the curve we selected a range that still shows a straight line with the Nernst factor approaching 29.5 mV/decade and linearity approaching 1. From the selected range of concentrations, we then determined the linear regression equation and used it as the glucose standard curve. The data on the potential measurement from glucose solution with a concentration of 10-8-10-2 M using E4 can be seen in Table 7. The curve of the relationship between log glucose and the measured potential can be seen in Figure 10, and the glucose standard curve can be seen in Figure 11.
Performance test results of electrodes and validity of analysis method The determination results of electrode response time The electrode response time is the average time that is taken by the electrodes to respond to analytes at a constant potential [18]. In this study, the response time was measured using E4 with the 10 -5-10-2 M glucose solution. The response time measurement aims to determine the sensitivity of the electrode. With a lower electrode response time to the analyte, the electrode is more sensitive. The data on electrode response time measurement using E4 can be seen in Table 8.
19
20 From Table 8, we can see that the response time of the electrodes becomes faster with higher glucose concentration. This occurs because the higher concentration of glucose solution causes the number of molecules contained in the solution to increase and quickly attach to the electrodes’ surface due to frequent collisions between molecules.
The determination results of measurement range The measurement range is the concentration range of the curve with the relationship between potential (E) against log concentration (log C), which results in a straight line and still meets the Nernst equation [17]. The potentiometric method can be said to be good if it produces a wide measurement range, has a Nernst factor approaching 0.0592 mV/decade and has linearity (r) close to 1 [11]. In this study, we conducted a comparison of the measurement range for E3, E4 and E5 while considering the Nernst factor and linearity (r). The data on the measurement range for E3, E4 and E5 can be seen in Table 9.
The determination results of Nernst factor Nernst factor is the slope of the glucose standard curve. In this research, we obtained the standard curve of glucose with the following linear regression equation: y = 28.80x + 380.8. Thus, in this study, the Nernst factor is 28.80 mV/decade.
The determination results of detection limit The limit of detection is the minimum or maximum amount of analyte that can be detected by an analytical method [19]. The limit of detection can be determined by marking the intersection between the linear line and nonlinear line of the standard curve. In this research, the lower detection limit is 5.87×10-5 M. Based on the calculation of the limit of
20
21 detection, it can be seen that the carbon paste/MIP electrode produced has quite a low detection limit so that it can be applied to the analysis of glucose in the blood.
The determination results of selectivity The determination of the selectivity of a method aims to determine the ability of the method to measure a specific species (analyte) carefully in a sample without interference from other species that may exist together in the analyte. In this study, the electrode selectivity is determined by adding urea solution to the glucose solution because the developed method is intended to analyze blood samples in which glucose molecules are found along with urea molecules. The selectivity of electrodes for glucose in a urea matrix was determined by adding urea solution to 10-3 M glucose solution to a final urea concentration of 5×10-3 M, 6×10-3 M, 7×10 -3 M and 10 -2 M. The 10-3 M glucose solution was selected because generally glucose is found in the blood at these concentrations, whereas the urea concentrations selected are below normal, normal and above normal. Potential measurements were conducted using E4, and then Kij was calculated. The calculation result of Kij for glucose concentrations 10-3 M with the addition of urea matrix can be seen in Table 10. According to Table 10, the addition of urea solution to the glucose solution gives a value of Kij <1. Thus, it can be concluded that the method used in this study is more selective for glucose and is not interfered with by urea.
The determination results of accuracy Accuracy is the conformity between the value of the measurement results and the real value and can be expressed as the recovery value [20]. Recovery is expressed by the ratio between the value of the standard solution concentrations that are obtained and the actual
21
22 concentration of standard solution. In determining accuracy, the working solution of glucose used a concentration of 10 -5-10-2 M because this concentration range is the measurement range of the electrode. From the concentration range, we obtained the linear regression equation from the calibration curve; the potential value of each concentration then was inserted into the equation to obtain the concentration of the glucose solution. The concentration of glucose solution from the calculation result was then used to calculate the accuracy; results for the 10-5-10-2 M glucose solution using E4 can be seen in Table 11. Based on Table 11, it can be seen that the accuracy obtained for the 10-5-10-2 M glucose solution is 70.7-129%, which indicates that the accuracy range is far from the real range [21]. This may be due to the electrode used being saturated with glucose solution so that the resulting potential is varied. It shows that with the greater of the accuracy value so the used method is more accurate to apply on the measurement of glucose in the blood.
The determination results of precision Precision is repeatability, which represents the similarity of measurement results between two or more repeated measurements made using the same method and in the same sample [19]. The amount of precision is expressed by the coefficient of variation (CV). In this study, precision was determined on the 10-5-10-2 M glucose solution using E4; the measurements were performed repeatedly on the same sample. The calculation results of the precision can be seen in Table 12. According to Taylor (1994) [22], precision is good if the value of CV is ≤3%. The smaller the precision value, the more conscientious the method used. Based on Table 12, it can be seen that the potentiometric method using carbon paste/MIP electrodes gives a small value of CV, and thus this method has good precision (accuracy).
22
23
The determination results of electrode lifetime The lifetime of the electrode is the time range during which the electrode has good performance [18]. The determination of the lifetime of the electrode can be done by measuring the potential of the electrode for the measurement of standard solutions, which then can be used to determine the Nernst factor. If the measurement range and the value of the Nernst factor stray away from the characteristics value of the electrode, the electrode is not eligible for use [23]. The calculation of lifetime was conducted in this study by counting the number of times the electrodes were used while still giving good performance for the analysis of glucose solution; one measurement is counted as one usage. In certain application ranges, the electrodes were used to measure the potential of the working solution of 10 -5-10-2 M glucose, then used to determine the Nernst factor and measurement range. The measurement range and Nernst factor used in determining the lifetime of electrodes can be seen in Table 13. Based on Table 13, it can be seen that with 145 usages, the electrode still gives good values for Nernst factor and measurement range. However, at 149 usages, there is a decrease in the Nernst factor, away from the theoretical value, so it can be said that the performance of the electrode has decreased. Use of the electrode affects the performance of the electrode itself; when the electrodes are used often it allows a change of the mold (binding site) on the electrode material. The decrease in the electrode’s performance can also be caused by long usage time, which causes the electrode material to dissolve in the solution so that the surface becomes uneven. The lifetime of the electrode depends on the durability of the electrode material against organic compounds, oxidizing agent, pH and solubility and the flexibility of the electrode material [23].
23
24
The performance comparison of carbon paste/MIP electrodes and validity of potentiometric method The results of this study include the validity of the method and the performance of carbon paste/MIP electrode by potentiometric measurement compared with the results of previous studies, such as analysis of glucose using carbon paste/zeolite electrodes [24] and electrodes with carbon paste/MIP with melamine chloranil as monomer [9]. The results of the method’s validity and the performance of the electrode can be seen in Table 14. From Table 14, it can be seen that the potentiometric method with different components of MIP has some advantages, such as wide measurement range, precision and lifetime. The measurement range of carbon paste/polymethacrylic acid electrodes is wider (10-5-10-2 M) than that of the carbon paste/zeolite electrodes, precision is quite good and the lifetime is quite long (145 times of usage). Based on the development of electrodes of carbon paste/MIP with methacrylic acid as monomer, the optimum electrode is the fourth electrode (E4) with a Nernst factor of 28.80 and a measurement range of 10 -5-10-2 M. In this study, the value of Kij>1, so it can be said that urea does not interfere in the analysis of glucose.
Conclusion The optimum composition of activated carbon, paraffin and MIP in producing the carbon paste/MIP electrodes is 50:35:15 (%, by weight). The measurement of glucose solution is conducted potentiometrically using carbon paste/MIP electrodes gives optimum results at pH 5 (without pH adjustment). The analysis of glucose using carbon paste/MIP electrodes gives a Nernst factor of 28.80 mV/decade with a linearity (r) of 0.9917. The resulting measurement range is 10-5-10 -2 M, while the glucose detection limit is 5.87.10-5 M.
24
25 The selectivity is stated with Kij and gives a value for 10-2 M urea solution of 0.086. From the value of Kij, it can be concluded that the electrode is selective toward glucose. The resulting accuracy of the 10 -5-10-2 M glucose solution is 70.7-129%. The precision stated by the coefficient of variation for the 10-5-10 -2M glucose solution is 0.06-0.18%. This electrode has an average response time of less than two minutes, and the lifetime of the electrode is up to 145 usages.
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Noryani, I., 2015, Development of electrodes carbon paste/molecularly imprinted polymer (MIP) with melamine and chloranil as monomer for potentiometry sensor of glucose (in Bahasa Indonesia), Thesis, Faculty of Science and Technology, Airlangga University
10. Safitri, B.A., 2011, Electrodecarbon paste/molecularly imprinted polymer (MIP) withmethacrylate acidas monomer for potentiometry sensor of melamine (in Bahasa Indonesia),Thesis, Faculty of Science and Technology, Airlangga University 11. Cattrall, R.W., 1997, Chemical Sensors, Vol 1, Oxford University Press, New York 12. Liang, R., Zhang, R., Qin, W., 2009, Potentiometric sensor based on molecularly imprinted polymer for determination of melamine in milk, Sensor and Actuators B: Chemical, 141(2), 544-550 13. Odian, G., 2004, Principles of polymerization, Fourth Edition, John Willey and Sons, Inc., New York 14. Prasad, B.B., Lakshmi, D., 2004, Barbituric acid sensor based on molecularly imprinted polymer-modified hanging mercury drop electrode, Electroanalysis, 17(14), 1260-1268 15. Nakamoto, K., 2008, Infrared and Raman Spectra of Inorganic and Coordination Compounds: Part B: Applications in Coordination, Organometallic, and Bioinorganic Chemistry, 7th Edition, John Willey and Sons, Inc., New York 16. Ariyanto, T., Prasetyo, I., Rochmadi, 2012, Effect of Pore Structure on the capacitance of supercapacitor electrodes made fromnanoporous carbon (in Bahasa Indonesia), Reactor, 14(1), 25-32
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27 17. Bakker, E., Buhlmann, P., Pretsch, E., 1997, Carrier-based ion-selective electrode and bulk optodes. 1. General characteristics, Chemical Reviews, 97(8), 3083-3132 18. Javanbakht, M., Fard, S.E., Mohammadi, A., Abdouss, M., Ganjali, M.R., Norouzi, P., Safaraliee, L., 2008, Molecularly imprinted polymer based potentiometric sensor for the determination of hydroxyzine in tablets and biological fluids, Analytica Chimica Acta,612(1), 65-74 19. Skoog, D.A., Leary, J.J., 1992, Principles of instrumental analysis, 4th Edition, Saunders College Publishing, Philadelphia 20. Skoog, D.A., West, D., Holler, F.J., 1990, Analytical chemistry: An introduction, Fifth Edition, Saunders College Publishing, Philadelphia 21. Taverniers, I., Loosee, M.D., Bockstaele, E.V., 2004, Trends in quality in the analytical laboratory. II. Analytical method validation and quality assurance, TrAC Trends in Analytical Chemistry, 23(8), 535-552 22. Taylor, L.R., Papp, R.B., Pollard, B.D., 1994, Instrumental methods for determining elements, Weinheim/VCH Publishers, New York 23. Kembaren, A., 2013, 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 24. Ilmiyah, B., 2015, Modification of electrodes carbon paste-imprinted zeolite as potentiometry sensor of blood glucose (in Bahasa Indonesia),Thesis, Faculty of Science and Technology, Airlangga University.
Competing interests The authors declare no competing financial interests.
27
28
Table 1. The composition of paraffin, activated carbon and MIP in the preparation of electrodes carbon paste/MIP Comparison of composition (%, w/w) Electrode MIP
Activated carbon Paraffin
EI
0
65
35
E2
5
60
35
E3
10
55
35
E4
15
50
35
E5
20
45
35
Table 2. The wave number data from spectrum of glucose and NIP Wave number (cm-1) Functional groups Glucose
NIP
-C-O (1025-1200 cm-1)
1155.40
1178.55
-C-C (1400-1200 cm-1)
1377.22
1224.84
O-H (3200-3600 cm-1)
3271.38
3483.56
Table 3. The wave number data from spectrum of control polymer, NIP and MIP Wave number (cm-1) Functional groups Control polymer
NIP
MIP
-C=O
1728.28
1759.14
1732.13
-C=C
1637.62
1751.42
1639.55
-O-H
-
3446.42
3483.56
28
29
Table 4. The values of Nernst factor, measurement range and linearity (r) from the measurement of electrode carbon/MIP that made with variation of compositions Mass (%, by weight) Electrode Activated carbon
Measurement Paraffin MIP range (M)
Nernst factor (mV/decade)
Linearity (r)
E1
65
35
0
10-5 - 10 -2
10.90
0.9570
E2
60
35
5
10-5 - 10 -2
14.60
0.8956
E3
55
35
10
10-5 - 10 -2
17.10
0.9296
E4
50
35
15
10-5 - 10 -2
28.80
0.9917
E5
45
35
20
10-5 - 10 -2
11.40
0.9713
Table 5. The values of Nernst factor, measurement range and linearity (r) of glucose solution that measured by electrode modified by MIP, NIP and control polymer Mass (%, by weight) Electrode Activated carbon
Measurement Paraffin MIP range (M)
Nernst factor (mV/decade)
Linearity (r)
E4
50
35
15
10-5 - 10-2
28.80
0.9917
ENIP
50
35
15
10-5 - 10-2
22.90
0.9980
EPOL
50
35
15
10-5 - 10-2
18.40
0.9750
29
30
Table 6. The calculation results of Nernst factor, measurement range, linearity (r) of the glucose solution 10-8 - 10 -2 M using E4 with pH range 4-8 Nernst factors
pH
Measurement range (M)
4
10-5 - 10 -2
17.70
0.9730
5
10-5 - 10 -2
22.50
0.9830
6
10-5 - 10 -2
20.80
0.9840
7
10-5 - 10 -2
18.62
0.9720
8
10-5 - 10 -2
16,90
0.9610
(mV/decade)
Linearity (r)
Table 7. The data of potential measurement from glucose solution with concentration 10-8 10-2 M using E4 The concentration of glucose (M)
Potential (mV)
10-8
225
10-7
239
10-6
241
10-5
236
10-4
269
10-3
290
10-2
325
Table 8. The data of electrode response time measurement using E4 on the glucose solution with concentration 10 -5 - 10-2 M
30
31 The concentration of glucose (M)
Time (sec)
10 -5
62
10 -4
58
10 -3
51
10 -2
45
Table 9. The data of measurement range for E3, E4 and E5 Electrode E3
Measurement
The regression Nernst factor Linearity
range (M)
equation
10 -5 - 10-2
y
=
(mV/decade)
(r)
17.1x
+ 17.1
0.9296
28.8x
+ 28.8
0.9917
11.4x
+ 11.4
0.9713
321.1 10 -5 - 10-2
E4
y
=
380.8 10 -5 - 10-2
E5
y
=
338.4
Table 10. The calculation result of Kij for glucose concentrations with the addition of urea solution The concentration The concentration of Selectivity of glucose (M)
10
-3
urea (M)
(Kij)
5x10-3
-0.168
6x10-3
-0.040
7x10-3
0
10x10 -3
0.086
coefficient
Table 11. The calculation result of accuracy for the glucose solution 10 -5 - 10-2 M using E4 The actual concentration The
calculation
of glucose (M)
concentration of glucose (M)
10 -5
1.20 x 10 -5
Accuracy (%) 120
31
32 10 -4
1.29 x 10 -4
129
10 -3
7.07 x 10 -4
70.7
10 -2
1.17 x 10 -2
117
Table 12. The calculation results of precision for glucose solution with concentration 10-5 10-2 M using E4 The concentration Potential (mV)
Standard
CV
of glucose (M)
Replication 1
Replication 2
Replication 3
Deviation (s)
(%)
10 -5
831
834
833
1.52
0.18
10 -4
863
864
862
1.00
0.11
10 -3
893
891
892
1.00
0.11
10 -2
923
922
923
0.57
0.06
Table 13. The measurement range and Nernst factor in determining the life time of electrodes Usage
Measurement range Nernst factors (M)
(mV/decade)
29
10-5 - 10-2
28.8
125
10-5 - 10-2
22.5
129
10-5 - 10-2
30.6
133
10-5 - 10-2
29.1
137
10-5 - 10-2
30.0
141
10-5 - 10-2
27.8
145
10-5 - 10-2
27.0
149
10-5 - 10-2
18.8
152
10-5 - 10-2
17.1
32
33 10-5 - 10-2
156
11.1
Table 14. The comparison of the validity of some methods and the performance of the electrodes on the analysis of glucose by potentiometric Electrode
carbon Electrode
Electrode carbon paste/poly
Parameter
paste/zeolite [24]
carbon
melamine chloranil paste/poly [9]
methacrylic acid
Limit of detection
5.62x10-5 M
8.75x10-5 M
5.87x10-5 M
Measurement range
10-4 M - 10-2 M
10 -4 M - 10-1 M
10 -5 M - 10-2 M
Response time (sec)
-
85-17
62-45
Accuracy (%)
88.64 - 94.63
51.8 - 141
70.7 - 129
Precision (%)
0.42 - 2.20
0.52 - 0.86
0.06 - 0.18
More selective for glucose
Selectivity
than
ascorbic acid and uric acid
Life
time
amount of usage)
(the
> 63
33
More selective for More
selective
glucose than ascorbic for glucose from acid and uric acid
the urea
96
145
34
Figure Captions Figure 1. The construction of electrodes carbon paste/MIP Figure 2. The reduction viscosity of control polymer at various concentrations Figure 3. The estimation bond between glucose with methacrylic acid Figure 4. The estimation of NIP formation Figure 5. The estimation of formed mold on the MIP [12] Figure 6. The FTIR spectra of glucose and NIP Figure 7. The FTIR spectra of control polymer, NIP and MIP Figure 8. The curve with relationship between log glucose and potential at various of electrode composition Figure 9. The standard curve of glucose from the measurement results using E4, ENIP and EPOL Figure 10. The curve with relationship between log glucose and the measured potential from glucose solution with concentration 10-8 - 10 -2 M Figure 11. The glucose standard curve
34
35
Figure 1. The construction of electrodes carbon paste/MIP
0.12 0.1
y = -0.113x + 0.111 R² = 0.801
ηred
0.08 0.06 0.04 0.02 0 -0.02 0
0.2
0.4
0.6
0.8
1
1.2
C (g/L)
Figure 2. The reduction viscosity of control polymer at various concentrations
35
36
Figure 3. The estimation bond between glucose with methacrylic acid
Figure 4. The estimation of NIP formation
36
37
Figure 5. The estimation of formed mold on the MIP [12]
Figure 6. The FTIR spectra of glucose and NIP
37
38
Figure 7. The FTIR spectra of control polymer, NIP and MIP
E1
350
E2
E4
Potential (mV)
325
E3
300
E5 275 250 225 200 -6
-5
-4
-3
-2
-1
0
Log [glucose] Figure 8. The curve with relationship between log glucose and potential at various of electrode composition
38
39 y = 22.9x + 1361 R² = 0.998
1400
ENIP
1200
EPOL
1000
y = 18.4x + 1087 R² = 0.975
Potential (mV)
E4
800 600 400
y = 28.8x + 380.8 200 R² = 0.991 0 -6
-5
-4
-3
-2
-1
0
Log [glucose] Figure 9. The standard curve of glucose from the measurement results using E4, ENIP and EPOL
350
300
Po te nti al (m V)
y = 28.80x + 380.8 R2 = 0.9917
250
200
y = 11.5x2 + 136.5x + 631 R2 = 1
150
100 -8
-7
-6
-5
-4
-3
-2
-1
0
Log [glucose] Figure 10. The curve with relationship between log glucose and the measured potential from glucose solution with concentration 10 -8 - 10-2 M
39
40 350
y = 28.8x + 380.8 R² = 0.991
300
Potential (mV)
250 200 150 100 50 0 -6
-5
-4
-3
-2
-1
Log [glucose]
Figure 11. The glucose standard curve
40
0