- Email: [email protected]
RETRACTED: Development of carbon paste electrodes modified by molecularly imprinted polymer as potentiometry sensor of uric acid
Accepted Manuscript Development of Carbon Paste Electrodes Modified by Molecularly Imprinted Polymer as Potentiometry Sensor of Uric Acid Handoko Darmokoesoemo, Nesti Widayanti, Yassine Kadmi, Miratul Khasanah, Heri Septya Kusuma, Hicham Elmsellem PII: DOI: Reference:
S2211-3797(17)30635-6 http://dx.doi.org/10.1016/j.rinp.2017.05.013 RINP 698
To appear in:
Results in Physics
Received Date: Accepted Date:
15 April 2017 8 May 2017
Please cite this article as: Darmokoesoemo, H., Widayanti, N., Kadmi, Y., Khasanah, M., Kusuma, H.S., Elmsellem, H., Development of Carbon Paste Electrodes Modified by Molecularly Imprinted Polymer as Potentiometry Sensor of Uric Acid, Results in Physics (2017), doi: http://dx.doi.org/10.1016/j.rinp.2017.05.013
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Development of Carbon Paste Electrodes Modified by Molecularly Imprinted Polymer as Potentiometry Sensor of Uric Acid Handoko Darmokoesoemo1,*, Nesti Widayanti1, 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
Abstract The development of carbon paste electrodes modified by molecularly imprinted polymer (MIP) for the potentiometric analysis of uric acid was carried out in this study. The aim of the study was to determine the optimum composition of the electrode constituent material, the optimum pH of the uric acid solution, and the performance of the electrode, which was measured by its response time, measurement range, Nernst factor, detection limits, selectivity coefficient, precision, accuracy, and life time. MIP was made from methyl methacrylate as the monomer, ethylene glycol dimethacrylate as the cross-linker, and uric acid as the template. Electrodes that give optimum performance were produced from carbon, MIP, and paraffin with a ratio of 40:25:35 (% w/w). The obtained results show that the measurement of uric acid solution gives optimum results at pH 5, Nernst factor of 30.19 mV/decade, and a measurement range of 10 -6-10 -3 M. The minimum detection limit of this method was 3.03.10 -6 M, and the precision and accuracy toward uric acid with concentration of 10-6-10-3 M ranged between 1.36%–2.03% and 63.9%–166%. The selectivity coefficient value was less than 1, which indicated that the electrode was selective against uric acid and not interfered with by urea. This electrode has a response time of less than 2 min; its life time is 8 weeks with 104 usage times.
Keywords: carbon paste electrodes, molecularly imprinted polymer, potentiometric, uric acid
2
Introduction A potentiometric is an electrochemical method wherein a cell potential indicator electrode is measured against the reference electrode at zero current [1]. The potentiometric method has several advantages: it can be used for the determination of electroactive and nonelectroactive compounds, sample preparation, and instrument operation. Many potentiometric methods have been developed by researchers for the analysis of compounds such as uric acid whose presence in the body should always be controlled. Normal levels of uric acid in the blood of men are in the range of 3.4 to 7.0 mg/dL, while in woman the range is from 2.4 to 5.7 mg/dL [2]. In general, with increasing age, the levels of uric acid in the blood also increase. Uric acid levels that exceed normal limits in the body can cause various diseases, including hyperuricemia, gout, leukemia, and pneumonia [3]. High uric acid levels can cause kidney damage, liver damage, and cardiovascular disease [4]. Controlling uric acid levels should be done early in order to prevent a dangerous disease. In general, the methods used for the analysis of uric acid levels are colorimetric with chemical reagents or by enzymatic reactions. Chen et al. (2005) [4] analyzed the levels of uric acid in the blood by spectrophotometry using a fosfotungstat acid reagent or enzyme uricase (uric oxidase). The analysis method of uric acid by spectrophotometry requires an approximately 2–3 mL blood sample, low sensitivity, and a high detection limit (mM). Another method developed for the analysis of uric acid is high-performance liquid chromatography (HPLC). George et al. (2006) [5] determined the levels of uric acid in the urine of cattle using the HPLC method. From the research, the detection limit is low (6.5x10 -7 M), and the recovery is high. However, this method requires long analysis time, as
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sample preparation is relatively complicated, and operational costs of instruments are expensive. In addition to spectrophotometric and HPLC methods, the voltammetry method has also been developed for the analysis of uric acid, such as using gold electrodes with the addition of mercury [6] and graphite [7]. The main drawback of using the voltammetry method for analysis of uric acid is that any interference from other compounds have close oxidation potential due to the different types of electrodes that are used [8]. The required methods for the analysis of uric acid levels, which has high sensitivity and selectivity because of uric acid in the sample combined with an other electroactive matrix [9]. Therefore, the development of uric acid analysis methods with high selectivity and sensitivity include the potentiometric method through the development of the working electrode. Selection of the proper working electrode is important to note upon potentiometric analysis. The shape and composition of the material making up the working electrode should be considered, as it will affect the performance of the potentiometric method. In recent years, the modified electrode has done much to improve the selectivity and sensitivity of the analysis results of uric acid. Saada (2015) [10] modified carbon paste electrodes with imprinted zeolite (IZ). Andayani (2014) [11] analyzed uric acid with carbon paste electrodes modified in nanopori moleculary imprinted polymer (MIP), which is made from methacrylic acid monomer. The reason for the use of MIP as a material for electrode modification is because MIP is stable in extreme conditions such as pH and temperature [12]. In this research, a carbon paste electrode modified with MIP was used as the working electrodes in potentiometric analysis of uric acid. Carbon paste electrodes are used because they are easily modified, inexpensive, and easily regenerated [13]. The polymer used as the MIP
is
polymethyl
methacrylate
(PMMA)
with
methyl
methacrylate
as
the
monomer. Selection of a methyl methacrylate monomer is due to the ability of functional
4
groups on the monomer to react with the functional groups on the template to form a hydrogen bond. Wijayani et al. (2014) [14] characterized the MIP as an achloramphenicol adsorbent using methyl methacrylate as the monomer and chloramphenicol as its template. The results showed that, in the formed MIP, hydrogen bonds occur on the -NH and -OH groups of the templatewith the group -C=O of PMMA. In this study, MIP made from methyl methacrylate is used as the monomer, uric acid as the template, ethylene glycol dimethacrylate as the cross-linker, and benzoyl peroxide as the initiator. The used mole ratio among the template, monomer, cross-linker, and initiator is 0.25: 1: 5: 0.5 [14]. The MIP technique is expected to be obtained by an electrode with a mold, which is used selectively against uric acid. The parameters studied in this research include MIP optimum composition, carbon, and paraffin on making the electrode and optimum pH of the uric acid solution. We further studied the performance of modified electrodes covering the range of measurements, detection limits, accuracy, precision, Nernst factor, electrode response time, selectivity, and life time of the electrode.This research is expected to result in selective electrodes for measurement of uric acid with low levels by potentiometry, so that the potentiometric method using carbon paste electrodes/MIP can be used as an alternative method for the analysis of uric acid levels in body fluids.
Materials and methods Materials and chemicals The materials used in this study are uric acid, methyl methacrylate, ethylene glycol dimethacrylate, benzoyl peroxide, chloroform, sodium hydroxide, glacial acetic acid, methanol,
sodium
acetate
trihydrate,
sodium
hidrogenfosfat
dihydrate,
sodium
dihidrogenfosfat dihydrate, urea, carbon powder, silver wire, solid paraffin, hydrochloric
5
acid, n-hexane, ethanol, and ammonium acetate. All chemicals used have a purity degree of pro analysis (p.a). The water used is distilled water.
Research Procedure Preparation of carbon Carbon was immersed in HCl 4N for 24 h, aided by stirring, so that it becomes homogeneous; then we did decantation and washing with distilled water and filtration using a Buchner funnel. The carbon was dried in an oven at ±150ºC. The dried carbon was soaked with n-hexane overnight and then evaporated over a water bath and heated in furnace for 2 h at 500ºC. Carbon activation results were characterized using Brunauer-Emmett-Teller (BET) and Barrett-Joyner-Halenda (BJH) to determine the surface area and pore diameter of carbon.
Preparation of polymethyl methacrylate (PMMA) First, methyl methacrylate was weighed at 0.1001 g, dissolved in 5 mL of chloroform, and heated. Next, ethylene glycol dimethacrylate (EGDMA) was weighed at 0.9905 g, put into a glass beaker, and then added with 0.1210 g benzoyl peroxide, which was dissolved in 1 mL of chloroform. The solution mixture was heated at 60°C for ± 2 h without stirring. The next stage was drying in the open air and washing with ethanol three times. Then the dried solids were heated in oven to obtain a dry powder. Furthermore, polymer molecular weight determination was done using an Ostwald viscometer. The synthesized PMMA was weighed at 0.0375 g, dissolved in chloroform, diluted with chloroform in a 25 mL flask to mark boundaries, and shaken until homogeneous. The obtained concentration is C, and the next dilution processes was performed to obtain concentrations of 0.1 C; 0.3 C; 0.5 C and 0.7 C. A total of 5 mL of each solution was determined at flow time (t) using an Ostwald viscometer. The flow time of the
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solvent (chloroform) is expressed as t0. The obtained data can determine specific viscosity and reduction viscosity. Further, the curves of PMMA concentration against the reduction viscosity can be determined. The intercept value obtained from the linear regression equation curve included in the Mark-Houwink Sakurada equation is η = K [Mv]a to obtain the molecular weight of PMMA. Its characterization was carried out by FTIR and SEM of the synthesized PMMA.
Preparation ofnon-imprinted polymer (NIP) NIP is made by mixing the monomer, initiator, cross-linker, and template with a mole ratio of 1: 0.5: 5: 0.25 [14]. Methyl methacrylate monomer was weighed at 0.1001 g and dissolved in 5 mL of chloroform. Uric acid was weighed at 0.0420 g as a template and added to a methyl methacrylate solution and allowed to stand for 1 h. In a different glass beaker, we inserted 0.9905 g EGDMA as a cross-linker and 0.1210 g benzoyl peroxide as an initiator, which has been diluted with 1 mL of chloroform. In the mixture of methyl methacrylate, uric acid is added with the mixture of cross-linker and initiator and then heated at 60°C without stirring to form solids. The formed solids were then dried in the open air. The solid was subsequently crushed and sieved with a 200 mesh sieve size. The formed NIP is washed using a mixture of acetic acid and methanol in a ratio of 1:1.
Preparation of molecularly imprinted polymer (MIP) MIP is obtained by extracting uric acid from half of the synthesized powders nonimprinted polymer (NIP) using 10 mL of ammonium acetate 1 M (in ethanol, acetic acid and water with ratio of 40: 25: 35) by centrifugation for 15 min. The extraction is performed three times. The solids that formed were washed with water and then filtered and dried in an
7
oven. Further characterization was carried out using FTIR and SEM to synthesize NIP and MIP powder.
Preparation of carbon paste electrodes/MIP Electrodes are made by filling three-quarters of the micropipette tube with molten paraffin in which Ag wire has been installed. An Ag wire electrode serves as liaison with the potentiometer. The remaining portion in a micropipette tube filled with a mixture consisting of solid paraffin, carbon, and MIP. The mixture previously had been heated to form a paste, then the paste was inserted into the remaining portion of the micropipette tubewith emphasis to be solid and full filled. An electrode tip is rubbed with HVS paper to make it smooth. Construction of carbon paste electrodes/MIP is shown in Figure 1. In this research, the preparation of electrodes with variations of the composition of mixture of carbon, solid paraffin, and MIP, in order to obtain an electrode that is able to work at its optimum. The composition of carbon, MIP, and the solid paraffin can be seen in Table 1. To discover the optimum performance of the electrode, the measurement potential of the uric acid solution with a concentration of 10-8 M to 10-2 M using electrodes made with the composition is shown in Table 1. Subsequently made curve with relationship between log concentration and the potential (E). The electrodes with optimum composition showoptimum performance. Having obtained the optimum electrode composition, the composition is then used as a basis for making modified carbon paste electrodes NIP and carbon paste electrodes modified polymethyl methacrylate. Furthermore, the two electrodes are used to measure the potential of the uric acid solution with a concentration of 10 -8 M to 10 -2 M as a comparison to determine the performance of carbon paste electrodes/MIP.
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Optimization of pH uric acid solution Optimization of pH uric acid solution was conducted to determine whether pH has an effect on the electrode potential measurement. Optimization of pH uric acid solution is done by measuring the potential of uric acid solution 10-8-10-3 M, which was added with the buffer solution. To make uric acid solution 10-3 M, we inserted 5.0 mL of uric acid solution 10-2 M into five 50 mLvolumetric flasks. Then we added 2 ml of pH buffer solution 4, 5, 6, 7, and 8, diluted with water up to the mark and shook to flasks until the solution became homogeneous. The same method was used to make a uric acid solution at 10 -8-10-4 M. After the solution was completed, each solution was analyzed using a working electrode carbon paste/MIP and reference electrode Ag/AgCl. We subsequently made a curve showing the relationship between log uric acid concentration with potential; curves were created for each pH variation.
Preparation of standard curve of uric acid Concentrations of uric acid solution 10 -8-10-3 M with pH optimum were measured for their potential using carbon paste electrodes/MIP. From the data of potential differences obtained, we made curves showing the relationship between potential and log concentration (log C) of uric acidsolution. The curve is a straight line (linear), which is the standard curve of a uric acid solution.
Test of electrode performance and method validity Determination of the electrode response time The response time of the carbon paste electrode/MIP against uric acid is obtained from the potential measure of standard solutions of various concentrations of uric acid. The determination ofresponse time is done using a uric acid solution concentration of 10-6-10 -
9
3
M. Measurements were made using working electrode carbon paste/MIP and reference
electrode Ag/AgCl while stirring with a magnetic stirrer. Potential was measured at specified intervals to obtain a constant potential [15].
Determination of measurement range The measurement range is realized by measuring the potential of the uric acid solution with a concentration of 10-8-10 -3 M at pH optimum. From the data showing potential differences of measurement results, we created a curve showing the relationship between potentialand the log concentration (log C) of uric acid solution, along with urther resulting linear regression equations and nonlinear lines. Measurement range was obtained from the curve in the concentration range, which still shows a linear line.
Determination of Nernst factor The Nernst factor is determined by measuring the potential of uric acid standard solution and creating the curve with a relationship between potential and the log concentration (log C) of a uric acid solution in order to obtain equations such as Equation (1). The slope of the curve represents the magnitude of the Nernst factor: y = a + bx,
(1)
where y isthe generated potential, x is the log concentration (log C) of uric acid solution, and a and b are the intercept and slope, respectively.
Determination of the detection limit The limit of detection is determined by determining the linear regression equation and nonlinear lines on the curve of potential against the log concentration (log C) of uric acid, then determining the point of intersection between the two lines. If the point of intersection of
10
the two lines is extrapolated to the abscissa, then we obtain the log concentration detection limit.
Determination of selectivity coefficient The coefficient of selectivity in this study was determined using the mixing solution method, in which uric acid is the main molecule and urea as the matrix, which is suspected to interfere with the analysis of uric acid. The determination is done by measuring the potential of uric acid solution 10 -4 M. Then we measured the potential of the uric acid solution 10-4M, which contains each urea solution 10 -2 M, 10-3 M, and 10-4 M. Then we prepared four flasks, each filled with 5 mL of uric acid 10-3 M. Into the second flask was added 5 mL of urea solution 10-2 M; the third flask added 5 mL of urea solution 10 -3 M; and the fourth flask added urea solution 10-4 M. Then each flask added 2 ml of buffer solution pH optimum and were further diluted with water up to the mark. Furthermore, the electrode potential was measured in each solution. The obtained potential value was then substituted into Equation (2) so that the value of ki,j can be obtained: =
⁄
,
(2)
where ai is the activity of ionic analytes, aj is the activity of the ions that interfere in the mixture, E1 is the potential before the addition of the ion that interferes, E2 is the potential after the addition of the ion that interferes, and S is the slope of the calibration curve of ionic analytes [16]. If ki,j> 1, then the electrodes are more selective to urea instead of uric acid. This shows that urea can interfere with the performance of carbon paste electrodes/MIP, whereas if ki,j <1, then the electrode is more selective to uric acid rather than the urea.
Determination of precision
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Precision is determined by measuring the potential of the solution with a concentration of uric acid in the range of potentiometric measurement using carbon paste electrodes/MIP, with each concentration measured three times in replication. The determination of precision is done by calculating the standard deviation (standard deviation/SD) and coefficient of variation (CV) using Equations (3) and (4): ∑ ̅
= =
̅
,
(3)
100%,
(4)
where SD is the standard deviation, CV is the coefficient of variation, Xi is the value of each measurement, ̅ is the average value of the measurement results, and n is the number of measurements.
Determination of accuracy Accuracy was determined by measuring the potential of uric acid solution with concentration
in the
range
of potentiometric
measurement using carbon paste
electrodes/MIP. Furthermore, the potential value can be an analogy of the y value substituted into the linear regression equation of the calibration curve, so that we obtain the value of log uric acid concentration for measurement results. The concentration of uric acid in the measuring range was measured in regards to the real concentration. The accuracy value can be calculated by Equation (5): $=
%&' (
100%,
(5)
where R is the recovery or accuracy, Csp is the concentration of uric acid from the analysis result, and KS is the actual concentration of uric acid (which is measured).
Determination of electrode life time 12
The life time of the electrode is determined by measuring the potential of the uric acid solution in the measurement range using carbon paste electrodes/MIP. The measurements were taken at once per week to show deviation slope values of the curve from the allowed Nernst factors limit. The life time of the electrodes is the time span measured because the electrodes are used and perform well up to a significant decline in performance.
Results and Discussion Preparation results of PMMA, NIP, and MIP PMMA acts as a control polymer, which is made by mixing the monomer alone without the addition of templates; NIP is a polymer made by adding a template into the mix, while the MIP is polymer made by adding a template and then extraction of the template, thus leaving mold on the polymer chain. In this study, the monomer used is methyl methacrylate, while the template used is uric acid. The selection of methyl methacrylate monomer is due to the ability of functional groups on the monomer to interact with the functional groups on the template. Methyl methacrylate has functional group C=O, which is assumed to be able to form a hydrogen bond with the functional groups -NH on the uric acid molecule.
Preparation results of PMMA PMMA was prepared by mixing methyl methacrylate, benzoyl peroxide, and ethylene glycol dimethacrylate (EGDMA) in chloroform. The mixture was heated at 60°C, and the heating was stopped when solids are formed. The solids formed are colorless and resemble plastic. Furthermore, the solids are washed using ethanol and put into oven at 60°C. The synthesized solids are crushed and sieved to produce powder. The final result of this preparation of PMMA is white powder. Furthermore, the molecular weight of PMMA is determined using an Oswald viscometer and characterization using FTIR and SEM.
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Preparation results of NIP NIP is prepared by reacting methyl methacrylate as the monomer, EGDMA as the crosslinker, uric acid as the template, and benzoyl peroxide as the initiator with a mole ratio of 1: 5: 0.25: 0.5 [14]. The template, monomer, cross-linker, and initiator were mixed in chloroform. The mixture was heated at 60°C, and heating is stopped when the form is awhite solid. The synthesized white solid is crushed and sieved to produce powder and then washed using mixture of methanol and acetic acid in a ratio of 1:1 (v/v) with each volume of 10 mL. NIP formed into white powder. The powder is further characterized by FTIR and SEM.
Preparation results of MIP The preparation of MIP is basically a continuation of the preparation of NIP. In this study, MIP is obtained by extracting uric acid trapped in the NIP. The solution used to extract is ammonium acetate 1 M (in ethanol, acetic acid, and water with ratio of 40:25:35). Extraction is done by centrifugation at a rotational speed of 5000 rpm for 15 min, performed five times with volume of ammonium acetate is 10 mL. Further washing with water three times removed residual ammonium acetate, which is used to extract uric acid. The extraction process is done to remove uric acid from the series of polymer to form printed polymer of uric acidmolecules. The bond between methyl methacrylate monomer and uric acid is hydrogen bonding. Estimates of hydrogen bonding that occurs between methyl methacrylate and uric acid is shown in Figure 2. The mold expected to form after extraction of uric acid from NIP is shown in Figure 3.
Results of the molecular weight determination of PMMA
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In this study, the molecular weight of the polymer is determined by the viscometry method (done by calculating the viscosity). Based on the curve obtained linear regression equation: y = -0,1334x + 0.2753, the value of the intrinsic viscosity of polymer is equal to the intercept of the regression equation of curve with a relationship between concentration against reduction viscosity. Thus, the obtained intrinsic viscosity value of PMMA synthesis result is 0.2753. The calculation of molecular weight is determined by the Mark-Houwink Sakurada equation, with K and a values for this system at 4.88x10 -6 and 0.82, respectively [17]. Based on this calculation, the molecular weight of PMMA is 622, 835.912 g/mole. The molecular weight of PMMA is obtained in accordance with an average molecular weight of commercial PMMA ranging from 500,000–1,000,000 g/mole.
Characterization using Fourier transform infrared (FTIR) A PMMA, NIP, and MIP synthesis product is characterized using FTIR, which aims to determine differences in functional groups contained in PMMA, NIP, and MIP. The spectral peak wavenumber data of PMMA, NIP, MIP, and uric acid are shown in Table 2, while the spectra of PMMA, NIP, MIP, and uric acid are shown in Figure 5. FTIR results of PMMA, NIP, MIP, and uric acid shows some spectra peaks at wavenumbers, indicating the presence of certain functional groups. Based on Table 2 and Figure 5, PMMA, NIP, MIP, or uric acid has bonds of C=O and C=C in the range of wavenumbers, respectively, from 1760 to 1690 cm-1 and 1500 to 1700 cm-1. The bond NH stretch, which is one of the clusters in uric acid, should only be contained in the NIP and uric acid. But the results of this characterization also are seen the spectra of N-H in the MIP, which is possible because the N-H bond in the ammonium acetate at the time of extraction process has not been completely washed out by distilled water, so MIP powder remains inside. The hydrogen bond is the bond between methyl methacrylate and uric acid contained
15
only in the NIP at a wavelength of 2993.62 cm-1. The C-O bond is on polymethyl methacrylate contained in the MIP, NIP, and PMMA.
Characterization using Scanning Electron Microscope (SEM) The characterization results of SEM for PMMA, NIP, and MIP, respectively, are shown in Figures 6 (a), (b), and (c). The three figures show the difference in surface morphology. Based on the assumption, the surface morphology of the NIP is denser than the surface morphology of the MIP. This is because, in the MIP, the analyte has been extracted and is expected to form a mold that forms cavity on the surface of MIP. The characterization results using SEM on the surface of PMMA and MIP can be seen in the presence of cavities while the NIP surface looks denser.
Preparation results of carbon In this research, the preparation of carbon is through the reactivation of the carbon. Preparation of carbon aims to expand and improve the surface conductivity of carbon. First, carbon is soaked in a hydrochloric acid solution (HCl) 4N while stirring with a magnetic stirrer for 24 h. HCl serves as activator because this acid has properties of a strong dehydrating agent, so it can refine the pores in the carbon structure while the immersion process aims to enlargen the carbon pore surface area. Furthermore, carbon is washed with distilled water and filtered using a Buchner funnel until there is no HCl content in the filtrate. The filtrate washery is tested with AgNO3 to determine the presence of HCl in the filtrate. The washing process is done repeatedly until there are no white deposits on the filtrate when tested with an AgNO3 solution, and we obtain neutral pH in the filtrate. Carbon that has been free of HCl is then dried in an oven at 150°C.
16
Carbon from drying results is then soaked in n-hexane for 24 h while stirring with a magnetic strirrer. Immersion in n-hexane aims to dissolve the nonpolar organic impurities, which are not soluble in HCl. After immersion, the carbon was dried over a water bath and then heated in a furnace at a temperature of 500°C for 2 h. Carbon from activation results were characterized using BET and BJH to determine the surface area and pore diameters of carbon. The results of characterization showed that the surface area of the carbon is 877.463 m2/g and pore diameter of the carbon is 3.835 nm. According to an experiment by Ariyanto et al. (2012) [18], the pore diameter size of the carbon has potential as an electrode material ranging from 2.1 to 6.5 nm. Thus, the carbon from activation results in this study can be used as the electrode material because it has the potential for high density and good pore accessibility.
Electrode Optimization Before performing potentiometric analysis of uric acid, we required optimization of the carbon paste electrodes/MIP. This optimization is expected to produce an electrode with optimum performance. In this research, we conducted optimization of the composition of the constituent material on the electrode preparation and pH optimization of uric acid solution.
Optimization results of the composition in the preparation of carbon paste electrodes/MIP Carbon paste electrodes/MIP were made from a mixture of MIP, activated carbon, and solid paraffin. MIP is expected to increase the selectivity and sensitivity of carbon paste electrodes. Activated carbon has high conductivity and is inert, so it does not react with the analyte at measurement time. Paraffin is useful to glue the carbon and MIP when inserted into the micro-pipette tube.
17
In this study, seven electrodes were made with different compositions in accordance with Table 3. MIP and carbon composition is varied, while the composition of paraffin is fixed. Before being used for measurement, an electrode soaked in uric acid solution 10-3 M is intended for conditioning. The optimum composition of carbon paste electrode/MIP is obtained by measuring the potential of uric acid solution 10-8-10 -2 M. From the measurement results of each electrode, we obtained Nernst factor value, measurement range, and linearity,which are shown in Table 3. The selection of optimum electrode composition is based on the good Nernst factor value, linearity value approaching 1, and a broad range of measurements. According to Cattrall (1997) [16], the potentiometric method meets the Nernst equation if the Nernst factor has a value of 59.2/n (± 1-2 mV), where n is the charge of ionic analytes or valence of molecules. In this study, the measured analyte is uric acid, which is a divalent molecule, so the Nernstnya factor was 59.2 mV/2 or 29.6 mV/decade. Table 3 shows that the electrode that produces the Nernst factor approached 29.6 mV/decade is E7 (the Nernst factor is 23.46 mV/decade). The linearity value of the calibration curve is good if the regression equation’s correlation coefficient (r) approaches 1. In this study, E7 has a fairly good correlation coefficient at 0.9680. Based on the results shwon in Table 3, E1 has better correlation coefficient than E7, which is equal to 0.9946, but E1 has a Nernst factor 14.66, which is far from the theoretical value.
Optimization results of pH Optimization pH of the uric acid solution is done to determine the range of pH that can producestable potential value. Determination ofoptimumpH is done by measuring the potential of uric acid solution 10 -8-10 -3 M with a pH range of 4–8 using E7 and E1 as
18
reference. The measurement results of each electrode obtained Nernst factor value, measurement range, and linearity are shown in Table4. The data in Table 4 shows that the pH has an effect on potential measurement of uric acid solution. Measurements using E1 and E7 show thatthe best Nernst factor is at pH 5 compared with Nernst factors values at the otherpH. But, for E7, the Nernst factor value is better than the Nernst factor value of E1. In this study, pH 5 is then used for analysis of uric acid using carbon paste electrodes/MIP.
Standard curve of uric acid The standard curve of uric acid was created by measuring the potential of uric acid solution 10-8-10-3 M at optimum pH by using E7. From the obtained data, we can create a curve with the relationship between log concentration of uric acid against the measured potential. The standard curve includes data that givesstraight line in the concentration range measured by the Nernst factor value approaching 29.6 mV/decade and the linearity approaching 1. Potential measurement data are shown in Table 5; the curve with the relationship between the log concentration of uric acid against the measured potential of uric acid solution is shown in Figure 7; and the standard curve of uric acid is shown in Figure 8.
Test results of electrode performance and validity of analysis method Determination results of electrode response time The electrode response time measurement aims to determine the sensitivity of an electrode to respond to the analyte. An electrode is said to be sensitive if the time it takes to respond to analytes is faster [19]. In this study, the measurement of electrode response time is done by measuring the potential of uric acid solution 10-6-10 -3 M and calculated the time
19
needed to produce constant potential at each concentration. The measurement of electrode response time data to uric acid solution 10-6-10 -3 M using E7 is shown in Table 6. The data in Table 6 shows that the higher concentration of uric acid can speed up the response time of the electrode. In theory,the higher concentration of the analyte then the amount of analyte molecule in solution are also increasing, so that it will quickly attach to the surface of the electrode. In this study, it can be said that the carbon paste electrodes/MIP (E7) has fairly high sensitivity.
Determination results of measurement range The measurement range is the range of concentrations that can still be measured properly and gives straight line. The measurement range is obtained by measuring the potential of uric acid solution 10 -8-10 -3 M and creating a curve of that shows the relationship between the log concentration of uric acid against the potential; then we conducted data collection that gives straight line. Based on the standard curve of uric acid in Figure 8, the obtained measurement range is the concentration of uric acid solution 10-6-10 -3 M with a Nernst factor value of 30.19 mV/decade and linearity of 0.9812.
Determination results of Nernst factor The Nernst factor is the slope of curve on the standard curve of uric acid. In this study, we obtained the standard curve of uric acid from linear regression equation y = 30.19x + 230.13. Thus, in this study, the Nernst factor was 30.19 mV/decade.
Determination results ofdetection limit The limit of detection was determined by establishing the intersection between the linear line and nonlinear lines of the curve with relationship between log concentration of uric acid
20
against the potential that can be seen in Figure 9. In this study,we determine that the detection limit is the minimum detection limit (the smallest concentration of analyte that can still be measured). The smaller concentration of the detection limit indicates tha tthe method is more sensitive. In this study, the obtained detection limits amounted to 3.03x10-6 M. Based on the obtained detection limits with normal levels of uric acid in the blood, the carbon paste electrodes/MIP (E7) can be used for analysis of uric acid in the blood sample (10-4 M) because it has a detection limit for the analysis of uric acid at a concentration of 3.03x10-6 M.
Determination results ofselectivity coefficient The determination of selectivity aims to determine the ability of methods in measuring compounds or components that were carefully analyzed in the presence of other components that may exist together in the sample. In this study, the selectivity test is done through a mixing method that is with the addition of urea solution in the uric acid solution because both components are located together in the blood. The urea solution is added to a uric acid solution to a final concentration of uric acid 10 -4M, while the final concentration of urea solution is 10 -3 M, 10 -4 M, and 10 -5 M. The selection of uric acid concentration is 10-4 M because of uric acid in the blood at these concentrations, whereas urea are selected at concentrations below normal, normal, and above normal in the sample. Our solution is measured for potential using E7 and E1 as a reference. The calculation of selectivity coefficient (Ki,j) is performed by inserting the potential value into the equation. The calculation result data of Ki,j for the uric acid solution concentration of 10-4 M with the addition of urea solution can be seen in Table 7. Data in Table 7 show that measurements using E1 or E7 have a value of Ki,j <1; the higher concentration of urea is added, and then the selectivity coefficient value becomes
21
higher. In the calculation of Ki,j if the obtained value of Ki,j <1, it can be said that the used methods include more selectively against the analyte. It can be concluded that the measurement of carbon paste electrodes/MIP (E7) is more selective against uric acid and is not interfered by urea.
Determination results of precision Precision is expressed as the amount of conformity or deviation of any reported measurement results. In this study, the precision is determined by measuring the potential of uric acid solution 10 -6-10-3 M using E7. Potential measurements at each concentration are done with three times replication. To measure the precisions it is first necessary to calculate the standard deviation (SD) and then calculate the coefficient of variation (CV). The calculation precision data of uric acid solution 10 -6-10-3 M using E7 are shown in Table 8. According to Taverniers et al. (2004) [20], at concentration of 10 -6 M and 10 -5 M, the acceptable coefficient of variation is 22.6% and 16%. While the coefficient of variation value for the concentration of 10-4 M and 10 -3 M is 11.3% and 8%. Thus, it can be said that, with the higher concentration, the coefficient of variation become smaller. The data in Table 8 concludes that the developed method has high precision.
Determination results of accuracy Accuracy is the ratio of the measured concentration with the actual concentration. In this study, the accuracy is calculated via the concentration of uric acid in the measurement range of 10 -6-10 -3 M. The potential value of the measurement results of each concentration incorporated into the linear regression equation of the standard curve is used to obtain the concentration of uric acid solution. Furthermore, accuracy is determined by comparing the concentration of uric acid solution from the calculation results with the actual concentration
22
of uric acid. The calculation accuracy data of uric acid solution 10-6-10 -3 M using E7 are shown in Table 9. The average accuracy value that is acceptable for the concentration range of 10 -6-10-4 M is 80%-110%, while for concentration of 10-3 M, the averageaccuracy value that is acceptable is 90%-107% [20]. The data in Table 9 conclude that the measured concentrations in this study did not meet the required range of accuracyvalue. Thus, the accuracy of the research can be said to be less than ideal. This is possible because the electrode is in a saturated state with uric acid solution; thus, the measurement is not optimal, and the obtained accuracy results are less than ideal.
Determination results ofelectrode life time The life time is a condition where, at certain times, the electrode’s ability to analyze analytes is still quite good. In this study, the electrode life time is determined by counting the number of times the electrode can be used for analysis with good performance, where one measurement is counted as one usage. In the span of particular use, the electrode is used to measure the potential of the uric acid solution in the measurement range; then, we determine the Nernst factor. The results of determination of the electrode life timecan be seen in Table 10. Table 10 shows that, over the usage of 104 times, the electrodes still give good Nernst factor value. The usage of 116 times shows that the obtained Nernst factor value is lower when compared with the theoretical value; thus, it can be said that the performance of the electrode has decreased. Instability measurement can occur due to shifting the equilibrium, thereby reducing the level of selectivity and response of an electrode [21].
Comparison of electrodes performance test
23
In this research, we conducted the comparison of performance test of electrode modified PMMA, NIP, and MIP. This test is performed to determine the effect of uric acid mold in the MIP. The preparation of electrode-modified polymer and electrode-modified NIP in accordance with the optimum composition of previous electrode is EMIP (E7) with composition carbon 0.120 g, paraffin 0.105 g, and polymer (MIP or NIP or PMMA) 0.075 g. Furthermore, the three electrodes are used to measure the potential of uric acid solution 10 6
-10-3 M, which were conditioned at pH 5. The measurement results of each electrode-
obtained Nernst factor value, measurement range, and linearity can be seen in Table 11 and the curve profile in Figure 10. Table 11 shows that the Nernst factor value of E7 is better than ENIP and EPMMA. This is because E7 has the specific binding sides to the uric acid molecules. However, if viewed from the linearity value, which is expressed as correlation coefficient (r), E7 has a correlation coefficient that is quite good: 0.9745. The results in Table 10 show that EPMMA has a better correlation coefficient than E7, which is equal to 0.9907, but EPMMA has a Nernst factor that is far away from the theoretical value of 15.20 mV/decade.
Comparison of potentiometric method using carbon paste electrodes/MIP with the methods used in the previous study for analysis of uric acid The results of this study include the validity of the method and the performance of carbon paste electrode/MIP by potentiometrycompared with the methods used in previous studies such as analysis of uric acid using the HPLC method [5], analysis of uric acid using the glassy carbonelectrode/MIP by stripping voltammetry [22], and analysis of uric acidusing carbon
paste
electrodes/MIP
with
methacrylic
acid
monomer
by potentiometry
[11]. Comparison results of validity of the some methods are presented in Table 12.
24
Table 12 shows that the potentiometric method with different components of MIP have advantages in the limit of detection and life time of the electrode. Measurements using carbon paste electrodes/polymethyl methacrylate resulted in a lower limit of detection than measurements using carbon paste electrodes/polymethacrylic acid. The life time of carbon paste electrodes/polymethyl methacrylate is eight weeks (the usage is 104 times) longer than the life time of carbon paste electrodes/poly methacrylic acid, which is six weeks (the usage is 56 times). When compared with other methods, it can be seen that the potentiometric method has limit of detection higher than HPLC and voltammetry methods. But the potentiometric method has a wider measurement range than HPLC and the voltammetry methods.
Conclusion The optimum composition of carbon, MIP, and paraffin, which gives optimum performance, carbon paste electrodes/MIP was at ratio of 40:25:35 (% w/w) in a uric acid solution with pH 5. Measurement of uric acid with carbon paste electrodes/MIP produced a Nernst factor of 30.19 mV/decade in the measurement range of concentration 10-6-10-3 M witha correlation coefficient (r) from calibration curve 0.9812, minimum detection limit 3.03.10 -6 M, accuracy and coefficient of variation (CV) of uric acid with concentration 10 -6-10-3 M ranged from 63.9 to 166% and 1.36 to 2.03%. The selectivity coefficient value was less than one, indicating that urea did not interfere with the results of the uric acid analysis. The response time of electrodes toward the uric acid with a concentration of 10-6-10 -3 M was 32-60 seconds, and the electrode’s life time was eight weeks (the usage is 104 times).
References
25
1.
Brett, C.M.A, Oliveira-Brett, A.M., 2011, Electrochemical sensing in solution-origins, applications and future perspectives, Journal of Solid State Electrochemistry, 15, 14871494
2.
Puig, J.G., Ruilope, L.M., 1999, Uric acid as a cardiovascular risk factor in arterial hypertension, Journal of Hypertension, 17(7), 869-872
3.
Ren, W., Luo, H.Q., Li, N.B., 2006, Simultaneous Voltammetric Measurement of Ascorbic Acid, Epinephrine and Uric Acid at a Glassy Carbon Electrode Modified with Caffeic Acid, Biosensors and Bioelectronics, 21(7), 1086-1092
4.
Chen, J.C., Chung, H.H., Hsu, C.T., Tsai, D.M., Kumar, A.S., Zen, J.M., 2005, A disposable single-use electrochemical sensor for the detection of uric acid in human whole blood, Sensors and Actuators B-Chemical, 110(2), 364-369
5.
George, S.K., Dipu, M.T., Mehra, U.R., Singh, P., Verma, A.K., Ramgaokar, J.S., 2006, Improved HPLC method for the simultaneous determination of allantoin, uric acid, and creatinin in cattle urine, Journal of Chromatography B, 832(1), 134-137
6.
Ermawati, A.D., 2013, Analysis ofuric acid in serum by voltammetry using gold electrode (in Bahasa Indonesia), Thesis, Faculty of Science and Technology, Airlangga University
7.
Handaru, A.Z.P., 2010, Analysis ofuric acid in serum bystripping voltammetry using graphite electrode (in Bahasa Indonesia), Thesis, Faculty of Science and Technology, Airlangga University
8.
Premkumar, J., Khoo, S.B., 2005, Electrocatalytic oxidations of biological molecules (ascorbic acid and uric acids) at highly oxidized electrodes, Journal of Electroanalytical Chemistry, 576(1), 105-112
26
9.
Khasanah, M., Mudasir, Kuncaka, A., Sugiharto, E., 2012, Development of uric acid sensor based on molecularly imprinted polymethacrylic acid-modified hanging mercury drop electrode, Journal of Chemistry and Chemical Engineering, 6, 209-214
10. Sa’ada, A.C., 2015, Analysis of uric acid using electrodes carbon paste-imprinted zeolite by potentiometriy (in Bahasa Indonesia), Thesis, Faculty of Science and Technology, Airlangga University 11. Andayani, S.N., 2014, The development of electrodes carbon paste nanoporous/MIP as uric acid sensor by potentiometry (in Bahasa Indonesia), Thesis, Faculty of Science and Technology, Airlangga University 12. 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 13. 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 14. Wijayani, F., Supriyanto, S., Suyanto, 2015, The characterization of molecularly imprinted polymer (MIP) from precipitation polymerization results as adsorbent of chloramphenicol (in Bahasa Indonesia), Journal of Mathematics and Natural Sciences, 17(2), 1-10 15. Purwanto, A., Ernawati, F., Sajima, 2011, Characterization of cadmium ion selective electrode and Cd test in zirconium (in Bahasa Indonesia), Proceeding, Research and Processing of Nuclear Devices, Yogyakarta, 249-257 16. Cattrall, R.W., 1997, Chemical Sensors, Vol 1, Oxford University Press, New York 17. Brandrup, J., Immergut, E.H., 1989, Polymer Handbook, 3rd Edition, John Willey and Sons, New York
27
18. 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 19. Gea, S., Andriyani, Lenny, S., 2005, Preparation of selective electrode for ion Cu(II) from chitosan polyethylene oxide (in Bahasa Indonesia), Thesis, Universitas Sumatera Utara 20. 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 21. Bakker, E., Buhlmann, P., Pretsch, E., 1997, Carrier-based ion-selective electrode and bulk optodes. 1. General characteristics, Chemical Reviews, 97(8), 3083-3132 22. Arwindah, P.R., 2010, Development of uric acid sensor using glassy carbon electrode modified with molecularly imprinted polymer and anilin as monomer by stripping voltammetry (in Bahasa Indonesia), Thesis, Faculty of Science and Technology, Airlangga University
Competing interests The authors declare no competing financial interests.
28
Table 1. Composition of carbon, MIP, and solid paraffin in the preparation of carbon paste electrodes/MIP Mass (g)
Mass ratio of carbon,
Electrode Carbon
MIP
Solid paraffin
MIP, and solid paraffin
E1
0.195
0
0.105
65 : 0 : 35
E2
0.180
0.015
0.105
60 : 5 : 35
E3
0.174
0.021
0.105
58 : 7 : 35
E4
0.165
0.030
0.105
55 : 10 : 35
E5
0.150
0.045
0.105
50 : 15 : 35
E6
0.135
0.060
0.105
45 : 20 : 35
E7
0.120
0.075
0.105
40 : 25 : 35
Table 2. The spectral peak wave number data of PMMA, NIP, MIP and uric acid
29
No 1
Functional
Range of wave Wave number (cm-1)
groups
numbers (cm-1)
C
PMMA
NIP
MIP
Uric acid
= 1800-1690
1730.21 1726.35
1718.63 1677.95
= 1500-1700
1635.69 1639.55
1643.41 1438.8
O stretch 2
C C stretch
3
NH stretch
3000-3500
3446.91 3433.41
3448.84 3080.11
4
NH bend
1650-1580
-
1450.52
-
1591.16
5
OH 3000-2500
-
2993.62
-
-
1300-1000
1172.76 1151.54
hydrogen bonds 6
CO stretch
1139.97 -
Table 3. The Nernst factor value, measurement range and linearity from the measurement results of uric acid solution using carbon paste electrodes/MIP with variety of compositions Mass (%, by weight) Electrode
Measurement
Carbon MIP Paraffin range (M)
Nernst factor (mV/decade)
Linearity (r)
E1
65
0
35
10-6 - 10-3
14.66
0.9946
E2
60
5
35
10-6 - 10-3
14.18
0.9932
E3
58
7
35
10-6 - 10-3
13.90
0.9682
E4
55
10
35
10-6 - 10-3
14.35
0.9774
E5
50
15
35
10-6 - 10-3
15,16
0.9853
10-6 - 10-2
18.94
0.9975
10-6 - 10-3
19.33
0.9960
E6
45
20
35
30
E7
40
25
35
10-6 - 10-3
23.46
0.9680
Table 4. The Nernst factor value, measurement range and linearity form the measurement results of uric acid solution using E1 and E7 Measurement
Nernst factor
Linearity
range (M)
(mV/decade)
(r)
4
10-6 - 10-3
14.31
0.9862
5
10-6 - 10-3
20.05
0.9471
6
10-6 - 10-3
12,90
0.9911
7
10-6 - 10-3
12.29
0.9771
8
10-6 - 10-3
15.37
0.9803
4
10-6 - 10-3
19.42
0.9829
5
10-6 - 10-3
27.02
0.9745
10-7 - 10-4
20.51
0.9992
10-6 - 10-3
14.16
0.8616
10-6 - 10-3
18.44
0.9970
10-6 - 10-4
23.80
0.9637
10-6 - 10-3
17.40
0.8942
Electrode pH E1
E7
6 7 8
Table 5. The result of potential measurement of uric acid solution 10-8 - 10-3 M using E7 Uric acid concentration (M)
Potential (mV)
10-8
62.2
10-7
65.9
10-6
50.7
10-5
73.3
10-4
116.0
10-3
137.1
Table 6. The measurement of electrode response time data to uric acid solution using E7 31
Uric acid concentration (M)
Time (sec)
10-6
60
10-5
52
10-4
46
10-3
32
Table 7. The data of Ki,j for the uric acid solution with concentration of 10 -4 M with the addition of urea solution Concentration of E1 urea
E7
solution Potential (mV)
Ki,j
Potential (mV)
Ki,j
(M) 0
527
0
540
0
10 -5
533
0.30
538
-0.15
10 -4
543
0.45
552
0.17
10 -3
557
0.66
578
0.22
Table 8. The calculation precision data of uric acid solution 10 -6 - 10 -3 M using E7 Concentration Potential (mV)
Standard Coefficient
of uric acid Replication- Replication- Replication- deviation of variation solution (M)
1
2
3
(SD)
(CV) (%)
10 -6
57.6
58.7
56.8
0.95
1.65
10 -5
75.0
76.9
76.9
1.09
1.44
10 -4
113.8
117.0
118.4
2.36
2.03
10 -3
134.9
138.1
134.9
1.85
1.36
Table 9. The calculation accuracy data of uric acid solution 10-6 - 10 -3 M using E7 Actual
uric
acid Calculation uric acid Accuracy
32
concentration (M)
concentration (M)
(%)
10-6
1.14x10 -6
114.0
10-5
6.39x10 -6
63.9
10-4
1.66x10 -4
166.0
10-3
8.29x10 -4
82.9
Table 10. The results of determination the electrode life time Measurement
Usage
range Nernst factor
(M)
(mV/decade)
50
10-6 - 10-3
27.02
74
10-6 - 10-3
30.19
78
10-6 - 10-3
27.07
82
10-6 - 10-3
27.83
86
10-6 - 10-3
27.58
90
10-6 - 10-3
28.57
96
10-6 - 10-3
29.30
100
10-6 - 10-3
27.40
104
10-6 - 10-3
27.80
116
10-6 - 10-3
19.9
120
10-6 - 10-3
17.3
124
10-6 - 10-3
15.4
Table 11. The Nernst factor values, measurement range and linearity from measurement results of uric acid solution using electrode modified MIP, NIP and PMMA Polymer
Electrodes
Carbon (%)
(MIP or NIP or PMMA)
Paraffin Measurement (% b)
range (M)
(%)
33
Nernst factor (mV/decade)
Linearity (r)
EMIP (E7)
40
25
35
10 -6 - 10 -3
27.02
0.9745
ENIP
40
25
35
10 -6 - 10 -3
20.60
0.9846
EPMMA
40
25
35
10 -6 - 10 -3
15.20
0.9907
Table 12. The comparison results of validity of the some methods to analysis of uric acid Potentiometry
Parameter
HPLC [5]
Voltammetry [22]
Measurement 1.19x10-6 – 5.95x10-9
–
Carbon
paste Carbon
electrodes/poly
electrodes/polymethyl
methacrylic
methacrylate
acid11
study)
10 -5 - 10 -2 M
10-6 - 10 -3 M
range
8.92x10-5 M
2.97x10-8 M
Linearity
-
0.9979
0.9999
0.9812
6.5x10 -7 M
1.8x10 -9 M
1.35x10-5 M
3.03x10-6 M
Accuracy
96-109%
84.75%
96.95-102.35%
63.9-166%
Precision
6.17-6.25%
0.49-7.83%
2.01-14.52%
1.36-2.03%
Limit detection
of
paste
(this
more selective Selectivity
-
against acid
uric rather
than ascorbic
more
selective more selective against
against uric acid uric acid rather than rather than urea
urea
acid Life time
-
six
-
weeks
(the eight weeks (the usage
usage is 56 times)
34
is 104 times)
Figure Captions Figure 1. Construction of carbon paste electrodes/MIP Figure 2. The estimation of inter-molecular hydrogen bonding that occurs between methyl methacrylate and uric acid Figure 3. The estimation of the mold that formed after the extraction of the uric acid Figure 4. The curve with relationship between concentration of PMMA solution against reduction viscosity of PMMA Figure 5. FTIR spectra of PMMA, NIP, MIP, and uric acid Figure 6. The characterization results of SEM (a) PMMA, (b) NIP, (c) MIP with magnification of 4000 times Figure 7. The curve with relationship between log concentration of uric acid with potential
35
Figure 8. The standard curve of uric acid Figure 9. The intersection of the linear line and non-linear in the curve with relationship between log concentration of uric acid against potential Figure 10. The curve with relationship between log concentration of uric acid against potential from measurements results using EMIP, ENIP and EPMMA
Figure 1. Construction of carbon paste electrodes/MIP
36
Figure 2. The estimation of inter-molecular hydrogen bonding that occurs between methyl methacrylate and uric acid
O CH2
O
CH2
O
O O
C
CH2
O
OCH3
O H2C
CH
CH3
CH2
CH 2
H2C O
OCH3
CH2
O O
OCH3
C
C
CH
O
O
O
O
CH2
H3CO
CH3 HC 2
OCH3
H3CO
CH2
O
HC 2
CH2
O O
O
2
C H2
H2 C
O
C
CH
O
CH3
H2 C
CH2
O
H C
C
CH3
O
Figure 3. The estimation of the mold that formed after the extraction of the uric acid
37
0.3 0.25
η red
0.2 0.15
y = -0,1334x + 0,2753 R² = 0,9188
0.1 0.05 0 0
0.2
0.4
0.6
0.8
1
1.2
C (g/L) Figure 4. The curve with relationship between concentration of PMMA solution against reduction viscosity of PMMA
Figure 5. FTIR spectra of PMMA, NIP, MIP, and uric acid
38
(a)
(b)
(c) Figure 6. The characterization results of SEM (a) PMMA, (b) NIP, (c) MIP with magnification of 4000 times
160 140
Potential (mV)
120 100 80 60 40 20 0
-9
-8
-7
-6
-5
-4
-3
-2
-1
0
Log concentration of uric acid
Figure 7. The curve with relationship between log concentration of uric acid with potential
39
160 140
Potential (mV)
120 100 80 60 40 20 0
-7
-6
-5
-4
-3
-2
-1
0
Log concentration of uric acid Figure 8. The standard curve of uric acid
160
Potential (mV)
140 120
y = 4.725x2 + 63.235x + 268.52
y = 30.19x + 230.13 100 80 60 40 20 0
-10
-8
-6
-4
-2
0
Log concentration of uric acid Figure 9. The intersection of the linear line and non-linear in the curve with relationship between log concentration of uric acid against potential
40
yEMIP (E7)
700
yENIP
600
yEPMMA
500
Potential (mV)
400 300 200 100 0 -7
-6
-5
-4
-3
-2
-1
0
Log concentration of uric acid Figure 10. The curve with relationship between log concentration of uric acid against potential from measurements results using EMIP, ENIP and EPMMA
41
S2211-3797(17)30635-6 http://dx.doi.org/10.1016/j.rinp.2017.05.013 RINP 698
To appear in:
Results in Physics
Received Date: Accepted Date:
15 April 2017 8 May 2017
Please cite this article as: Darmokoesoemo, H., Widayanti, N., Kadmi, Y., Khasanah, M., Kusuma, H.S., Elmsellem, H., Development of Carbon Paste Electrodes Modified by Molecularly Imprinted Polymer as Potentiometry Sensor of Uric Acid, Results in Physics (2017), doi: http://dx.doi.org/10.1016/j.rinp.2017.05.013
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Development of Carbon Paste Electrodes Modified by Molecularly Imprinted Polymer as Potentiometry Sensor of Uric Acid Handoko Darmokoesoemo1,*, Nesti Widayanti1, 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
Abstract The development of carbon paste electrodes modified by molecularly imprinted polymer (MIP) for the potentiometric analysis of uric acid was carried out in this study. The aim of the study was to determine the optimum composition of the electrode constituent material, the optimum pH of the uric acid solution, and the performance of the electrode, which was measured by its response time, measurement range, Nernst factor, detection limits, selectivity coefficient, precision, accuracy, and life time. MIP was made from methyl methacrylate as the monomer, ethylene glycol dimethacrylate as the cross-linker, and uric acid as the template. Electrodes that give optimum performance were produced from carbon, MIP, and paraffin with a ratio of 40:25:35 (% w/w). The obtained results show that the measurement of uric acid solution gives optimum results at pH 5, Nernst factor of 30.19 mV/decade, and a measurement range of 10 -6-10 -3 M. The minimum detection limit of this method was 3.03.10 -6 M, and the precision and accuracy toward uric acid with concentration of 10-6-10-3 M ranged between 1.36%–2.03% and 63.9%–166%. The selectivity coefficient value was less than 1, which indicated that the electrode was selective against uric acid and not interfered with by urea. This electrode has a response time of less than 2 min; its life time is 8 weeks with 104 usage times.
Keywords: carbon paste electrodes, molecularly imprinted polymer, potentiometric, uric acid
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Introduction A potentiometric is an electrochemical method wherein a cell potential indicator electrode is measured against the reference electrode at zero current [1]. The potentiometric method has several advantages: it can be used for the determination of electroactive and nonelectroactive compounds, sample preparation, and instrument operation. Many potentiometric methods have been developed by researchers for the analysis of compounds such as uric acid whose presence in the body should always be controlled. Normal levels of uric acid in the blood of men are in the range of 3.4 to 7.0 mg/dL, while in woman the range is from 2.4 to 5.7 mg/dL [2]. In general, with increasing age, the levels of uric acid in the blood also increase. Uric acid levels that exceed normal limits in the body can cause various diseases, including hyperuricemia, gout, leukemia, and pneumonia [3]. High uric acid levels can cause kidney damage, liver damage, and cardiovascular disease [4]. Controlling uric acid levels should be done early in order to prevent a dangerous disease. In general, the methods used for the analysis of uric acid levels are colorimetric with chemical reagents or by enzymatic reactions. Chen et al. (2005) [4] analyzed the levels of uric acid in the blood by spectrophotometry using a fosfotungstat acid reagent or enzyme uricase (uric oxidase). The analysis method of uric acid by spectrophotometry requires an approximately 2–3 mL blood sample, low sensitivity, and a high detection limit (mM). Another method developed for the analysis of uric acid is high-performance liquid chromatography (HPLC). George et al. (2006) [5] determined the levels of uric acid in the urine of cattle using the HPLC method. From the research, the detection limit is low (6.5x10 -7 M), and the recovery is high. However, this method requires long analysis time, as
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sample preparation is relatively complicated, and operational costs of instruments are expensive. In addition to spectrophotometric and HPLC methods, the voltammetry method has also been developed for the analysis of uric acid, such as using gold electrodes with the addition of mercury [6] and graphite [7]. The main drawback of using the voltammetry method for analysis of uric acid is that any interference from other compounds have close oxidation potential due to the different types of electrodes that are used [8]. The required methods for the analysis of uric acid levels, which has high sensitivity and selectivity because of uric acid in the sample combined with an other electroactive matrix [9]. Therefore, the development of uric acid analysis methods with high selectivity and sensitivity include the potentiometric method through the development of the working electrode. Selection of the proper working electrode is important to note upon potentiometric analysis. The shape and composition of the material making up the working electrode should be considered, as it will affect the performance of the potentiometric method. In recent years, the modified electrode has done much to improve the selectivity and sensitivity of the analysis results of uric acid. Saada (2015) [10] modified carbon paste electrodes with imprinted zeolite (IZ). Andayani (2014) [11] analyzed uric acid with carbon paste electrodes modified in nanopori moleculary imprinted polymer (MIP), which is made from methacrylic acid monomer. The reason for the use of MIP as a material for electrode modification is because MIP is stable in extreme conditions such as pH and temperature [12]. In this research, a carbon paste electrode modified with MIP was used as the working electrodes in potentiometric analysis of uric acid. Carbon paste electrodes are used because they are easily modified, inexpensive, and easily regenerated [13]. The polymer used as the MIP
is
polymethyl
methacrylate
(PMMA)
with
methyl
methacrylate
as
the
monomer. Selection of a methyl methacrylate monomer is due to the ability of functional
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groups on the monomer to react with the functional groups on the template to form a hydrogen bond. Wijayani et al. (2014) [14] characterized the MIP as an achloramphenicol adsorbent using methyl methacrylate as the monomer and chloramphenicol as its template. The results showed that, in the formed MIP, hydrogen bonds occur on the -NH and -OH groups of the templatewith the group -C=O of PMMA. In this study, MIP made from methyl methacrylate is used as the monomer, uric acid as the template, ethylene glycol dimethacrylate as the cross-linker, and benzoyl peroxide as the initiator. The used mole ratio among the template, monomer, cross-linker, and initiator is 0.25: 1: 5: 0.5 [14]. The MIP technique is expected to be obtained by an electrode with a mold, which is used selectively against uric acid. The parameters studied in this research include MIP optimum composition, carbon, and paraffin on making the electrode and optimum pH of the uric acid solution. We further studied the performance of modified electrodes covering the range of measurements, detection limits, accuracy, precision, Nernst factor, electrode response time, selectivity, and life time of the electrode.This research is expected to result in selective electrodes for measurement of uric acid with low levels by potentiometry, so that the potentiometric method using carbon paste electrodes/MIP can be used as an alternative method for the analysis of uric acid levels in body fluids.
Materials and methods Materials and chemicals The materials used in this study are uric acid, methyl methacrylate, ethylene glycol dimethacrylate, benzoyl peroxide, chloroform, sodium hydroxide, glacial acetic acid, methanol,
sodium
acetate
trihydrate,
sodium
hidrogenfosfat
dihydrate,
sodium
dihidrogenfosfat dihydrate, urea, carbon powder, silver wire, solid paraffin, hydrochloric
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acid, n-hexane, ethanol, and ammonium acetate. All chemicals used have a purity degree of pro analysis (p.a). The water used is distilled water.
Research Procedure Preparation of carbon Carbon was immersed in HCl 4N for 24 h, aided by stirring, so that it becomes homogeneous; then we did decantation and washing with distilled water and filtration using a Buchner funnel. The carbon was dried in an oven at ±150ºC. The dried carbon was soaked with n-hexane overnight and then evaporated over a water bath and heated in furnace for 2 h at 500ºC. Carbon activation results were characterized using Brunauer-Emmett-Teller (BET) and Barrett-Joyner-Halenda (BJH) to determine the surface area and pore diameter of carbon.
Preparation of polymethyl methacrylate (PMMA) First, methyl methacrylate was weighed at 0.1001 g, dissolved in 5 mL of chloroform, and heated. Next, ethylene glycol dimethacrylate (EGDMA) was weighed at 0.9905 g, put into a glass beaker, and then added with 0.1210 g benzoyl peroxide, which was dissolved in 1 mL of chloroform. The solution mixture was heated at 60°C for ± 2 h without stirring. The next stage was drying in the open air and washing with ethanol three times. Then the dried solids were heated in oven to obtain a dry powder. Furthermore, polymer molecular weight determination was done using an Ostwald viscometer. The synthesized PMMA was weighed at 0.0375 g, dissolved in chloroform, diluted with chloroform in a 25 mL flask to mark boundaries, and shaken until homogeneous. The obtained concentration is C, and the next dilution processes was performed to obtain concentrations of 0.1 C; 0.3 C; 0.5 C and 0.7 C. A total of 5 mL of each solution was determined at flow time (t) using an Ostwald viscometer. The flow time of the
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solvent (chloroform) is expressed as t0. The obtained data can determine specific viscosity and reduction viscosity. Further, the curves of PMMA concentration against the reduction viscosity can be determined. The intercept value obtained from the linear regression equation curve included in the Mark-Houwink Sakurada equation is η = K [Mv]a to obtain the molecular weight of PMMA. Its characterization was carried out by FTIR and SEM of the synthesized PMMA.
Preparation ofnon-imprinted polymer (NIP) NIP is made by mixing the monomer, initiator, cross-linker, and template with a mole ratio of 1: 0.5: 5: 0.25 [14]. Methyl methacrylate monomer was weighed at 0.1001 g and dissolved in 5 mL of chloroform. Uric acid was weighed at 0.0420 g as a template and added to a methyl methacrylate solution and allowed to stand for 1 h. In a different glass beaker, we inserted 0.9905 g EGDMA as a cross-linker and 0.1210 g benzoyl peroxide as an initiator, which has been diluted with 1 mL of chloroform. In the mixture of methyl methacrylate, uric acid is added with the mixture of cross-linker and initiator and then heated at 60°C without stirring to form solids. The formed solids were then dried in the open air. The solid was subsequently crushed and sieved with a 200 mesh sieve size. The formed NIP is washed using a mixture of acetic acid and methanol in a ratio of 1:1.
Preparation of molecularly imprinted polymer (MIP) MIP is obtained by extracting uric acid from half of the synthesized powders nonimprinted polymer (NIP) using 10 mL of ammonium acetate 1 M (in ethanol, acetic acid and water with ratio of 40: 25: 35) by centrifugation for 15 min. The extraction is performed three times. The solids that formed were washed with water and then filtered and dried in an
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oven. Further characterization was carried out using FTIR and SEM to synthesize NIP and MIP powder.
Preparation of carbon paste electrodes/MIP Electrodes are made by filling three-quarters of the micropipette tube with molten paraffin in which Ag wire has been installed. An Ag wire electrode serves as liaison with the potentiometer. The remaining portion in a micropipette tube filled with a mixture consisting of solid paraffin, carbon, and MIP. The mixture previously had been heated to form a paste, then the paste was inserted into the remaining portion of the micropipette tubewith emphasis to be solid and full filled. An electrode tip is rubbed with HVS paper to make it smooth. Construction of carbon paste electrodes/MIP is shown in Figure 1. In this research, the preparation of electrodes with variations of the composition of mixture of carbon, solid paraffin, and MIP, in order to obtain an electrode that is able to work at its optimum. The composition of carbon, MIP, and the solid paraffin can be seen in Table 1. To discover the optimum performance of the electrode, the measurement potential of the uric acid solution with a concentration of 10-8 M to 10-2 M using electrodes made with the composition is shown in Table 1. Subsequently made curve with relationship between log concentration and the potential (E). The electrodes with optimum composition showoptimum performance. Having obtained the optimum electrode composition, the composition is then used as a basis for making modified carbon paste electrodes NIP and carbon paste electrodes modified polymethyl methacrylate. Furthermore, the two electrodes are used to measure the potential of the uric acid solution with a concentration of 10 -8 M to 10 -2 M as a comparison to determine the performance of carbon paste electrodes/MIP.
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Optimization of pH uric acid solution Optimization of pH uric acid solution was conducted to determine whether pH has an effect on the electrode potential measurement. Optimization of pH uric acid solution is done by measuring the potential of uric acid solution 10-8-10-3 M, which was added with the buffer solution. To make uric acid solution 10-3 M, we inserted 5.0 mL of uric acid solution 10-2 M into five 50 mLvolumetric flasks. Then we added 2 ml of pH buffer solution 4, 5, 6, 7, and 8, diluted with water up to the mark and shook to flasks until the solution became homogeneous. The same method was used to make a uric acid solution at 10 -8-10-4 M. After the solution was completed, each solution was analyzed using a working electrode carbon paste/MIP and reference electrode Ag/AgCl. We subsequently made a curve showing the relationship between log uric acid concentration with potential; curves were created for each pH variation.
Preparation of standard curve of uric acid Concentrations of uric acid solution 10 -8-10-3 M with pH optimum were measured for their potential using carbon paste electrodes/MIP. From the data of potential differences obtained, we made curves showing the relationship between potential and log concentration (log C) of uric acidsolution. The curve is a straight line (linear), which is the standard curve of a uric acid solution.
Test of electrode performance and method validity Determination of the electrode response time The response time of the carbon paste electrode/MIP against uric acid is obtained from the potential measure of standard solutions of various concentrations of uric acid. The determination ofresponse time is done using a uric acid solution concentration of 10-6-10 -
9
3
M. Measurements were made using working electrode carbon paste/MIP and reference
electrode Ag/AgCl while stirring with a magnetic stirrer. Potential was measured at specified intervals to obtain a constant potential [15].
Determination of measurement range The measurement range is realized by measuring the potential of the uric acid solution with a concentration of 10-8-10 -3 M at pH optimum. From the data showing potential differences of measurement results, we created a curve showing the relationship between potentialand the log concentration (log C) of uric acid solution, along with urther resulting linear regression equations and nonlinear lines. Measurement range was obtained from the curve in the concentration range, which still shows a linear line.
Determination of Nernst factor The Nernst factor is determined by measuring the potential of uric acid standard solution and creating the curve with a relationship between potential and the log concentration (log C) of a uric acid solution in order to obtain equations such as Equation (1). The slope of the curve represents the magnitude of the Nernst factor: y = a + bx,
(1)
where y isthe generated potential, x is the log concentration (log C) of uric acid solution, and a and b are the intercept and slope, respectively.
Determination of the detection limit The limit of detection is determined by determining the linear regression equation and nonlinear lines on the curve of potential against the log concentration (log C) of uric acid, then determining the point of intersection between the two lines. If the point of intersection of
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the two lines is extrapolated to the abscissa, then we obtain the log concentration detection limit.
Determination of selectivity coefficient The coefficient of selectivity in this study was determined using the mixing solution method, in which uric acid is the main molecule and urea as the matrix, which is suspected to interfere with the analysis of uric acid. The determination is done by measuring the potential of uric acid solution 10 -4 M. Then we measured the potential of the uric acid solution 10-4M, which contains each urea solution 10 -2 M, 10-3 M, and 10-4 M. Then we prepared four flasks, each filled with 5 mL of uric acid 10-3 M. Into the second flask was added 5 mL of urea solution 10-2 M; the third flask added 5 mL of urea solution 10 -3 M; and the fourth flask added urea solution 10-4 M. Then each flask added 2 ml of buffer solution pH optimum and were further diluted with water up to the mark. Furthermore, the electrode potential was measured in each solution. The obtained potential value was then substituted into Equation (2) so that the value of ki,j can be obtained: =
⁄
,
(2)
where ai is the activity of ionic analytes, aj is the activity of the ions that interfere in the mixture, E1 is the potential before the addition of the ion that interferes, E2 is the potential after the addition of the ion that interferes, and S is the slope of the calibration curve of ionic analytes [16]. If ki,j> 1, then the electrodes are more selective to urea instead of uric acid. This shows that urea can interfere with the performance of carbon paste electrodes/MIP, whereas if ki,j <1, then the electrode is more selective to uric acid rather than the urea.
Determination of precision
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Precision is determined by measuring the potential of the solution with a concentration of uric acid in the range of potentiometric measurement using carbon paste electrodes/MIP, with each concentration measured three times in replication. The determination of precision is done by calculating the standard deviation (standard deviation/SD) and coefficient of variation (CV) using Equations (3) and (4): ∑ ̅
= =
̅
,
(3)
100%,
(4)
where SD is the standard deviation, CV is the coefficient of variation, Xi is the value of each measurement, ̅ is the average value of the measurement results, and n is the number of measurements.
Determination of accuracy Accuracy was determined by measuring the potential of uric acid solution with concentration
in the
range
of potentiometric
measurement using carbon paste
electrodes/MIP. Furthermore, the potential value can be an analogy of the y value substituted into the linear regression equation of the calibration curve, so that we obtain the value of log uric acid concentration for measurement results. The concentration of uric acid in the measuring range was measured in regards to the real concentration. The accuracy value can be calculated by Equation (5): $=
%&' (
100%,
(5)
where R is the recovery or accuracy, Csp is the concentration of uric acid from the analysis result, and KS is the actual concentration of uric acid (which is measured).
Determination of electrode life time 12
The life time of the electrode is determined by measuring the potential of the uric acid solution in the measurement range using carbon paste electrodes/MIP. The measurements were taken at once per week to show deviation slope values of the curve from the allowed Nernst factors limit. The life time of the electrodes is the time span measured because the electrodes are used and perform well up to a significant decline in performance.
Results and Discussion Preparation results of PMMA, NIP, and MIP PMMA acts as a control polymer, which is made by mixing the monomer alone without the addition of templates; NIP is a polymer made by adding a template into the mix, while the MIP is polymer made by adding a template and then extraction of the template, thus leaving mold on the polymer chain. In this study, the monomer used is methyl methacrylate, while the template used is uric acid. The selection of methyl methacrylate monomer is due to the ability of functional groups on the monomer to interact with the functional groups on the template. Methyl methacrylate has functional group C=O, which is assumed to be able to form a hydrogen bond with the functional groups -NH on the uric acid molecule.
Preparation results of PMMA PMMA was prepared by mixing methyl methacrylate, benzoyl peroxide, and ethylene glycol dimethacrylate (EGDMA) in chloroform. The mixture was heated at 60°C, and the heating was stopped when solids are formed. The solids formed are colorless and resemble plastic. Furthermore, the solids are washed using ethanol and put into oven at 60°C. The synthesized solids are crushed and sieved to produce powder. The final result of this preparation of PMMA is white powder. Furthermore, the molecular weight of PMMA is determined using an Oswald viscometer and characterization using FTIR and SEM.
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Preparation results of NIP NIP is prepared by reacting methyl methacrylate as the monomer, EGDMA as the crosslinker, uric acid as the template, and benzoyl peroxide as the initiator with a mole ratio of 1: 5: 0.25: 0.5 [14]. The template, monomer, cross-linker, and initiator were mixed in chloroform. The mixture was heated at 60°C, and heating is stopped when the form is awhite solid. The synthesized white solid is crushed and sieved to produce powder and then washed using mixture of methanol and acetic acid in a ratio of 1:1 (v/v) with each volume of 10 mL. NIP formed into white powder. The powder is further characterized by FTIR and SEM.
Preparation results of MIP The preparation of MIP is basically a continuation of the preparation of NIP. In this study, MIP is obtained by extracting uric acid trapped in the NIP. The solution used to extract is ammonium acetate 1 M (in ethanol, acetic acid, and water with ratio of 40:25:35). Extraction is done by centrifugation at a rotational speed of 5000 rpm for 15 min, performed five times with volume of ammonium acetate is 10 mL. Further washing with water three times removed residual ammonium acetate, which is used to extract uric acid. The extraction process is done to remove uric acid from the series of polymer to form printed polymer of uric acidmolecules. The bond between methyl methacrylate monomer and uric acid is hydrogen bonding. Estimates of hydrogen bonding that occurs between methyl methacrylate and uric acid is shown in Figure 2. The mold expected to form after extraction of uric acid from NIP is shown in Figure 3.
Results of the molecular weight determination of PMMA
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In this study, the molecular weight of the polymer is determined by the viscometry method (done by calculating the viscosity). Based on the curve obtained linear regression equation: y = -0,1334x + 0.2753, the value of the intrinsic viscosity of polymer is equal to the intercept of the regression equation of curve with a relationship between concentration against reduction viscosity. Thus, the obtained intrinsic viscosity value of PMMA synthesis result is 0.2753. The calculation of molecular weight is determined by the Mark-Houwink Sakurada equation, with K and a values for this system at 4.88x10 -6 and 0.82, respectively [17]. Based on this calculation, the molecular weight of PMMA is 622, 835.912 g/mole. The molecular weight of PMMA is obtained in accordance with an average molecular weight of commercial PMMA ranging from 500,000–1,000,000 g/mole.
Characterization using Fourier transform infrared (FTIR) A PMMA, NIP, and MIP synthesis product is characterized using FTIR, which aims to determine differences in functional groups contained in PMMA, NIP, and MIP. The spectral peak wavenumber data of PMMA, NIP, MIP, and uric acid are shown in Table 2, while the spectra of PMMA, NIP, MIP, and uric acid are shown in Figure 5. FTIR results of PMMA, NIP, MIP, and uric acid shows some spectra peaks at wavenumbers, indicating the presence of certain functional groups. Based on Table 2 and Figure 5, PMMA, NIP, MIP, or uric acid has bonds of C=O and C=C in the range of wavenumbers, respectively, from 1760 to 1690 cm-1 and 1500 to 1700 cm-1. The bond NH stretch, which is one of the clusters in uric acid, should only be contained in the NIP and uric acid. But the results of this characterization also are seen the spectra of N-H in the MIP, which is possible because the N-H bond in the ammonium acetate at the time of extraction process has not been completely washed out by distilled water, so MIP powder remains inside. The hydrogen bond is the bond between methyl methacrylate and uric acid contained
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only in the NIP at a wavelength of 2993.62 cm-1. The C-O bond is on polymethyl methacrylate contained in the MIP, NIP, and PMMA.
Characterization using Scanning Electron Microscope (SEM) The characterization results of SEM for PMMA, NIP, and MIP, respectively, are shown in Figures 6 (a), (b), and (c). The three figures show the difference in surface morphology. Based on the assumption, the surface morphology of the NIP is denser than the surface morphology of the MIP. This is because, in the MIP, the analyte has been extracted and is expected to form a mold that forms cavity on the surface of MIP. The characterization results using SEM on the surface of PMMA and MIP can be seen in the presence of cavities while the NIP surface looks denser.
Preparation results of carbon In this research, the preparation of carbon is through the reactivation of the carbon. Preparation of carbon aims to expand and improve the surface conductivity of carbon. First, carbon is soaked in a hydrochloric acid solution (HCl) 4N while stirring with a magnetic stirrer for 24 h. HCl serves as activator because this acid has properties of a strong dehydrating agent, so it can refine the pores in the carbon structure while the immersion process aims to enlargen the carbon pore surface area. Furthermore, carbon is washed with distilled water and filtered using a Buchner funnel until there is no HCl content in the filtrate. The filtrate washery is tested with AgNO3 to determine the presence of HCl in the filtrate. The washing process is done repeatedly until there are no white deposits on the filtrate when tested with an AgNO3 solution, and we obtain neutral pH in the filtrate. Carbon that has been free of HCl is then dried in an oven at 150°C.
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Carbon from drying results is then soaked in n-hexane for 24 h while stirring with a magnetic strirrer. Immersion in n-hexane aims to dissolve the nonpolar organic impurities, which are not soluble in HCl. After immersion, the carbon was dried over a water bath and then heated in a furnace at a temperature of 500°C for 2 h. Carbon from activation results were characterized using BET and BJH to determine the surface area and pore diameters of carbon. The results of characterization showed that the surface area of the carbon is 877.463 m2/g and pore diameter of the carbon is 3.835 nm. According to an experiment by Ariyanto et al. (2012) [18], the pore diameter size of the carbon has potential as an electrode material ranging from 2.1 to 6.5 nm. Thus, the carbon from activation results in this study can be used as the electrode material because it has the potential for high density and good pore accessibility.
Electrode Optimization Before performing potentiometric analysis of uric acid, we required optimization of the carbon paste electrodes/MIP. This optimization is expected to produce an electrode with optimum performance. In this research, we conducted optimization of the composition of the constituent material on the electrode preparation and pH optimization of uric acid solution.
Optimization results of the composition in the preparation of carbon paste electrodes/MIP Carbon paste electrodes/MIP were made from a mixture of MIP, activated carbon, and solid paraffin. MIP is expected to increase the selectivity and sensitivity of carbon paste electrodes. Activated carbon has high conductivity and is inert, so it does not react with the analyte at measurement time. Paraffin is useful to glue the carbon and MIP when inserted into the micro-pipette tube.
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In this study, seven electrodes were made with different compositions in accordance with Table 3. MIP and carbon composition is varied, while the composition of paraffin is fixed. Before being used for measurement, an electrode soaked in uric acid solution 10-3 M is intended for conditioning. The optimum composition of carbon paste electrode/MIP is obtained by measuring the potential of uric acid solution 10-8-10 -2 M. From the measurement results of each electrode, we obtained Nernst factor value, measurement range, and linearity,which are shown in Table 3. The selection of optimum electrode composition is based on the good Nernst factor value, linearity value approaching 1, and a broad range of measurements. According to Cattrall (1997) [16], the potentiometric method meets the Nernst equation if the Nernst factor has a value of 59.2/n (± 1-2 mV), where n is the charge of ionic analytes or valence of molecules. In this study, the measured analyte is uric acid, which is a divalent molecule, so the Nernstnya factor was 59.2 mV/2 or 29.6 mV/decade. Table 3 shows that the electrode that produces the Nernst factor approached 29.6 mV/decade is E7 (the Nernst factor is 23.46 mV/decade). The linearity value of the calibration curve is good if the regression equation’s correlation coefficient (r) approaches 1. In this study, E7 has a fairly good correlation coefficient at 0.9680. Based on the results shwon in Table 3, E1 has better correlation coefficient than E7, which is equal to 0.9946, but E1 has a Nernst factor 14.66, which is far from the theoretical value.
Optimization results of pH Optimization pH of the uric acid solution is done to determine the range of pH that can producestable potential value. Determination ofoptimumpH is done by measuring the potential of uric acid solution 10 -8-10 -3 M with a pH range of 4–8 using E7 and E1 as
18
reference. The measurement results of each electrode obtained Nernst factor value, measurement range, and linearity are shown in Table4. The data in Table 4 shows that the pH has an effect on potential measurement of uric acid solution. Measurements using E1 and E7 show thatthe best Nernst factor is at pH 5 compared with Nernst factors values at the otherpH. But, for E7, the Nernst factor value is better than the Nernst factor value of E1. In this study, pH 5 is then used for analysis of uric acid using carbon paste electrodes/MIP.
Standard curve of uric acid The standard curve of uric acid was created by measuring the potential of uric acid solution 10-8-10-3 M at optimum pH by using E7. From the obtained data, we can create a curve with the relationship between log concentration of uric acid against the measured potential. The standard curve includes data that givesstraight line in the concentration range measured by the Nernst factor value approaching 29.6 mV/decade and the linearity approaching 1. Potential measurement data are shown in Table 5; the curve with the relationship between the log concentration of uric acid against the measured potential of uric acid solution is shown in Figure 7; and the standard curve of uric acid is shown in Figure 8.
Test results of electrode performance and validity of analysis method Determination results of electrode response time The electrode response time measurement aims to determine the sensitivity of an electrode to respond to the analyte. An electrode is said to be sensitive if the time it takes to respond to analytes is faster [19]. In this study, the measurement of electrode response time is done by measuring the potential of uric acid solution 10-6-10 -3 M and calculated the time
19
needed to produce constant potential at each concentration. The measurement of electrode response time data to uric acid solution 10-6-10 -3 M using E7 is shown in Table 6. The data in Table 6 shows that the higher concentration of uric acid can speed up the response time of the electrode. In theory,the higher concentration of the analyte then the amount of analyte molecule in solution are also increasing, so that it will quickly attach to the surface of the electrode. In this study, it can be said that the carbon paste electrodes/MIP (E7) has fairly high sensitivity.
Determination results of measurement range The measurement range is the range of concentrations that can still be measured properly and gives straight line. The measurement range is obtained by measuring the potential of uric acid solution 10 -8-10 -3 M and creating a curve of that shows the relationship between the log concentration of uric acid against the potential; then we conducted data collection that gives straight line. Based on the standard curve of uric acid in Figure 8, the obtained measurement range is the concentration of uric acid solution 10-6-10 -3 M with a Nernst factor value of 30.19 mV/decade and linearity of 0.9812.
Determination results of Nernst factor The Nernst factor is the slope of curve on the standard curve of uric acid. In this study, we obtained the standard curve of uric acid from linear regression equation y = 30.19x + 230.13. Thus, in this study, the Nernst factor was 30.19 mV/decade.
Determination results ofdetection limit The limit of detection was determined by establishing the intersection between the linear line and nonlinear lines of the curve with relationship between log concentration of uric acid
20
against the potential that can be seen in Figure 9. In this study,we determine that the detection limit is the minimum detection limit (the smallest concentration of analyte that can still be measured). The smaller concentration of the detection limit indicates tha tthe method is more sensitive. In this study, the obtained detection limits amounted to 3.03x10-6 M. Based on the obtained detection limits with normal levels of uric acid in the blood, the carbon paste electrodes/MIP (E7) can be used for analysis of uric acid in the blood sample (10-4 M) because it has a detection limit for the analysis of uric acid at a concentration of 3.03x10-6 M.
Determination results ofselectivity coefficient The determination of selectivity aims to determine the ability of methods in measuring compounds or components that were carefully analyzed in the presence of other components that may exist together in the sample. In this study, the selectivity test is done through a mixing method that is with the addition of urea solution in the uric acid solution because both components are located together in the blood. The urea solution is added to a uric acid solution to a final concentration of uric acid 10 -4M, while the final concentration of urea solution is 10 -3 M, 10 -4 M, and 10 -5 M. The selection of uric acid concentration is 10-4 M because of uric acid in the blood at these concentrations, whereas urea are selected at concentrations below normal, normal, and above normal in the sample. Our solution is measured for potential using E7 and E1 as a reference. The calculation of selectivity coefficient (Ki,j) is performed by inserting the potential value into the equation. The calculation result data of Ki,j for the uric acid solution concentration of 10-4 M with the addition of urea solution can be seen in Table 7. Data in Table 7 show that measurements using E1 or E7 have a value of Ki,j <1; the higher concentration of urea is added, and then the selectivity coefficient value becomes
21
higher. In the calculation of Ki,j if the obtained value of Ki,j <1, it can be said that the used methods include more selectively against the analyte. It can be concluded that the measurement of carbon paste electrodes/MIP (E7) is more selective against uric acid and is not interfered by urea.
Determination results of precision Precision is expressed as the amount of conformity or deviation of any reported measurement results. In this study, the precision is determined by measuring the potential of uric acid solution 10 -6-10-3 M using E7. Potential measurements at each concentration are done with three times replication. To measure the precisions it is first necessary to calculate the standard deviation (SD) and then calculate the coefficient of variation (CV). The calculation precision data of uric acid solution 10 -6-10-3 M using E7 are shown in Table 8. According to Taverniers et al. (2004) [20], at concentration of 10 -6 M and 10 -5 M, the acceptable coefficient of variation is 22.6% and 16%. While the coefficient of variation value for the concentration of 10-4 M and 10 -3 M is 11.3% and 8%. Thus, it can be said that, with the higher concentration, the coefficient of variation become smaller. The data in Table 8 concludes that the developed method has high precision.
Determination results of accuracy Accuracy is the ratio of the measured concentration with the actual concentration. In this study, the accuracy is calculated via the concentration of uric acid in the measurement range of 10 -6-10 -3 M. The potential value of the measurement results of each concentration incorporated into the linear regression equation of the standard curve is used to obtain the concentration of uric acid solution. Furthermore, accuracy is determined by comparing the concentration of uric acid solution from the calculation results with the actual concentration
22
of uric acid. The calculation accuracy data of uric acid solution 10-6-10 -3 M using E7 are shown in Table 9. The average accuracy value that is acceptable for the concentration range of 10 -6-10-4 M is 80%-110%, while for concentration of 10-3 M, the averageaccuracy value that is acceptable is 90%-107% [20]. The data in Table 9 conclude that the measured concentrations in this study did not meet the required range of accuracyvalue. Thus, the accuracy of the research can be said to be less than ideal. This is possible because the electrode is in a saturated state with uric acid solution; thus, the measurement is not optimal, and the obtained accuracy results are less than ideal.
Determination results ofelectrode life time The life time is a condition where, at certain times, the electrode’s ability to analyze analytes is still quite good. In this study, the electrode life time is determined by counting the number of times the electrode can be used for analysis with good performance, where one measurement is counted as one usage. In the span of particular use, the electrode is used to measure the potential of the uric acid solution in the measurement range; then, we determine the Nernst factor. The results of determination of the electrode life timecan be seen in Table 10. Table 10 shows that, over the usage of 104 times, the electrodes still give good Nernst factor value. The usage of 116 times shows that the obtained Nernst factor value is lower when compared with the theoretical value; thus, it can be said that the performance of the electrode has decreased. Instability measurement can occur due to shifting the equilibrium, thereby reducing the level of selectivity and response of an electrode [21].
Comparison of electrodes performance test
23
In this research, we conducted the comparison of performance test of electrode modified PMMA, NIP, and MIP. This test is performed to determine the effect of uric acid mold in the MIP. The preparation of electrode-modified polymer and electrode-modified NIP in accordance with the optimum composition of previous electrode is EMIP (E7) with composition carbon 0.120 g, paraffin 0.105 g, and polymer (MIP or NIP or PMMA) 0.075 g. Furthermore, the three electrodes are used to measure the potential of uric acid solution 10 6
-10-3 M, which were conditioned at pH 5. The measurement results of each electrode-
obtained Nernst factor value, measurement range, and linearity can be seen in Table 11 and the curve profile in Figure 10. Table 11 shows that the Nernst factor value of E7 is better than ENIP and EPMMA. This is because E7 has the specific binding sides to the uric acid molecules. However, if viewed from the linearity value, which is expressed as correlation coefficient (r), E7 has a correlation coefficient that is quite good: 0.9745. The results in Table 10 show that EPMMA has a better correlation coefficient than E7, which is equal to 0.9907, but EPMMA has a Nernst factor that is far away from the theoretical value of 15.20 mV/decade.
Comparison of potentiometric method using carbon paste electrodes/MIP with the methods used in the previous study for analysis of uric acid The results of this study include the validity of the method and the performance of carbon paste electrode/MIP by potentiometrycompared with the methods used in previous studies such as analysis of uric acid using the HPLC method [5], analysis of uric acid using the glassy carbonelectrode/MIP by stripping voltammetry [22], and analysis of uric acidusing carbon
paste
electrodes/MIP
with
methacrylic
acid
monomer
by potentiometry
[11]. Comparison results of validity of the some methods are presented in Table 12.
24
Table 12 shows that the potentiometric method with different components of MIP have advantages in the limit of detection and life time of the electrode. Measurements using carbon paste electrodes/polymethyl methacrylate resulted in a lower limit of detection than measurements using carbon paste electrodes/polymethacrylic acid. The life time of carbon paste electrodes/polymethyl methacrylate is eight weeks (the usage is 104 times) longer than the life time of carbon paste electrodes/poly methacrylic acid, which is six weeks (the usage is 56 times). When compared with other methods, it can be seen that the potentiometric method has limit of detection higher than HPLC and voltammetry methods. But the potentiometric method has a wider measurement range than HPLC and the voltammetry methods.
Conclusion The optimum composition of carbon, MIP, and paraffin, which gives optimum performance, carbon paste electrodes/MIP was at ratio of 40:25:35 (% w/w) in a uric acid solution with pH 5. Measurement of uric acid with carbon paste electrodes/MIP produced a Nernst factor of 30.19 mV/decade in the measurement range of concentration 10-6-10-3 M witha correlation coefficient (r) from calibration curve 0.9812, minimum detection limit 3.03.10 -6 M, accuracy and coefficient of variation (CV) of uric acid with concentration 10 -6-10-3 M ranged from 63.9 to 166% and 1.36 to 2.03%. The selectivity coefficient value was less than one, indicating that urea did not interfere with the results of the uric acid analysis. The response time of electrodes toward the uric acid with a concentration of 10-6-10 -3 M was 32-60 seconds, and the electrode’s life time was eight weeks (the usage is 104 times).
References
25
1.
Brett, C.M.A, Oliveira-Brett, A.M., 2011, Electrochemical sensing in solution-origins, applications and future perspectives, Journal of Solid State Electrochemistry, 15, 14871494
2.
Puig, J.G., Ruilope, L.M., 1999, Uric acid as a cardiovascular risk factor in arterial hypertension, Journal of Hypertension, 17(7), 869-872
3.
Ren, W., Luo, H.Q., Li, N.B., 2006, Simultaneous Voltammetric Measurement of Ascorbic Acid, Epinephrine and Uric Acid at a Glassy Carbon Electrode Modified with Caffeic Acid, Biosensors and Bioelectronics, 21(7), 1086-1092
4.
Chen, J.C., Chung, H.H., Hsu, C.T., Tsai, D.M., Kumar, A.S., Zen, J.M., 2005, A disposable single-use electrochemical sensor for the detection of uric acid in human whole blood, Sensors and Actuators B-Chemical, 110(2), 364-369
5.
George, S.K., Dipu, M.T., Mehra, U.R., Singh, P., Verma, A.K., Ramgaokar, J.S., 2006, Improved HPLC method for the simultaneous determination of allantoin, uric acid, and creatinin in cattle urine, Journal of Chromatography B, 832(1), 134-137
6.
Ermawati, A.D., 2013, Analysis ofuric acid in serum by voltammetry using gold electrode (in Bahasa Indonesia), Thesis, Faculty of Science and Technology, Airlangga University
7.
Handaru, A.Z.P., 2010, Analysis ofuric acid in serum bystripping voltammetry using graphite electrode (in Bahasa Indonesia), Thesis, Faculty of Science and Technology, Airlangga University
8.
Premkumar, J., Khoo, S.B., 2005, Electrocatalytic oxidations of biological molecules (ascorbic acid and uric acids) at highly oxidized electrodes, Journal of Electroanalytical Chemistry, 576(1), 105-112
26
9.
Khasanah, M., Mudasir, Kuncaka, A., Sugiharto, E., 2012, Development of uric acid sensor based on molecularly imprinted polymethacrylic acid-modified hanging mercury drop electrode, Journal of Chemistry and Chemical Engineering, 6, 209-214
10. Sa’ada, A.C., 2015, Analysis of uric acid using electrodes carbon paste-imprinted zeolite by potentiometriy (in Bahasa Indonesia), Thesis, Faculty of Science and Technology, Airlangga University 11. Andayani, S.N., 2014, The development of electrodes carbon paste nanoporous/MIP as uric acid sensor by potentiometry (in Bahasa Indonesia), Thesis, Faculty of Science and Technology, Airlangga University 12. 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 13. 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 14. Wijayani, F., Supriyanto, S., Suyanto, 2015, The characterization of molecularly imprinted polymer (MIP) from precipitation polymerization results as adsorbent of chloramphenicol (in Bahasa Indonesia), Journal of Mathematics and Natural Sciences, 17(2), 1-10 15. Purwanto, A., Ernawati, F., Sajima, 2011, Characterization of cadmium ion selective electrode and Cd test in zirconium (in Bahasa Indonesia), Proceeding, Research and Processing of Nuclear Devices, Yogyakarta, 249-257 16. Cattrall, R.W., 1997, Chemical Sensors, Vol 1, Oxford University Press, New York 17. Brandrup, J., Immergut, E.H., 1989, Polymer Handbook, 3rd Edition, John Willey and Sons, New York
27
18. 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 19. Gea, S., Andriyani, Lenny, S., 2005, Preparation of selective electrode for ion Cu(II) from chitosan polyethylene oxide (in Bahasa Indonesia), Thesis, Universitas Sumatera Utara 20. 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 21. Bakker, E., Buhlmann, P., Pretsch, E., 1997, Carrier-based ion-selective electrode and bulk optodes. 1. General characteristics, Chemical Reviews, 97(8), 3083-3132 22. Arwindah, P.R., 2010, Development of uric acid sensor using glassy carbon electrode modified with molecularly imprinted polymer and anilin as monomer by stripping voltammetry (in Bahasa Indonesia), Thesis, Faculty of Science and Technology, Airlangga University
Competing interests The authors declare no competing financial interests.
28
Table 1. Composition of carbon, MIP, and solid paraffin in the preparation of carbon paste electrodes/MIP Mass (g)
Mass ratio of carbon,
Electrode Carbon
MIP
Solid paraffin
MIP, and solid paraffin
E1
0.195
0
0.105
65 : 0 : 35
E2
0.180
0.015
0.105
60 : 5 : 35
E3
0.174
0.021
0.105
58 : 7 : 35
E4
0.165
0.030
0.105
55 : 10 : 35
E5
0.150
0.045
0.105
50 : 15 : 35
E6
0.135
0.060
0.105
45 : 20 : 35
E7
0.120
0.075
0.105
40 : 25 : 35
Table 2. The spectral peak wave number data of PMMA, NIP, MIP and uric acid
29
No 1
Functional
Range of wave Wave number (cm-1)
groups
numbers (cm-1)
C
PMMA
NIP
MIP
Uric acid
= 1800-1690
1730.21 1726.35
1718.63 1677.95
= 1500-1700
1635.69 1639.55
1643.41 1438.8
O stretch 2
C C stretch
3
NH stretch
3000-3500
3446.91 3433.41
3448.84 3080.11
4
NH bend
1650-1580
-
1450.52
-
1591.16
5
OH 3000-2500
-
2993.62
-
-
1300-1000
1172.76 1151.54
hydrogen bonds 6
CO stretch
1139.97 -
Table 3. The Nernst factor value, measurement range and linearity from the measurement results of uric acid solution using carbon paste electrodes/MIP with variety of compositions Mass (%, by weight) Electrode
Measurement
Carbon MIP Paraffin range (M)
Nernst factor (mV/decade)
Linearity (r)
E1
65
0
35
10-6 - 10-3
14.66
0.9946
E2
60
5
35
10-6 - 10-3
14.18
0.9932
E3
58
7
35
10-6 - 10-3
13.90
0.9682
E4
55
10
35
10-6 - 10-3
14.35
0.9774
E5
50
15
35
10-6 - 10-3
15,16
0.9853
10-6 - 10-2
18.94
0.9975
10-6 - 10-3
19.33
0.9960
E6
45
20
35
30
E7
40
25
35
10-6 - 10-3
23.46
0.9680
Table 4. The Nernst factor value, measurement range and linearity form the measurement results of uric acid solution using E1 and E7 Measurement
Nernst factor
Linearity
range (M)
(mV/decade)
(r)
4
10-6 - 10-3
14.31
0.9862
5
10-6 - 10-3
20.05
0.9471
6
10-6 - 10-3
12,90
0.9911
7
10-6 - 10-3
12.29
0.9771
8
10-6 - 10-3
15.37
0.9803
4
10-6 - 10-3
19.42
0.9829
5
10-6 - 10-3
27.02
0.9745
10-7 - 10-4
20.51
0.9992
10-6 - 10-3
14.16
0.8616
10-6 - 10-3
18.44
0.9970
10-6 - 10-4
23.80
0.9637
10-6 - 10-3
17.40
0.8942
Electrode pH E1
E7
6 7 8
Table 5. The result of potential measurement of uric acid solution 10-8 - 10-3 M using E7 Uric acid concentration (M)
Potential (mV)
10-8
62.2
10-7
65.9
10-6
50.7
10-5
73.3
10-4
116.0
10-3
137.1
Table 6. The measurement of electrode response time data to uric acid solution using E7 31
Uric acid concentration (M)
Time (sec)
10-6
60
10-5
52
10-4
46
10-3
32
Table 7. The data of Ki,j for the uric acid solution with concentration of 10 -4 M with the addition of urea solution Concentration of E1 urea
E7
solution Potential (mV)
Ki,j
Potential (mV)
Ki,j
(M) 0
527
0
540
0
10 -5
533
0.30
538
-0.15
10 -4
543
0.45
552
0.17
10 -3
557
0.66
578
0.22
Table 8. The calculation precision data of uric acid solution 10 -6 - 10 -3 M using E7 Concentration Potential (mV)
Standard Coefficient
of uric acid Replication- Replication- Replication- deviation of variation solution (M)
1
2
3
(SD)
(CV) (%)
10 -6
57.6
58.7
56.8
0.95
1.65
10 -5
75.0
76.9
76.9
1.09
1.44
10 -4
113.8
117.0
118.4
2.36
2.03
10 -3
134.9
138.1
134.9
1.85
1.36
Table 9. The calculation accuracy data of uric acid solution 10-6 - 10 -3 M using E7 Actual
uric
acid Calculation uric acid Accuracy
32
concentration (M)
concentration (M)
(%)
10-6
1.14x10 -6
114.0
10-5
6.39x10 -6
63.9
10-4
1.66x10 -4
166.0
10-3
8.29x10 -4
82.9
Table 10. The results of determination the electrode life time Measurement
Usage
range Nernst factor
(M)
(mV/decade)
50
10-6 - 10-3
27.02
74
10-6 - 10-3
30.19
78
10-6 - 10-3
27.07
82
10-6 - 10-3
27.83
86
10-6 - 10-3
27.58
90
10-6 - 10-3
28.57
96
10-6 - 10-3
29.30
100
10-6 - 10-3
27.40
104
10-6 - 10-3
27.80
116
10-6 - 10-3
19.9
120
10-6 - 10-3
17.3
124
10-6 - 10-3
15.4
Table 11. The Nernst factor values, measurement range and linearity from measurement results of uric acid solution using electrode modified MIP, NIP and PMMA Polymer
Electrodes
Carbon (%)
(MIP or NIP or PMMA)
Paraffin Measurement (% b)
range (M)
(%)
33
Nernst factor (mV/decade)
Linearity (r)
EMIP (E7)
40
25
35
10 -6 - 10 -3
27.02
0.9745
ENIP
40
25
35
10 -6 - 10 -3
20.60
0.9846
EPMMA
40
25
35
10 -6 - 10 -3
15.20
0.9907
Table 12. The comparison results of validity of the some methods to analysis of uric acid Potentiometry
Parameter
HPLC [5]
Voltammetry [22]
Measurement 1.19x10-6 – 5.95x10-9
–
Carbon
paste Carbon
electrodes/poly
electrodes/polymethyl
methacrylic
methacrylate
acid11
study)
10 -5 - 10 -2 M
10-6 - 10 -3 M
range
8.92x10-5 M
2.97x10-8 M
Linearity
-
0.9979
0.9999
0.9812
6.5x10 -7 M
1.8x10 -9 M
1.35x10-5 M
3.03x10-6 M
Accuracy
96-109%
84.75%
96.95-102.35%
63.9-166%
Precision
6.17-6.25%
0.49-7.83%
2.01-14.52%
1.36-2.03%
Limit detection
of
paste
(this
more selective Selectivity
-
against acid
uric rather
than ascorbic
more
selective more selective against
against uric acid uric acid rather than rather than urea
urea
acid Life time
-
six
-
weeks
(the eight weeks (the usage
usage is 56 times)
34
is 104 times)
Figure Captions Figure 1. Construction of carbon paste electrodes/MIP Figure 2. The estimation of inter-molecular hydrogen bonding that occurs between methyl methacrylate and uric acid Figure 3. The estimation of the mold that formed after the extraction of the uric acid Figure 4. The curve with relationship between concentration of PMMA solution against reduction viscosity of PMMA Figure 5. FTIR spectra of PMMA, NIP, MIP, and uric acid Figure 6. The characterization results of SEM (a) PMMA, (b) NIP, (c) MIP with magnification of 4000 times Figure 7. The curve with relationship between log concentration of uric acid with potential
35
Figure 8. The standard curve of uric acid Figure 9. The intersection of the linear line and non-linear in the curve with relationship between log concentration of uric acid against potential Figure 10. The curve with relationship between log concentration of uric acid against potential from measurements results using EMIP, ENIP and EPMMA
Figure 1. Construction of carbon paste electrodes/MIP
36
Figure 2. The estimation of inter-molecular hydrogen bonding that occurs between methyl methacrylate and uric acid
O CH2
O
CH2
O
O O
C
CH2
O
OCH3
O H2C
CH
CH3
CH2
CH 2
H2C O
OCH3
CH2
O O
OCH3
C
C
CH
O
O
O
O
CH2
H3CO
CH3 HC 2
OCH3
H3CO
CH2
O
HC 2
CH2
O O
O
2
C H2
H2 C
O
C
CH
O
CH3
H2 C
CH2
O
H C
C
CH3
O
Figure 3. The estimation of the mold that formed after the extraction of the uric acid
37
0.3 0.25
η red
0.2 0.15
y = -0,1334x + 0,2753 R² = 0,9188
0.1 0.05 0 0
0.2
0.4
0.6
0.8
1
1.2
C (g/L) Figure 4. The curve with relationship between concentration of PMMA solution against reduction viscosity of PMMA
Figure 5. FTIR spectra of PMMA, NIP, MIP, and uric acid
38
(a)
(b)
(c) Figure 6. The characterization results of SEM (a) PMMA, (b) NIP, (c) MIP with magnification of 4000 times
160 140
Potential (mV)
120 100 80 60 40 20 0
-9
-8
-7
-6
-5
-4
-3
-2
-1
0
Log concentration of uric acid
Figure 7. The curve with relationship between log concentration of uric acid with potential
39
160 140
Potential (mV)
120 100 80 60 40 20 0
-7
-6
-5
-4
-3
-2
-1
0
Log concentration of uric acid Figure 8. The standard curve of uric acid
160
Potential (mV)
140 120
y = 4.725x2 + 63.235x + 268.52
y = 30.19x + 230.13 100 80 60 40 20 0
-10
-8
-6
-4
-2
0
Log concentration of uric acid Figure 9. The intersection of the linear line and non-linear in the curve with relationship between log concentration of uric acid against potential
40
yEMIP (E7)
700
yENIP
600
yEPMMA
500
Potential (mV)
400 300 200 100 0 -7
-6
-5
-4
-3
-2
-1
0
Log concentration of uric acid Figure 10. The curve with relationship between log concentration of uric acid against potential from measurements results using EMIP, ENIP and EPMMA
41