Article                                                                                                                   ...
Analytical Chemistry                                                                                                      ...
Analytical Chemistry                                                                                                      ...
Analytical Chemistry                                                                                                      ...
Analytical Chemistry                                                                                                      ...
Analytical Chemistry                                                                                                      ...
Analytical Chemistry■                                                                                                     ...
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Quasi real time quantification of uric acid in urine using borondoped diamond microelectrode with in situ cleaning


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Quasi real time quantification of uric acid in urine using borondoped diamond microelectrode with in situ cleaning

  1. 1. Article Time Quantification of Uric Acid in Urine Using BoronDoped Diamond Microelectrode with in Situ CleaningRaphael Kiran,*,† Emmanuel Scorsone,† Pascal Mailley,‡ and Philippe Bergonzo††CEA-LIST, Diamond Sensors Laboratory, 91191 Gif-sur-Yvette, France‡CEA-INES, Laboratoire de Stockage de l’Electricité, 73377, Le Bourget du Lac, France * Supporting Information S ABSTRACT: We report herein an innovative electrochemical (EC) technique based on boron doped diamond (BDD) microelectrodes which enable the fast determination of uric acid (UA) concentrations in urine. On the basis of fast cyclic voltammetry (CV), the technique was assessed in human urine samples and compared successfully using routine spectrophotometric diagnosis. The approach relies on the use of BDD’s superior properties such as low background current, low adsorption of species, long-term stability, and antifouling capabilities using electrochemical reactivation. Moreover, the article also describes an in situ activation technique, where the electrodes were reactivated within human urine, thereby opening the way toward automatic quantification of UA with in situ cleaning. The time taken to quantify UA concentration and cleaning remains below 0.5 s. Two analytic models were derived, based on different concentrations of ascorbic acid (AA) and uric acid, consisting of 2 s order calibration curves. Solving the second order equation enables the direct estimation of UA concentration, and values demonstrated good accuracy when compared with spectrophotometric measurements.U ric acid (UA) is the principal breakdown product of purine metabolism,1 and the normal UA concentration inhuman urine is around 2 mM.2 Hyperuricemia, associated with without any electrode modification.18 However the analyte must be diluted to prevent the surface from fouling, using this technique. We report on a simple electrochemical method forrenal disease, can cause gout,3 cardiovascular disease,4 kidney selective quantification of UA using boron doped diamondstones,5 etc., whereas hypouricemia can be due to Fanconi (BDD) microelectrode, without any special modification or usesyndrome,6 nephritis,7 and other kidney disorders. Hence it is of any specific reagents or analyte dilution. The technique canimportant to monitor UA level in bodily fluids such as blood be performed directly in urine and is combined with an in situand urine. Nowadays the determination of uric acid in urine is cleaning/activation technique which brings back the reactivityperformed in medical laboratories using mainly spectrophoto- of the electrode that has been lost due to fouling. Thus it opensmetric analysis methods. However there is also an interest for the possibility to automate the urine UA monitoring techniquecontinuous monitoring of UA in urine in particular for patients with minimal power consumption especially when compared toadmitted in intensive care units (ICU), where the early in situ cleaning by sonoelectrodes.diagnostic of acute renal failure (ARF), observed in up to 25% The BDD electrode possesses unique electrochemicalof the patients admitted in ICUs, can have a major impact on properties viz. low capacitive background current, widethe survival rate of those patients.8 In this context, electro- potential window in aqueous media, poor adsorption of polarchemical (EC) detection techniques are seen as a promising molecules, and corrosion resistance in harsh environmentsalternative to conventional optical methods due to their good making them highly promising as an electrochemical sensorsensitivity, fast measuring time, portability, low power when compared to other conventional electrode materials.19−21consumption, and cost effectiveness, thus enabling direct Bioinertness and long-term stability of BDD material makesbedside monitoring. Various electrochemical approaches such them ideal candidates for biomedical applications.22 Because ofas polymer modified electrode,9−11 chemically modified its carbon nature, diamond opens the way to immobilization ofelectrode,12−15 enzyme modified electrode,16 and electro- functional groups such as DNA, proteins, enzymes, etc. ontochemical pretreatment17 were developed to detect UA. the electrode surface,23 thereby bringing high selectivity toHowever UA coexists with ascorbic acid (AA) in biological sensing process. Popa et al. has achieved UA and AA peakfluids and has got nearly the same oxidation potential.10 separation by anodizing the diamond surface.2 However, at highAlthough modified electrodes show good selectivity to UA, pH values the peak separation was diminished thus making UAcomplications like adsorption, fouling, etc. are associated withthose techniques.13 Simultaneous determination of dopamine, Received: May 3, 2012AA, and UA were also investigated using fast cyclic Accepted: November 5, 2012voltammetry (CV) and differential pulse voltammetry (DPV) Published: November 5, 2012 © 2012 American Chemical Society 10207 | Anal. Chem. 2012, 84, 10207−10213
  2. 2. Analytical Chemistry Articlequantification difficult in the presence of AA. Moreover, the quantification directly in urine without dilution. Otheranalyte has to be diluted by several thousand folds to obtain approaches based on high-performance liquid chromatography,reliable results. Fast CV demonstrates the production of a very solid phase extraction, or permselect membranes techniquesreducible electro-active species that has been produced as a require sample preparation further to result in the contami-result of oxidation of UA.24 nation of the detection module (membrane). Here our The typical CV response of UA shows one broad irreversible innovative detection technique directly enables UA quantifica-peak at slow potential sweeping (down to 1 V s−1) on classical tion without particular sample preparation and thus could bemacro-electrodes. By using fast CV (1 V s−1), Dryhurst implemented easily in automatic systems, e.g., for home patientdemonstrated the existence of a weak reduction peak resulting monitoring. ■from UA oxidation.24 Indeed, the oxidation of C4C5 bond ofUA gives readily reducible bis-imine (on C4 atom) that may EXPERIMENTAL SECTIONundergo further irreversible chemical hydration reaction if notquickly electrochemically reduced (Figure 1). Conversely, AA BDD BioMEMS Fabrication. A novel technological processelectrochemical oxidation is known to be highly irreversible.25 was developed for the fabrication of diamond microelectrodesHence fast CV may be used to selectively determine the which involves the selective growth of diamond over siliconconcentration of UA in the presence of AA. substrates, followed by the deposition of metal contacts and passivation layers. This process includes initially the fixing of detonation diamond nanoparticles over a preoxidized 4 in. silicon wafer using a protocol described elsewhere.27 Then an aluminum hard mask consisting of a disk of 100 μm in diameter was selectively deposited, over the areas where the BDD microelectrodes have to be grown. Photolithography enabled to partially pattern the Al mask thus used to protect areas where diamond nanoparticles were protected from being etched in a reactive ion etching (RIE) process in oxygen. The Al hard mask was finally removed and the diamond electrode was grown. The dimensions of the resulting diamond disk electrodes were typically of 300 nm in thickness and 100 μm in diameter. Then an assembly of Ti (50 nm)/Pt (150 nm) metal tracks was deposited over the substrate with a metal ring going around the edge of the diamond disk in order to take electrical contact from the diamond electrode. Then a silicon nitride (Si3N4) passivation layer was deposited by CVD over the substrate. Finally an opening of 40 μm was made over the diamond electrodes by using local etching of the Si3N4 layer by RIE with SF6 gas. Apparatus, Chemicals and EC Measurement. An Autolab PGSTAT 302 potentiostat was used to perform all the EC characterizations and experiments. The prepared microelectrodes were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). AFigure 1. Proposed schematics for UA oxidation in the presence/absence of AA where (I) is UA, (II) bis-imine compound, (III) imine- three electrode setup consisting of the BDD microelectrode asalcohol compound, and (IV) uric acid-4,5-diol. the working electrode, a platinum wire mesh as the counter electrode, and an Ag/AgCl (3 M KCl) reference electrode was employed for the CV experiments. The Ag/AgCl electrode was The present study reports on the fabrication and character- replaced by a Pt pseudoreference for the EIS experiment. EISization of the BDD microelectrode sensors for the selective and was recorded over a frequency range of 50 kHz−0.1 Hz withsensitive detection of UA in the presence of low and high logarithmic point spacing and potential amplitude of 0.01 Vquantities of AA. Since the technique used for quantification is rms, while the BDD electrode was maintained at an openfast CV, the low double layer capacitance of diamond reduces circuit potential. The electron transfer rate k0 was determinedthe background current and increases the signal to background from the Nyquist plot fit using the ZSimWin 3.21 software.ratio. Microelectrodes show a decreased ohmic drop, hemi- Ultrapure deionized (DI) water (Millipore Direct Q3) was usedspherical diffusion layer, and fast establishment of a steady-state to prepare all the solutions. EIS was performed in an aqueoussignal when compared to macro-electrodes.26 Moreover, the solution containing 0.5 M potassium chloride (Acrosdouble layer capacitance and the volume required to analyze is Organics), 1 mM of potassium ferricyanide(III) and offurther decreased by microstructuring such an electrode. The potassium hexacyanoferrate(II)·trihydrate (both from Acroslong-term goal of our work is to develop BDD bioMEMS Organics). The 0.5 M lithium perchlorate (Sigma Aldrich)platforms for quantifying different constituents in urine that aqueous solution was used as the electrolyte for thecould be directly incorporated in bedside monitoring devices. measurement of the potential window. The electrodes were“As grown” BDD electrodes were used for the quantification rinsed in DI water and dried under a flow of argon gas prior totaking advantage of diamond’s high electrochemical reactivity. each experiment. The steady state limiting current plateau wasWhen compared to other electrochemical detection techniques, observed in a 0.5 M potassium chloride aqueous solutionno groups have yet reported the possibility to perform UA containing 1 mM Fe(CN)64− ion while the electrodes were 10208 | Anal. Chem. 2012, 84, 10207−10213
  3. 3. Analytical Chemistry Articlescanned at 100 mV s−1 from 0.05 to 0.45 V vs Ag/AgCl. Theelectrode electron transfer rate k0 is defined as k 0 = RT /n2F 2SRTC0 (1)where R is the universal gas constant, T the absolutetemperature (K), S the surface area of the electrode (cm2), FFaraday’s constant (96 500 C mol−1), RT the electron transferresistance of the electrode (ohm), C0 the concentration ofredox couple (mol cm−3), and n the number of electronstransferred. The limiting current ilim recorded at the BDDelectrode is given by the following equation: ilim = 4nrFDC0 (2)where r the radius of the electrode, D the diffusion coefficientof Fe(CN)64− (6.67 × 10−6 cm2 s−1),28 and C the bulkconcentration of the species. Unless stated otherwise, thepotential is given versus an Ag/AgCl reference electrodethroughout the paper. UA and AA solutions were prepared daily by dissolving in aphosphate buffer saline (PBS) solution of pH 7.2 (all threechemicals are from Sigma Aldrich). Oxidation and reductionpeak were obtained from fast CV (scan rate = 20 V s−1) fordifferent concentration of UA and AA. The electrode wasscanned from −0.3 to 1.4 V vs Ag/AgCl. Calibration curves ofUA were plotted from the obtained value. Urine samples werecollected from volunteers and were used without anypretreatment or dilution.■ RESULTS AND DISCUSSION EC Characterization of BDD Film. CV in LiClO4 solutionhas demonstrated that the accessible potential window of the Figure 2. Cyclic voltammograms of uric acid (dashed line) andBDD film is about 3.4 V with a background current of 30 pA at ascorbic acid in phosphate buffer solution scanned at (a) 0.1 V s−10.2 V s−1 and a steady state limiting current ilim of 4.7 nA in a indicating overlapping oxidation potential and at (b) 20 V s−1.0.5 M potassium chloride aqueous solution containing 1 mM Although peak A is observed for both uric acid and ascorbic acid, peakFe(CN)64− ion. The EIS results demonstrated that the B is observed only for uric acid reduction.electrodes exhibit a very fast electron transfer rate (k0) up to0.02 cm s−1. The potential window corresponds to that of primary oxidized products IIa or IIb can be electrochemicallydiamond and the fast electron transfer rate, and the low reduced owing to the reversibility of the first oxidation step duebackground current demonstrates that neither cracks nor to the instability of the imine compound. At slower scan rates,pinholes were detected in the passivation layer. oxidation products of UA are rapidly hydrated to irreversible CV of AA and UA. Figure 2a shows the CV of AA and UA uric acid-4,5-diol. Conversely to UA and as expected, norecorded at low scanning rate (0.1 V s−1) under steady state reduction wave of the electrochemically produced dehydroas-hemispherical diffusion in PBS buffer. Both AA and UA exhibit corbic acid (DHAA) was observed in the scanned potentialwell-defined oxidation waves with half-wave potentials around window (Figure 2b), even at the highest investigated scanning0.5 V vs Ag/AgCl that correspond to the irreversible exchange rate of 20 V s−1. However, DHAA is known to strongly adsorbof two electrons and two protons.24,25 These CVs clearly show at electrochemical interfaces and may induce diamond foulingthe overlapping of the electrochemical oxidation waves of both along UA determination. Hence, according to the aforemen-species at the same potential range. More precisely, the EC tioned electrochemical behavior, fast CV can be used tooxidation of UA gives, through a reversible two electron-two selectively determine the concentration of UA.proton exchange, an unstable bis-imine that could exist in two Elsewhere, one can distinguish from Figure 2a for UA, thetautomeric forms (species IIa and IIb, Figure 1).29 This bis- existence of a second flat wave (oxidation half-wave potential ofimine compound is then decomposed, at the used experimental 0.8 V vs Ag/AgCl). Indeed, uric acid-4,5-diol undergoespH of 7.2, into uric acid-4,5-diol (species IV, Figure 1), through subsequent chemical rearrangement at pH 7 leading to thetwo fast irreversible hydration steps. By increasing the scanning formation of allantoin and urea as the main products. However,rate from 0.1 to 20 V s−1, one can see, as already mentioned by parabanic acid can be also produced from uric acid-4,5-diol,Dryhurst, the appearance of a weak reduction peak (for which through complex chemical rearrangement to dihydroxyimida-the amplitude increases with the scan rate) at a potential of zole and subsequent two electron-two proton electrochemical−0.1 V vs Ag/AgCl (Figure 2b). Studies have been carried out oxidation. Indeed, Dryhurst has clearly shown that such latterto evaluate the signal to background ratio (S/B) as a function of decomposition follows a minority path which increases in yieldthe scan rate. The S/B was found to increase with the scan rate with acidity of the media.24,29 Moreover, the yield of thisup to an optimal value of 20 V s−1 and to decrease beyond this secondary path strongly depends on the electrode material asvalue. Indeed, provided the sweep rate is fast enough, the reported by Struck et al.30 who detected parabanic acid 10209 | Anal. Chem. 2012, 84, 10207−10213
  4. 4. Analytical Chemistry Articlereduction using polarography following UA oxidation at a with concentrations of UA. AA can deactivate the BDDspectroscopic graphite electrode in place of pyrolitic graphite. electrode due to deposition of its oxidation product. It wasIn such a way, according to the local pH decrease at the vicinity observed from Nyquist plots that the reactivity of the activeof the electrode owing to UA oxidation and to the acidic BDD electrode was reduced by 10% after a few CVs incomportment and the nature of hydrogenated diamond solutions containing AA. This could be one explanation for theelectrodes, parabanic acid may be produced with a yield of decrease in iB values when the concentration of AA is increased.around 6% (obtained from ratio of the plateau limiting currents The other assumption is derived from the antioxidant nature ofthat involve both two electrons and two protons). One can AA. AA is known to reduce quinone imines.31 In the presencenotice the disappearance of such a second electrochemical step of AA, the bis-imines might have been attacked or quicklyin fast CV (Figure 2b), and this behavior can be ascribed to the reduced to UA chemically. Hence, as the concentration of AA isenhanced electrochemical recycling of UA that decreases the increased, the amplitude of iB is decreased.yield of production of parabanic acid which is produced When the concentration of AA was increased beyond 1 mM,through a slow chemical and electrochemical pathway. the peak B has disappeared completely for UA concentrationsMoreover, this second electrochemical oxidation peak is hardly below 2 mM. Hence model 1 cannot be used to determine thevisible due to the Cottrell evolution of the anodic current, itself concentration of UA. However, a third peak (peak C) appearsbeing attributed to the primary oxidation wave of UA. at 0.8 V vs Ag/AgCl as seen in Figure 4. Thus, a second model Calibration Curves for UA Concentration. CV of UA (1mM), at the BDD electrode in a solution that contains varyingconcentrations of AA, is shown in Figure 3. In order to plot theFigure 3. Cyclic voltammogram of 1 mM uric acid and ascorbic acid(0, 250, 500 μM) in phosphate buffer solution scanned at 20 V s−1. Figure 4. Cyclic voltammograms of 1.5 mM uric acid and ascorbic acidPeak A increases with rising concentrations of both uric acid and (0, 2, 4 mM) in phosphate buffer solution scanned at 20 V s−1. Peaksascorbic acid, whereas peak B decreases with rising concentrations of A and C increase with rising concentrations of both uric acid andascorbic acid. ascorbic acid.calibration curve, the concentration of the UA solution was (model 2) is proposed using the two peaks (peaks A and C)varied from 0, 500, 1000, 1500, 2000 to 2500 μM and AA and their corresponding oxidation peak currents (iA and iC).concentration from 0, 250 to 500 μM. CVs of different For different concentrations of UA and AA mixtures, the peakcombinations of UA and AA mixtures were done, and the peak oxidation current (iA) and second peak oxidation current (iC)oxidation current (iA) and peak reduction current (iB) were were extracted from the CV measurements at 20 V s−1. Theobserved at 20 V s−1. Two 3D curves were plotted with UA and concentration of the UA solution was varied from 0, 500, 1000,AA concentrations (CUA and CAA) on the X and Y axes and iA or 1500, 2000 to 2500 μM and AA concentration from 1, 3 to 5iB values on the Z axis, respectively. Two second order mM. Like model 1, two 3D curves were plotted with UA andequations were plotted from the curve which corresponds to AA concentrations (CUA and CAA) on the X and Y axes and iA ormodel 1: iC values on the Z axis, respectively. The equations of model 2 are iA = 4.24 + 0.07C UA + 0.06CAA − (2.9 × 10−6C UA 2) iA = 27.93 + 0.02C UA + 0.07CAA + (1.36 × 10−6C UA 2) − (2.36 × 10−5CAA 2) − {(1.82 × 10−5)(C UACAA )} (3) − (3.54 × 10−9CAA 2) − {(3.14 × 10−6)(C UACAA )} (5) iB = 8.2 + 0.02C UA − 0.009CAA − (1.9 × 10−6C UA 2) iC = 25.2 + 0.017C UA + 0.03CAA + (3.89 × 10−6C UA 2) − (2.67 × 10−7CAA 2) − {(8.52 × 10−6)(C UACAA )}) (4) + (2.43 × 10−6CAA 2) + {(2.41 × 10−6)(C UACAA )} By solving the eqs 3 and 4, the concentration of UA and AA (6)(CUA and CAA) can be obtained. It was observed that iA The concentration of UA and AA (CUA and CAA) can thus beincreases with the concentration of UA as well as that of AA, obtained by solving eqs 5 and 6. Both iA and iC increase withwhereas iB decreases with concentrations of AA but increases the concentration of UA and that of AA. For higher 10210 | Anal. Chem. 2012, 84, 10207−10213
  5. 5. Analytical Chemistry Articleconcentrations of AA (>5 mM), it was observed that the value and the time lag between the spectrophotometric and ECiA does not depend much on UA concentration. As proposed measurement might have also influenced the difference in theearlier, the AA catalyzes the production of imine-alcohol, and results. The other parameter which could affect the measure-no peak B is observed at higher concentrations of AA. On BDD ment is the variation in electron transfer rate k0 of the electrode.electrodes, the peak C was not observed during the CV in Only one electrode was used to verify the comparisonsolely AA even at higher concentrations. The simultaneous between the spectroscopic and electrochemical (using theoxidation of UA in the presence of high concentrations of AA presented models) results. It was observed that iA and iB/iCpresents a complex mechanism. At a higher concentration of values were the same for any BDD electrode prepared using theAA, the adsorbed oxidation products of AA influence the same technique (or from the same batch) after electrochemicalelectro-kinetics of UA oxidation. A possible explanation is activation. It was also observed that after activation, the CVrelated to the fouling properties of DHAA. Thereby, because of curves overlap and iA and iB/iC values with a maximumthe blockade of electroactive sites by DHAA, UA oxidation may deviation less than 1%. Furthermore, the reactivity of thetake place through two different pathways: one via still electrode (k0) has an influence on the peak current values. Itelectroactive DHAA-free diamond-surface sites and one via has been already shown by our group that electrodes from aDHAA blocked sites through the adsorbed species. Oxidation microelectrode array exhibit different k0 values because of theof UA at the active sites and at the fouled surface could cause relative variations in boron intake in the diamond grains as wellthe peak separation. On the other hand, one can note that peak as the associated difference in surface area due to the diamondC potential fits quite well with the potential of the second roughness.32 The fouling of the electrode is another parameterplateau observed at slow scan rate, thus suggesting the presence that also affects the reactivity of the electrode. As the analyte isof parabanic acid. Thereby, the increase in the peak C a complex biological fluid, electrodes were systematically andamplitude with both UA and AA can be explained first by a automatically cleaned between each measurement.higher generation of parabanic acid with UA concentration and Automation of Quantification Procedure and in Situsecond to the possible recycling of parabanic acid owing to the Cleaning. When electrodes are used continuously in aantioxidant nature of AA. Indeed, some complementary studies biological fluid, they lose their reactivity because of fouling.33are on the way to examine more in depth the contributions of Electrode fouling can be due to adsorption or adhesion ofparabanic acid and/or DHAA fouling. biomolecules such as proteins, enzymes, cells, intermediate The pH of the UA solution was varied from 2 to 13, and the products of oxidation of organic compounds, etc.34 Althoughmodel 1 was tested in a solution containing 1 mM UA and 0.5 hydrogen terminated BDD exhibit high reactivity, continuousmM AA. It was observed that the pH variation does not impact use in urine leads to deactivation of electrode reactivity becauseon the peak values of iA and iB. Also there was no interference of fouling. This would lead to difficulty in automation ofobserved with other compounds and including glucose, NaCl, quantification process. Earlier, we have reported on an in situand KCl. activation process of BDD electrode which has been tried on Proposed Model vs Spectrophotometric Quantifica- several biological and synthetic fluids.34 Human urine samplestion. Human urine samples were selected as real samples for were diluted 2-fold in PBS solution. From this solution, fiveanalysis by the proposed models and were compared with the other solutions were prepared by adding different quantities ofspectrophotometric method. In spectrophotometric method, UA (400, 800, 1200, 1600, and 2000 μM). CVs at 20 V s−1uric acid is oxidized to allantoin in the presence of the enzyme were done in solution 1 (diluted urine) through 6 (diluteduricase, which leads to formation of H2O2, which reacts with 4- urine + 2000 μM UA). iA and iB values were obtained from eachamino phenazone in the presence of peroxidase to form scan, and from the CV of solution 1 the UA concentration wasquinone-diimine. The intensity of the color of the quinone estimated to be 2012 μM. It was observed that the values of iAdiimine is directly proportional to the concentration of uric and iB were not increased as expected, after each scan (solutions 1−6) despite the increase in concentration of UA (Figure 5).acid. Human urine samples were neither diluted nor pretreated This is due to fouling of the electrode. The electrodes were ECfor EC quantification by the proposed models. CV was done on cleaned and CVs were done from solutions 1 (diluted urine)each urine sample and based on the nature of the peaks, using through 6 (diluted urine + 2000 μM UA). Between each CV,model 1 or model 2; the equations were solved to identify the the electrodes were activated in the previous solution. The ECUA value. The results are presented in Table 1. The measured activation was done directly in the solution containing urine, byvalues using the proposed model were observed to be very applying a train of negative current pulses consisting of 3 pulsesclose to the spectrophotometric results with a maximum where each pulse has amplitude of −100 mA cm−2 and durationdifference of 13%. The spectrophotometric measuring of 100 ms and duty cycle of 90%. The values of iA and iB weretechnique, although widely used in the field of UA estimation, increased as expected, after each scan (Figure 6). Values of iAhas an error percentage of 5%. UA and AA are fast antioxidants, and iB were extracted from these graphs and are termed as iAO1, iBO1 and iAO2, iBO2 for trails with and without activation betweenTable 1. Comparison of Uric Acid Concentration Measured CVs. The concentration of UA was estimated using eqs 3 and Different Urine Sample Using Models 1 and 2 and the Also the theoretical values of iA and iB were calculated using theSpectrophotometric Technique models and are termed iAC and iBC. The close comparison between the calculated value iAC and observed value iAO1 (trialssample no. spectrophotometer (mM) proposed model (mM) % error with activation) and iAO2 (trials without activation) demon- 1 3.66 4.15 13 strated that there was a negligible difference between iAC and 2 4.76 5.10 7.3 iAO1 when compared with the difference between iAC and iAO2. A 3 3.01 2.68 −11 detailed comparison between the different values is depicted in 4 5.90 5.15 −12 Table 2 and Figure 7, where Table 2 shows the expected and 5 2.78 2.74 −1.4 measured concentration of UA. Because of heavy fouling of the 10211 | Anal. Chem. 2012, 84, 10207−10213
  6. 6. Analytical Chemistry ArticleFigure 5. Cyclic voltammogram of urine diluted by 2-fold and that ofdiluted urine containing added uric acid (250−1250 μM) scanned at20 V s−1. Both peaks A and B are expected to increase steadily, but dueto fouling of the electrode the amplitudes of peaks A and B are notincreased as expected. Figure 7. Comparison between the calculated value (iAC, iBC), observed value with activation between CVs (iAO1, iBO1), and observed value without activation between CVs (iAO2, iBO2) for the peak currents iA and iB extracted from CV in urine solutions 1−6.Figure 6. Cyclic voltammogram of urine diluted by 2-fold and that of ■ CONCLUSIONS Selective determination of UA in the presence of AA wasdiluted urine containing added uric acid (250−1250 μM) scanned at achieved using a BDD microelectrode without any further20 V s−1. The electrodes were activated electrochemically in the same modification. Comparison of the EC quantification techniquesolution in between two successive scans. and that of the spectrophotometric technique shows that an accurate measurement can be done using the two proposedTable 2. Comparison between the Measured Concentration models. This technique highlights the potential of BDDand Expected Concentration for Uric Acid for Both electrodes as a biosensor owing to its low double layerActivated and Nonactivated Trials capacitance, robustness at high current density, and corrosion measured measured resistance. The EC treatment retrieves the lost reactivity of an expected concentration % concentration (no % electrode either aged in air or fouled by a medium withoutconcentration (activation) recovery activation) recovery using any specific reagent or solution. The advantage of this 2012 2012 100.00 2012 100.00 technique is to enhance the reusability of the BDD electrode by 2412 2496 103.48 2250 93.28 activating in urine itself. This demonstrates the possibility of 2812 2924 103.98 2375 84.46 automation of UA quantification as the electrode can be 3212 3330 103.67 2479 77.18 activated directly in urine and hence it can be used for 3612 3566 98.72 2751 76.16 continuous monitoring for a long period of time. The time 4012 4010 99.95 2708 67.50 taken for activation is 300 ms, and the time taken for CV at 20 V s−1 is less than 200 ms. ■electrode, the electrode was deactivated and hence thepercentage recovery was as low as 67.5%, whereas the ASSOCIATED CONTENTpercentage recovery for activated trials were close to 100%. Itis clearly demonstrated that a simple EC activation procedure * Supporting Information Scan restore the loss of reactivity of the electrode for the Second order curve fitting results and cyclic voltammogram ofelectrode to be reused in the sample solution for consecutive uric acid. This material is available free of charge via themeasurements. Internet at 10212 | Anal. Chem. 2012, 84, 10207−10213
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