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Available online at www.sciencedirect.com

Biosensors and Bioelectronics 23 (2008) 857–864

An amperometric bilirubin biosensor based on a conductivepoly-terthiophene–Mn(II) complex

Md. Aminur Rahman a, Kyung-Sun Lee a, Deog-Su Park a,Mi-Sook Won b, Yoon-Bo Shim a,∗

a Department of Chemistry and Center for Innovative BioPhysio Sensor Technology,Pusan National University, Pusan 609-735, South Korea

b Korea Basic Science Institute, Pusan 609-735, South Korea

Received 15 May 2007; received in revised form 29 August 2007; accepted 7 September 2007Available online 19 September 2007

bstract

An amperometric bilirubin biosensor was fabricated by complexing the Mn(II) ion with a conducting polymer and the final biosensor surfaceas coated with a thin polyethyleneimine (PEI) film containing an enzyme, ascorbate oxidase (AsOx). The complexation between poly-5,2′-5′,2′′-

erthiophene-3-carboxylic acid (PolyTTCA) and Mn(II) through the formation of Mn–O bond was confirmed by XPS. The PolyTTCA–Mn(II)omplex was also characterized using cyclic voltammetry. The PolyTTCA–Mn(II)/PEI–AsOx biosensor specifically detect bilirubin through theediated electron transfer by the Mn(II) ion. To optimize the experimental condition, various experimental parameters such as pH, temperature,

nd applied potential were examined. A linear calibration plot for bilirubin was obtained between 0.1 �M and 50 �M with the detection limitf 40 ± 3.8 nM. Interferences from other biological compounds, especially ascorbate and dopamine were efficiently minimized by coating theiosensor surface with PEI–AsOx. The bilirubin sensor exhibited good stability and fast response time (<5 s). The applicability of this bilirubinensor was tested in a human serum sample.

2007 Elsevier B.V. All rights reserved.

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eywords: Amperometric biosensor; Ascorbate oxidase; Bilirubin; PolyTTCA

. Introduction

Bilirubin is a tetrapyrrole compound that is formed fromhe breakdown of heme in red blood cells and present in thelood in an unconjugated (free) form (With, 1968). The bro-en down heme travels to the liver where it is transformed towater-soluble conjugated form and secreted into bile. The

etermination of bilirubin in blood serum samples is consid-red as a true test of liver function. Normal level of bilirubin inerum of adult ranges from 10−5 M to 10−6 M (With, 1968). Theigh concentration of bilirubin is associated with liver diseases,epatocellular diseases such as cirrhosis or hepatitis, jaundice,rain damage or even death especially in newborns (Hargreaves,

968; Maisels, 1989). On the other hand, a low level of bilirubinoncentration is associated with iron deficiency (Kanada andnishi, 1981) and coronary artery disease (Schiff and Schiff,

∗ Corresponding author. Tel.: +82 51 510 2244; fax: +82 51 514 2430.E-mail address: ybshim@pusan.ac.kr (Y.-B. Shim).

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956-5663/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.bios.2007.09.005

II) complex

993). Thus, the accurate determination of bilirubin is clinicallymportant and there is a strong demand to develop an inexpensivenalytical method for the determination of bilirubin.

Various methods have been developed for the detection ofilirubin in clinical samples, and the most common detectionethods are the direct spectroscopic measurement (Doumas et

l., 1973) and the diazo reaction (Bergmeyer, 1985). However,he direct spectroscopic measurement of bilirubin suffers fromnterference from other heme proteins and the accuracy in theetermination of the bilirubin concentration with the methodased on the diazo reaction is compromised, partly as the reac-ion rate is pH dependent (Li and Rosenzweig, 1997). Othernalytical methods, such as polarography (Wang et al., 1985),uorometry (Koch and Oakingbe, 1981), etc., have also beennown for the bilirubin analysis. These methods are also lesselective compared to the diazo reaction. On the other hand,

here have been extensive attempts to obtain more accurate andimple routine analytical methods (Li and Rosenzweig, 1997;alilis et al., 1996; Vidal et al., 1996), including various enzy-atic systems (Mullon and Langer, 1987; Kurosaka et al., 1998;

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58 Md.A. Rahman et al. / Biosensors

uo and Dong, 1997) for the determination of bilirubin. Ofhese enzymes, bilirubin oxidase (BOx) have been used byany investigators that catalyzes the oxidation of bilirubin to

iliverdin by molecular oxygen, which results in the formation ofater. Electrochemical amperometric biosensors based on BOxave been fabricated and applied for bilirubin analysis throughhe measurement of the decreasing level of molecular oxygenWang and Ozsoz, 1990) or oxidation of hydrogen peroxideFortuney and Guibault, 1996). However, the biosensor based onhe measurement of the decreasing oxygen content suffers fromther electroactive species and is characterized in a relativelyong response time. On the other hand, the biosensor based onhe oxidation of hydrogen peroxide also suffers from a problemn that hydrogen peroxide is not formed stoichimetrically withespect to bilirubin but depends on the oxygen concentration inhe system (Shoham et al., 1995). Moreover, BOx is an unstablenzyme due to very easy denaturation (50% activity lost within7 h at 37 ◦C) (Sung et al., 1986) thus, limiting the applicationf this biocatalyst in a biosensor device. Furthermore, BOx isxpensive and the search for substitutes for BOx would be morettractive. Thus, it is necessary to develop a stable biosensoror bilirubin without BOx. We expected that a conducting poly-er, CP (Park, 1997; Shim and Park, 1997; Guiseppi-Elie et al.,

997)-based bilirubin biosensor will be an alternative for a BOx-ased bilirubin biosensor. For this, we introduced a functionalroup grafted conducting polymer (Lee et al., 2002).

Until now, there are few reports of the metal ion complexith CPs, due to the weak interaction of a ligating atom or CPith metal ions. However, the functional groups, such as amine,

mine, and carboxylic acid can be used as ligands for the metalon complexation (Cotton and Wilkinson, 1988; Mehrotra andohra, 1983). We have previously studied the complexationsf metal ions with CPs having amine groups (Lee et al., 1992;oopathi et al., 2002). CP having a carboxylic acid functionalroup can coordinate with a metal ion to form the coordina-ion complex. Until now, there is no report on the complexationetween a carboxylic acid functionalized CP and manganese ionnd its application towards a biological compound detection.

In the present study, we have fabricated an amperomet-ic bilirubin biosensor based on PolyTTCA–Mn(II) complexoated with a thin film of polyethyleneimmine (PEI) containingscorbate oxidase (AsOx) to eliminate interferences from otherlectroactive biological compounds, such as ascorbic acid andopamine. The advantage of using AsOx in this bilirubin biosen-or is to avoid interference from ascorbic acid that can oxidizescorbic acid to dehydroxy ascorbic acid, which is electrochem-cally inactive. The PolyTTCA–Mn complex was characterizedy XPS and cyclic voltammetry. Various experimental param-ters, which affect the bilirubin detection were optimized andpplied to a real serum sample for the detection of bilirubin.

. Experimental

.1. Materials

Bilirubin and ascorbate oxidase, AsOx (EC. 1.10.3.3,02.3 units mg−1) from cucurbitas, polyethyleneimmine (PEI),

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ioelectronics 23 (2008) 857–864

nd dichloromethane (99.8%, anhydrous, sealed under N2 gas)ere purchased from Sigma Co. (USA). The human serum

ample was also obtained from Sigma Co. and was usedithout dilution. Tetrabutylammonium perchlorate (TBAP, elec-

rochemical grade) was received from Fluka (USA), purified,nd then dried under vacuum at 10−5 Torr. A terthiopheneonomer bearing a carboxylic acid group, 5,2:5,2-terthiophene-

-carboxylic acid (TTCA), was synthesized according to arevious report (Lee et al., 2002). A phosphate buffer saline solu-ion (PBS) was prepared by modifying 0.1 M disodium hydrogenhosphate (Aldrich) with the admixture of 0.1 M sodium dihy-rogen phosphate (Aldrich) with 0.9% sodium chloride. Theilirubin stock solution was prepared by dissolving 2.0 mg ofilirubin in a 0.1 ml of 0.1 M NaOH, diluting it with a 6.0 mlhosphate buffer solution at pH 8.0 and was stored in amberlass vials. Lower concentrations of bilirubin solution wereade by diluting the stock solution daily before each experi-ent. All other chemicals were of extra pure analytical grade

nd used without further purification. All aqueous solutions wererepared in doubly distilled water, which was obtained from ailli-Q water purifying system (18 M � cm).

.2. Apparatus

A PolyTTCA–Mn(II) complex film on the gold electrodearea = 7 mm2), an Ag/AgCl (in saturated KCl), and a Ptire were used as working, reference, and counter elec-

rodes, respectively. Cyclic voltammograms and amperogramsere recorded using Potentiostat/Galvanostat, Kosentech modelST-P2 (South Korea). A quartz crystal microbalance (QCM)

xperiment was performed using a SEIKO EG & G model QCA17 and a PAR model 263A potentiostat/galvanostat (USA). Anu working electrode (area: 0.196 cm2; 9 MHz; AT-cut quartz

rystal) was used for the QCM experiment. XPS experimentsere performed using a VG Scientific ESCALAB 250 XPS

pectrometer with monochromated Al K� source with chargeompensation at KBSI (Busan). XPS experiments with goldisk electrodes were performed by fixing the gold disks (3 mmiameter and 3 mm long) into a 3 mm diameter hole in a Teflonheet having a thickness of 2 mm. For the electrochemical exper-ments, the one side of the disk was connected with a copper wiresing silver conductive paste and the connected part was coveredith an epoxy resin and with a special tape (Nitto Co., Japan).o control the temperature, the electrochemical cell was ther-ostated by circulating an ethylene glycol and water mixture

hrough a water jacket.

.3. Preparation of the PolyTTCA–Mn(II) complex on thelectrode and PEI–AsOx coating

At first, the PolyTTCA film was grown on gold electrodeshrough electropolymerization and then the PolyTTCA coatedlectrode was incubated in a Mn(II) solution of an weak acid,

H 5.5. Prior to electropolymerization, gold electrodes were pol-shed with 0.05 �m alumina/water slurry on a polishing cloth tomirror finish, followed by sonicating and rinsing with distilledater. The polished electrodes were then electrochemically

Md.A. Rahman et al. / Biosensors and Bioelectronics 23 (2008) 857–864 859

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Fig. 2. (A) Cyclic voltammograms recorded for the electropolymerization ofTTCA monomer in a 0.1 M TBAP/CH2Cl2 for three consecutive potential cycles.(B) Cyclic voltammograms recorded with a (a) PolyTTCA (dotted line), (b) aPa(

eiFcsaocrPo+Tacaot

ig. 1. Schematic representation of the preparation of PolyTTCA–Mn(II) com-lex on the gold electrode.

leaned by cycling the potential between −0.2 V and 1.5 V in.05 M H2SO4 at a scan rate of 100 mV/s until the cyclic voltam-ogram (CV) pattern of a clean gold electrode was observed.he PolyTTCA film at the cleaned gold electrodes was grown

hrough electropolymerization of TTCA monomer (1.0 mM)n a 0.1 M TBAP/CH2Cl2 solution by cycling the potentialrom 0 V to 1.6 V for three times. The resultant electrode wasashed with dichloromethane for the removal of excess adheredTCA monomer. The PolyTTCA–Mn(II) complex was madey immersing the electrode in a 0.1 M KCl solution (pH 5.5)ontaining 1.0 mM MnSO4 for 10 min with continuous shaking.fter that the PolyTTCA–Mn(II) complex-modified electrodeas washed carefully with distilled water. The schematic repre-

entation of the preparation of the PolyTTCA–Mn(II) complexn the electrode is shown in Fig. 1. The PolyTTCA–Mn(II) mod-fied electrode was coated with a film of PEI containing AsOx.he PEI–ascorbate oxidase (AsOx) coating was performedy dipping the PolyTTCA–Mn(II) complex-modified electrodehree times in a 1% solution of PEI containing 102.3 units/mlsOx. We tested dipping time and three times dipping was best.he modified electrode was completely dried after PEI–AsOxoating.

. Results and discussion

.1. Electrochemical characterization of theolyTTCA–Mn(II) complex film

The PolyTTCA film obtained during the electropolymeriza-ion was characterized using CV. Fig. 2A shows CVs recordedor electropolymerization of 1.0 mM TTCA monomer in a 0.1 MBAP/CH2Cl2 solution for three consecutive potential cycles.t the first anodic scan, the CV exhibited one oxidation peak at

round 1.3 V where the monomer oxidized and form polymermmediately. A polymer reduction peak was observed at around.1 V in the reverse cathodic scan. The peak currents at 1.3 Vnd 1.1 V increased as the potential cycle numbers increased,ndicating the formation and the growth of the PolyTTCA film.he thickness of the PolyTTCA film grown after three potentialycles was about 200 nm from scanning electron microscopeSEM) image as similar to previous experiment (Lee and Shim,

001). From the EQCM study, the amount of PolyTTCA grownfter three cycles was determined to 1.02 ± 0.21 �g from arequency change of about 0.95 kHz. The surface coverage ofolyTTCA was calculated to be (3.5 ± 0.4) × 10−9 mol/cm2.

steo

olyTTCA–Mn(II) complex-modified electrodes (solid line) in a PBS of pH 7.4,nd a (c) PolyTTCA-coated electrode in 1.0 mM of Mn(II) solution in 0.1 M KCldashed line).

After the complexation of Mn(II) ion with PolyTTCA coatedlectrode, the PolyTTCA–Mn(II) complex electrode was placedn a phosphate buffer solution and the CV was recorded.ig. 2 shows the CV recorded for (b) a PolyTTCA–Mn(II)omplex-modified electrode (solid line) in a phosphate bufferolution (PBS) of pH 7.4. A redox peak was clearly observedt +317/+262 mV versus Ag/AgCl. The redox peak was notbserved when the CV was recorded for (a) a mere PolyTTCA-oated electrode (Fig. 2, dotted line). This indicates that theedox peak was come from the Mn species complexed witholyTTCA. The anodic peak at +317 mV corresponded to thexidation of Mn(II) to Mn(III), whereas the cathodic one at262 mV corresponded to the reduction of Mn(III) to Mn(II).he MnO2 film-modified CPE (Beyene et al., 2004) showedreduction signal starting at +300 mV and increased signifi-

antly below −800 mV (in the cathodic scan). The signal wasttributed to formation of lower oxidation state manganesexides (MnO and Mn2O3). Above 400 mV, re-oxidation ofhese oxides to MnO2 occurred. The oxide system (+400 mV)

howed a similar oxidation potential to ours (+317 mV) buthe reduction potential of our system (+262 mV) was differ-nt from the oxide system. This indicates that the oxidationf Mn(II) to Mn(III) is similar but the reduction of oxidized

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60 Md.A. Rahman et al. / Biosensors

n species is little different due to the different coordina-ion environment in our PolyTTCA–Mn(II) complex system.he CV was also recorded for a PolyTTCA-coated electrode

n a 0.1 M KCl solution containing 1.0 mM of Mn(II) (Fig. 2,ashed line (c)). The redox peaks of Mn(II) in the solution phaseere observed at the similar potentials as those observed forPolyTTCA–Mn(II) complex-modified electrode. The formal

otential of the PolyTTCA–Mn(II) complex was determined toe +289 mV. The anodic and cathodic peak currents were pro-ortional to the scan rate indicating that the redox reaction wasnvolved with a surface confined process (Murray, 1984). Theeak potential of the redox peaks should be independent of scanate for a surface-bound electroactive species for a reversibleystem. However, in our case, we confirmed that surface-boundrocess of PolyTTCA–Mn(II/III) was irreversible because peakeparation was found to be increased with the scan rate. The peakeparation may be larger than the theoretical value and the peakhift takes place due to the diffusion of counter ion in the polymerlm. The transfer coefficient (α) and electron transfer rate con-tant (ks) of the Mn(II)/Mn(III) redox couple were determinedsing the method for a surface confined electrochemical sys-em (Laviron, 1979). The α and ks values were determined to be.48 s−1 and 1.18 s−1, respectively, at the scan rate of 100 mV/s.

The maximum surface coverage of the complexed Mn(II) onhe PolyTTCA film at the optimized condition (concentrationf Mn(II) in the complexing solution was selected as 1.0 mM,H of the complexing solution was maintained at 5.5, and theptimum time for the complexation of Mn(II) with PolyTTCAas chosen as 10 min) was calculated by using the following

quation (Bard and Faulkner, 1980):

p = n2F2νAΓ

4RT

TTaT

ig. 3. XPS analysis of PolyTTCA-coated (solid line) and PolyTTCA–Mn(II) compp peaks before application of any potential, and (d) Mn 2p peaks of the PolyTTCAolyTTCA–Mn(II) surface after reduced at +262 mV (solid line).

ioelectronics 23 (2008) 857–864

here Ip is the peak current, n the number of electron, F the Fara-ay constant, R the gas constant, T the temperature, ν the scanate, A the area of the electrode and Γ is the surface coveragef Mn species. The surface coverage of the complexed Mn(II)pecies was determined to be (4.16 ± 0.15) × 10−11 mol/cm2

rom the oxidation process of Mn(II) to Mn(III).

.2. XPS characterization of the PolyTTCA–Mn(II) complex

To characterize the modified surfaces, XPS analyses weretudied and are shown in Fig. 3. Fig. 3a shows the sur-ey spectra obtained for PolyTTCA-coated (solid line) andolyTTCA–Mn(II) complex-modified surfaces (dashed line).he C 1s spectrum for the PolyTTCA-coated surface exhibited

wo peaks at 284.3 eV and 289.2 eV. The peak at 284.3 eV did notffect upon complexation. However, the peak at 289.2 eV shiftedlightly to a higher energy of 290.1 eV for a PolyTTCA–Mn(II)omplex-modified surface. The O 1s spectrum (Fig. 3b) for theolyTTCA-coated surface exhibited two peaks at 531.0 eV and32.0 eV, which corresponded to C O and C–O bonds, respec-ively (Ng et al., 1998). Both peaks shifted to higher energiesf 532.5 eV and 534.0 eV after complexation. Two S 2p peakst 163.5 eV (2p3/2) and 164.63 eV (2p1/2) were observed for theolyTTCA-coated surface. After complexation, S 2p3/2 and Sp1/2 peaks were almost overlapped and slightly shifted to aigher energy, indicating that S atoms may involve the complex-tion. However, the degree of binding energy shifted was largern the case of O 1s peak than that observed in the S 2p peakndicated that the complex formation between Mn(II) and Poly-

TCA occurred through the direct formation of Mn–O bonds.he XPS spectra of Mn 2p peaks (Fig. 3c) for PolyTTCA-coatednd PolyTTCA–Mn(II) complex-coated surfaces were recorded.he PolyTTCA-coated surface did not show any peak for

lex-modified (dashed line) surfaces; (a) survey spectra, (b) O 1s peaks, (c) Mn–Mn(II) modified surface after oxidized at +317 mV (dashed line), oxidized

and Bioelectronics 23 (2008) 857–864 861

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Fig. 4. (A) Cyclic voltammograms recorded for a (a) bare gold electrode(dotted line) and (b) a PolyTTCA-coated electrode (dashed line) in a PBSsolution (pH 7.0) containing 0.65 mM bilirubin. (B) Cyclic voltammogramsrecorded for PolyTTCA–Mn(II) complex (a; dotted line, d; dashed line) andPlb

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Md.A. Rahman et al. / Biosensors

n, whereas the PolyTTCA–Mn(II) complex-modified surfacehowed two Mn 2p peaks at 640.8 eV and 652.7 eV correspondedo 2p3/2 and 2p1/2 environments, respectively, which indicateshat Mn(II) species present in the PolyTTCA–Mn(II) complex-

odified surface (Zaw and Chiswell, 1995). To identify theifferent oxidation states of Mn species, XPS spectra wereaken for a PolyTTCA–Mn(II) complex-modified surface afterxidized at +317 mV and for an oxidized PolyTTCA–Mn(III)omplex-modified surface after reduced at +262 mV. As shownn Fig. 3d, the Mn 2p3/2 peak after oxidized and reducedhe PolyTTCA–Mn(III) complex-modified surface shifted to42 eV and 641 eV, which corresponded to a Mn(III) and Mn(II)pecies (Zaw and Chiswell, 1995), respectively.

Electrochemistry can provide the evidence for immobiliza-ion of surface-bound electroactive species but cannot providehe exact type of chemical bonding. The XPS measurement isery useful to check the presence of any chemical bonding at thelectrode surface. In our study, XPS spectra clearly showed thathe PolyTTCA–Mn(II) complex was formed through the Mn–Oond formation. Thus, Mn(II) was chemically but not physicallydsorbed on PolyTTCA.

.3. Response of PolyTTCA–Mn(II)/PEI–AsOx modifiedlectrode to the detection of bilirubin

Fig. 4A shows the CVs recorded for (a) a bare gold elec-rode (dotted line) and (b) a PolyTTCA-coated electrode (dashedine), in a PBS solution (pH 7.0) containing 6.5 × 10−4 Milirubin. A small anodic peak due to the oxidation of biliru-in was observed at +0.35 V versus Ag/AgCl for a bare goldlectrode. Because of the inherent electroactive nature of biliru-in, irreversible oxidation of bilirubin was observed at a bareold electrode. This oxidation is accompanied by poisoninghe electrode surface with deposition of a dark colored poly-

er, which resulted in an alteration of the electrode responseowards bilirubin (Sung et al., 1986). The oxidation of bilirubinFig. 4A-b) at the PolyTTCA-coated electrode was observed at0.38 V versus Ag/AgCl, which the anodic peak current wasigher than that obtained with a bare gold electrode. Ascorbiccid, dopamine, and other biological compounds can inter-ere in the bilirubin detection because they also oxidized atimilar potentials. Thus, we conducted experiments after coat-ng the biosensor surface with a PEI film containing AsOx.ig. 4B shows CVs recorded for PolyTTCA–Mn(II) (a and) and PEI–AsOx coated PolyTTCA–Mn(II) complex-modifiedb and c) electrodes in the absence (a and b) and presencec and d) of bilirubin. CVs recorded for PolyTTCA–Mn(II)omplex (Fig. 4B-a) and PEI–AsOx coated PolyTTCA–Mn(II)omplex (Fig. 4B-b) electrodes in the absence of bilirubin inBS solution only showed the redox peak of Mn(II)/Mn(III).he peak current and peak potential of the Mn(II)/Mn(III)

edox couple obtained for a PolyTTCA–Mn(II) complex andEI–AsOx coated PolyTTCA–Mn(II) complex electrode in a

BS solution were almost similar. On the other hand, largenodic peaks at +0.42 V and +0.40 V versus Ag/AgCl dueo the oxidation of bilirubin were observed when CVs wereecorded in biliribin solution with PolyTTCA–Mn(II) complex

wop

olyTTCA–Mn(II)/PEI–AsOx electrodes (b, dashed dotted line and c, solidine) in a PBS solution (pH 7.0) without (a and b) or with (c and d) 0.65 mMilirubin.

nd PEI–AsOx coated PolyTTCA–Mn(II) complex electrodes,espectively. The anodic peak current obtained for a PEI–AsOxoated PolyTTCA–Mn(II) complex-modified electrode wasittle smaller than that obtained for a PolyTTCA–Mn(II)omplex-modified electrode. However, the anodic peak obtainedor a PEI–AsOx coated PolyTTCA–Mn(II) complex-modifiedlectrode was seven and five times larger than that obtainedt a bare Au and a PolyTTCA-coated electrode, respectively.his means that the oxidation of biliubin might have beenccurred through the mediated electron transfer of Mn(II)pecies complexed with the PolyTTCA. The anodic peak currentas proportional to the bilirubin concentration, which con-rms that the anodic peak solely came from the oxidation ofilirubin.

.4. Optimization of experimental parameters for bilirubinetection

The experimental parameters for the detection of bilirubinith a PolyTTCA–Mn(II)/PEI–AsOx modified electrode wasptimized in terms of pH, the temperature, and the appliedotential. The effect of pH on the oxidation of bilirubin was

862 Md.A. Rahman et al. / Biosensors and Bioelectronics 23 (2008) 857–864

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ig. 5. (A) Optimization of experimental parameters: effects of pH (a) and tempilirubin. (B) Chronoamperomeric measurements for the interference effectlectrodes with or without AsOx and PEI coating.

tudied over the pH range 2.0–10 in PBS buffer solutionsontaining 0.65 mM bilirubin. Fig. 5A-a shows the oxidationurrent gradually increased from pH 2.0 to 7.0. The oxidationurrent decreased rapidly as the pH increased over 7.0. Theapid decrease of current with the increase of pH may bettributed to the hydroxide formation of manganese at a higherH. The maximum oxidation current was observed at the pH of.0. Thus, the optimum pH was chosen as 7.0.

Fig. 5A-b shows the effect of temperature for the oxidationf bilirubin in the range of 10–70 ◦C. The response was found toe increased as the temperature increased from 10 ◦C to 70 ◦C.he response versus temperature curve exhibits three different

egions. In the first region (10–25 ◦C), the oxidation currentas slightly increased, however, the current response rapidly

ncreased from 30 ◦C to 60 ◦C, where as in the third region60–70 ◦C), the current response did not change significantly.his high temperature dependency of the bilirubin oxidationight be related to the temperature depending activity of then(II) complex. However, due to the biological significance of

he bilirubin detection, the subsequent experiment was carriedut at 30 ◦C.

The effect of applied potential on the oxidation of.5 × 10−4 M bilirubin was also studied with chronoamperom-try (data not shown). The current response increased as thepplied potential varied from +0.1 V to the more positive direc-ion. The maximum response was observed at +0.4 V and the

pplication of the more positive potential up to +0.6 V did notncrease the current response. Thus, the PolyTTCA–Mn(II)/EI–AsOx complex-modified electrode was polarized at +0.4 V

n the subsequent amperometric experiments.

Pa4

e (b) on the oxidation of bilirubin in a PBS buffer solution containing 0.65 mMascorbic acid and (b) dopamine with PolyTTCA–Mn(II) complex-modified

.5. Interference effects

In order to assess the possibility of interference fromome other bio-compounds, the current response was measuredmperometrically with the presence of other bio-compounds.he effects of some common interfering substances such asscorbic acid (AA), uric acid (UA), glutamic acid, creatine, glu-ose, dopamine, and others in the determination of bilirubin werevaluated. The sensor responses did not affect by the presencef l-glutamic acid, uric acid, creatine, and glucose in normalhysiological concentration even without coating a PEI film con-aining AsOx. However, ascorbic acid and dopamine were foundo interfere with the bilirubin detection when present in 1.0 mMnd 10 �M concentrations, respectively, without PEI and AsOxoating. Fig. 5B shows interference effects from ascorbic acida) and dopamine (b) of the PolyTTCA–Mn(II) complex elec-rode with or without AsOx and PEI coating. After coating withPEI film containing AsOx, PEI protected positively charged

iomolecules, such as dopamine to reach at the electrode sur-ace and ascorbate oxidase oxidized AA to dehyroxyascorbiccid, which is not an electroactive compound. Thus, AA andopamine interference completely eliminated, and the modifiedlectrode became highly selective for bilirubin detection.

.6. The calibration plot

Fig. 6a shows the current–time plot obtained with aolyTTCA–Mn(II)/PEI–AsOx modified electrode during theddition of 0.5 mL of 1 �M concentration of bilirubin in a.5 mL of PBS solution. The successive additions of bilirubin

Md.A. Rahman et al. / Biosensors and Bioelectronics 23 (2008) 857–864 863

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ig. 6. (a) Chronoamperometric measurements by successive addition of bilirubpplied potential: +0.4 V. Inset in (a) shows the blank noise level response.

t concentrations between 1 �M and 100 �M were contin-ed. The oxidation current rose steeply to a stable value asoon as the bilirubin solution was introduced. The modifiedlectrode achieved 95% of steady-state currents within 10 s.ig. 6b shows the calibration plot for the bilirubin detec-

ion. Under the optimized condition, the steady-state currentshowed a linear relationship with the bilirubin concentrationn the range of 0.1–50 �M. This linear dependency of theilirubin concentration yielded the regression equation of Ip�A) = −(0.080 ± 0.017) + (0.028 ± 0.001) [C] (�M) with cor-elation coefficient of 0.997. The detection limit for bilirubinas determined to be 40 ± 3.8 nM based on three times mea-

urements for the standard deviation (0.4 nA) of the blank noiseignal (5 nA) (95% confidence level, k = 3, n = 5). The repro-ucibility expressed in terms of the relative standard deviationR.S.D.) was about 5.3% at a bilirubin concentration of 1.0 �M.he detection limit was much lower than previously describedilirubin oxidase-based electrochemical bilirubin biosensorsWang and Ozsoz, 1990; Fortuney and Guibault, 1996) and fiberptic sensor (Li and Rosenzweig, 1997; Li et al., 1996). The pro-osed biosensor exhibited a good hydrodynamic range. The highensitivity of this biosensor is an additional advantage over BOx-ased electrochemical biosensors and fiber optic-based sensors.he low detection limit of this bilirubin biosensor made it possi-le to diagnosis iron deficiency (Kanada and Onishi, 1981) andoronary artery disease (Schiff and Schiff, 1993), where a lowevel of bilirubin was found. The biosensor could still be usefuln cases of acute or chronic jaundice, where the concentrations ofilirubin levels can give rise to concentration higher than 30 �M.

.7. Stability of the PolyTTCA/Mn(II)/PEI–AsOx biosensor

The further studies showed that this biosensor exhibited highensitivity and stability. When it was stored in a phosphate bufferolution at 4 ◦C for a period of 2 month, the biosensor retained

ore than 93.5% of its initial response to the oxidation of biliru-

in. At the same time, the stability of the biosensor to multipleses was assessed by repetitively using one modified electrode in0.2 M phosphate solution containing bilirubin. The electrode

fttb

lution into PBS at pH 7.4 and (b) the calibration plot for the bilirubin detection.

ost only 3.4% of the initial response in about 30 continuouseasurements. The superior stability of the modified electrodeay be ascribed to the stable complex formation and PEI coating

ver the complex at the electrode surface.

.8. Real sample analysis

To investigate the potential application of the proposediosensor, it was used for bilirubin assay in a human serum sam-le. The determination of bilirubin content in a serum sampleas carried out by the standard addition method for reducing

he matrix effect of the serum sample. The current–time plotas obtained with a PolyTTCA–Mn(II)/PEI–AsOx biosensoruring the addition of a 500 �l of this human serum sample in a.5 ml PBS buffer solution first followed by additions of varyingmounts of standard bilirubin solution. The real sample analysisf bilirubin in a serum sample was repeated for five times. Thenalytical performance of this bilirubin sensor was satisfactory.he bilirubin content in the human serum sample was deter-ined to be 5.2 ± 0.7 �M, which is consistent with the result

btained from a spectrophotometric method (4.9 ± 0.2 �M).

. Conclusion

An amperometric bilirubin biosensor was fabricated by com-lexing Mn(II) ion with PolyTTCA followed by coating theiosensor surface with a PEI film containing AsOx. XPS stud-es confirmed the formation of Mn–O bonds in the complex.he PolyTTCA–Mn(II)/PEI–AsOx-based-biosensor exhibited aide dynamic range, low detection limit, short response time,

nd long lifetime stability. The easy and straight forward methodf the fabrication of this bilirubin biosensor is an additionaldvantage over BOx-based conventional biosensors. This biliru-in sensor got rid of interference produced by the ascorbic acidnd dopamine present in blood. The proposed sensor was applied

or the detection of bilirubin in the serum sample and satisfac-ory results were obtained. The above observations showed thathe developed sensor might be promising in the detection ofilirubin in clinical samples.

8 and B

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WWang, J., Luo, D.B., Farias, P.A.M., 1985. J. Electroanal. Chem. 185, 61–

64 Md.A. Rahman et al. / Biosensors

cknowledgements

The financial support for this work from the Ministry ofealth and Welfare (grant nos. A020605 and A050426) is grate-

ully acknowledged.

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