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Biosensors and Bioelectronics 23 (2008) 1595–1601

An amperometric immunosensor for osteoproteogerin based ongold nanoparticles deposited conducting polymer

Kanika Singh a, Md. Aminur Rahman b, Jung Ik Son b, Kyung Chun Kim a,∗, Yoon-Bo Shim b,∗a School of Mechanical Engineering and MEMS/Nano Technology Center, Pusan National University, Pusan 609-735, South Korea

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

Received 17 October 2007; received in revised form 13 December 2007; accepted 16 January 2008Available online 24 January 2008

bstract

An amperometric immunosensor was fabricated for the detection of osteoproteogerin (OPG) by covalently immobilizing a monoclonal OPGntibody (anti-OPG) onto the gold nanoparticles (AuNPs) deposited functionalized conducting polymer (5,2′:5′,2′′-terthiophene-3′-carboxyliccid). AuNPs were electrochemically deposited onto the conducting polymer using cyclic voltammetry. The particle size of deposited AuNPs wasontrolled by varying the scan rate and was characterized by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS).he immobilization of anti-OPG was also confirmed using XPS. The principle of immunosensor was based on a competitive immunoassay between

ree-OPG and labeled-OPG for the active sites of anti-OPG. HRP was used as a label that electrochemically catalyzes the H2O2 reduction. Theatalytic reduction was monitored amperometrically at −0.4 V vs. Ag/AgCl. The immunosensor showed a linear range between 2.5 and 25 pg/mlnd the detection limit was determined to be 2 pg/ml. The proposed immunosensor was successfully applied for real human samples to detect OPG.

2008 Elsevier B.V. All rights reserved.

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eywords: Ampereometry; Gold nanoparticles; Immunosensor; Osteoproteoge

. Introduction

Osteoprotegerin (OPG) is a glycoprotein, which is a mem-er of the tumor necrosis factor (TNF) receptor superfamilyHofbauer and Schoppet, 2004) related to bone growth. OPGas been shown to reduce or prevent increased bone resorptionBlair et al., 2006), which increases the bone mineral densitynd bone volume (Hofbauer and Schoppet, 2004). OPG is alsodentified as a biomarker for lytic bone metases (Boyle et al.,003). In addition, OPG is directly related to osteoporosis (OP),bone disease in which the bone becomes porous and fragile

Atkinson, 1964; Trueta, 1966; Singh et al., 2006). The level ofPG in serum is very low, which difficults screening, diagnosis

nd prognosis of the OP disease (Boyle et al., 2003). Thus, aensitive method is demanded for monitoring OPG concentra-

ion in biofluids. Enzyme linked immunosorbent assays (ELISA)re commonly used for detection of OPG (Chen et al., 2001).owever, this is time consuming, tedious, requires qualified pro-

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

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956-5663/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.bios.2008.01.016

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essionals, and an adequate environment. To overcome theseisadvantages, immunosensors (Turner, 1997) could be a substi-ute to the ELISA method for OPG detection. Receptor activatorf NF-KB ligand (RANKL)-based-sensors and real-time piezo-lectric immunosensors are also available for OPG detectionSkladal et al., 2005). Although the piezoelectric immunosen-or can be regenerative, a long response time is usually neededor a single analysis, which makes this technique less suitableor practical applications. On the other hand, an electrochemicalEC) immunosensor is an alternative to other detection tech-iques because of its simplicity, low cost, quick measurements,nd portability (Heineman and Halsall, 1985). In particular, anmperometric immunosensor with an enzyme labeling is a sim-le method for detecting biomolecules due to its fast responsend high sensitivity (Darain et al., 2003).

Conducting polymers (CPs) have attracted wide attentionwing to their applicability in optical devices, energy conversionevices, sensors, etc. (Park, 1997; Doblhofer and Rajeshwar,

998). CPs are also widely used in the development of biosen-ors (Cosnier, 1999; Gerard et al., 2002). Of these, CPs havingunctional groups, such as –COOH or –NH2 are attractive forhe fabrication of biosensors because biomolecules can be cova-

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596 K. Singh et al. / Biosensors and

ently immobilized onto CPs (Cosnier et al., 1991; Lee andhim, 2001; Rahman et al., 2005; Ban et al., 2004; Kwon etl., 2006). Stable immobilization of biomolecules is crucialo obtain a sensitive biosensor. Some immobilization methodsre frequently used in the fabrication of conducting polymer-ased biosensors for obtaining a stable and sensitive biosensor.hey are the direct electropolymerization-deposition methods,

he entrapment of biomolecules, and covalent binding methods,ollowed by subsequent polymerization. However, the directlectropolymerization in an enzyme containing organic solu-ion might cause damage in protein activity, and the physicalntrapment of enzyme gave a less stable sensor due to the lossf antibody during measurement (Gerard et al., 2002). Thus,he biomolecule immobilization through covalent bonding is anfficient way to achieve a stable biosensor system. Previously,COOH group’s functionalized terthiophene CP was used forabricating a DNA sensor (Lee and Shim, 2001), immunosen-or (Darain et al., 2003), and for studying the DNA–proteinnteraction (Ban et al., 2004).

In the present study, we have fabricated an amperometricPG immunosensor by covalently immobilizing an antibodynto AuNPs deposited polyTTCA (5,2′:5′,2′′-terthiophene-3′-arboxylic acid, TTCA) modified electrode. Since AuNPsKatz and Willner, 2004; Ivnitski and Rishpon, 1996; Wangt al., 2006) deposited CP layers show the unique properties,uch as high electrocatalytic activity, increasing conductivity,ensitivity, etc. (Shiddiky et al., 2007). The surface of anti-PG/AuNPs/polyTTCA modified electrodes was characterizedy SEM, XPS, cyclic voltammetry, and chronoamperom-try. The amperometric experiment was performed withhe HRP–OPG/anti-OPG/AuNPs/polyTTCA electrode. Experi-

ental parameters affecting the response of the immunosensorere optimized in terms of anti-OPG amount, incubation time

or the immunoreaction, pH, and applied potential in chronoam-erometric measurements. The proposed immunosensor waspplied in real human serum samples for the detection ofPG.

. Experimental

.1. Materials

Anti-OPG (monoclonal, developed in mouse) was sup-lied by R&D Systems (Minneapolis, MN, USA). OPG fromhe human serum was supplied by Peprotech (USA) andorseradish peroxidase (HRP) (EC.1.11.1.7, 180 U/mg) fromigma. A terthiophene monomer bearing carboxylic acid group,,2′:5′,2′′-terthiophene-3′-carboxylic acid (TTCA) was syn-hesized according to the previous report (Lee and Shim,001). Tetrabutylammonium perchlorate (TBAP, electrochem-cal grade) was obtained from Fluka (USA) and purifiedccording to the general method, followed by drying underacuum at 10−5 Torr. 1-ethyl-3-(3-dimethylamino-propyl) car-

odiimide (EDC), casein (bovine milk), glutaraldehyde andysine were obtained from Sigma Co. (USA). Dichloromethane99.8%, anhydrous, sealed under N2 gas) was purchased fromldrich and was used as received. A phosphate buffer saline

sms4

ectronics 23 (2008) 1595–1601

olution (PBS) (pH 5.5–8.4) was prepared with 0.01 M ofisodium hydrogen phosphate (Aldrich), 0.01 M of sodium dihy-rogen phosphate (Aldrich) and 0.9% sodium chloride (Sigma).Sephadex G-25 medium was received from Pharmacia (Swe-

en). The HRP–OPG conjugate was prepared by using thereviously described method (Shiddiky et al., 2007). Test solu-ions were freshly prepared before use by diluting the stockolution with 10 mM of PBS. All other chemicals were of extraure analytical grade and used without further purification. Allqueous solutions were prepared with doubly distilled water,hich was obtained from a Milli-Q water purifying system

18 M� cm). The human serum samples were obtained fromhe Pusan National University Hospital in Busan, South Korea.

.2. Apparatus

The antibody-immobilized AuNPs/polyTTCA coated on alassy carbon electrode (GCE) (area = 0.07 cm2), Ag/AgClin saturated KCl), and a platinum (Pt) wire were useds working, reference, and counter electrodes, respectively.he measurement was performed in batch system. Cyclicoltammograms and amperograms were recorded employingpotentiostat/galvanostat, Kosentech Model KST-P2 (Southorea). Scanning electron microscopy (SEM) images werebtained using a Cambridge Stereoscan 240. XPS experi-ents were performed using a VG scientific ESCA lab 250PS spectrometer with a monochromated Al K� source with

harge compensation at the Korea Basic Science InstituteBusan).

.3. Fabrication of an immunosensor

The polyTTCA film was grown on the electrode accordingo the previously described method (Lee and Shim, 2001). Goldanoparticles (AuNPs) were then electrochemically depositedn the polyTTCA modified electrode by sweeping the poten-ial between −0.1 and +1.29 V vs. Ag/AgCl in a 0.1 M H2SO4olution containing 1 mM HAuCl4 at different scan rates rang-ng between 20 and 200 mV/s. The AuNPs/polyTTCA modifiedlectrode was used to immobilize anti-OPG as shown in Fig. 1.he AuNPs/polyTTCA modified electrode was immersed forh in a 10 mM phosphate buffer solution (PBS) (pH 7.0)ontaining 10 mM EDC to activate the carboxylic groups ofolyTTCA. Then, AuNPs/polyTTCA treated with EDC wasashed with PBS and subsequently incubated for 24 h in�g of anti-OPG in 5 ml of PBS (pH 7.4) at 4 ◦C. By this

tep, anti-OPG was immobilized onto the AuNPs/polyTTCAayer through the formation of the covalent bond between car-oxylic acid groups of the polyTTCA and amine groups of thentibody (here after anti-OPG/AuNPs/polyTTCA/GCE). Thenti-OPG/AuNPs/polyTTCA/GCE surface was then blockedsing a 0.3% (w/v) casein solution for 1 h at room temperature tovoid non-specific adsorption followed by rinsing with a buffer

olution. The HRP–OPG conjugate was then interacted with theodified electrode by incubating the electrode in a conjugate

olution for 30 min. The final modified electrode was stored at◦C until use.

K. Singh et al. / Biosensors and Bioelectronics 23 (2008) 1595–1601 1597

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.4. Electrochemical measurements

Chronoamperometric experiments were carried out bypplying a potential of −0.4 V to the HRP–OPG/anti-PG/AuNPs/polyTTCA to carry out the reduction of H2O2

dded to the cell. The steady-state current upon the additionf 1.0 mM H2O2 into a 9 ml of 10 mM phosphate buffer (pH.4) solution was monitored with stirring. Chronoamperomet-ic experiments were continued with the addition of varyingmounts of free-OPG added consecutively into a measuringolution and the decrease of the steady-state currents was mea-ured. 25 �L of 1.0 ng/ml of OPG was first added to 10.0 mLBS. Then, 50, 80, 122, 185, and 265 �L of OPG were added to

he cell. Prior to measurements, solutions were deaerated withhe nitrogen gas for 15 min and maintained under a nitrogentmosphere during measurements.

.5. Immunosensor response to OPG

The principle of the competitive immunosensing protocolsed in the present study is shown in Fig. 1. The added H2O2as reduced by the labeled HRP generating a cathodic current,hich was found to be decreased with the addition of free-OPG.his was due to the fact that the competition between free- and

abeled-OPG for the active site of antibody occurred, whichesulted in a decrease concentration of the labeled-OPG on thentibody. Eventually, the enzymatic activity can be assessed byhe decrease of amperometric or voltammetric reduction cur-ents.

. Results and discussions

.1. Electrochemical characterization

During the growth of polyTTCA film through the potentialycling method, the CV exhibited an anodic peak at around.38 V and a cathodic peak at 1.03 V, which were due to the

xidation of TTCA monomer and the reduction of polymer,espectively (Lee and Shim, 2001). The peak currents increaseds the potential cycle numbers increased indicating that therogressive growth of the polyTTCA film on the electrode.

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rication of an OPG immunosensor.

he thickness of polyTTCA after three cycles was around50–200 nm (Lee and Shim, 2001). The surface morphology wastudied for AuNPs deposited on polyTTCA with SEM images.

The electrochemically deposited AuNPs on the surface ofolyTTCA modified electrode were characterized by recordinghe CV in a 0.1 M H2SO4 solution. The gold oxidation peakas observed at 0.9 V, indicating that AuNPs were deposited

t the polyTTCA surface. Fig. 2 shows SEM images of AuNPsbtained at various scan rates. As shown in Fig. 2(a), the electro-hemical deposition of AuNPs at a scan rate of 20 mV/s was notomogeneous and the aggregation of NPs was observed, wherehe particle size of AuNPs was determined to be 90–120 nm. Ashe scan rate increased to 50 mV/s, the particle size of AuNPsas decreased to be 30–50 nm. The smaller size of AuNPsbtained at 50 mV/s might have been due to the short depo-ition time at a relatively high scan rate. On the other hand,he deposition of AuNPs at the scan rate of 100 mV/s showso aggregation and the particle size of AuNPs (10–20 nm) wasetermined to be much smaller than that obtained at lower scanates of 20–50 mV/s. However, faster scan rate over 100 mV/sid not make much smaller AuNPs than 10–20 nm. Thus, a scanate of 100 mV/s was used for the electrochemical deposition ofuNPs.Fig. 2(b) shows XPS spectra for C1s, Au4f, and N1s peaks

or polyTTCA, AuNPs, and anti-OPG/AuNPs/polyTTCA. Two1s peaks for polyTTCA exhibited at 284.6 and 289.2 eV, whichas due to the formation of C–H, C–S, or C–C bonds of COOHroup, respectively. O1s peaks for polyTTCA layers exhibitedt 534.5 and 532.5eV, which corresponded to C O and C–O,espectively. After the deposition of AuNPs, the modified sur-ace showed five additional peaks at 568.11, 346.46, 343.90, 86.9nd 82.9 eV corresponded to Au4p3, Au4d3, Au4d5, Au4f3,nd Au4f7, respectively. After immobilization of anti-OPG, a1s peak was observed at 396.70 eV. The C1s peak at 284.6 eVas shifted to a higher energy at 286.2 eV due to the formationf a covalent bond between carboxylic acid groups of poly-TCA and amine groups of anti-OPG. The peak observed at

86.2 eV for anti-OPG/AuNPs/polyTTCA corresponded to the–N bond. The N1s spectrum exhibited a clear peak at 396.7,fter the immobilization of anti-OPG, which was due to –N–Cond. The atomic percents of the N1s peak were 0 and 3.54

1598 K. Singh et al. / Biosensors and Bioelectronics 23 (2008) 1595–1601

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In order to determine the optimum quantity of anti-OPG at theelectrode surface, the EDC treated AuNPs/polyTTCA modifiedelectrode was incubated in an anti-OPG solution with variousdilution ratios in the range between 2000 and 50. Fig. 4a shows

ig. 2. (a) SEM images of AuNPs obtained at various scan rates in the electrocnd O1s peak (d) for polyTTCA/GCE, AuNPs/polyTTCA/GCE and HRP–OPG

efore and after immobilization of anti-OPG, respectively. Thisonfirms that anti-OPG was successfully immobilized onto poly-TCA.

.2. Activity of the HRP–OPG label

The labeling of HRP to OPG was confirmed by recordingV for a HRP–OPG/anti-OPG/polyTTCA modified electrode

n a PBS solution. A pair of redox peak was observed at 0 Vnd −0.1 V vs. Ag/AgCl, which corresponded to the oxidationnd the reduction of heme group present in HRP. The peak cur-ents were proportional to the scan rate indicating that the redoxeaction of HRP was a surface-confined process. The separationetween anodic and cathodic peaks increased with increasing thecan rate, indicating that the redox reaction was quasi-reversible.fter, confirming the HRP redox peak in HRP–OPG conju-ate, the activity of HRP labels on the OPG bound surface wasvaluated using the amperometric technique. The steady-stateesponse was obtained at a potential of −0.40 V vs. Ag/AgClfter adding various aliquots of H2O2 into a buffer solution ashown in Fig. 3. The HRP–OPG/anti-OPG/polyTTCA modified

lectrode exhibited a linear response between 0.1 and 2.0 mM.hese results clearly indicated that HRP was successfully con-

ugated with OPG and conjugated HRP has catalytic activityowards the reduction of H2O2.

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al deposition using cyclic voltammetry. XPS survey spectra (b), C1s peak (c),Ps/polyTTCA/GCE surfaces.

.3. Optimization of experimental parameters

ig. 3. Chronoamperometric response of HRP–OPG/AuNPs/polyTTCA modi-ed electrode towards different concentration of H2O2 at −0.40 V vs. Ag/AgCl.nset shows the corresponding calibration plot.

K. Singh et al. / Biosensors and Bioelectronics 23 (2008) 1595–1601 1599

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ig. 4. The effects of (a) dilution time of anti-OPG, (b) incubation time for im

2O2 and OPG concentrations were used to be 1 mM and 8 pg/ml, respectively

he effect of the anti-OPG amount on the reduction currentf H2O2. The H2O2 and OPG concentrations were used to bemM and 8 pg/ml, respectively. The dilution ratio of anti-OPGas varied from 2000 to 50. The concentration of anti-OPG

ncreased as the dilution ratio decreased. The current differ-nce (�I) increased as the concentration of anti-OPG increased.n increase of anti-OPG-HRP quantity at a low dilution would

mprove the efficiency of the electrocatalytic reduction of H2O2.owever, the reduction current did not change significantly at

he lower dilution ratio of anti-OPG than 250 due to the satura-ion of the active sites of polyTTCA for immobilizing anti-OPG.he maximum response was found at the dilution ratio of 250.hus, the dilution ratio of anti-OPG solution was selected as 250

or its immobilization onto a polyTTCA layer.The influence of the immunochemical incubation time (i.e.,

hen the antigen–antibody reaction occurs) on the responseignals (�I) was also investigated. Fig. 4b displays the effectf incubation time ranging from 5 to 35 min on immunoas-ay, using the same concentration of conjugate. The currentesponses obtained in this study increased with the incubationime rapidly up to 30 min and after that the current did not changeignificantly. After 30 min, the response reaches a saturationevel, which would imply that the competitive reaction betweenabeled and unlabeled antigens was complete after 30 min andhe surface was saturated with labeled antigens. The maximumesponse time was at the incubation time of 30 min. Thus, anncubation time of 30 min was used for the present study.

The effect of pH on the immunosensing response toward

0 pg/ml of OPG was studied between pH 5.5 and 8.5 as shownn Fig. 4c. The response gradually increased from pH 5.5 to 7.4nd then decreased over pH 7.4. The decreased response over pH.4 might have been due to the loss of anti-OPG or HRP activity.

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reaction, (c) pH and (d) applied potential on the immunosensor response. The

n our previous work (Rahman et al., 2004), we also observedhat over pH 7.4, the electrocatalytic reduction of H2O2 by HRPas found to be decreased due to the poor enzyme activity. Theaximum response was observed at the pH of 7.4. Thus, a pH

f 7.4 was used in all subsequent experiments.The effect of applied potential on the amperometric current

esponse was studied for anti-OPG/AuNPs/polyTTCA/GCEFig. 4d). The current response increased as the applied poten-ial went from −0.2 V to the more negative direction and the

aximum response was obtained at −0.4 V. The application ofore negative potential up to −0.5 V did not improve the current

esponse. Hence, −0.4 V was used for the final analysis.

.4. Interference effect and stability

The specificity of the present OPG immunosensor was eval-ated by measuring the response with an anti-OPG/AuNPs/olyTTCA electrode after blocking with a 0.3% casein solutionor other proteins, such as human IgG, human serum albu-in, bovine serum albumin, and human thrombin. The OPG

mmunosensor did not response to the above proteins even at aignificantly high concentration. Some electrochemically activeompounds, such as ascorbic acid, dopamine, uric acid, etc., inlood serum can be oxidized at positive potentials. However, thepplied potential was used as −0.4 V in this experiment and thus,he above biological compounds cannot be oxidized at this lowotential. Electrochemically reducible compounds, such as dis-olved oxygen can be reduced at the similar potential. However,

n this experiment, oxygen reduction was observed at more neg-tive potential (−0.55 V) and cannot be interfered. This resultuggests that the present OPG immunosensor is highly selectiveor the OPG detection.

1600 K. Singh et al. / Biosensors and Bioelectronics 23 (2008) 1595–1601

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ig. 5. (a) Chronoamperometric responses towards various concentration of freebtained for the HRP–OPG/anti-OPG/AuNPs/polyTTCA immunosensor.

The regeneration of the immunosensor surface is a key factoror developing a practical immunosensor. The immunosensorurface was regenerated by simply immersing it in a 0.1 Mlycine–hydrochloric acid solution for 5 min with stirring fol-owed by washing with water. The glycine–HCl solution wasrepared by mixing 0.1 M glycine and 0.1 M HCl solution inistilled water. A relative standard deviation (R.S.D.) of 4.8%as obtained when the immunosensor was repeatedly used six

imes in consecutive measurements. The long-term stability ofhe OPG immunosensor was also examined for every 5 days.fter each experiment, the immunosensor was washed with PBS

nd kept at 4 ◦C. The sensitivity of the immunosensor retained5% of its initial sensitivity over 1 month. The good stabilityight be due to the fact that anti-OPG molecules were attachedrmly onto the surface of AuNPs/polyTTCA.

.5. Calibration plot

The HRP–OPG/anti-OPG/AuNPs/polyTTCA electrode wasnally examined as an immunosensing probe for the competitive

mmunoassay of OPG as shown in Fig. 5a. As the concentrationf free-OPG in the solution increased, the activity of HRP–OPGonjugate decreased on the electrode surface. Thus, the cur-

ent response for the reduction of H2O2 was observed to beecreased. A reduction signal was first observed in the pres-nce of 1 mM H2O2 in a PBS solution. A gradual decrease inhe current was observed with the addition of free-OPG. The

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Fig. 6. Standard addition plots for real sample analysis: (a) healthy

. Inset shows the effect of interference from other proteins. (b) Calibration plot

ecrease in the current response could be attributed to the for-ation of the immunocomplex between anti-OPG and OPG

antibody/antigen). The result indicates that the immunosensors capable of determining OPG concentrations ranging from 2.5o 25 pg/ml. The reproducibility expressed in terms of the rel-tive standard deviation (R.S.D.) was 4.7% (n = 5) at the OPGoncentration of 5 pg/ml. The calibration plot was constructedy taking the difference in the current response before and afterhe addition of OPG as shown in Fig. 5b. The calibration plotas found to be linear between 2.5 and 25 pg/ml for the com-etitive analysis. This linear range yielded a regression equationf I (nA) = (128 ± 5.0) + (9.44 ± 0.35) × [OPG] (pg/ml), with aorrelation co-efficient of 0.997 (n = 6). The detection limit wasetermined to be 2.0 pg/ml.

.6. Real sample analysis

To examine the practical application, the HRP–OPG/anti-PG/AuNPs/polyTTCA sensor was applied for the detectionf OPG in healthy human (n = 5) and OP patient’s (n = 5)erums samples. The standard addition method was followedor determining the OPG content in serum samples. Fig. 6hows standard addition plots for the real sample analysis.

he average OPG content was determined to be 79 ± 7 and40 ± 11 pg/ml for healthy and OP patient’s serum samples,espectively. The OPG content in the healthy human serum sam-le determined by the present method can be compared with

human serum and (b) osteoporosis patient’s serum samples.

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Skladal, P., Jı́lkova, Z., Svoboda, I., Kolar, V., 2005. Biosens. Bioelectron. 20,

K. Singh et al. / Biosensors and

he result obtained from an ELISA method (Kruk et al., 2002)here OPG level was determined in the range between 49 and30 pg/ml (mean 77 ± 22 pg/ml, median 74 pg/ml). This indi-ates that our immunosensor method is comparable to ELISAethod for the detection of OPG. The OPG content in an OP

atient’s serum was found to be higher than that obtained in aealthy human serum. This result is consistent with a previouslyeported result using ELISA method (Grigorie et al., 2003).

. Conclusions

An OPG immunosensor has been fabricated by cova-ently immobilizing anti-OPG on the AuNPs depositedolyTTCA layer. The OPG–HRP/anti-OPG/AuNPs/polyTTCAmmunosensor gave high sensitivity with a dynamic rangeetween 2.5 and 25 pg/ml. The detection limit was determinedo be 2.0 pg/ml, which is lower than that obtained in a previousork (Skladal et al., 2005). The present immunosensor was suc-

essfully applied to the healthy human’s and OP patient’s serumamples for the detection of OPG.

cknowledgements

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

ully acknowledged. One of the authors, Kanika Singh, ishankful to Korean Research Foundation grant funded by Koreanovernment (KRF-2005-211-D00203) and for the permissionrom IGNOU, New Delhi, India.

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