Biosensors and Bioelectronics 42 (2013) 439–446

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Biosensors and Bioelectronics

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Highly sensitive immunosensing of prostate-specific antigen based onionic liquid–carbon nanotubes modified electrode: Application ascancer biomarker for prostate biopsies

Abdollah Salimi a,b,n, Begard Kavosi a, Farden Fathi c, Rahman Hallaj a

a Department of Chemistry, University of Kurdistan, P.O. Box 416, Sanandaj, Iranb Research Center of Nanotechnology, University of Kurdistan, P.O. Box 416, Sanandaj, Iranc Cellular and Molecular Research Center, Kurdistan University of Medical Sciences, Sanandaj, Iran

a r t i c l e i n f o

Article history:

Received 7 July 2012

Received in revised form

12 October 2012

Accepted 15 October 2012Available online 23 October 2012

Keywords:

Tumor marker

Electrochemical immunosensor

Ionic liquid

Carbon nanotube

Prostate specific antigen

63/$ - see front matter & 2012 Elsevier B.V. A

x.doi.org/10.1016/j.bios.2012.10.053

esponding author at: Department of Chemis

416, Sanandaj, Iran. Tel.: þ98 871 6624001;

ail addresses: absalimi@uok.ac.ir, absalimi@ya

a b s t r a c t

A novel and highly sensitive electrochemical immunosensor was developed for detection of prostate

specific antigen (PSA) based on immobilization of PSA-antibody (anti-PSA) onto robust nano composite

containing multiwalled carbon nanotubes (MWCNTs) and ionic liquid (IL)1-buthyl-methylpyrolydi-

nium bis (trifluromethyl sulfonyl) imide [C4mpyr][NTf2]. Thionine was used as redox system for

electrochemical probe. The adsorbed thionine displayed a surface-controlled electrode process with

electron transfer rate constant of 6.5 s�1 and surface coverage of 3.1�10�11 mol cm�2. The PSA

antibody (anti-PSA) was immobilized or entrapped in the nanocomposite and used in a sandwich type

complex immunoassay with anti-PSA labeled with horseradish peroxidase (HRP) as secondary antibody

and H2O2 as the substrate. Under optimal conditions, the DPV peak current of the immunosensor

increased linearly with increasing PSA concentration in two concentration ranges, 0.2–1.0 and 1–

40 ng ml�1 with detection limit 20 pg ml�1. The intra-assay and inter-assay coefficients of variation

(CVs) for five replicate determinations were 2% and 1.35% at 5 ng ml�1 PSA. The immunosensor has

been used for PSA detection in serum samples and the accuracy of the method was demonstrated by

comparison to the standard ELISA assay. Furthermore, the immunosensor can be used for detection of

PSA in prostate tissue samples. The simple one-step fabrication method, high sensitivity, good

reproducibility and repeatability as well as acceptable accuracy for PSA detection in human serum

samples are the main advantages of this immunosensor. Furthermore, the results indicate that

differences in PSA concentration can be detected with the proposed immunosensor for cancer tissue

samples which holds great promise in clinical screening of cancer biomarkers.

& 2012 Elsevier B.V. All rights reserved.

1. Introduction

Detection of tumor markers plays an important role in screen-ing, diagnosing and evaluating the prognosis of the diseases (Wuet al., 2007). Prostate specific antigen (PSA) levels have beenidentified as the most reliable tumor marker for the earlydiagnostics of prostate cancer (Panini et al., 2008); therefore,developing a simple, rapid and non-invasive method with lowcost for PSA detection is a challenge for cancer diagnosis.

Immunoassays with high selectivity and affinity of antibodymolecules to their corresponding antigens have been widely used inclinical diagnosis (Okuno et al., 2007). Electrochemical immunoassaysand immunosensors based on antigen–antibody interaction have

ll rights reserved.

try, University of Kurdistan,

fax: þ98 871 6624008.

hoo.com (A. Salimi).

recently attracted considerable interest due to their high sensitivity,low cost, simple instrumentation, ease of miniaturization, highsensitivity, fast response, low detection limit, wide dynamic rangeand good compatibility (Wang et al., 2006a; Okuno et al., 2007;Mantzila et al., 2008; Zhou et al., 2009; Du et al., 2010; Huang et al.,2010; Tang and Ren, 2010). They provide a sensitive and selective toolfor the determination of immunoreagents by detecting the changes ineither potential, current, capacitance, conductance or impedance thatare induced by a specific biorecognition reaction (Hayes et al., 2007).The immobilization of immunoreagent onto the electrode surface is acrucial step for the construction of the electrochemical immunosen-sors. Furthermore, sensitivity of an electrochemical immunosensorfor antigen detection at very low concentration range can beincreased by increasing the loading amount of antibodies as well ascontrolling the orientation of immobilized antibodies on electrodesurfaces (Darain and Shim, 2003).

Different materials including organically modified sol–gel, titaniasol–gel and gold alumina derived sol–gel (Tan et al., 2006), self

A. Salimi et al. / Biosensors and Bioelectronics 42 (2013) 439–446440

assembled monolayer of thiol and dendrimer (Namgung et al., 2009),colloidal Au particles modified electrode (Wang et al., 2004) andpolymeric film (Dong et al., 2006) have been used for immobilizationof antibodies. Low reproducibility, high detection limit and lack ofsensitivity are the disadvantages of proposed immunosensors.

Due to biocompatibility and large specific surface area ofnanomaterials, the nanoparticle-based amplification process hasdramatically enhanced the intensity of the electrochemical signal.Therefore, the combination of nanostructured materials withelectrochemical immunoassay opens new horizons for highlysensitive detection of biomarkers (Daniel and Astruc, 2004;Tang and Ren, 2010). Different nanomaterials, such as carbonnanotubes (CNTs) (Chen et al., 2009), gold nanoparticles (Zhouet al., 2009; Tang and Ren, 2010), Pt nanoparticles (Zhang et al.,2010), Au–Fe3O4 nanoparticles (Wei et al., 2010), silica nanopar-ticles (Wang et al., 2006b), carbon nanoparticles (Ho et al., 2009),carbon nanospheres (Du et al., 2010), CNTs/gold nanoparticles( Huang et al., 2010; Mao et al., 2010) and In2O3 nanowires(Li et al., 2005) have been widely used for the immobilization ofantibodies and construction of electrochemical immunosensors.

Carbon nanomaterials, especially CNTs, have attracted consid-erable attention due to their extraordinary physical properties,good chemical stability and remarkable conductivity (Katz andWillner, 2004). It has also been demonstrated that the electro-catalytic activity of CNTs strongly depends on the dispersingagent used to immobilize them on the electrode surface(Pauliukaite et al., 2009). Due to nonheterogeneity of distributedCNTs on the electrode surfaces, the reproducibility of fabricationprocess and electrode response is decreasing. Therefore, thesolvent for solubilization and homogenization of CNTs is extre-mely important. The composites of CNTs with chitosan (Huanget al., 2010) and nafion (Chen et al., 2009) exhibited improvedrobustness and facilitated immobilization of antibodies. Anotherimportant class of novel materials for various electrochemicalapplications is ionic liquids (IL), due to high stability and electricalconductivity, very low vapor pressure, good conductivity andwide electrochemical window (Buzzeo et al., 2004; Maleki et al.,2006). Furthermore, due to p–p or cation–p interaction betweenionic liquids and carbon nanotubes, the CNTs–IL composite hadshown potential application in the fabrication of electrochemicalsensors and biosensor (Zhu et al., 2010). The combination of theroom temperature ionic liquid (RTIL) and CNTs can create uniquematerials for fabrication of electrochemical sensors and biosen-sors. In the present study the nanocomposite containing MWCNTsand IL was used for entrapment of antibody and also forimmobilization of thionine as electron transfer mediator. Here,PSA was used as a model cancer biomarker and demonstrated theamplification process in sandwich detection. The resulted elec-trode exhibits high stability and reproducibility. The proposedimmunosensor was applied to determine PSA in human serumsamples and prostate cancer cells with satisfactory results usingdifferential pulse voltammetry technique.

2. Experimental

2.1. Materials

Primary anti-human prostate specific antigen (anti-PSA anti-body), tracer secondary anti-PSA antibody labeled with horse-radish peroxidase (HRP) and PSA were purchased from CanAgcompany (Gothenburg, Sweden). Multiwall carbon nanotubeswith purity 95% (10–20 nm diameter), 1 mm length and surfacespecific area of 480 m2/g were obtained from Nanolab (Brighton,MA). Ionic liquid, 1-buthyl-methylpyrolydinium bis(trifluro-methyl sulfonyl)imide [C4mpyr][NTf2], was obtained from Sigma.

H2O2, thionine (TH), K3Fe(CN)6 and K4Fe(CN)6 and all otherreagents of analytical grade were from Merck or Fluka. Phosphatebuffer solution with pH 7 was used for all electrochemicalmeasurements. Solutions were deaerated by bubbling high purity(99.99%) argon gas through them prior to the experiments.

2.2. Apparatus and procedures

Electrochemical experiments were performed with a computercontrolled m-Autolab modular electrochemical system (Eco Che-mie Ultecht, The Netherlands), driven with GPES software (EcoChemie). A conventional three-electrode cell was used with a Ag/AgCl/(sat KCl) as the reference electrode, a Pt wire as the counterelectrode and a disk glassy carbon electrode (GCE) (modified andunmodified) as the working electrode. Voltammetry on ILs/MWCNTs/TH coated electrode was done in buffers free of thio-nine. The morphologies of the surface were observed in a Vega-Tescan electron microscope. The differential pulse voltammetrymeasurements were performed by scanning the potential from�0.1 to �0.5 V with pulse amplitude of 50 mV and pulse width of50 ms.

2.3. Preparation of MWCNTs–ILs–thionine nanocomposite

modified electrode

The GCE was first carefully polished with alumina on polishingcloth. The electrode was placed in an ethanol container and bathultrasonic cleaner was used in order to remove adsorbed parti-cles. The nanocomposite was prepared by mixing 10 ml of IL and3 mg of CNT. By casting nanocomposite onto the surface of a GCEand drying under room temperature, the nanocomposite modifiedGCE was fabricated. By immersing the modified electrode in PBS(pH¼7) containing 1 mM of thionine and applying 10 consequentpotential cycling at potential range from 0.5 to �0.7 V, a stablethin film of thionine deposited onto nanocomposite. After rinsingwith water, modified electrode can be used for electrochemicalexperiments or it was stored at ambient condition when notin use.

2.4. Fabrication of immunosensor and detection of PSA

The GCE modified with IL/MWCNTs/TH was washed with PBSand immersed in buffer solution containing anti-PSA (0.5 mg/ml)and 3% BSA at 4 1C for 2 h. The reaction steps for the preparationof the immunosensor were shown (Scheme 1 of Supplement).The prepared immunosensor was stored at 4 1C when not in use.The buffer solutions containing 4 pM ml�1 of HRP-anti-PSA anti-body and different concentrations of PSA were prepared andincubated at 25 1C for 30 min. The GCE/IL/MWCNTs/TH/anti-PSAimmunosensor was incubated with 10 ml of buffer solution con-taining HRP labeled-anti-PSA and different concentrations of PSAfor 20 min in order to form the sandwich immunocomplex. Theimmunosensor was then placed in electrochemical cell containing1 ml PBS, pH 7 and hydrogen peroxide with concentration of2.5 mM. Detection of PSA level was performed by measuring thedecrease of catalytic peak current.

2.5. Preparation of prostate cancer cells for PSA detection

The human prostate cancer cell line LNCaP and normal humanprostate cell were obtained from the national cell bank of Iran(Pasteur institute of Iran, Tehran) and were cultured in RPMI-1640 (Gibco), supplemented with penicillin/streptomycin (100 U/ml and 100 mg/ml, respectively) and 10% fetal bovine serum, at37 1C in a humidified atmosphere of 5% CO2. Trypsin-separatedLNCaP and normal human prostate cell were fixed in 70% ethanol

A. Salimi et al. / Biosensors and Bioelectronics 42 (2013) 439–446 441

for 20 min at 4 1C. The cells were then washed two times withdistilled water and were used for PSA analysis. For each sampleapproximately 5000, 10000 and 15000 cells were captured andsubsequently processed for protein extraction. Briefly, 100 mlof T-PER (tissue protein extraction buffer) was placed in eachtube with the enclosed caps, the tubes were inverted to ensurethat the buffer was adequately dispersed to cover the surface ofthe caps and vortexed for 10 s, followed by incubation on ice for10 min. The extraction solution was then recovered by centrifu-gation at 14000 rpm for 2 min, then stored at �80 1C until readyfor analysis. 2 ml of antibody-labeled HRP was added to10 ml ofextraction solution. After incubation at 25 1C for 20 min, themixture was rinsed onto the nano-composite modified electrode.Fresh tissue biopsies were obtained from patients who had beenreferred to Besat Hospital Medical Center (Sanandaj, Iran).The samples were carried to the lab in Hanks’ balanced saltsolution. Tissue specimens were mechanically and enzymaticallydissociated by treatment with DNAse and collagenase. The 5000isolated cells were washed with PBS and PSA extracted with100 ml of T-PER.

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Fig. 1. (A) Cyclic voltammograms of (a) GC, (b) GC/ILs/MWCNTs modified

electrode, (c) GC/ILs/MWCNTs/anti-PSA and (d) GC/ILs/MWCNTs/anti-PSA/PSA/

anti-PSA-labeled HRP in PBS pH 7 containing 2.5 mM of Fe(CN)63�/4� at scan rate

50 mV s�1. (B) Cyclic voltammograms of (a) GC, (b) GC/ILs/MWCNTs modified

electrode, (c) GC/ILs/MWCNTs/TH modified electrode, (d) GC/ILs/MWCNTs/TH/

anti-PSA immunosensor, (e) GC/ILs/MWCNTs/TH/anti-PSA/PSA and (f) GC/ILs/

MWCNTs/TH/anti-PSA/PSA/anti-PSA labeled HRP in pH 7 PBS at scan rate

100 mV s�1.

3. Results and discussion

3.1. Characterization of IL/MWCNTs/thionine modified glassy

carbon electrode

The typical SEM images of GCE modified with MWCNTs andMWCNTs/ILs are shown (Fig. 1 of Supplement). It can be seen thatMWCNTs were cross-linked with each other to form a highlyporous structure while for MWCNTs/IL nanocomposite, a uniforminterface appeared with ionic liquids inside the porous structureof MWCNTs. Cyclic voltammograms of IL/MWCNTs/thionine mod-ified GCE at different scan rates were shown (Fig. 2 ofSupplement). As illustrated, a well defined redox couple withEo0¼ �0.22 V vs. Ag/AgCl electrode is observed for immobilized

thionine. For GC/MWCNTs/IL modified electrode no obviouselectrochemical peak is found at a potential range from 0.2 to�0.45 V. As shown in the inset of Fig. 2 of Supplement the peakcurrents linearly increased with increase in the scan rate from 10to 1000 mV s�1 as expected for redox surface controlled process.Based on the Laviron theory (Lavion, 1979) the electron transferrate constant (ks) and charge transfer coefficient (a) can bedetermined by measuring the variation of peak potential withscan rate. The a and ks were estimated as 0.53 and 6.5 s�1,respectively, suggesting that IL/MWCNTs nanocomposite has highability for promoting electron between thionine and electrodesurface. The effect of thionine concentration on the sensorresponse was evaluated by immersing the GC/MWCNTs/IL mod-ified electrode in PBS (pH¼7) containing different concentrationsof thionine (0.1–2 mM) and applying 10 consequent potentialcycling at a potential range from 0.5 to �0.7 V at scan rate 100mV s�1. The results indicate that a significant increase of peakcurrent was observed between 0.1 and 1 mM, then peak currentwas leveled off for higher concentration. Therefore, 1 mM ofthionine solution was used as optimum concentration value forelectrode modification.

The stability of the modified electrode was examined byrepetitive recording of the cyclic voltammograms in pH 7 PBS(Fig. 3 of Supplement). After the modified electrode was succes-sively scanned for 200 cycles at a scan rate of 100 mV s�1 inworking buffer, no changes were observed for peak current andpeak potential separation. In addition, it was found that thevoltammetric response of the modified electrode decreased by5% when it was stored in ambient condition for four weeks.The high stability of immobilized thionine against desorption

in aqueous solution is related to chemical and mechanicalstability of CNTs–IL film, the strong interaction of aromatic groupof thionine complex with p-staking of carbon nanotubes orp–p interaction between IL–thionine and CNTs.

3.2. Electrochemical characterization of the immunosensor

Cyclic voltammograms of different modified electrodes inbuffer solution containing 2.5 mM of K3Fe(CN)6 and K4Fe(CN)6

are presented in Fig. 1A. At the bare GCE, the Fe(CN)63�/4� redox

label revealed reversible cyclic voltammogram (Fig. 1A, voltam-mogram ‘‘a’’). After modification of GCE with CNTs/ILs nanocom-posite, the peak current increased greatly due to increasingeffective surface area in the presence of carbon nanotubes(Fig. 1A, voltammogram ‘‘b’’). When anti-PSA was immobilizedon the electrode surface, the peak current clearly decreased.This result indicates the immobilization of anti-PSA antibody onthe electrode surface reducing the effective surface area andavailable active sites for electron transfer process (Fig. 1A, vol-tammogram ‘‘c’’). After the immunosensor was incubated in anincubation solution containing 2 ng ml�1 PSA, it could be foundthat the voltammetric peak current was further decreased (Fig. 1A,voltammogram ‘‘d’’), which indicated the immunocomplex for-mation between anti-PSA and PSA. Therefore, the insulating anti-PSA and PSA layers on the electrode surface hindered the electrontransfer. The immobilization of anti-PSA and immunocomplex

A. Salimi et al. / Biosensors and Bioelectronics 42 (2013) 439–446442

formation between PSA and anti-PSA were investigated byrecording cyclic voltammogram of IL/MWCNTs/TH modified elec-trode at different experimental condition. For IL/MWCNTs/THmodified electrode, a well-defined redox couple was observed(Fig. 1B). After immersing the modified electrode in anti-PSAsolution for 2 h, the peak current clearly decreased, whichindicated the immobilization of anti-PSA on the electrode surface.The associated bulky antibody provides an effective barrier toelectron transfer of redox probe. Subsequently, the modifiedimmunosensor was incubated in PSA and HRP labeled anti-PSAfor 30 min. As illustrated the oxidation and reduction peakcurrents decreased obviously which could be attributed tothe immunocomplex formation between antibody and antigen.The produced immunocomplex shielded the active centers ofmediator and decreased its ability in electron transfer process(Li et al., 2005).

The immunosensor fabrication process and the formation ofantigen–antibody complex were also characterized by electro-chemical impedance spectroscopy (EIS). Fig. 2 shows the Nyquistplots during stepwise construction of immunosensor. The straightline at low frequency is related to the diffusion process known asWarburg element, while the high frequency semicircle is relatedto the electron transfer resistance (Ret), which controls theelectron transfer kinetics of the redox probe at the electrodeinterface. For bare GCE and GCE/IL/MWCNTs nanocomposite andGCE/IL/MWCNTs/TH modified electrode, it can be seen that analmost straight line is exhibited, which was characteristic of thediffusion controlled electrochemical processes (Fig. 2, plots ‘‘a, b,c’’). These results indicate very low resistance of nanocompositemodified electrode for redox probe. Subsequently, when the anti-PSA was loaded on the nanocomposite modified electrode, Ret

increased dramatically, 4000 O (Fig. 2, plot ‘‘d’’), suggesting thatthe antibody was immobilized on the electrode surface. Whenanti-PSA adsorbed, a hydrophobic layer of the protein insulatesthe conductive support and perturbs the interfacial electrontransfer. After the resulting immunosensor was incubated in10 ng ml�1 PSA, the Ret is increased to 5000 O (Fig. 2, plot ‘‘e’’).The interaction between antibody–antigen decreased the electrontransfer kinetics of the redox probe due to insulating proper-ties of formed immunocomplex. Ret is increased (7000 O) inthe same way after anti-PSA labeled HRP attached on the result-ing electrode surface (Fig. 2, plot ‘‘f’’). The attachment ofmore hydrophobic protein layer on the electrode surfacedecreases the electron transfer kinetics. Therefore, the EIS and

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GC (a)GC/Composite (b)GC/Composite/Th (c)GC/Composite/Th/Ab1 (d)GC/Composite/Th/Ab1/Anti-PSA (e)GC/COmposite/Th/Ab1/Anti-PSA/Ab2 (f )

Fig. 2. Electrochemical impedance spectroscopy of (a) GC, (b) GC/ILs/MWCNTs,

(c) GC/ILs/MWCNTs/TH (d) GC/ILs/MWCNTs/TH/anti-PSA, (e) GC/ILs/MWCNTs/TH/

anti-PSA/PSA and (f) GC/ILs/MWCNTs/TH/anti-PSA/PSA/anti-PSA-labeled HRP in

PBS pH 7 containing 2.5 mM of Fe(CN)63�/4 and 0.1 M KCl at the frequency range

from 0.1 Hz to 10 kHz.

CV measurements indicate the successful stepwise assembly ofthe immunosensor.

3.3. Amplification of biocatalyzed anti-PSA labeled with HRP

HRP can catalyze the oxidation reaction of thionine by H2O2.The cyclic voltammogram of GCE/IL/MWCNTs/TH/anti-PSAimmunosensor in buffer solution containing 2.5 mM of H2O2

shows a well-defined cyclic voltammogram, which correspondsto the electrochemical response of thionine (Fig. 4 of Supplement,voltammogram ‘‘a’’). As shown, no catalytic current is observedfor H2O2 reduction at the proposed immunosensor in the absenceof PSA. Same results have been observed for PSA and AFPimmunosensors (Du et al., 2010). After incubating the immuno-sensor with 10 ml of buffer solution containing 2 ng ml�1 PSA andanti-PSA labeled HRP, the oxidation peak current decreased andthe reduction peak current increased due to electrocatalyticactivity of immobilized HRP toward H2O2 reduction (Fig. 4 ofSupplement, voltammogram ‘‘b’’). With increasing of PSA concen-tration to 4 ng ml�1, the electrocatalytic current increased sig-nificantly (Fig. 4 of Supplement, voltammogram ‘‘c’’). Theseresults indicate that the HRP attached to the immunosensorsurface retains high catalytic activity and that the thionine is asuitable electron transfer mediator. The following mechanismshows the enzymatic catalytic reduction of H2O2:

HRP ðredÞþH2O2-HRP ðoxÞþH2O ð1Þ

Thionine ðredÞþHRP ðoxÞ-HRP ðredÞþThionine ðoxÞ ð2Þ

Thionine ðoxÞþ2e�-Thionine ðredÞ ð3Þ

3.4. Optimizing conditions for immunoassay detection

3.4.1. pH optimizing

Since electrochemical activity of thionine and biocatalyticactivity of HRP are pH dependent, the effect of pH on theimmunosensor response was investigated. The immunosensorresponse for 10 ng ml�1 of PSA in buffer solutions with differentpH values containing 2.5 mM of H2O2 was evaluated by recordingdifferential pulse voltammograms. As shown in Fig. 3A, the peakcurrents increase with increasing of pH value from 5 to 7 anddecreases when pH value increases further. Since maximumcurrent response occurs at pH 7, this value was chosen as theoptimum value for PSA measurement.

3.4.2. Optimization of H2O2 concentration

The performance of the electrochemical enzyme-catalyzedanalysis was related to the concentration of H2O2 in the measur-ing system. DPV response of the resulting GCE/IL/MWCNTs/anti-PSA/PSA/anti-PSA labeled HRP immunosensor for 10 ng ml�1 ofPSA in pH 7 buffer solution containing different concentrations ofhydrogen peroxide was recorded. As shown in Fig. 3C, the DPVpeak current of the immunosensor increased with increasingH2O2 concentration from 1 to 2.5 mM, and then started to leveloff. Thus, 2.5 mM of H2O2 was chosen as optimum concentrationfor the determination of PSA.

3.4.3. Optimization of incubation time

The incubation time is an important parameter for immuno-complex formation between antibody and antigen. For optimiza-tion of incubation time between anti-PSA and PSA, the GCE/IL/MWCNTs–anti-PSA immunosensor was incubated for differenttimes in the presence of PSA (10 ng ml of PSA). As shown inFig. 3B, the DPV peak current response of the immunosensor wasrapidly increased with increasing incubation time from 5 to

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anti-PSA labeled HRP after incubation with (a) 0.0, (b) 0.2, (c) 0.4, (d) 0.6, (e) 0.8, (f)

1, (g) 2, (h) 4, (i) 6, (j) 8 (k) 10, (l) 15, (m) 20, (n) 25, (o) 30, (p) 35 and Q (40) ng

ml�1 of PSA in PBS, pH 7 containing 2.5 mM of H2O2. Insets, plots of the

immunosensor response vs. PSA concentration.

A. Salimi et al. / Biosensors and Bioelectronics 42 (2013) 439–446 443

20 min and then leveled off slowly. Therefore, 20 min was used asthe optimum incubation time for PSA detection. As shown inFig. 3D the optimized incubation time between PSA and anti-PSAlabeled HRP is 30 min.

3.5. Electrochemical response of the immunosensor to PSA

Under optimal conditions, the DPV of the immunosensor in thepresence of different concentrations of PSA was recorded.As shown in Fig. 4, the bioelectrocatalytic current of the immu-nosensor increased with increasing PSA concentration in twoconcentration ranges. For concentration range 0.2–1 ng ml�1,the regression equation was I (mA)¼0.95[PSA]ng ml�1

�0.083 mAand the correlation coefficient was 0.997. The sensitivity was0.95 mA ng ml�1 and detection limit was 20 pg ml�1 basedon signal to noise ratio of 3. For higher concentration rangeof PSA, 1–40 ng ml�1, the regression equation was I (mA)¼0.1143[PSA] ng ml�1

þ0.923 mA with correlation coefficient andsensitivity 0.998 and 0.1143 mA ng ml�1, respectively. The reasonthat calibration curve present two linear concentration rangeswith different slopes is that the amount of antibody immobilizedon the nanocomposite surface is some certain. When the immu-nosensor is used to detect antigen (PSA), PSA can bind with activesites of antibody which was immobilized on the electrode surface.With increasing of antigen concentration, active sites of antibodyon the immunosensor become fewer. Therefore, the sensitivitydeclines when the immunosensor is used to determine a higherconcentration of PSA and calibration curves with different slopeswere observed. The same phenomena were reported for otherantigen immunosensors (Yuan et al., 2009; Dai et al., 2005; Yuanet al., 2007). The analytical parameters of the present immuno-sensor for PSA detection were comparable to or better than otherPSA immunosensors (Table 1A). Furthermore, the simple and one

step fabrication process is a major advantage compared todifferent PSA immunosensors using special reagents and multi-step amplification methods.

3.6. Reproducibility, stability and regeneration of the immunosensor

The reproducibility of the immunosensor was evaluated byfabricating six modified electrodes independently at the same

Table 1AAnalytical parameters for detection of PSA with different immunosensors.

Electrode Electrochemical technique Detection limit (ng/ml) Concentration range (g/ml)

PGa/SWCNTsb (Yu et al., 2006) Amperometry 0.004 –

GCEc/GSd (Wei et al., 2010) 0.005 0.01–10

OECTe/AuNPf (Zhang et al., 2010) CVn 0.001 0.1–100

GEg/gold colloids (Liu, 2008) CV 3.4 4.0–13

Pt/SWCNTs (Okuno et al., 2007) DPVo 0.25 –

Graphene-modified GC (Shoujiang et al., 2011) ECLp 0.008 0.010–8

DMEh/DBAi (Shusheng and and Ping,2007) CV 0.10 0.20–16.0

SPEj (Priyabrata et al., 2002) Amperometry 0.25 –

SPE/Polymer GE/FCk-peptide (Ning et al., 2010) DPV 0.2 0.5–40

GE/silica nanoparticles (Bo et al., 2008) LSV 0.76 1–35

SPE-nAu (Vanessa et al., 2009) CV 1 1–10

GCE/MWCNTsl/polytyrosine (Yuan and Robin, 2010) CV 0.0022 –

GS/Quantum dot (Minghui et al., 2011) SWVq 0.003 0.005–10

GE/thiol (Songqin and Zhang, 2008) CV 1.1 –

GCE/ILsm/CNTs (this study) DPV 0.020 0.2–40

a Pyrolytic graphite.b Single walled carbon nanotubes.c Glassy carbon electrode.d Graphene sheet.e Organic electrochemical transistor.f Gold nanoparticle.g Gold electrode.h dropping mercury electrode.i 3,4-diaminobenzoic acid.j Screen-printed electrode.k Ferrocene.l Multi walled carbon nanotubes.m Ionic liquids.n Cyclic voltammetry.o Differential pulse voltammetry.p Electrochemical luminescence.q Square wave voltammetry.

Table 1BDetection of PSA in serum samples with two methods.

Sample Proposed method (ng/ml) Reference method (ELISA) (ng/ml)

1 570.15 5.2

2 2.570.10 2.6

3 3.8770.2 4

4 1.970.2 2

A. Salimi et al. / Biosensors and Bioelectronics 42 (2013) 439–446444

experimental condition, and using them for detection of 5 and0.4 ng ml�1 PSA. As shown in Fig. 5 of Supplement, the same DPVwas observed for six immunosensors. The relative standarddeviations were 2% and 2.6% for detection of 5 and 0.4 ng ml�1

PSA, respectively. These results suggested the acceptable fabrica-tion reproducibility and precision of the immunosensor. The longtime stability of the immunosensor was also investigated bystoring it in 4 1C in refrigerator. The response of the immunosen-sor for 5 ng ml�1 of PSA after three weeks was about 97(72)% ofits initial response. For 0.4 ng ml�1 of PSA the same stability wasobserved and after one week the immunosensor response wasabout 98.5% of its initial response. Regeneration is a key factor fordeveloping a practical immunosensor. After the immunosensorwas used to detect PSA, the modified electrode was treated with4 M urea solution for 30 min to dissociate antigen–antibodylinkage. After rinsing with distilled water, the regenerated immu-nosensors were used to detect the same PSA concentration.The RSD of 1.3% was obtained for five regenerated immunosen-sors at the PSA concentration of 5 ng ml�1. The effect of PSAconcentration on reproducibility of the system was also investi-gated. In the presence of 1 and 20 ng ml�1 of PSA for 5 regener-ated immunosensors the RSD were 1.5% and 1.8% respectively.This result indicates that the antibody has the same activity at awide concentration range of PSA.

3.7. Application of immunosensor for PSA detection in clinical

serum samples

The immunosensor was used for detection of PSA in fourhuman serum samples containing different concentrations ofPSA, using the standard addition method. The amount of PSAwas examined by the proposed immunosensor and ELISA method

(Table 1B). As indicated, good accuracy was observed betweenELISA method and the proposed immunosensor.

3.8. Detection of PSA in human prostate cancer biopsies

The proposed immunosensor was also used for detection ofPSA in prostate cancer biopsies. The normal prostate tissue wasused as control signal for no cancer cell. Micrograph of prostatecancer biopsies is shown in Fig. 5A. We can see the tumor andnormal cells. Fig. 5B shows the DPV response of the immunosen-sor in the presence of extracted PSA from various numbers ofcancer cells. As can be seen, the voltammetric response increasedwith increasing cancer cells number (500, 1000 and 1500 cells).Furthermore, as illustrated, PSA was easily determined inapproximately 500 cancer cells, which indicates the high sensi-tivity of the proposed immunosensor. In another experiment fordetection of PSA in cancer cells, 10 ml of T-PER containingextracted PSA from approximately 500 cancer cells of biopsyprostate cancer (two different patients) and normal humanprostate cells were used (Fig. 5C). As illustrated, in comparisonto normal human prostate cells (Fig. 5C voltammogram ‘‘b’’) forprostate cancer samples the immunosensor response increased(Fig. 5C voltammograms ‘‘c and d’’). It can be seen that for normalprostate tissue immunosensor shows response, which indicates

4

5

6

7

8

9

10

11

-0.35 -0.25 -0.15 -0.05Potential(v)vs.Ag/AgCl

Cur

rent

(μA

)

Probe(GC/IL/Th/Anti Body)Extracted PSA from 500 cellsExtracted PSA from 1000 cells

Extracted PSA from 1500 cells

3

5

7

9

11

13

-0.35 -0.3 -0.25 -0.2 -0.15 -0.1Potential(v)vs.Ag/AgCl

Cur

rent

(μA

)

a

b

ca:Probe(GC/IL/Th/Anti Body)b:Normal human prostate cells

c:1000 cancer prostate cellsfor patient 1

d: 1000 cancer prostate cellsfor patient 2

Fig. 5. (A) Micrograph of prostate cancer biopsy. (B) Differential pulse voltammograms of GC/ILs/MWCNTs/TH/anti-PSA/PSA/anti-PSA labeled HRP in the absence of

prostate cells(a), (b–d) after incubation with extracted PSA from 500, 1000 and 1500 prostate cancer cells in pH 7 PBS containing 2.5 mM of H2O2. (C) DPV of GC/ILs/

MWCNTs/TH/anti-PSA/PSA/anti-PSA labeled HRP in the absence of prostate cells (a), after incubation with extracted PSA from 1500 normal human prostate cells (b), (c)

and (d) after incubation with extracted PSA from 1000 prostate cancer cells for two patients.

A. Salimi et al. / Biosensors and Bioelectronics 42 (2013) 439–446 445

that PSA is normally present in healthy men. Results obtained withcomparison to PSA standard in serum were as follows, sample c, 14(ag) PSA/cells and sample d, 40 (ag)/cells. These results indicate thatdifferences in PSA concentration can be detected with MWCNTs/ILsimmunosensor for cancer tissue samples.

4. Conclusion

Herein, a novel immunosensor based on combination of IL andMWCNTs for detection of PSA is reported. The combination ofCNTs with high active surface area and biocompatibility of ILsincreased the entrapment of electron transfer mediator (thionine)and anti-PSA in proposed nanocomposite, which enhanced thesensitivity of the method significantly. The results indicated thatthe ILs/CNTs based immunosensor can be used for detection ofPSA at low concentration range pg ml�1, and it has enough sensitivityfor PSA detection in human serum samples with good precisionand accuracy. Furthermore, the proposed immunosensor can beused successfully for quantitatively measuring PSA in prostatetissue samples. Excellent stability, simplicity of immunosensor

preparing without special reagents and complex-multistep process,low detection limit, high sensitivity and good reproducibility are themain advantages of the proposed system in comparison to other PSAimmunosensors. The sensitivity was 0.95 mA ng ml�1 and detectionlimit was 20 pg ml�1 based on signal to noise ratio of 3. For higherconcentration range of PSA, 1–40 ng ml�1, the correlation coefficientand sensitivity were 0.998 and 0.1143 mA ng ml�1, respectively. Weanticipate that ILs/CNTs based immunosensors can be expandedrapidly for detection of other tumor markers and the proposedimmunosensor has the potential for reliable point of care diagnosticsof cancers.

Acknowledgments

The financial supports of Iranian Nanotechnology initiativeand Research Office of University of Kurdistan are gratefullyacknowledged. The authors thank Dr. Verdi Medical Laboratoryfor PSA measurement in serum samples with ELISA method,Dr. Heshmatollah Sofemajidpour for preparing biopsy sampleand Dr. Fathollah Moradi for prostate cancer biopsy micrographs.

A. Salimi et al. / Biosensors and Bioelectronics 42 (2013) 439–446446

Appendix A. Supporting information

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.bios.2012.10.053.

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