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Sensors and Actuators B 142 (2009) 316–320

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical

journa l homepage: www.e lsev ier .com/ locate /snb

lpha-1-fetoprotein antibody functionalized Au nanoparticles: Catalytic labelsor the electrochemical detection of �-1-fetoprotein based on TiO2 nanoparticlesynthesized with ionic liquid

in Tana, Yaqian Chena, Hua Yanga, Ya Shia, Jianfei Si a, Guangming Yanga, Zaisheng Wub,ing Wanga, Xuxiao Lua, Huiping Baia, Yunhui Yanga,∗

College of Chemistry and Chemical Engineering, Yunnan Normal University, Yunnan, Kunming 650092, PR ChinaState Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082, PR China

r t i c l e i n f o

rticle history:eceived 4 June 2009eceived in revised form 12 August 2009ccepted 13 August 2009vailable online 20 August 2009

a b s t r a c t

A novel strategy for the preparation of amperometric immunosensor for rapid determination of �-1-fetoprotein (AFP) in human serum has been developed. TiO2 nanoparticles (NPs) were prepared bysolvothermal reaction using TiCl4 as raw materials and the mixture of ionic liquids and doubly dis-tilled water as solvent. �-1-fetoprotein antibody (AFP Ab) was mixed with TiO2 NPs/chitsotan (CHIT)

eywords:onic liquidsiO2 nanoparticlesFP

solution and immobilized onto the surface of a glassy carbon electrode. AFP (Ab) functionalized Au NPswere used as catalytic labels for the amperometric detection of AFP by means of the electrocatalyzedreduction of Au NPs to H2O2. The electrochemical behavior of the immunosensor was studied. Otherexperimental conditions such as pH, immunoreactions temperature and time were also studied. The pre-pared immunosensor offers an excellent amperometric response for AFP ranging from 1.0 to 160.0 ng/mLwith a detection limit of 0.1 ng/mL. The result shows that the immunosensor displays rapid response, high

cibilit

u-labeled AFP (Ab)

mmunosensor sensitivity, good reprodu

. Introduction

Alpha-1-fetoprotein (AFP) is an embryo-specific glycoproteinssociated with the normal growth of the mammalian fetus. It ishe most reliable and widely used tumor marker for the diagnosis ofepatocellular carcinoma (HCC) and yolk sac tumor [1,2]. The con-entration of serum AFP in healthy adults is very low, typically at theange of 4–13 ng/mL [3]. Higher levels of serum AFP were associatedith the diagnosis of liver diseases. For example, the AFP concen-

ration of a liver cancer patient can attain as high as 10 mg/mL. Aimple method with high sensitivity is thus greatly needed for thelinical diagnosis and early detection of liver carcinoma.

Electrochemical immunosensors are valuable analytical toolsnd have been developed [4,5]. Attempts have been reported intudies to fabricate the immunosensors with various enzymes6–12]. However, most of enzyme-linked immunosensors needome mediator molecules to achieve the electron-transfer [13,14],

hich leads to a more complex immunoassay system and increases

peration time and analytical expense. Metal nanoparticles cane used as labels to substitute enzymes labels. Compared withnzyme-labeled methods, metal nanoparticles might have a longer

∗ Corresponding author.E-mail address: yyhui2002@yahoo.com.cn (Y. Yang).

925-4005/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.snb.2009.08.011

y and favorable stability.© 2009 Elsevier B.V. All rights reserved.

lifetime because they overcome some of the problems associ-ated with the thermal and environmental variables. Furthermore,this method did not need the addition of mediators or non-immunoreagents. Conductivity measurements [15] and strippingvoltammetry [16] were commonly employed methods for thedetection the deposited metal labels, which make the detectionmore complex. It is well known that Pt can catalyze the reduc-tion of H2O2 [17–19]. Polsky et al. [20] have developed an aptamersensor based on nucleic acid-functionalized Pt NPs which act ascatalytic labels for the amplified electrochemical detection of DNAhybridization and aptamer/protein recognition by employing PtNPs labels as catalysts for the reduction of H2O2. But only a fewstudies reported that Au NPs can also electro catalyze the reductionof H2O2 [21].

This paper reports the results of studies relating to using TiO2nanoparticles prepared by our previous work [22] to construct anAFP amperometric immunosensors. TiO2 nanoparticles, exhibit-ing a large specific surface area, high surface-activity and strongadsorption, might increase the amount and stability of immobi-lizing AFP antibodies on the surface of the sensor, and improve

the sensitivity of the sensor at the same time. Chitosan (CHIT) isa promising matrix for biomaterial immobilization because of itsexcellent film forming and adhesion ability, together with non-toxicity and good biocompatibility. So �-1-fetoprotein antibody(AFP Ab) was mixed with TiO2 NPs/chitsotan (CHIT) solution and

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was incubated in AFP (Ag) solution with various concentrations at33 ◦C. Next, the immunosensor was incubated in Au-labeled AFP(Ab) solution with the same concentrations for 40 min at 33 ◦C. Theimmunosensor was rinsed thrice thoroughly with 0.5 M NaCl anddouble distilled water after each incubation.

L. Tan et al. / Sensors and A

mmobilized onto the glassy carbon electrode. AFP antibody func-ionalized Au nanoparticles were used as catalytic labels for themperometric determination of �-1-fetoprotein (AFP) in humanerum. The determination of AFP was established by chronoam-erometry recording the reduction current response of H2O2 byeans of the direct catalysis of Au nanoparticles. In contrastith the common methods of using metal nanoparticles as labels

or electrochemical detection with stripping voltammetry, thehronoamperometry using the metal nanoparticles as catalystabels is analogous to the way enzymes traditionally are used.

eanwhile, metal nanoparticles might have a longer lifetime thanhat of enzymes. To the best of our knowledge, there is no othereport using Au NPs as catalytic labels for electrocatalyzed reduc-ion of H2O2 to fabricate the amperometric immunosensor. Therepared immunosensor displays rapid response, high sensitivity,ood reproducibility, favorable stability and better detection limit.he proposed method significantly simplifies the immunoassayrocedure, shortens assay times and would be valuable for clinical

mmunoassay.

. Experimental

.1. Apparatus and reagents

Cyclic voltammetric and amperometric measurements werearried out on XJP-821(C) polarograph (Jiangsu, China). Trans-ission electron microscopy (TEM) analysis was performed using-800 electron microscope (Hitachi Instrument, Japan). A three-lectrode cell (10 mL) with the modified glassy carbon (GC)lectrode as the working electrode, a saturated calomel electrodeSCE) as reference electrode and a platinum foil electrode as counterlectrode was used. All potentials were measured and reportedersus the SCE.

Chloroauric acid tetrahydrate (HAuCl4·4H2O) was purchased byinopharm Chemical Reagent Co., Ltd. (Shanghai, China); diagnos-ic kit for �-1-fetoprotein (AFP) and human chorionic gonadotropinHCG) were produced by Beijing Biosynthesis Biotechnology Co.,td. (Beijing, China); bovine serum albumin (BSA, 96–99%) was sup-lied by Sigma (St. Louis, MO, USA). Chitosan (CHIT, MW ∼ 1 × 106;5–80% deacetylation) was supplied by Sigma. All other chemicalssed were of analytical reagent grade and used as received withouturther purification.

The supporting electrolyte was phosphate buffer solution0.1 M), which was prepared with NaH2PO4·2H2O, Na2HPO4·12H2Ond NaCl; all other reagents were of analytical grade, and dou-ly distilled water was used throughout. Serum specimens werebtained from the second affiliated hospital of Kunming medicalollege and stored at 4 ◦C.

.2. Material synthesis

.2.1. Synthesis of colloidal Au solutionAll glassware used in the following procedures was cleaned in a

ath of aqua regia, rinsed thoroughly in double distilled water andried in air.

Colloidal gold solutions were prepared by the well-establishedrens method by reducing HAuCl4 aqueous solution with citrate.n this method it is possible to control the size of the synthesizedarticles over a wide size range (1–100 nm) by varying the [AuIII)]/[citrate] ratio during the reduction step [23]. In brief, 1 mLf trisodium citrate (1%) was added into 50 mL of HAuCl4 aque-

us solution (0.01%), stirred and heated to 100 ◦C for 17 min [24].fter cooling, the synthesized gold colloids were stored at 4 ◦C in aark bottle. Particle sizes were confirmed by transmission electronicroscopy (TEM), and the size of the prepared Au nanoparticlesas approximately 10 nm.

ors B 142 (2009) 316–320 317

2.2.2. Synthesis of Au-labeled AFP (Ab)The Au NPs-labeled antibody was prepared according to Ref. [25]

with a slight modification. In brief, �-1-fetoprotein antibody solu-tion (AFP, Ab) was dialyzed with 0.005 M of NaCl and left overnightat 4 ◦C in order to remove the additional salt ion. The pH of colloidalAu solution was adjusted to 5.0 with 0.1 M HCl. 0.1 mL of �-1-fetoprotein antibody solution was added to 0.1 mL of the colloidalAu solution (pH 5.0) and stirred at the same time. Then, 0.05 mLof 5% (w/w) BSA solution was added to stabilize the colloidal goldsolution and the mixture was incubated for an additional 10 min.Upon centrifugation at 13,000 × g for 1 h at 10 ◦C, the precipitatewas reserved and diluted by 0.01 M of PB (pH 7.5) solution con-taining 1% BSA. Then the centrifugation was repeated and the finalprecipitate was suspended to 0.2 mL by the PB solution containing1% BSA. The prepared Au-colloid labeled �-1-fetoprotein antibodywas stored in a refrigerator.

2.3. Preparation of immunosensor

Glassy carbon electrode (GCE, 3 mm diameter) was first pol-ished with emery paper and alumina slurry, successively rinsedthoroughly with nitric acid (1:1), absolute alcohol and doublydistilled water in ultrasonic bath for 10 min, and dried in air natu-rally.

Chitosan (CHIT), a natural polymer product, is derived fromchitin via deacetylation with alkali [26]. A 0.5-wt% CHIT solutionwas prepared by 0.05 M acetic acid and stirred for 3 h at room tem-perature until complete dissolution. TiO2 nanoparticles (0.1 mg)prepared by our previous work were dispersed in CHIT solutions(2 mL) for 30 min sonication. Subsequently, 5 �L of AFP (Ab) solu-tion and 5 �L of TiO2 NPs/CHIT solutions were mixed. Then, 5 �L ofthe mixture was dropped onto the surface of GC electrode and dried.The evaporation of water resulted in a thin and robust compositefilm containing AFP (Ab)/TiO2 NPs/CHIT. At last the modified elec-trode was incubated in 0.1 M glycin solution for 30 min at 25 ◦C inorder to block possible remaining active sites of the TiO2 nanopar-ticles and avoid the nonspecific adsorption. The immunosensorwas stored at 4 ◦C when not in use. The stepwise assembly of theimmunosensor is shown in Scheme 1.

2.4. Experimental measurements

The electrochemical measurement was based on a sandwichimmunoassay method. Before measurement, the immunosensor

Scheme 1. Schematic diagram of the stepwise immunosensor fabrication process:(a) coating TiO2 NPs/CHIT/AFP (Ab), (b) loading AFP (Ag), and (c) loading Au-labeledAFP (Ab).

3 ctuators B 142 (2009) 316–320

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The cyclic voltammetric behavior of the modified electrode indifferent solution was investigated in 10 mL of 0.1 M PB at room

emperature. The amperometric detection is based on the changen the amperometric response (�i) before and after adding 9.9 mM

2O2 at −0.10 V.

. Results and discussion

.1. Cyclic voltammetry characterization

Fig. 1 displays the cyclic voltammograms of the modified elec-rode in 0.1 M PB (pH 6.35) solution without (a) and with 9.9 mM2O2 (b). Only background current could be observed in Fig. 1(a).hen PB contains 9.9 mM H2O2, the cathode peak current of elec-

rode obviously increased, indicating that Au exhibits excellentlectrocatalytic activity toward H2O2.

.2. Optimization of experimental variables

.2.1. Effect of pHThe influence of the pH of the PB solution on the ampero-

etric response was investigated in the pH range 5.68–7.56 inhe presence of 9.9 mM H2O2. Fig. 2 shows the current response

ig. 1. Cyclic voltammograms of the immunosensor at different solution: (a) 0.1 MB (pH 6.35); (b) 0.1 M PB (pH 6.35) containing 9.9 mM H2O2.

ig. 2. The effect of pH on the immunosensor response in 0.1 M PB containing.9 mM H2O2.

Fig. 3. The effect of the incubation temperature on immunoreaction.

arrives at a maximum value at pH 6.35. In order to obtain max-imum sensitivity and bioactivity, pH 6.35 was chosen for thewhole of the experiments, which is also a general pH value inimmunology.

3.2.2. Effect of the incubation temperature and timeThe incubation temperature has been reported to have a very

important effect on the activity of the antibody and antigen [27],so the influence of the incubation temperature was studied. Theincubation temperatures were 20, 25, 30 and 36 ◦C, using the sameanalyte concentrations in this study. The results are shown in Fig. 3.We simultaneously took into account the activity of biomoleculesand the stability of composite membrane. Therefore, temperature33 ◦C was fixed for the rest of the experiments.

The effect of the incubation time on the amperometric responsewas also investigated. In the incubating solution, when the ana-lyte antigens reach the antibodies at the electrode surface of theimmunosensor, it takes some times for the contacting species toform immunocomplexes. The results are shown in Fig. 4. Withincreasing incubation time, the current responses increased and

then stabilized when the incubation time was longer than 40 min.The flat showed that combined capacity of the antigen on the sensorgradually tends to saturation. Thus, an incubation time of 40 minwas adopted in the subsequent work.

Fig. 4. The effect of the incubation time on immunoreaction.

L. Tan et al. / Sensors and Actuators B 142 (2009) 316–320 319

Table 1Determination of AFP in human serum samples.

Sample number Measured by immunosensor (n = 5) (ng mL−1) RSD (%) Determined by radioimmunoassay (ng mL−1) Relative error (%)

1 3.74 3.6 3.75 0.32 9.49 4.3 10.05 5.6

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.3. Immunosensor response characteristics

Under the optimal conditions, the calibration curve for AFPetection with the proposed immunosensor is illustrated. Fig. 5epicts that the response signal increased with the increase of AFPoncentration. The immunosensor is linear in the range from 1o 160.0 ng/mL with a regression equation of �i (�A) = 3.12 + 3.48

10−3C (ng/mL) and correlation coefficient of 0.992. The detectionimit is 0.1 ng/mL at 3�, which is lower than the report values inefs. [7,28,29].

.4. Selectivity

In order to investigate the specificity of the immunosensors, BSAnd HCG were used to evaluate the selectivity of the electrode inhis study. The immunosensor was separately incubated with 2%wt) BSA and 60 mIU of HCG for 40 min, and then incubated inu-labeled AFP antibody solution for 40 min at 33 ◦C. The inter-

erential degree of the substance can be judged from the value ofhe amperometric response (�i). The response of the immunosen-or was 0.06 �A incubating with BSA and 0.05 �A incubating withCG. The test results show that BSA and HCG did not cause obvious

nterference to the electrode.

.5. Regeneration and stability

The repeated use of the same immunosensor can significantlyeduce the cost of analysis compared to disposable immunosen-ors. After completing each assay, the immunosensor was simplymmersing in a stirred 4 M urea solution for about 30 min to dis-ociate the antigen–antibody complex after each determination.

hen the immunosensor was rinsed thoroughly with 0.5 M NaClnd doubly distilled water.

The stability of the successive assays was studied. After 200Vs measurements in 0.1 M PB (pH 6.35) containing 9.9 mM H2O2,

ig. 5. The calibration curves of immunosensor in different AFP concentration.

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the current decrease of 1.47% was acquired. The variation coeffi-cient was 6.5% for five successive assays. The long-time stability ofthe immunosensor was also investigated. When the immunosen-sor was stored at 4 ◦C and measured intermittently (once aweek), no apparent change in the working buffer was found over45 days.

3.6. Real sample analysis

The applicability of the proposed immunosensor was assessedby the determination of AFP concentration in the real serum sam-ples. Results were compared with those determined by the secondaffiliated hospital of Kunming medical college using radioim-munoassay (RIA) analysis method which utilizes radioisotopes125I-labeled AFP antigen (Ag*) to determine the content of anti-gen through competitive binding to their common specific AFPantibody (Ab). The results are shown in Table 1. The investigationof the statistically significant difference of those two techniqueswas accomplished using F-test. The calculated results show thatthe results determined by the immunosensor were in satisfac-tory agreement with those given by radioimmunoassay. Thus, theproposed method could be satisfactorily applied to the clinicaldetermination of AFP in human serum for clinical diagnosis.

4. Conclusions

In this paper, TiO2 nanoparticles were simply synthesized usingionic liquids. An amperometric immunosensor for rapid determi-nation of AFP based on AFP (Ab)/TiO2 NPs/CHIT film was prepared.A sandwich immunoassay format was employed to detect AFP withAu nanoparticles as catalytic labels to catalyze the reduction ofH2O2. As far as we are aware, this is the first report using Au NPsas catalytic labels for electrocatalyzed reduction of H2O2 to fabri-cate the amperometric AFP immunosensor. The detection limit waslower than the value which has been reported. The analytical resultsby the proposed method were in satisfactory agreement with thoseby the radioimmunoassay analysis method. This strategy would bevaluable for clinical immunoassay and could be extended readily topreparation of other amperometric immunosensors and detectionof other clinically important antigens.

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (Grants No. 20865006), the Foundation of Sci-ence Commission of Yunnan Province (No. 2006B0028M) and theFoundation of Department of Education of Yunnan Province (No.07Z10087).

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Biographies

Lin Tan graduated from Chongqing Three Gorges University in 2002, majoring inChemistry. She obtained her M.S from Yunnan Normal University in 2009. Herresearch interests also cover chemical and biosensors.

Yaqian Chen graduated from Yunnan Normal University in 2009.

Hua Yang is currently undergraduate students of Chemistry at Yunnan NormalUniversity.

Ya Shi is currently undergraduate students of Chemistry at Yunnan Normal Univer-sity.

Jianfei Si is currently undergraduate students of Chemistry at Yunnan Normal Uni-versity.

Guangming Yang graduated from Yunnan Normal University in 2002, majoring inChemistry. He got his M.S from Yunnan Normal University. His research also focuseson chemical and biosensors.

Zaisheng Wu obtained his Ph.D from Hunan University in 2008, majoring in Ana-lytical Chemistry. He is currently a lecturer of Chemistry at Hunan University. Hisresearch focuses on biosensors.

Ping Wang graduated from Sichuan University in 1988, majoring in Chemistry.She is currently an associate professor at Yunnan Normal University. Her researchinterests are regarding physical chemistry.

Xuxiao Lu obtained his M.S. from Yunnan Normal University in 2008, majoring inChemistry. His research interests cover chemical and biosensors.

Huiping Bai obtained his M.S. from Yunnan Normal University in 2008, majoring inChemistry. Her research interests cover chemical and biosensors.

Yunhui Yang obtained her Ph.D from Hunan University in 2005, majoring in Ana-lytical Chemistry. She is currently a professor of Chemistry at Yunnan NormalUniversity. Her research interests cover chemical and biosensors.