Electroanalytical Determination of Carcinoembryonic Antigen at a Silica Nanoparticles/Titania Sol–Gel Composite Membrane-Modified Gold Electrode

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Electroanalytical Determination of Carcinoembryonic Antigen at aSilica Nanoparticles/Titania Sol –Gel CompositeMembrane-Modified Gold ElectrodeYan Liu,* Hong Jiang

Department of Chemistry, Fuling Normal College, Chongqing 408003, P. R. China*e-mail: [email protected]

Received: January 03, 2006Accepted: February 21, 2006

AbstractA highly hydrophilic and nontoxic colloidal silica nanoparticle/titania sol – gel composite membrane was prepared ona gold electrode via a chemical vapor deposition method. With carcinoembryonic antigen (CEA) as a model antigenand encapsulation of carcinoembryonic antibody (anti-CEA) in the composite architecture, this membrane could beused for reagentless electrochemical immunoassay. The presence of silica nanoparticles provided a congenialmicroenvironment for adsorbed biomolecules. The formation of immunoconjugate by a simple one-step immuno-reaction between CEA in sample solution and the immobilized anti-CEA introduced the change in the potential. Themodified procedure was further characterized by electrochemical impedance spectroscopy and cyclic voltammetry.Compared to the commonly applied methods, i.e., the TiO2 direct embedding procedure, this strategy could allow forantibodies immobilized with higher loading amount and better retained immunoactivity. The resulting immunosensorexhibited high sensitivity, good precision, acceptable stability, accuracy, reproducibility and wide linear range from 1.5to 240 ng mL�1 with a detection limit of 0.5 ng mL�1 at 3s. Analytical results of clinical samples show that thedeveloped immunoassay is comparable with the enzyme-linked immunosorbent assays (ELISAs) method, implying apromising alternative approach for detecting CEA in the clinical diagnosis. Furthermore, this composite membranecould be used efficiently for the entrapment of other biomarkers and clinical applications.

Keywords: Carcinoembryonic antigen, Electrochemial immunosensor, Silica nanoparticles, Sol – gel compositemembrane, Titania

DOI: 10.1002/elan.200603479

1. Introduction

Carcinoembryonic antigen (CEA) is a glycoprotein mostoften associated with colorectal cancer, and used to monitorpatients with this type of cancer [1]. Its most popular use is inearly detection of relapse in individuals already treated forcolorectal cancer. After surgery, serial measurements in-dicate the surgery@s success and are used to detect early signsof recurrence. It has recently been found to be useful whenmeasured during surgery for colorectal cancer to helpdetermine prognosis and who will benefit from adjuvanttreatment [2]. CEA is measured in the blood plasma. It isvery nonspecific and can be increased in many types ofcancer: gastrointestinal, colorectal, ovarian, bladder, cer-vical, stomach, kidney, lung, pancreatic, liver, prostate,thyroid, melanoma, lymphoma, and breast [3]. People withnoncancerous conditions, such as cirrhosis or peptic disease,or inflammatory intestinal conditions such as colitis ordiverticulitis, may also have increased levels [4]. CEA levelscan be elevated in elderly patients and in those who smoke[4].

Recently, some diagnostic procedures, such as enzyme-linked immunosorbent assays (ELISAs), piezoelectric im-

munoassay, RT-PCR, radio immunoassays (RIAs), flowinjection chemiluminescence, etc., have been adapted toclinical analyses [5 – 14]. Most methods, unfortunately,require multiple washing, separation steps, highly qualifiedpersonnel, tedious assay time, or sophisticated instrumen-tation. Electrochemical method would offer a straightfor-ward route to in vivo monitoring because of its high time-resolution and capability for in vivo measurement as well ashigh sensitivity [15 – 21]. In electrochemical immunoassays,however, development of specific immobilization of captureantibody that maintains its biological integrity and bioreac-tivity has been a critical issue in immunoassay technology,because its underlying principles are based on specificinteractions between the immobilized ligands and theircounterpart [22 – 24]. Thus, search for new immobilizationmethod with improved electrochemical response character-istics is of considerable interest.

Herein we introduce a new method to immobilize anti-CEA on a gold electrode surface based on 3-mercapto-propionie acid (3-MPA), silica nanoparticles and titania solgel as matrixes. 3-MPA containing �COO� group wasinitially assembled on a gold electrode surface to absorb the�NHþ

3 group of anti-CEA molecule via the opposite-

1007

Electroanalysis 18, 2006, No. 10, 1007 – 1013 C 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

charged adsorption technique, in the meantime, anti-CEAwas adsorbed onto the surface of silica nanoparticles, thenanti-CEA and silica nanopartilces were entrapped intotitania sol – gel composite membrane via a chemical vapordeposition technique. The detection is based on the changein the potentiometric response before and after the anti-gen – antibody reaction. The immunosensors studied exhibithigh sensitivity, rapid response, good reproducibility, andacceptable stability towards CEA. Subsequently, the im-munoassay system was evaluated by analyzing severalpractical samples with results compared with those obtainedby the ELISA method.

2. Experimental

2.1. Apparatus and Reagents

Cyclic voltammetric (CV) measurements were carried outon PAR 273 potentiostat/galvanostat and model 270 soft-ware (EG & G Princeton Applied Researchm Princeton,NJ, USA) and Model XJP-821 polarographic analyzer(Jiangsu Electroanalytical Instruments, Jiansu, China). ACimpedance of the electrodes was measured with a ModelIM6e (ZAHNER Elektrick Co., Germany). A three-compartment electrochemical cell contained a Pt wireauxiliary electrode, a saturated calomel reference electrode(SCE) and a modified gold electrode as working electrode.The size of SiO2 nanoparticles was estimated from trans-mission electron microscopy (TEM) (H600, Hitachi Instru-ment Co., Japan). All potentiometric and pH measurementswere made with a pH meter (MP 230, Mettler-Toledo Co,Switzerland) and a digital ion analyzer (Model PHS-3C,Dazhong Instruments, Shanghai, China).

Carcinoembryonic antigen (CEA) and carcinoembryonicantibody (anti-CEA) were purchased from Bosai Bioengin.Co. (Zhengzhou, China). 3-Mercapto-propionie acid (3-MPA), tetraethoxysilane (TEOS), titanium propoxide andbovine serum albumin (BSA, 96 – 99%) were obtained fromSigma (USA). Ammonjum hydroxide (30 %), bis-(2-ethyl-hexyl) sodium sulfosuccinate or AOT was the products ofTiantai Fine Chemical (Tianjing, China). In the measuringsystem, 0.1 M pH 7.0 phosphate buffer solution (PBS) wasused as the electrolyte. The dilute solution of CEA standardor sample solution was 0.1 M pH 7.0 PBS. All other reagentswere of analytical grade. All solutions were made up withdeionized water of 18 MW purified from a Milli-Q purifi-cation system.

2.2. Fabrication of CEA Immunosensors

All substrates used were new and before their modificationthey were submitted to a cleaning treatment. To be sure thatthe surfaces of the electrodes (4 mm in diameter) wereclean, CV was carried out in 0.5 M H2SO4 solution. Then, theelectrodes were washed with ethanol and twice distilledwater. Finally, they were dried with an argon gas flow. The

cleaned gold electrodes were submerged in 1 mM watersolutions of 3-MPA for 6 h. Afterwards, the substrates werewashed with ethanol, dried under an argon gas flow andmaintained in a desiccator for further characterization.

Silica nanoparticles were synthesized by using a water-in-oil microemulsion procedure [25, 26], where the hydrolysisand polycondensation of TESO precursor occurred in theAOT/cyclohexane system. Further separations of the prod-ucts were conducted using acetone, followed by centrifugingand washing in the ultrasonication cleaner with ethanol andwater to remove any “oil” and surfactant molecules from theparticles@ surface. The size of the SiO2 nanoparticlesprepared is about 30 nm, which was confirmed by trans-mission electron microscopy. 50 mL of the standard anti-CEA water solution (500 ng mL�1) was mixed with 50 mL ofcolloidal silica water solution (w/v, 5%) in a beaker in icewater. Two hours later, a solution of anti-CEA-adsorbedsilica nanoparticles (anti-CEA�SiO2) was obtained. Fol-lowing that, 10 mL of anti-CEA�SiO2 solution was droppedon 3-MPA-modified gold electrode surface, and the elec-trode was suspended vertically above titanium isopropoxidein a sealed conical flask and kept at a temperature of 25 8Cfor 4 h to form a anti-CEA�SiO2 encapsulated titania sol –gel membrane (anti-CEA�SiO2/SG) [27 – 29]. After rinsingthoroughly with twice distilled water, the immunosensorwas incubated in 0.25 % BSA for 60 min at 37 8C to block theremaining active groups and eliminate nonspecific bindingeffect. Then the immunosensor was formed and the finishedimmunosensor was stored at 4 8C when not in use. Theschematic diagram of immunosensor and the structure ofthe electrode coating are shown in Figure 1.

2.3. Electrochemical Immunoassay

The one-step immunoreaction was accomplished on theimmunosensor surface by a potentiometer with an immu-nosensor as working electrode and saturated calomel

Fig. 1. An schematic diagram of the immunosensor and thestepwise immunosensor fabrication process.

1008 Y. Liu, H. Jiang

Electroanalysis 18, 2006, No. 10, 1007 – 1013 www.electroanalysis.wiley-vch.de C 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.electroanalysis.wiley-vch.de

electrode as the reference electrode. The detection is basedon the change in the potential before and after the antigen –antibody reaction: (i) Prior to the measurement, theimmunosensor was immersed in a 5 mL stirring zerophosphate buffer solution (pH 7.0), and the steady-statepotentiometric value (E) was recorded; (ii) various concen-trations of CEA standard or sample solution was added intothe stirring zero PBS solution, and the steady-state poten-tiometric value (E@) was obtained. In order to avoid theerror resulting from additions of analytes, the potentiomet-ric responses to each of the analytes were recorded as theimmunoreaction proceeded from 5 min (after the additionof analytes) until equilibrium was reached. All experimentswere carried out in triplicate measurements. The potentio-metric changes in all of the experiments referred to thepotentiometric responses of immunoreaction otherwisebeing remarked.

2.4. ELISAs Procedure

Sandwich scheme ELISA procedure was performed withstandard polystyrene 48-well plates (Bosai Bioengin. Co.Zhengzhou, China). In order to modify each individual wellwith the primary antibody, 100 mL of a 4.0 mg mL�1 solutionof antibody against CEA diluted in 0.1 mol L�1 phosphatebuffer solution, pH 7.0 (PBS) was passively adsorbed to thepolystyrene cuvettes (wells) at 37 8C for 1 h, and the cuvetteswere rinsed three times (3 min each) with 0.1 mol L�1

phosphate buffer solution (pH 7.0) containing 0.5 mol L�1

NaCl and 0.1% Tween 20, (washing buffer). Aliquot of50 mL of serum sample suspension was incubated in thecuvettes at 37 8C for 30 min and the cuvettes were rinsedthree times (3 min each) with washing buffer. Then 50 mL ofthe conjugate solution was added and incubated for 1 h. Thecuvettes were again rinsed as previously described and atlast 50 mL of dye-reagent was added and incubated at 37 8Cfor 10 min. The enzymatic reaction was stopped by adding50 mL of 2.0 mol L�1 H2SO4 to each well. The results ofELISA were measured by a spectrophotometric ELISA-reader at a wavelength of l¼ 450 nm. The positive ornegative results of the serum samples are evaluated asfollowing the equation:

ODsample/OD(�) � 2.1 positive result

otherwise

ODsample/OD(�) < 2.1 negative result

where ODsample is the OD value of serum sample (vs. the ODvalue at a wavelength of l¼ 450 nm), OD(�) represents themean OD value of the standard negative serum (vs. the ODvalue at a wavelength of l¼ 450 nm).

3. Results and Discussion

3.1. Characteristics Performance of TiO2 Sol–GelModified Composite Membrane

Titania sol – gel composite architecture formed via a vapordeposition method has an efficient scaffold of nanoscaledimension with an orderly structural organization of a largevariety of nanoparticles [27 – 29]. It not only provides a veryhydrophilic interface for retaining the bioactivity andimproving the stability of the immobilized biomelcules,but also promoted the electrical communication [27 – 29].Moreover, titania sol gel has high surface area, goodbiocompatibility, relatively good conductivity and opticaltransparency, and they have been utilized in the immobili-zation of protein or enzyme on electrode surface for eithermechanistic study of the protein or fabricating electro-chemical biosensor [30].

Figure 2 shows cyclic voltammograms (CVs) of differ-ently modified electrode in 2.5 mM ferricyanide solution.Well-defined CVs, characteristic of the Fe2þ/Fe3þ redoxcouple are observed at the bare gold electrode (Fig. 2a).When the electrode was modified with 3-MPA, an obviousdecrease in the anodic and cathodic peaks was observed(Fig. 2b). The reason is that the 3-MPA hinders the diffusionof ferricyanide toward the electrode surface. When silicananoparticles, anti-CEA and TiO2 were immobilized on theelectrode surface, the peak currents of the redox couple of[Fe(CN)6]

4�/[Fe(CN)6]3� decreased again (Fig. 2c). Espe-

cially, after CEA were reacted with anti-CEA on theimmunosensor, a considerable decrease of the peak currentand increase of the potential separation between thecathodic and anodic peaks of the redox probe were obtained(Fig. 2d). This illustrates that the antigen – antibody com-plex insulates the electrode and perturbs the interfacialelectron transfer considerably. It is consistent with theenhanced electron transfer barriers introduced upon theassembly of these layers.

3.2. Electrochemical Impedance Investigation for Anti-CEA Immobilized on MPA-Modified Gold Electrode

Figure 3 shows the AC impedance spectra of Fe(CN)3�=4�6 at

bare gold electrode, MPA-modified gold electrode, TiO2/SiO2/MPA-modified gold electrode and TiO2/anti-CEA�SiO2/MPA-modified gold electrode, respectively,obtained in 2.5 mM Fe(CN)4�=3�

6 PBS, pH 7.0. The dataanalysis of these impedance spectra gave the electrontransfer resistance of Fe(CN)3�=4�

6 to be 106� 5, 1214� 21,1923� 37 and 3178� 54 W, respectively. It was clearlyobserved that the formation of SG layer increased remark-ably the impedance, which further increased upon theentrapment of anti-CEA conjugate (curves b, c and d inFig. 2). Compared with the bare GCE, the presence oftitania SG membrane or entrapping colloidal silica nano-particles in the membrane obviously decreased the value ofcontact angle. As well known, the smaller the contact angle

1009Determination of Carcinoembryonic Antigen



value is, the better the surface hydrophilicity are. Titania SGmembrane greatly improved the surface hydrophilicity, thusmade the immobilized anti-CEA conjugate more stable.The impedance change of the modification process alsoshowed that anti-CEA had attached to the electrodesurface.

3.3. Optimization of Prepared Methods of theImmunosensors

The potentiometric responses of the immunoreaction forthe anti-CEA-bound probe prepared using the TiO2/anti-

CEA�SiO2/MPA conjugating procedure were monitoredwith the aforementioned TiO2/anti-CEA/MPA direct em-bedding procedure (Fig. 4). As can be seen from Figure 4,the potential shifts of two probes showed almost the trend asimmunoreaction proceeded towards the equilibrium. Theoverall potential shifts of immunoreaction are, however,obviously different. The TiO2/anti-CEA�SiO2/MPA conju-gating procedure presents the highest potential change andthe TiO2/anti-CEA/MPA direct embedding procedure thelowest potential change. The difference may be explained bythe fact that the immune reaction between the immobilizedantibody and the antigen leads to the binding of antigenmolecule on the sensing surface after the antibody-immo-bilized electrode is immersed in the antigen solution. Thebinding amount of this complex is correlated to the amountof SiO2 nanoparticles on the surface, then also correlated tothe amount of the analyte. The process of amplifiedimmunoassay of the analyte is represented in Figure 1.

3.4. Kinetic Study of the Reaction Between CEA andAnti-CEA

Kinetic study of potentiometric responses of the immuno-sensor in the presence and absence of CEA antigen atpH 7.0 in a phosphate buffer solution at room temperaturewere illustrated in Figure 5. The potentiometric responsesincreased with the increment of reaction time and started tolevel off after four minutes. The result indicates that thereaction between immobilized antibody and free antigen isan equilibrium process. After anti-CEA combined withCEA, electrical charge of the resulting complex will bedifferent from that of anti-CEA or CEA alone. If anti-CEAis immobilized on the gold electrode, the surface charge ofthe immunosensor will depend on the net charge of the

Fig. 2. Cyclic voltammograms (CVs) in a 2.5 mM Fe(CN)4�=3�6

solution (10 mM PBSþ 0.1 M KCl, pH 7.0) after different steps ofmodification, a) bare gold electrode, b) 3-MPA-modified goldelectrode, c) TiO2/anti-CEA-SiO2/3-MPA-modified gold elec-trode, and d) TiO2/anti-CEA�SiO2/3-MPA-modified gold elec-trode incubated with 100 ng mL�1 CEA. Scanning rate: 50 mV s�1.

Fig. 3. Electrochemical impedance spectroscopy (EIS) of (a)bare gold electrode, (b) 3-MPA-modified gold electrode, (c) TiO2/SiO2/3-MPA-modified gold electrode, and (d) TiO2/anti-CEA�SiO2/3-MPA-modified gold electrode in PBS (pH 7.0)þ0.1 M KClþ 2.5 mM Fe(CN)4�=3�

6 solution. The frequency rangeis at 1� 10�2� 1� 106 Hz at 20 8C (Z’ vs. Z’’ at 220 mV vs. SCE).

Fig. 4. Immunoreaction potentiometric response to differentconcentrations of CEA for the probes prepared using TiO2/anti-CEA�SiO2/3-MPA conjugating procedure (a) and TiO2/anti-CEA/3-MPA conjugating procedure (b) at room temperature inpH 7.0 PBS.




immobilized anti-CEA. When CEA is present in thesolution, the immunochemical reaction will take place atthe interface with a resulting change of the surface charge.

3.5. Determination of CEA

Figure 6 shows the calibration curve obtained using CEAstandards. The curve is not a linear one, as commonlyobserved for an immunoassay. A curve-fitting procedurecould be used for the calibration procedure. A pseudolinearrelationship between the electric potential and the loga-rithm of the concentration of CEA, however, can be fitted tothe experimental points from 1.5 to 240 ng mL�1. The linearregression equation is DE (mV)¼�8.03þ 37.5 log CCEA

with a correlation coefficient of 0.999. A detection limit of0.5 ng mL�1 was estimated to be 3� the standard deviationof zero-dose response, which coincides with the lower boundof the pseudolinear part of the calibration curve.

3.6. Selectivity and Analytical Performance of theImmunosensors

To investigate the selectivity of the immunosensor, thesensor was incubated in a phosphate buffer solution(pH 7.0) containing separately diphtheria toxoid (DT),hepatitis B surface antigen (HBsAg), BSA, glycine (Gly)and lysine (Lys). The inhibition potentiometric responseobtained for each interfering substance presented at aconcentration of 0.05 mg mL�1 (unless otherwise stated) wascompared to that of 0.05 mg mL�1 CEA, and this ratio is usedas a criterion for the selectivity of the sensor. The relativestandard deviation (RSD) of CEA containing interferingagents is between 1.05% and 2.16% (Table 1). So theselectivity of the proposed immunosensor was satisfactory.Furthermore, after the immunosensor was incubated in50 ng mL�1 CEA followed by rinsing with stripping buffer ofpH 2.8 glycine-HCl [31 – 33] to remove the CEA from theAg�Ab immunocomplex, the obtained potentiometricsignal restored the 93% of the initial value. Thus, theimmunosensor had a good selectivity to CEA as well as aacceptable regeneration efficiency.

Fig. 5. Real trace of potentiometric response of the immuno-sensor vs. reaction time before (a) and after (b) the addition 50 ngmL�1 CEA into the pH 7.0 PBS at room temperature.

Fig. 6. Calibration curves of the relationship between thepotentiometric shift of immunoreaction and the log concentrationof CEA for the TiO2/anti-CEA�SiO2/3-MPA-modified immuno-sensor at room temperature in pH 7.0 PBS.

Table 1. The selectivity of the proposed immunosensor in incubation solution containing 0.05 mg mL�1 CEA and 0.05 mg mL�1

interfering agents.

Incubation solution Electrode number and DE (mV) DE� SD RSD (%)

1 2 3

CEA 55.1 54.7 56.2 55.3� 0.8 1.45CEAþDT 56.7 55.8 54.6 55.7� 1.1 1.97CEAþHBsAg 56.4 56.1 57.3 56.6� 0.6 1.06CEAþBSA 57.3 56.7 56.2 56.7� 0.6 1.05CEAþGly 55.3 55.7 56.4 55.8� 0.6 1.07CEAþLys 56.5 55.9 54.1 55.5� 1.2 2.16




In order to investigate the possibility of applying theproposed immunosensor for practical analysis, severalserum samples, which were obtained from two clinicallydiagnosed patients with colorectal carcinomas and sepa-rated from the cell without hemolysis, were analyzed byusing different immunosensors under the optimized con-ditions. The results are compared with those obtained by theELISA method. Figure 7 describes the correlation betweenthe results obtained by the developed potentiometryimmunoassay and by the ELISA method. The regressionequation of the line given by the ELISA method versus thedeveloped potentiometry immunoassay is y¼ 0.9938 xþ0.5432 with a correlation coefficient of 0.999 (p> 0.05). Itobviously indicates that there is no significant differencebetween the results given by two methods, that is, thedeveloped immunoassay may provide a feasible alternativetool for determining CEA in human serum in clinicallaboratory.

The intra-assay precision of the immunosensor wasexamined by successively assaying the CEA levels of twosera for five times. The variation coefficients of the resultswith this method were 3.8% and 6.1% at the CEAconcentrations of 10 and 150 ng mL�1 in serum samples,respectively. The inter-assay precision, or the fabricationreproducibility, was estimated by determining in duplicatethe CEA level in one serum sample with three immuno-

sensors made at the same electrode independently. Theinter-assay precision of this method was 4.8% at the CEAconcentration of 50 ng mL�1, showing acceptable fabrica-tion reproducibility.

The performance stability of the biosensor was examinedby storage in air and in pH 7.0 PBS, separately. Thebiosensor lost its sensitivity rapidly if stored in air. However,when the immunosensor was stored in pH 7.0 PBS at 4 8C, itretained 90% of its initial potential after a storage period of17 days (Table 2).

4. Conclusions

In the current work, silica nanoparticles/titania sol – gelcomposite membrane has been initially incorporated todevelop a new biomolecular immobilization strategy forpotentiometric immunosensors. The immobilization inter-face developed could allow a large amount of antibodies tobe immobilized with well-retained immunoactivity andcould be readily regenerated by 0.2 M, pH 2.8 glycine – hy-drochloric acid (Gly�HCl) buffer solution. Such an immo-bilization interface protocol may be extended as a generalalternative for biosensors since its fabrication processvirtually removes all constraints on substrate size and shapeof transducers. In particular, the new immunosensing systemis much more rapid and cost-effective and simpler toperform than the commonly used ELISA and RIA methods.It may potentially meet the increasing need of developinggreater sensitivity and more rapid detection rates for bothautomated and miniaturized clinical analysis.

5. Acknowledgements

Financial support of this work was provided by the NaturalScience Foundation of Chongqing City and the EducationCommittee Foundation of Chongqing City, China(KJ051304).

6. References

[1] S. P. Prete, D. Cappelletti, S. Baier, P. Nasuti, F. Guadagni, L.De Vecchis, J. W. Greiner, E. Bonmassar, G. Graziani, A.Aquino, Int. Immunopharm. 2002, 2, 641.

[2] N. Sawabata, M. Ohta, S. Takeda, H. Hirano, Y. Okumura, H.Asada, H. Maeda, Ann. Thoracic Surgery 2002, 74, 174.

[3] P. Thomas, H. Hayashi, R. Zimmer, R. Forse, Cancer Lett.2004, 209, 251.

Fig. 7. Comparison of the titer results of determining samplesbetween the ELISA method and the potentiometry immunoassaymethod. A regression equation of the line: y¼ 0.9938 xþ 0.5432was obtained with a correlation coefficient of 0.999 (p> 0.05).

Table 2. Potentiometric response results (DE (mV)) of different immunosensors to the same concentration of CEA.

Time (day) Electrode number Mean RSD(%)

1 2 3 4 5 6 7 8

1th day 26.3 27.1 27.9 27.1 26.9 25.4 28.1 26.1 26.7 3.48th day 25.3 26.5 24.7 25.6 24.9 23.7 26.5 24.9 25.3 3.7

17th day 24.5 25.2 24.1 23.7 24.6 24.7 23.9 24.3 24.5 3.1




[4] M. Okada, W. Nishio, T. Sakamoto, K. Uchino, T. Yuki, A.Nakagawa, N. Tsubota, Ann. Thoracic Surgery 2004, 78,1004.

[5] F. Yan, J. Zhou, J. Lin, H. Ju, X. Hu, J. Immun. Methods 2005,305, 120.

[6] C. Sadahira, K. Yoneda, Y. Kubota, J. Am. Acad. Dermatol-ogy 2005, 53, 531.

[7] K. W. Hance, H. E. Zeytin, J. W. Greiner, Mutation Res./Fundamental and Molecular Mechanisms of Mutagenesis2005, 576, 132.

[8] G. Shen, H. Wang, T. Deng, G. Shen, R. Yu, Talanta 2005, 67,217.

[9] T. Okamoto, T. Nakamura, J. Ikeda, R. Maruyama, F. Shoji,T. Miyake, H. Wataya, Y. Ichinose, Europ. J. Cancer 2005, 41,1286.

[10] J. Wang, S. Lin, C. Lu, C. Chen, D. Wu, C. Chai, F. Chen, J.Hsieh, T. Huang, Cancer Lett. 2005, 223, 129.

[11] Z. Dai, J. Chen, F. Yan, H. Ju, Cancer Det. Prev. 2005, 29, 233.[12] L. Zhao, S. Xu, G. Fjaertoft, K. Pauksen, L. Hakansson, P.

Venge, J. Immun. Methods 2004, 293, 207.[13] Z.Dai , F. Yan , H. Yu, X. Hu, H. Ju, J. Immun. Methods

2004, 287, 13.[14] J. Lin, F. Yan, H. Ju, Clin. Chim. Acta 2004, 341, 109.[15] J. Wang, Electroanalysis 2005, 17, 1341.[16] J. Wang, Electroanalysis 2005, 17, 1133.

[17] N. S. Lawrence, J. Wang, Electrochem. Commun. 2006, 8,7176.

[18] J. Wang, Analyst 2005, 130, 421.[19] J. Wang, Stripping Analysis, VCH, New York 1985.[20] J. Wang, J. Pharm. Biomed. Anal. 1999, 19, 53.[21] J. Wang, G. Liu, A. Merkoci, J. Am. Chem. Soc. 2003, 125,

3214.[22] J. Wang, Biosens. Bioelectr. 2005, in press (doi:10.1016/

j.bios.2005.10.027).[23] J. Wang, Anal. Electrochem. Wiley, New York, 2000.[24] J. Wang, D. Xu, A. Kawde, R. Polsky, Anal. Chem. 2001, 73,

5567.[25] F. Q. Tang, X. W. Men, D. Chen, J. G. Ran, L. Gou, C. Q.

Zhen, Sci. China (ser. B) 2000, 30, 119.[26] H. Wang, Z. Y. Wu, G. L. Shen, R. Q. Yu, J. Chem. Chin.

Univ. 2001, 22, 1305.[27] J. H. Yu, H. X. Ju, Anal. Chem. 2002, 74, 3579.[28] Y. Saih, K. Segawa, Catalysis Today 2003, 86, 61.[29] J. Chen, J. Tang, F. Yan, J. Ju, Biomaterials 2006, 27, 2313.[30] Q. Li, G. Luo, J. Feng, Electroanalysis 2001, 13, 359.[31] J. L. Boitieux, R. Groshemy, Anal. Chim. Acta 1987, 197, 229.[32] F. V. Bright, T. A. Betts, K. S. Litwiler, Anal. Chem. 1990, 62,

1065.[33] S. N. Buhl, K. Y. Jackson, R. Lubinski, R. E. Vanderlinde,

Clin. Chem. (Washington, D. C.) 1976, 22, 1872.




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Electroanalytical Determination of Carcinoembryonic Antigen at a Silica Nanoparticles/Titania Sol–Gel Composite Membrane-Modified Gold Electrode