electrochemical immunoassay for carbohydrate antigen-125 based on polythionine and gold hollow...

Full Paper

Electrochemical Immunoassay for Carbohydrate Antigen-125Based on Polythionine and Gold Hollow Microspheres ModifiedGlassy Carbon ElectrodesXiao-Hong Fu*

School of Chemistry and Chemical Engineering, Yibin University, Yibin 644000, P. R. China*e-mail: [email protected]

Received: May 25, 2007Accepted: June 22, 2007

AbstractA new electrochemical immunosensor for the detection of carbohydrate antigen-125 (CA125), a carcinoma antigen,was developed by immobilization CA125 antibody (anti-CA125) on gold hollow microspheres and porouspolythionine (PTH) modified glassy carbon electrodes (GCE). The gold hollow microspheres provided abiocompatible microenvironment for proteins, and greatly amplified the coverage of anti-CA125 molecules on theelectrode surface. The performance and factors influencing the immunosensor were investigated in detail. Thedetection is based on the current change before and after the antigen-antibody interaction. Under optimal conditions,the amperometric changes were proportional to CA125 concentration ranging from 4.5 to 36.5 U/mL with a detectionlimit of 1.3 U/mL (at 3s). The CA125 immunosensor exhibited good precision, high sensitivity, acceptable stability,accuracy and reproducibility. The as-prepared immunosensors were used to analyze CA125 in human serumspecimens. Analytical results suggest that the developed immunoassay has a promising alternative approach fordetecting CA125 in the clinical diagnosis.

Keywords: Electrochemical immunoassay, Gold hollow microspheres, Carbohydrate antigen-125, Porous polythionine

DOI: 10.1002/elan.200703943

1. Introduction

Biomarkers play an essential role in modern medicine [1].This includes classical diagnostic applications such asdetection, diagnosis, prognosis and monitoring of diseases.Biomarkers can also significantly contribute to our under-standing of a disease at the molecular level and help us todefine new drug targets, better develop new drugs, predictundesirable side-effects, target new medicines to likelyresponders, etc. [2]. Great efforts have been made world-wide to develop and improve immunoassays for thedetection of biomarkers with the aim of making portableand affordable devices. In despite of many advances in thisfield, it is still a challenge to exploit new approaches that canimprove the simplicity, selectivity, and sensitivity of clinicalimmunoassay, to meet the requirements of modern medicaldiagnostics and biomedical research applications [3 – 6].New enabling technologies, such as electrochemical immu-noassay methods, have strongly fostered efforts to detectnovel biomarkers in the recent years because of their lowdetection limit, simplicity of the equipment required and theability to analyze heterogeneous and colored samples [7 – 9].

In electrochemical immunoassay systems, the key step isthe immobilization of sensing biomolecules, which shouldbe simple, fast and leads to robust materials with stable andhighly active immobilized reagents which do not leach fromthe substrate [10]. The present immobilization methods are

mainly based on adsorption, sandwich, entrapment, cova-lent binding, or cross-linking technique [11 – 14]. Anotherimportant issue is to enhance the coverage of the biomol-ecules on the transducer surface owing to directly influenc-ing the sensitivity of the immunosensors. As interest inpatterning has increased, several different techniques, suchas immobilization of biomolecules to DNA, nanowires,nanorods, nanoclusters and nanotubes, have been devel-oped [15 – 22]. However, there are little reports focusing onnanostructured hollow microspheres for the biomoleculeimmobilization of electrochemical immunosensors. Thesenanostructured hollow microspheres with porous shellstructure are preferable to other carriers, such as mono-disperse nanoparticles, membranes, microcapsules, andnanowires, when used to immobilize proteins, becausethey provide the regularly spherical surface that couldmake the distribution of immobilized proteins average,reduce the diffusion limitation of both reactants andproducts, and facilitate fast biochemical reactions andspecific combination of bioactive molecules and theircounterparts [23]. Liu et al. [24] developed a gold hollowball made of NH2-polydiacetylene for the enhancement ofimmobilization. Kumar et al. [25] utilized hollow goldnanoparticles for the immobilization of horseradish perox-idase.These results suggested that goldhollowmicrospherescould improve the extent of ligand binding and detectionsensitivity.

1831

Electroanalysis 19, 2007, No. 17, 1831 – 1839 D 2007 WILEY-VCH Verlag GmbH& Co. KGaA, Weinheim

Carbohydrate antigen-125 (CA125) is a membrane mu-cin-like glycoprotein greater than 200 kDa with a thresholdvalue of 35 U/mL, high levels of which have been found inovarian cancer and has been used for monitoring the courseof epithelial ovarian tumors [26]. TheCA125 is also elevatedin other cancers including endometrial, pancreatic, lung,breast, and colon cancer, and in menstruation, pregnancy,endometriosis, and other gynecologic and non gynecologicconditions [27].Various electrochemical immunosensors forthe detection of CA125 have been reported [28 – 32]. Theseimmunoassays, however, have some shortcomings such astime-consuming, high-cost, low sensitivity and complicatedoperation with several separation steps. Thus the develop-ment of fast, low-cost, high sensitivity and easy-to-usemethods is important for health protection of medicaltreatment. Considering the advantages of gold hollownanospheres and the biocompatibility of metal gold, wesynthesized nanostructured gold hollow microspheres withporous shell structure, and fabricated an electrochemicalimmunosensor for the detection of CA125, as a modelbiomarker, on the gold hollow microspheres-functionalizedglassy carbon electrode (GCE). Cyclic voltammetry (CV),electrochemical impedance spectroscopy (EIS), and quartzcrystal microbalance (QCM) techniques were used toinvestigate the interaction between the as-prepared goldhollow microspheres and anti-CA125. Experimental resultsindicated that the gold hollow microspheres with porousshell structure exhibited a very high surface area and roomfor protein adsorption. Moreover, the developed immuno-sensors exhibit a highly electrochemical response in thedynamic range of 4.5 – 36.5 U/mL relative to CA 125concentration. In addition, the performance and factorsinfluencing the performance of the resulting immunosensorhave been investigated.

2. Experimental

2.1. Materials

Carbohydrate antigen-125 (CA125, 0 – 100 U/mL), CA125monoclonal antibody (anti-CA125), HAuCl4, sodium cit-rate, bovine serum albumin (BSA) and thionine werepurchased from Sigma. Ammonium hydroxide, 2-ethyl-1-hexanol, n-butanol, hydroxylamine, 3-(mercaptopropyl)tri-methoxysilane (MPTS), silicon dioxide (SiO2, LUDOX),and Span-80 (C24H44O6) were obtained from Aldrich. Allother reagents were of analytical grade. Doubly distilledwater was used for all experiments. The serum samples wereobtained from two clinically diagnosed patients withovarian cancer. The sera were separated from the cell,without hemolysis. Gold nanoparticles with 16 nm indiameter were prepared according to the literature [33].The particle sizes were confirmed by transmission electronmicroscopy (TEM, H600, Hitachi Instrument Co., Japan)(data not shown).

2.2. Preparation of Gold Hollow Microspheres (GHM)

Prior to prepare the gold hollow nanospheres, the ceramichollow spheres were initially synthesized according to theliterature [34]. Briefly, the mixture solution containing20 mL aqueous LUDOX solution (10%, w/v) and 100 mL 2-ethyl-1-hexanol was initially sonicated for 5 min at roomtemperature, and then 10 mL aqueous NH4OH (25 wt%)and 0.40 mL Span-80 were added in turn. At this stage, thecolloidal silica particles dispersed in water were surroundedby a 2-ethyl-1-hexanol. Following that, 200 mL n-butanolwas added to extract the water in LUDOX. After stirredover 5 min, the solution remaining after the extraction wasfiltered through filter paper and then dried for 24 h atambient temperature to achieve ceramic hollow spheres.

Subsequently, the gold hollow microspheres were synthe-sized via consulting the literatures [35] as follows(Scheme 1a): (i) the MPTS monolayers were self-assembledonto the surface of ceramic hollow spheres; (ii) goldnanoparticles were attached to theMPTS monolayer coatedceramic hollow spheres via Au�S covalent bonds; (iii)additional gold nanoparticles were grown, in situ, byimmersing the gold nanoparticle coated ceramic hollowspheres into an aqueous mixture of hydroxylamine and goldchloride; and (iv) the samples were dried, calcined, treatedwith sodium hydroxide to dissolve the ceramic shell andproduce thereby nanostructured gold hollow microspheres.

2.3. Fabrication of Immunosensors

The electropolymerization of thionine (PTH) was carriedout according to the literature [26] in two steps: (i) holdingthe cleaned GCE electrode (1-mm diameter) under aconstant potential of �1.5 V (vs. SCE) for 3 min in amixture solution containing 10% HNO3 and 2.5% K2CrO4

to make it negatively charged, and then (ii) voltammetriccycles between �1.0 and þ1.5 V at 50 mV/s for 10 min in0.1 M thionine solution of pH 2.8. The resulting electrodepolymerized with thionine was then removed from thesolution and rinsed with water. The electrode was dried inair with the humidity of 30% at room temperature for about1 h. Attachment of gold hollow microspheres (or pure 16-nm gold nanoparticles, as a comparison) onto the amine-modified surfaces was performed in aqueous gold hollowmicrospheres for 12 h at 4 8C. Then the modified electrodewas treated with anti-CA125 solution (0.1 M NaCl, 10 mMphosphate buffer, pH 7.4) overnight. The structures of thegold hollow microspheres-modified probe and pure goldnanoparticle-modified probe were schematically illustratedin Scheme 1b and Scheme 1c. For a comparison, anti-CA125/nanogold/PTH/GCE, anti-CA125/PTH/GCE andanti-CA125/GCE were prepared by immersing them intoanti-CA125 solution, respectively. After being stored forabout 24 h at 4 8C, the formed immunosensors wereincubated in 0.25 wt% BSA for 60 min at 37 8C to eliminatenon-specific binding effect and block the remaining active

1832 X.-H. Fu

Electroanalysis 19, 2007, No. 17, 1831 – 1839 www.electroanalysis.wiley-vch.de D 2007 WILEY-VCH Verlag GmbH& Co. KGaA, Weinheim

www.electroanalysis.wiley-vch.de

groups. The finished immunosensors were stored at 4 8Cwhen not in use.

2.4. Quartz Crystal Microbalance (QCM) Measurement

The quartz crystal microbalances (QCM) measurement wasperformed on 10-MHz QCM devices (PICBALANCE,Italy). Each amount of the immobilized peptide probes wasevaluated by the resonant frequency shift between beforeand after the immobilization reaction. The resonant fre-quency shift was converted to a mass increase (Dm) by theSauerbreyMs equation given in Equation 1 [36].

Df¼�2f0 Dm A�1 (mqrq)�1/2 (1)

where Df is the observed frequency shift (Hz), f0 is the basicfrequency of the crystal, A is the piezoelectric activity area,mq is the shear modulus of quartz, and rq is its density.

2.5. Electrochemical Measurement

Electrochemical measurements were carried out with anAutoLab (Eco Chemie, The Netherlands). The AC impe-dance of the electrodes was measured with an IM6e(ZAHNER Elektrick Co., Germany). A conventionaltwo-compartment three-electrode cell contained a platinumwire auxiliary electrode, a saturated calomel referenceelectrode (SCE) and the modified GCE as workingelectrode. Impedance measurements were performed inthe presence of a 2.5 mM K3[Fe(CN)6]/ K4[Fe(CN)6] (1 :1)

Scheme 1. a) Schematic procedure to prepare gold hollow microspheres. b) Fabrication procedure of gold hollow microspheres-modified immunosensors. c) Fabrication procedure of pure gold nanoparticles-functionalized immunosensors.

1833Electrochemical Immunoassay for Carbohydrate Antigen-125



mixture as a redox probe in PBS (containing 0.1 M KCl,pH 7.4) at the frequency range from 10�2 to 106 Hz at theformal potential of 220 mV, using alternating voltage of10 mV.After the immunosensor forCA125was incubated in50 mL incubation solution at 35 8C for 30 min and thenwashed carefully with doubly distilled water, the electro-chemical measurement was recorded in HAc-NaAc buffer,pH 6.5. The differential pulse voltammetric (DPV) analyseswere from �300 to 500 mV with pulse amplitude of 50 mVand width of 50 ms. All electrochemical measurements weredone in an unstirred electrochemical cell at 25� 0.5 8C.

2.6. Enzyme-Linked Immunosorbent Assay (ELISA) forCA125

Sandwich scheme ELISA procedure was performed withstandard polystyrene 96-well plates (Biocell Bioengin. Co.Zhengzhou, China). At first aliquot of 50 mL of serumsample suspension was incubated in the wells at 37 8C for30 min and the wells were rinsed three times (3 min each)with 0.1 mol/L phosphate buffer solution (pH 7.4) contain-ing 0.5 mol/L NaCl and 0.1% Tween 20, (washing buffer).Then 50 mL of the conjugate solution was added andincubated for 1 h. The wells were again rinsed as previouslydescribed and at last 50 mL of dye-reagent was added andincubated at 37 8C for 10 min. The enzymatic reaction wasstopped by adding 50 mL of 2.0 mol/L H2SO4 to each well.The results of ELISA were measured by a spectrophoto-metric ELISA-reader at a wavelength of l¼ 450 nm.

3. Results and Discussion

3.1. Construction and Analysis of the GHMs-ModifiedImmunosensors

The mechanism of thionine electropolymerization has beenproposed and confirmed [37]. It was commonly consideredthat initial adsorption of thionine monomer occurred on thegold electrode surface and the electropolymerization reac-tion proceeded and started at a fairly high potential at whichreactive cation-radical species formed. The monomer unitsin PTH linked through �NH-bridges in the aromaticposition a or b (both ortho with respect to �NH2) [37].The electroactivity of PTH lies in not only its electroactiveheterocyclic nitrogen atoms and nitrogen bridges, but alsoits free amine groups [37]. Thus, gold nanoparticles on thesurface of GHMs can be easily self-assembled to �NH2

groups on the PTH surface. To clarify the above conclusionmentioned, scanning electronmicroscopy (SEM, Japan)wasused to observe the morphology of differently modifiedsurface (Fig. 1). Figure 1a shows the SEM image of the bareGCE. When the thionine was electropolymerized on theGCE surface, the surface roughness was increased, andtherewere somedefects at themeantime (Fig. 1b).After thesubstrate was modified with gold hollow microspheres, amuch rougher surface was achieved (Fig. 1c). The reason is

that the GHMs are much bulky compared with couplingagent (PTH). These results suggested the GHMs couldassembled onto the surface of PTH film.

During the self-assembly procedure, cyclic voltammetrywas also used to investigate the electrochemical character-istics, because the electron transfer between the solutionspecies and the electrode must occur by tunneling eitherthrough the barrier or through the defects in the barrier[38 – 40]. Figure 2 shows the cyclic voltammograms (CVs) ofthe bare and modified GCE in pH 6.5 acetic buffer. Twoweaken and irreversible redox peaks were observed at bareGCE (curve a). When the thionine was electropolymerizedonto the GCE surface, a couple of redox peak, characteristicof a diffusion-limited redox process, was obtained (curve b),which suggested that the formed PTH film on the electrodesurface is a good mediator. When gold hollow microsphereswere self-assembled onto the PTH/GCE surface, the peakcurrents were increased to some extent (curve c), which wasin agreement with the fact that a compact film of goldnanoparticles has good conductivity [41]. The reason of theincrement for peak currents may be the fact that the goldhollow nanospheres were not only self-assembled on the

Fig. 1. SEM images of a) bare GCE, b) PTH/GCE, and c) GHM/PTH/GCE.

Fig. 2. Cyclic voltammograms of a) bare GCE, b) PTH/GCE, c)GHM/PTH/GCE, and d) anti-CA125/GHM/PTH/GCE in pH 6.5acetic buffer at 50 mV/s.

1834 X.-H. Fu



outer surface of the PTH, but also partly distributed withinthe film as tiny conduction centers and facilitated theelectron transfer. Curve d is the cyclic voltammogram of theresulting electrode immobilized with anti-CA125. Thedecrease in peak current might be caused by the hindranceof the macromolecular structure of anti-CA125 to theelectron transfer, and it also confirmed the successfulimmobilization of anti-CA125.

Typical cyclic voltammograms of anti-CA125/GHM/PTH/GCE in 0.1 M acetic buffer solution (pH 6.5) atdifferent scan rates are studied (data not shown). It wasclear that the potentials of the anodic peak and the cathodicpeak hardly changed with the scan rate, v, i.e., the peakpotential was independent of the scan rate in the rangebetween 10 and 500 mV/s. Both the anodic peak current andthe cathodic peak current were proportional to v1/2 at theabove scan rate range, suggesting that the electrode reactionis a diffusion-controlled process [42 – 44].

3.2. Electrochemical Behaviors of the DifferentImmunosensors

As shown in Figure 3a, a couple of reversible redox peakscould be obtained with anti-CA125/GHM/PTH/GCE inpH 6.5 acetic acid buffer. In contrast, only a small pair ofredoxwaves could be observedat the anti-CA125/nanogold/PTH/GCE (Fig. 3b) and anti-CA125/PTH/GCE (Fig. 3c).Two slight and irreversible redox peaks, however, wereobserved on the voltammogram in this potential range withthe anti-CA125/GCE (Fig. 2d), which was almost the sameas that of the bare GCE in pH 6.5 acetic acid buffer. Thereason for the slight peak mainly ascribed to the directlyelectrochemical behavior of the bare GCE in pH 6.5 aceticacid buffer according to the literature [45]. These resultsclearly suggested that these two peaks of the immunosen-sors could be improved with the aid of gold hollownanospheres.

To investigate the effect of various immobilizationmethods on the performance of the immunosensor, a seriesof experiments were performed to determine the electro-chemical responses of CA125 at anti-CA125/GHM/PTH/GCE, anti-CA125/nanogold/PTH/GCE, anti-CA125/PTH/GCE and anti-CA125/GCE (Table 1), respectively. Theresponseswere basedon the current changebefore and afterincubation with CA125 for 30 min at 35 8C. Seen from

Table 1, a large amperometric change for CA125 wasobtained at anti-CA125/GHM/PTH/GCE. A likely explan-ation for this feasibility is that the gold hollow microspheresprovided a biocompatible microenvironment for proteins,and greatly amplified the coverage of anti-CA125moleculeson the electrode surface. Moreover, the sensors with goldhollow microspheres exhibited more rapid current responsethan the sensors with pure gold nanoparticles in terms ofsteady-state current. The above results obviously suggestthat the immunosensors can generate better performancesfor the detectionofCA125, and exhibit fast electron transferreactivity as a result of the spatial effect of gold hollowmicrospheres. Thus, the anti-CA125/GHM/PTH/GCE elec-trodes were used to the following experiments.

3.3. EIS Characteristics of Different Immunosensors

In electrochemical impedance spectroscopy (EIS), thesemicircle diameter of EIS equals the electron transferresistance, Ret. This resistance controls the electron transferkinetics of the redox-probe at electrode interface, which isrelated to the amount of the immobilized antibody on theelectrode surface, then also related to the amount of theanalyte [46, 47]. Thus, Ret can be used to describe the

Fig. 3. Cyclic voltammograms of a) anti-CA125/GHM/PTH/GCE, b) anti-CA125/nanogold/PTH/GCE, c) anti-CA125/PTH/GCE, and d) anti-CA125/GCE in pH 6.5 acetic buffer at 50 mV/s.

Table 1. Amperometric changes of different immunosensors in pH 6.5 acetic buffer before and after incubation with various CA125levels for 30 min at 35 8C.

Immunosensors Current shift (mA) [a]

C[CA125] (U/mL)5 10 15 20 25 30 35

anti-CA125/GHM/PTH/GCE �2.4 �6.7 �13.2 �18.5 �24.6 �31.4 �37.9anti-CA125/nanogold/PTH/GCE �1.3 �3.7 �7.8 �12.1 �17.3 �22.9 �27.1anti-CA125/PTH/GCE �1.1 �1.3 �1.6 �3.5 �6.8 �9.4 �10.1anti-CA125/GCE �0.03 �0.04 �0.05 �0.07 �0.08 �0.08 �0.09

[a] Each value is the average of three measurements.




interface properties of the electrode. Its value varies whendifferent substances are adsorbed onto the electrode sur-face. Figure 4 exhibits the derived calibration plots thatcorresponds to the electron transfer resistance (Ret) at anti-CA125/GHM/PTH/GCE (Fig. 4a), anti-CA125/nanogold/PTH/GCE (Fig. 4b), anti-CA125/PTH/GCE (Fig. 4c) andanti-CA125/GCE (Fig. 4d) toward different concentrationsof the analyte CA 125. The change of electron transferresistance is calculated as following the equation:

DRet¼RAb�Ag�RAb

where RAb and RAb-Ag represent the value of electrontransfer resistance before and after the immobilized anti-CA125 binds to CA125 in the sample. Experimental resultsshow the anti-CA125/GHM/PTH/GCE immunosensor ex-hibits much higher DRet than others, indicating that theGHM/PTH/GCE electrode can load more anti-CA125 onthe electrode surface. On the basis of the CVs and EISresults, we might make a conclusion that anti-CA125 couldbe successfully immobilized on the surface of GCE elec-trode and the gold hollow nanospheres could enhance thesensitivity of the immunosensors than that with pure goldnanoparticles.

3.4. QCM Analysis of Different Immunosensors

To further examine the effect of pure gold nanoparticles andgold hollow microspheres on the immobilization of anti-CA125 and the antigen-antibody interaction, QCM tech-

nique was also used to investigate the abilities of differentprobe surfaces for the anti-CA125 adsorption (Fig. 5). Seenfrom Figure 5, one can find that the probe with gold hollowmicrospheres shows much greater frequency change(219.5� 3.2 Hz) than the probe with pure gold nanoparti-cles (165.7� 4.3 Hz). According to the SauerbreyMs equa-tion, the frequency shift corresponding to a mass increase of273.1 ng cm�2 was less optimism for the anti-CA125adsorption on the nanogold hollow balls than that for thegold nanoparticles conjugation (206.2 ng cm�2). Moreover,the probe with nanogold hollow balls exhibited more rapidQCM response than the probe with gold nanoparticles interms of frequency response rate, which is in accordancewith the CV response. The result reveals that the goldhollow microspheres-modified electrode could providemore binding sites and room for protein adsorption.

3.5. Optimization of Experimental Conditions

The conditions used for immunoreaction greatly affectedthe amperometric response for CA125 immunoassay. Theseconditions included incubation time and incubation temper-ature. With an increasing incubation temperature from 10 to55 8C, the immunosensor after incubation for 30 min showeda maximum current change at 35 8C as shown in Figure 6a.Thus 35 8C was used for immunoreaction, at which thecurrent shift of the anti-CA125/GHM/PTH/GCE to 20 U/mL CA125 increased with the increment of incubation timeand leveled off after 30 min. Longer incubation time did notimprove the response (Fig. 6b). Therefore, 30 min was

Fig. 4. The resistance shifts (DRet) of a) anti-CA125/GHM/PTH/GCE, b) anti-CA125/nanogold/PTH/GCE, c) anti-CA125/PTH/GCE,and d) anti-CA125/GCE in a 2.5 mM Fe(CN)6

4�/3� PBS (pH 7.4) before and after incubation with various concentrations of CA125.Inset: faradaic impedance spectra of anti-CA125/GHM/PTH/GCE to CA125.

1836 X.-H. Fu



chosen as the incubation time for the determination ofCA125 antigen using the immunosensor.

3.6. Amperometric Responses of the Immunosensors toCA125

For the measurement of CA125, a voltammetric measure-ment was applied under optimized conditions. The ampero-metric responses of the immunosensors observed, when noincubation in an analyte in the absence of CA125 wasperformed, would be the baseline signal, since there was noconjugate for antigen binding sites. Then from the baselinesignal, the standard solution of CA125 at a known concen-tration or one serum sample was added into the incubationsolution with a controlled volume ratio. To further verify theamplification of the immunosensor signal via immobiliza-tion of gold hollow nanospheres on the biorecognitioninterface, the detective capabilities of the probes preparedwith different conjugating procedures were studied incomparison. The DPV peak currents of the immunosensorsafter the antigen-antibody reaction showed a decrease withan increasing CA125 concentration in the incubationsolution. The curve is not a linear one, as commonlyobserved for an immunoassay. A curve-fitting procedurecould be used for the calibration procedure. Figure 7describes the calibration curves of the relationship betweenthe amperometric shift of immunoreaction and CA125concentrations. As shown in Figure 7 and Table 2, theimmunosensor with gold hollow nanospheres exhibitedhigher sensitivity and lower detection limit (which isestimated to be 3� the standard deviation of zero-doseresponse) than those of others. Higher or lower antigenconcentrations would deviate from the linear relationspresumably due to that the unbalance of the ratio of antigento antibody may result in a decreased formation of lattice-like immunocomplex, which is also termed as the prozonephenomenon in immunology.

Fig. 5. QCM responses of a) anti-CA125/GHM/PTH/GCE andb) anti-CA125/nanogold/PTH/GCE to 20 U/mL CA125.

Fig. 6. The effect of a) incubation temperature and b) incubationtime on the amperometric response of the anti-CA125/GHM/PTH/GCE.

Fig. 7. Calibration curves of the relationship between the currentchange of immunoreaction and CA125 concentration for a) anti-CA125/GHM/PTH/GCE, b) anti-CA125/nanogold/PTH/GCE, c)anti-CA125/PTH/GCE, and d) anti-CA125/GCE. Each datarepresents the average value of triplicate measurements.




3.7. Precision, Selectivity, and Stability of the anti-CA125/GHM/PTH/GCE

The intra-assay precision of the as-prepared immunosensorwas evaluated via assaying the CA125 levels of four sera forfive replicate measurements in the same run. The variationcoefficients of intra-assay with this method were 3.4, 7.1, 5.2,and 4.6% at the CA125 concentrations of 5.0, 15, 25, and35 U/mL, respectively, while the inter-assay variation co-efficient on six CA125 immunosensors used independentlywas 7.2% at 20 U/mL. Thus, the precision and reproduci-bility of the proposed immunosensor were satisfactory.Good reproducibility may be explained by the strongabsorption effects of hollow gold nanospheres, and anti-CA125 is firmly attached on the surface of the electrodesurface. The electrode-to-electrode reproducibility wasestimated from the response to 20 U/mL CA125 at 5different immunosensors. The results show the currentresponse characteristics of 5 different immunosensorsprepared in the presence of 20 U/ml CA125 in acetate acidbuffer, pH 7.0. The relative standard deviation (RSD) wasbetween 3.4% and 5.8%. Thus it may be preliminarilyapplied for the determination of CA125 in human serum forroutine clinical diagnosis.

To investigate the selectivity of the anti-CA125/GHM/PTH/GCE, the sensorwas incubated in incubation solutionscontaining separately carcinoembryonic antigen (CEA),cancer antigen 15-3 (CA153) or cancer antigen 19-9(CA199). The results shown in Table 3 indicated that theselectivity of the anti-CA125/GHM/PTH/GCE immuno-sensor based on the highly specific antigen-antibody immu-noreaction was satisfactory. The stability of the immuno-sensor was investigated by using the different immunosen-sors at the same batch. When these immunosensors werestored dry at 4 8C and measured intermittently (every 3 – 5days, measurement once for each immunosensor), noapparent change in the same concentration of CA125 was

found over 17 days, which attributed to the strong inter-actions between anti-CA125 and gold hollow microspheres.

3.8. Detection of Clinical Samples

In order to investigate the possibility of the newly developedtechnique to be applied for clinical analysis, 23 serumspecimens, which were gifted from Cancer and TumourHospital of Chengdu, China, were examined by thedeveloped immunoassay and the ELISA method. Themeasurement method of the proposed immunosensor is asfollowed: 30 mL serum sample was added into the 70 mLincubation solution, and then the as-prepared immunosen-sor was incubated in the incubation solution at 35 8C for30 min. After a washing step with doubly distilled water, thevoltammetric measurement was carried out in pH 6.5acetate acid buffer. These results are shown in Figure 8.The regression equation (linear) for these data is as follows:y¼ 0.6449þ 0.9825 x (R2¼ 0.973) (x-axis: by the as-pre-pared immunosensor; y-axis: by ELISA). These data showsthat there is no significant difference between the resultsgiven by two methods which are in concordance with theresults obtained using the standard methods proposed byELISA, that is, the developed immunoassays may provide apromising alternative tool for determining CA125 in humanserum in clinical laboratory.

4. Conclusions

This contribution introduced a new fabricated strategy ofelectrochemical immunosensor for the determination ofCA125 based on gold hollow microspheres and PTH asmatrixes. Although the similar immobilization method hasbeen reported, this study mainly focused on the effect ofgold hollow microspheres on the performance of electro-

Table 2. Comparison of analytical results for CA125 with the various immunosensors. The working ranges are obtained by fitting thecalibration curves for the detection of CA125 with the corresponding immunosensors. The detection limits are estimated according tothe s (standard deviation) rule.

Immunosensors Linear range (U/mL) Detection limits (U/mL)

anti-CA125/GHM/PTH/GCE 4.5 – 36.5 1.3anti-CA125/nanogold/PTH/GCE 5.0 – 35.0 2.1anti-CA125/PTH/GCE 15.0 – 28.5 6.5anti-CA125/GCE Almost no response

Table 3. Amperometric change of the anti-CA125/GHM/PTH/GCE in pH 6.5 acetic buffer after incubation in incubation solutioncontaining 20 U/mL CA125 and various concentrations of interfering agents.

Interfering agents Current shift (mA) [a] Mean (mA) RSD (%)

C[interfer agent] (U/mL)0 5 10 20 40

CEA (ng/mL) �18.6 �18.1 �19.5 �18.4 �20.7 �19.1 5.5CA153 (U/mL) �18.7 �17.4 �19.3 �21.6 �19.4 �19.3 7.9CA199 (U/mL) �18.5 �18.1 �19.5 �19.8 �20.6 �19.3 5.2

[a] Each value is the average of three measurements.

1838 X.-H. Fu



chemical immunosensor.Experimental results revealed thatthe large periphery at the external space of gold hollowmicrospheres could provide much room for accommodationof protein, and improve the extent of ligand binding anddetection sensitivity. Most importantly, the present method-ology could be easily extended to other protein assaysystem.

5. Acknowledgements

This work is supported by the Specialized Research Fundsfor the Excellent Young Teachers from Yibin University(grant no. 2005Z14), China.

6. References

[1] F. Apple, L. Pearce, A. Chung, R. Ler, M. Murakami, Clin.Chem. 2007, 53, 874.

[2] O. Bondar, D. Barnidge, E. Klee, B. Davis, G. Klee, Clin.Chem. 2007, 53, 673.

[3] J. Wang, A. Ibanez, and M. P. Chatrathi, J. Am. Chem. Soc.2003, 125, 8444.

[4] B. Munge, G. Liu, J. Wang and G. Collins, Anal. Chem. 2005,77, 4662.

[5] J. Wang, Analyst 2005, 139, 421.[6] J. Wang, M. Musameh, R. Laochoensuk, Electrochem.

Commun. 2005, 7, 652.[7] J. Wang, Electroanalysis 2007, 4, 415.[8] J. Wang, T. Tangkuaram, S. Loyprasert, T. Vazquez-Alvarez,

W. Veerasai, P. Kanatharana, P. Thavarungkul, Anal. Chim.Acta 2007, 581, 1.

[9] E. Palecek, J. Wang and F. Scheller, Electrochemistry ofNucleic Acids and Proteins, Elsevier, Amsterdam 2005.

[10] Nanoparticles for Electrochemical Assays, in Nanobiotech-nology, Vol. II (Eds: C. Mirkin, C. Niemeyer), Wiley-VCH,Weinheim, 2007, p. 125.

[11] X. Yao, H. Wu, J. Wang, S. Qu, G. Chen, Chem. A Eur. J.2007, 13, 846.

[12] S. Qu, J. Wang, J. Kong, P. Yang, G. Chen, Talanta 2007, 71,1096.

[13] T. Tangkuaram, J. Wang, M. C. RodrPguez , R. Laocharoen-suk, W. Veerasai, Sens, Actuators B 2007, 121, 277.

[14] J. Wang, M. Scamicchio, A. Blasco, A. Escarpa, Anal. Chem.2006, 78, 2060.

[15] O. A. Loaiza, R. Laocharoensuk, J. Burdick, M. C. Rodri-guez, J. M. Pingarron, M.Pedrero, J. Wang, Angew. Chem.Int. Ed. 2007, 46, 1508.

[16] Z. Dai, A. Kawde, Y. Xiang, J. T. La Belle, J. Gerlach, V. P.Bhavanandan, L. Joshi, J. Wang, J. Am. Chem. Soc. 2006, 128,100018.

[17] J. Wang, M. Scampicchio, R. Laocharoensuk, F. Valentini, O.Gonzalez-GarcPa, J. Burdick, J. Am. Chem. Soc. 2006, 128,4562.

[18] J. Hansen, J. Wang, A. Kawde, Y. Xiang, J. Am. Chem. Soc.2006, 128, 2228.

[19] J. Wang, J. Dai, T. Yarlagadda, Langmuir 2005, 21, 9.[20] J. Wang, G. Chen, M. P. Chatrathi, M. Musameh, Anal.

Chem. 2004, 76, 298.[21] J. Wang, R. Deo, P. Poulin, M. Mangey, J. Am. Chem. Soc.

2003, 125, 14706.[22] J. Wang, G. Liu, B. Munge, L. Lin, Q. Zhu, Angew. Chem. Int.

Ed. 2004, 43, 2158.[23] S. Sadasivan, G. Sukhorukov, J. Colloid. Interf. Sci. 2006, 304,

437.[24] S. Liu, Y. Li, J. Li, L. Jiang, Biosens. Bioelectron. 2005, 21,

789.[25] R. Kumar, A. Maitra, P. Patanjali, P. Sharma, Biomaterials

2005, 26, 6743.[26] D. Tang, R. Yuan, Y. Chai, Anal. Chim. Acta 2006, 564, 158.[27] J. Kang, W. Hudak, N. Keller, B. Criswell, Clin. Chem. 1988,

34, 1983.[28] Z. Dai, F.Yan, J. Chen, H. Ju, Anal. Chem. 2003, 75, 5429.[29] J. Wu, Z. Zhang, Z. Fu, H. Ju, Biosens. Bioelectron. 2007,

DOI: 10.1016/j.bios.2007.03.023.[30] G. Yan, H. Ju, Z. Liang, T. Zhang, J. Immun. Meth. 1999, 225,

1.[31] L. Wu, J. Chen, D. Du, H. Ju, Electrochim. Acta 2006, 51,

1208.[32] L. Wu, F. Yan, H. Ju, J. Immun. Meth. 2007, 322, 12.[33] N. Jane, L. Gearheart, C. Murphy, Langmuir 2001, 17, 6782.[34] K. R. Brown, M. J. Natan, Langmuir 1998, 14, 726.[35] S. Chah, J. Fendler, J. Yi, J. Colloid. Interf. Sci. 2002, 250, 142.[36] G. Sauerbrey, Z. Phys. 1959, 155, 2333.[37] J. Bauldreay, M. Archer, Electrochim. Acta 1983, 28, 1515.[38] J. Wang, G. Liu, M. Jan, J. Am. Chem. Soc. 2004, 126, 3010.[39] D. Tang, R. Yuan, Y. Chai, J. Phys. Chem. B 2006, 110, 11640.[40] D.Tang, R. Yuan, Y. Chai, Electroanalysis 2006, 18, 259.[41] Y. Liu, Y. Wang, R. Claus, Chem. Phys. Lett. 1998, 298, 315.[42] G. Zaitseva, Y. Gushike, E. Ribeiro, S. Rosatto, Electrochim.

Acta 2002, 47, 1469.[43] D. Tang, J. Ren. Electroanalysis 2005, 17, 2208.[44] D. Tang, R. Yuan, Y. Chai, L. Zhang, J. Dai, Y. Liu, X.

Zhong, Electroanalysis 2005, 17, 155.[45] M. Quintino, K. Araki, H. Toma, L. Angnes, Talanta 2006,

68, 1281.[46] R. Yuan, D. P. Tang, Y. Q. Chai, X. Zhong, Y. Liu, J. Y. Dai,

Langmuir 2004, 20, 7240.[47] D. Tang, R. Yuan, Y. Chai, J. Dai, X. Zhong, Y. Liu,

Bioeletrochemistry 2004, 65, 15.

Fig. 8. Comparison of the examining results of serum samplesbetween the ELISA method and the developed immunosensor.




electrochemical immunoassay for carbohydrate antigen-125 based on polythionine and gold hollow...

Documents