Amperometric immunosensor for carcinoembryonic antigen detection with carbon nanotube-based film decorated with gold nanoclusters

Analytical Biochemistry 414 (2011) 70–76

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Amperometric immunosensor for carcinoembryonic antigen detection with carbon nanotube-based film decorated with gold nanoclusters Xia Gao, Yiming Zhang, Huan Chen, Zhichun Chen, Xianfu Lin ⇑ Department of Chemistry, Zhejiang University, Hangzhou 310027, People’s Republic of China

a r t i c l e

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Article history: Received 23 November 2010 Received in revised form 3 March 2011 Accepted 4 March 2011 Available online 10 March 2011 Keywords: CEA Immunosensor Layer-by-layer assembly Carbon nanotubes Gold nanocluster

a b s t r a c t A new amperometric immunosensor for the determination of carcinoembryonic antigen (CEA) was constructed. First, the uniform nanomultilayer film was fabricated via layer-by-layer (LBL) assembly of positively charged carbon nanotubes wrapped by poly(diallyldimethylammonium chloride) and negatively charged poly(sodium-p-styrene-sulfonate), which could provide a high accessible surface area and a biocompatible microenvironment. Subsequently, gold nanoclusters were electrodeposited on the electrode to immobilize anti-CEA. The fabricated process and electrochemical behaviors of the immunosensor were characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and scanning electron microscopy (SEM). Under optimal conditions, the proposed immunosensor could detect CEA in two linear ranges from 0.1 to 2.0 ng mL 1 and from 2.0 to 160.0 ng mL 1, with a detection limit of 0.06 ng mL 1. Ó 2011 Elsevier Inc. All rights reserved.

The detection of tumor marker levels in human serum plays an important role in clinical diagnoses. It is well known that carcinoembryonic antigen (CEA),1 an acidic glycoprotein with a molecular weight of approximately 200 kDa, is a reliable and widely used tumor maker. For healthy individuals, the serum CEA level is below 5 ng mL 1. High serum CEA level is related to some familiar cancers, such as lung cancer [1,2], ovarian carcinoma [3,4], breast cancer [5], colon cancer [6–8], and cystadenocarcinoma [3]. Thus, monitoring the level of CEA is very helpful for clinical tumor diagnoses. Many strategies have been reported for the sensitive measurement of CEA, such as enzyme immunoassay [9], radioimmunoassay [10], and fluoroimmunoassay [11]. Traditional methods used in immunoassays are successful but involve tedious assay processes, long analytical time, and difficulties in automation. For example, enzyme-linked immunosorbent assay is time-consuming and it requires skillful personnel, and radioimmunoassay exposes laboratory workers to a significant safety hazard. Compared with conventional immunoassay techniques, the electrochemical immunosensor has recently attracted increasing interest because of the promising properties of specific and simple detection, short analysis time, inexpensive instrumentation, and suitable miniaturization [12–16]. Elec⇑ Corresponding author. Fax: +86 571 87951895. E-mail address: [email protected] (X. Lin). Abbreviations used: BSA, bovine serum albumin; CEA, carcinoembryonic antigen; CNTs, carbon nanotubes; EIS, electrochemical impedance spectroscopy; GCE, glassy carbon electrode; GNP/CNT/Ch, gold nanoparticles and carbon nanotube-doped chitosan; LBL, layer-by-layer; PBS, phosphate buffer solution; PDCNTs, poly(diallyldimethylammonium chloride)-wrapped CNTs; PDDA, poly(diallyldimethylammonium chloride); PSA, prostate specific antigen; PSS, poly(sodium-p-styrene-sulfonate). 1

0003-2697/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2011.03.005

trochemical immunosensors determine the analyte level through detecting changes in potential, current, conductance, or impedance caused by the immunoreaction. The amperometric immunosensor is especially promising for the relatively low detection limit and high sensitivity in those immunosensors [17,18]. Therefore, searching for an amperometric immunosensor with good sensitivity and selectivity but without a complicated labeling process is of considerable interest. Biomolecular immobilization on the surface of immunosensors is the key point. The layer-by-layer (LBL) assembly technique represents interesting methodology to obtain ultrathin multilayer films with a high order of molecular scale [19,20]. This technique involves the alternate electrostatic adsorption of oppositely charged components. It is a simple way to produce multilayer films with controlled composition, thickness, and structural morphology. This technique has been used to obtain films of various proteins and nanoparticles, combined with a uniform distribution of the deposited material. The simplicity and versatility of the LBL technique can pave the way to fabricate immunosensors of multilayer films with unique properties [21,22]. Nowadays, nanomaterials have been introduced to electrochemical biosensors to enhance the sensitivity of the electrochemical detection of bimolecules. Carbon nanotubes (CNTs), with excellent electron transfer rate, high chemical stability, and mechanical strength, have become the subject of intense investigation in sensing applications [23–25]. Moreover, CNTs are predominant materials for biomolecule immobilization due to the high accessible surface area. Gold nanoparticles have also been widely applied owing to their large specific surface area and satisfactory

Immunosensor for carcinoembryonic antigen / X. Gao et al. / Anal. Biochem. 414 (2011) 70–76

biocompatibility [26,27]. For example, Yuan and co-workers [28] developed an amperometric immunosensor based on an Azure I/ multiwalled carbon nanotube (Azure I/MWNT) composite membrane and gold nanoparticles. Zhang and co-workers [29] prepared a sensitive immunosensor by immobilizing AFP antigen onto a glassy carbon electrode modified by gold nanoparticles and carbon nanotube-doped chitosan (GNP/CNT/Ch) film. In recent years, deposited gold nanoclusters have also been employed to construct immunosensors [30,31]. The uniformly distributed gold nanoclusters could provide a large surface area to immobilize biomolecules, which resulted in high bioactivity and high electron-transfer efficiency. In addition, the deposited gold nanoclusters could have long stability and excellent reproducibility. In this study, an amperometric immunosensor for CEA detection was constructed by immobilizing carcinoembryonic antibody (anti-CEA) on the CNT-based film decorated with gold nanoclusters. The uniform nanomultilayer film fabricated via LBL assembly of positively charged poly(diallyldimethylammonium chloride)wrapped CNTs (PDCNTs) and negatively charged poly(sodium-pstyrene-sulfonate) (PSS) could provide a high accessible surface area and a good biocompatible microenvironment. The flower-like gold nanoclusters obtained by electrodeposition offered a large specific surface area for immobilizing antibody with fine bioactivity. The prepared amperometric immunosensor showed high sensitivity, low detection limit, and long-term stability. Compared with the results obtained by an enzyme-linked immunosorbent assay (ELISA), the developed immunosensor showed acceptable accuracy. Materials and methods Chemicals and reagents CEA and anti-CEA were purchased from Keyuezhongkai Biotech Co., Ltd. (Beijing, China) and stored at 4 °C. The multiwall carbon nanotubes (MCNTs, 95%) were purchased from Nanotech. Port. Co., Ltd. (Shenzhen, China). Prior to use, MCNTs were treated in order to introduce carboxylic acid groups according to Ref. [32]. Poly(diallyldimethylammonium chloride) (PDDA, 20%, w/w in

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water, MW 100,000–200,000) and poly(sodium-p-styrene-sulfonate) (MW 70,000) were obtained from Aldrich. Hydrogen tetrachloroaurate (HAuCl4
4H2O) and bovine serum albumin (BSA, 98%) were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Prostate specific antigen (PSA) was purchased from Keyuezhongkai Biotech Co., Ltd. (Beijing, China). CEA enzyme-linked immunosorbent assay kits were purchased from Autobio Co., Ltd. (Zhengzhou, China). All other chemicals were of analytical grade and used without pretreatments. Double distilled water was used throughout the work. Phosphate buffer solutions (PBS, 0.01 M) at various pH values were prepared by mixing the stock standard solutions of NaH2PO4 and Na2HPO4. Apparatus Electrochemical measurements were carried out with a CHI 650 C electrochemical workstation (Shanghai CH Instrument Company, China). A conventional three-electrode electrochemical cell was used with a platinum disk electrode as the auxiliary electrode, a saturated calomel electrode (SCE) as the reference electrode, and a modified glassy carbon (GC) electrode (U = 4 mm) as the working electrode. The scanning electron micrographs were performed on a scanning electron microscope (Zeiss Ultra 55). Preparation of soluble PDDA-CNTs (PDCNTs) Soluble PDDA-CNTs (PDCNTs) were prepared according to Ref. [33]. Briefly, 2 mg mL 1 carboxylated MCNTs was dispersed into a PDDA aqueous solution (1 mg mL 1, containing 0.5 M NaCl) and the dispersion was sonicated for 30 min to give a homogeneous suspension. Residual PDDA polymers were removed by high-speed centrifugation and the complex was rinsed with water for three times. Fabrication of the immunosensor The fabricated procedure of the immunosensor is shown in Fig. 1. The bare glassy carbon electrode was first polished with 1.0, 0.3, and 0.05 lm alumina slurry and consequently and rinsed

Fig.1. Schematic illustration of the fabrication process of the immunosensor.

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with double-distilled water, followed by successive sonications in double-distilled water, ethanol, and double-distilled water for 5 min. Then the electrode was allowed to dry at room temperature. PSS solutions (1 mg mL 1) containing 0.5 mol L 1 NaCl were prepared. Multilayer film of PDCNTs/PSS was assembled on GC electrode by alternately dipping the electrode into the PDCNTs and PSS solution for 40 and 20 min, respectively. After every adsorption step, the modified electrode was thoroughly rinsed with distilled water and dried with nitrogen. The gold nanoclusters were electrochemically deposited on the modified electrode in HAuCl4 solution (1.5 mM) between 0.2 and 1.0 V at a scan rate of 50 mV s 1 for 15 cycles to obtain the Au/PDCNTs/(PSS/ PDCNTs)2/GCE. Then, it was immersed in anti-CEA solution overnight at 4 °C. Finally, the obtained electrode was incubated in 0.2% BSA for 2 h in order to block possible remaining active sites of gold nanoclusters and MCNTs and avoid the nonspecific adsorption. The prepared immunosensor was stored at 4 °C when not in use. Au/PDDA/(PSS/PDDA)2/GC electrode was fabricated in a similar method for a comparison. Electrochemical measurements Electrochemical measurements were done in a conventional electrochemical cell at 37 °C. Cyclic voltammograms were performed in the presence of 5.0 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1/1) mixture as a redox probe in 0.01 M PBS (pH 6.5) containing 0.1 M KCl from 0.6 to 0.2 V (vs SCE) at a scan rate of 100 mV s 1. The change of oxidation current response (DI) before and after immunoreaction was evaluated as in the equation DI = I I0, where I0 represented the current response before the immunoreaction and I represented the current response after immunoreaction. The immunoreaction was performed by incubating the immunosensor in 0.01 M PBS (pH 6.5) containing various concentrations of CEA for 20 min at 37 °C and then electrochemical experiments were performed. Electrochemical impedance spectroscopy measurements were carried out in the presence of 5 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1/ 1) mixture as a redox probe in 0.01 M PBS (pH 6.5) containing 0.1 M KCl with the frequencies ranging from 104 to 10 1 Hz. Results and discussion SEM characterization of modified electrodes The morphologies and microstructures of the as-prepared different films were studied by the SEM observation (Fig. 2). When the first PDCNT layer was assembled, MCNTs were randomly oriented and solely dispersed without wrapping with each other (Fig. 2a). After the number of layers increased, a dramatic increase in MCNT coverage was observed (Fig. 2b). The obtained multilayer film was highly homogeneous, which would facilitate the fabrication of the immunosensor. After gold nanoclusters were deposited on the multilayer film, many different diameter clusters could be observed (Fig. 2c). Then the flower-like clusters could offer a large specific surface area to increase the amount of immobilized antiCEA [34,35]. Electrochemical characterization of modified electrodes Electrochemical impedance spectroscopy (EIS) is an effective tool for monitoring the interfacial properties of surface-modified electrodes. In the study, the impedance changes of the immunosensor surface in the fabrication process and the formation of an antigen–antibody complex were observed by EIS (Fig. 3). In the EIS, the linear part at the low frequencies corresponds to the diffu-

Fig.2. SEM images of (a) PDCNT film, (b) PDCNT/(PSS/PDCNT)2 film, and (c) Au/ PDCNT/(PSS/PDCNT)2 film.

sion-limited process and the semicircle portion at the high frequencies corresponds to the electron transfer-limited process. The semicircle diameter is equal to the interfacial electron transfer resistance (Ret) [31]. Bare GCE electrode exhibited an almost straight line (Fig. 3a), indicating a low Ret in electrolyte solution. The Ret of PDCNT/(PSS/PDCNT)2-modified electrode was remarkably high and was estimated to be 2479 X (curve b of inset). This increase was attributed to the nonconductive properties of PDDA and PSS, which would obstruct the electron transfer of the redox probe. After gold nanoclusters were electrodeposited on the mod-

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Zim/Ω

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Zre/Ω Fig.3. EIS of different modified electrodes measured in 5.0 mM [Fe(CN)6]4 /3 containing 0.1 M KCl: (a) bare GCE; (b) Au/PDCNTs/(PSS/PDCNTs)2/GCE; (c) antiCEA/Au/PDCNTs/(PSS/PDCNTs)2/GCE; (d) BSA/anti-CEA/Au/PDCNTs/(PSS/PDCNTs)2/ GCE; (e) CEA/BSA/anti-CEA/Au/PDCNTs/(PSS/PDCNTs)2/GCE. The inset shows the EIS of (a) bare GCE and (b) PDCNTs/(PSS/PDCNTs)2/GCE.

ified electrode, the Ret decreased obviously (Fig. 3b) owing to the good conductivity of gold nanoclusters. When anti-CEA was immobilized onto the modified electrode, the Ret presented an apparent increase and was estimated to be 516 X (Fig. 3c). The reason may be that anti-CEA acted as an inert electron layer and hindered the electron transfer. The Ret increased in a similar way after BSA was used to block nonspecific sites (Fig. 3d), which was estimated to be 557 X. After the immunosensor was incubated with the CEA antigen, the Ret further increased and was estimated to be 730 X (Fig. 3e), which indicated the formation of a hydrophobic immunocomplex layer hindering the electron transfer. The cyclic voltammograms of different modified electrodes in the potential range from 0.6 to 0.2 V in the presence of 5 mM [Fe(CN)6]4 /3 as a redox probe in 0.01 M PBS (pH 6.5) containing 0.1 M KCl are shown in Fig. 4. The redox-labeled [Fe(CN)6]4 /3 revealed a reversible CV at the bare GCE (Fig. 4a). After the pretreated GCE was modified with a PDCNT/(PSS/PDCNT)2 film, the peak current decreased (Fig. 4b) due to the polyelectrolyte film hindering the transfer of electrons. Then a pair of well-defined redox peaks was appeared again when gold nanoclusters were loaded onto the modified electrode (Fig. 4c). The peak current decreased clearly after anti-CEA was immobilized onto the modified electrode surface (Fig. 4d), which suggested that the big protein molecules hindered the electron transfer, in agreement with the results of EIS. When BSA was employed to block nonspecific sites, a further decrease of the peak currents was observed (Fig. 4e). After the immunosensor was incubated with CEA antigen, the peak current decreased again due to the immunocomplex blocking the tunnel for mass and electron transfer (Fig. 4f). Fig. 5 shows the CVs of the prepared immunosensor at different scan rates. It can be observed that the potentials and peak currents were dependent on the scan rate. Furthermore, both the anodic and the cathodic peak current were directly proportional to the square root of scan rates in the range of 10–200 mV s 1 (shown in the inset of Fig. 5), suggesting a diffusion-controllable redox process. Optimization of analytical conditions The factors influencing the performance of the immunosensor included the buffer pH, the incubation temperature, and the incu-

0.2

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Fig.4. CVs of different modified electrodes measured in 5.0 mM [Fe(CN)6]4 /3 containing 0.1 M KCl: (a) bare GCE; (b) PDCNTs/(PSS/PDCNTs)2/GCE; (c) Au/ PDCNTs/(PSS/PDCNTs)2/GCE; (d) anti-CEA/Au/PDCNTs/(PSS/PDCNTs)2/GCE; (e) BSA/anti-CEA/Au/PDCNTs/(PSS/PDCNTs)2/GCE; (f) CEA/BSA/anti-CEA/Au/PDCNTs/ (PSS/PDCNTs)2/GCE.

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bation time. The effect of pH on the detection solution on the immunosensor behavior was investigated over a pH range from 5.0 to 8.5 with 10 ng mL 1 CEA. As shown in Fig. 6a, the current responses increased from pH 5.0 to 6.5 to reach the maximum value and decreased from pH 6.5 to 8.5. Hence, pH 6.5 was chosen as the optimum pH of the detection solution throughout this study to obtain a high sensitivity. Temperature was an important factor for the activity of the antibody and antigen. The effect of temperature was studied over a range from 15 to 45 °C with 10 ng mL 1 CEA. Fig. 6b shows that the current response increased with increasing temperature to 37 °C and then decreased. The reason may be that the high temperature partly caused denaturation of proteins [36]. Thus, the temperature of 37 °C was selected as the optimal incubation temperature. The immunosensor was incubated in a constant concentration of CEA for different times. DI rapidly increased within the first 20 min and then tended to level off. Therefore, 20 min was chosen as the optimal incubation time.

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Performance of the immunosensor Calibration curve The calibration plots for CEA detection with the prepared immunosensor under optimal experimental conditions are shown in Fig. 7a. The changes of oxidation peak current response (DI) of the immunosensor were found to be proportional to the CEA concentration in two linear ranges from 0.1 to 2.0 ng mL 1 and from 2.0 to 160.0 ng mL 1, with a detection limit of 0.06 ng mL 1 (S/ N = 3). The linear slopes were 4.69 and 0.51 lA mL ng 1, and the correlation coefficients were 0.992 and 0.995, respectively. Comparative studies were carried out using the immunosensor without PDCNTs (Au/PDDA/(PSS/PDDA)2/GCE, Fig. 7b). The linear ranges were 0.5–5.0 ng mL 1 and 5.0–160.0 ng mL 1, with a detection limit of 0.1 ng mL 1 (S/N = 3). The proposed immunosensor exhibited higher sensitivity with a lower detection limit. The reason might be that the CNT-based film constructed by the LBL technique was highly homogeneous and stable. Therefore, the uniformly distributed CNTs exhibited excellent electronic conductivity and high surface area, which promoted the electron transfer and increased the amount of immobilized anti-CEA.

Selectivity The selectivity of the immunosensor was studied in the incubation solution with 50 ng mL 1 CEA in the presence of some possible 160

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interferences, such as glucose, cysteine, glycine, prostate specific antigen, bovine serum albumin. Here, the concentrations of cysteine and glycine were 1 lg mL 1, the concentrations of BSA and PSA were 50 ng mL 1, and the concentration of glucose was 1 mg mL 1. The values of the current ratio, which were calculated by comparing DI obtained in the presence of the interference and that without the interference, are shown in Table 1. The results showed that the interferences of relatively high concentrations only posed negligible effects on CEA detection, indicating that the selectivity of the proposed immunosensor was acceptable. Regeneration of the immunosensor Regeneration of immunosensors was an important factor. The prepared immunosensor could be regenerated by simply immersing it in regeneration solution for about 5 min followed by a rinse with double-distilled water. In this experiment, 4 M urea solution and 0.2 M glycine-hydrochloric acid (Gly-HCl) solution (pH 2.8) were chosen as the regeneration solutions with a relative standard deviation (RSD) of 3.4% and 4.2%, respectively (5 regenerations and measurement). Reproducibility, precision, and stability of the immunosensor The reproducibility of the immunosensor was evaluated by intraassay and interassay coefficients of variation (CVs). The intraassay precision was evaluated by assaying the CEA level for 30 determinations of one sample on the same immunosensor. The intraassay CVs of this method were 6.5% and 4.3% at CEA concentrations of 10 and 20 ng mL 1, respectively. The interassay precision was estimated by determining the CEA level in one sample with five immunosensors. The interassay CV was 5.6% at a CEA concentration of 10 ng mL 1. Thus, the precision and reproducibility of the proposed immunosensor were acceptable.

40 Table 1 Specificity of the immunosensor.

20

0 0

40

80

120

160

-1

CCEA /(ng mL ) Fig.7. Calibration plot of the oxidation peak current response of the immunosensor versus concentration of CEA under optimal conditions: (a) BSA/anti-CEA/Au/ PDCNTs/(PSS/PDCNTs)2/GCE, and (b) BSA/anti-CEA/Au/PDDA/(PSS/PDDA)2/GCE. Inset: Cyclic voltammograms of the immunosensor with different CEA concentrations of (a) 0, (b) 5, (c) 10, (d) 20, (e) 40, and (f) 60 ng mL 1. Scan rate 100 mV s 1.

Possible interferences

Current ratioa

Glucose Glycine Cysteine Prostate specific antigen Bovine serum albumin

1.04 1.01 1.00 1.06 1.05

a Current ratio = DI2/DI1, DI2 was the current change in the CEA solution with interference and DI1 was the current change in the CEA solution without interference.

Immunosensor for carcinoembryonic antigen / X. Gao et al. / Anal. Biochem. 414 (2011) 70–76 Table 2 Comparison of serum CEA levels determined using two methods. [8] Serum samples Immunosensor (ng mL ELISA (ng mL 1) Relative deviation (%)

1

)

1

2

3

4

37.4 36.7 1.9

55.2 51.7 6.8

64.8 67.6 4.1

109.0 111.6 2.3

[9]

[10]

The storage stability of immunosensor was also investigated over a 30-day period by detecting the current response to 10 ng mL 1 CEA. When stored, the immunosensor was suspended above 0.01 M PBS at 4 °C. The current response maintained about 97.8%, 95.6%, and 82.5% of the original value after the storage periods of 10 days, 20 days, and 30 days, respectively. The good stability may be ascribed to the fact that the anti-CEA molecules were absorbed firmly on the surface of gold clusters and PDCNTs which provided a good biocompatible microenvironment.

[11]

[12]

[13]

[14]

Preliminary application of the immunosensor

[15]

The proposed immunosensor was used for the detection of real serum samples and the results were compared with those obtained by ELISA. The results and the relative deviations are shown in Table 2. The results indicated that the two methods were in acceptable agreement. Thus, this method could be further developed for clinical detection of CEA.

[16]

[17]

[18]

Conclusions [19]

In this paper, we described a simple way for developing a reagentless amperometric immunosensor for CEA based on a Au/ PDCNT/(PSS/PDCNT)2 multilayer film modified glassy carbon electrode. The CNT-based film constructed by the LBL technique on the electrode surface was highly homogeneous and stable, which assured high reproducibility and sensitivity. The presence of gold nanoclusters increased the electron-transfer efficiency and the amount of immobilized anti-CEA. The proposed immunosensor offered several advantages including high sensitivity, low detection limit, long-term maintenance of bioactivity, and cost-effectiveness. Thus, the immobilized technique and the detection methodology could be further developed for clinically interested biospecies.

[20]

[21]

[22]

[23] [24]

Acknowledgments This research was supported by the National Natural Science Foundation of China (Nos. 30800247, 20805043).

[25]

[26]

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