New Antibody Immobilization Strategy Based on Gold Nanoparticles

J. Phys. Chem. C 2007, 111, 8443-8450

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New Antibody Immobilization Strategy Based on Gold Nanoparticles and Azure I/Multi-Walled Carbon Nanotube Composite Membranes for an Amperometric Enzyme Immunosensor Na Li, Ruo Yuan,* Yaqin Chai, Shihong Chen, Haizhen An, and Wenjuan Li Chongqing Key Laboratory of Analytical Chemistry, College of Chemistry and Chemical Engineering, Southwest UniVersity, Chongqing 400715, China ReceiVed: December 14, 2006; In Final Form: April 9, 2007

A convenient and effective strategy based on the unique characteristics of Azure I/multi-walled carbon nanotube (Azure I/MWNT) composite membranes and the amplification response of enzymes to the antigen-antibody reaction was used to develop a highly sensitive amperometric immunosensor for the detection of R-fetoprotein (AFP). A novel type of Azure I/MWNT composite membrane was fabricated by two steps: first, acid-pretreated MWNT was modified on the surface of glassy carbon electrode. Then, Azure I was coated on MWNT to obtain an Azure I/MWNT composite membrane. Subsequently, gold nanoparticles were adsorbed onto the Azure I/MWNT composite surface by electrostatic interactions between the negatively charged gold nanoparticle and the positively charged Azure I. Later, AFP antibodies were assembled onto the surface of gold nanoparticles. Finally, horseradish peroxidase (HRP) instead of bovine serum albumin was employed to block sites against nonspecific binding and amplified the current signal of the antigen-antibody reaction. Several techniques, including cyclic voltammetry, UV-vis absorption spectroscopy, scanning electron microscopy, transmission electron microscopy, atomic force microscopy, Raman spectroscopy, and X-ray photoelectron spectroscopy, have been employed to characterize the assembly process. Because of the electrocatalytic ability of HRP and MWNT for the reduction of H2O2 and the unique properties of gold nanoparticles, and especially the synergistic augmentation of Azure I and MWNT to facilitate electron-transfer processes, the immunosensor displayed a high sensitivity, fast analytical time, and broader linear response to AFP in two ranges from 0.1 to 8.0 and 8.0 to 250.0 ng/mL with a relatively low detection limit of 0.04 ng/mL at 3 times the background noise. Moreover, the studied immunosensor possessed good reproducibility and storage stability. The proposed methodology was economical, efficient, and potentially attractive for clinical immunoassays.

1. Introduction Immunoassays based on specific antigen-antibody recognition for analytical purposes have been an attractive subjected for clinical diagnosis,1,2 environmental analysis,3 and the food industry.4 R-Fetoprotein (AFP), an oncofetal glycoprotein with a molecular mass of about 68 kDa,5 is well-known as a tumor marker. In healthy human serum, the average concentration of AFP is typically below 25 ng/mL, and an elevated AFP concentration in adult plasma may be an early indication of some cancerous diseases including hepatocellular cancer, yolk sac cancer, liver metastasis from gastric cancer, testicular cancer, and nasopharyngeal cancer.6 Thus, it is necessary to detect AFP for clinical diagnosis. A number of methods and strategies has been reported for the determination of AFP such as fluorometry atomic studies,7 absorption spectrometry,8 and enzyme-linked immunoassay (ELISAs).9 Electrochemical immunoassays seem to be excellent candidates for the rapid and inexpensive diagnosis of genetic diseases and for the detection of pathogenic biological species of clinical interest, due to their advantages such as simple pretreatment procedure, fast analytical time, precise and sensitive current measurements, and inexpensive and miniaturizable instrumentation.10 Recently, numerous immunological methods for determining the concentration of AFP * Corresponding author. Tel: +86-23-68252277. Fax: +86-23-68254000. E-mail: [email protected].

by electrochemical imunoassays have been described.11,12 In the development of electrochemical immunosensing strategies, the stability or activity of the immobilized biocomponents and signal amplification of the immunoconjugates are two key factors.13 Attempts have been reported in the literature to improve the stability or activity of the immobilized biocomponents by using various novel materials such as organic-inorganic composite materials,14 carbon nanotubes,15 and other nanomaterials.16 Conventional signal amplification of the immunoconjugates often was achieved by enzyme labeling of the antigen or antibody, which was relatively expensive and time-consuming and required laborious sample pretreatment.17 Thus, the development of high sensitivity, faster, simpler, and label-free immunosensors has attracted widespread attention. Carbon nanotubes (CNTs) have been the focus of intense research in recent years because of their remarkable nanostructure, which combines a high surface area, high electrical conductivity, good chemical stability, and high mechanical strength.18 Previous studies showed that CNT-modified electrodes possessed a high electrocatalytic effect and a fast electrontransfer rate.19-21 It also was reported that CNTs could electrocatalyze many molecules such as H2O2, NADH, dopamine, etc.22-24 Just recently, the research interest has been extended to functionalize the surface of carbon nanotubes, so as to improve their solubility in physiological solutions and improve selective binding to biotargets. Functionalization of

10.1021/jp068610u CCC: $37.00 © 2007 American Chemical Society Published on Web 05/25/2007

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SCHEME 1: Schematic Illustration of AFP Immunosensor

carbon nanotubes can be achieved by covalent or noncovalent methodologies. In contrast to the covalent method, the noncovalent approach can immobilize functional molecules on the surface of CNTs while maintaining their geometric structures.25 Some strategies have been reported to functionlize CNTs with dyes by the noncovalent approach, which has been shown to be very simple and useful for further introducing other molecules onto the tube surfaces (e.g., nanoparticles, proteins, and enzymes) and enriching the application of carbon nanotubes. Xu et al. developed a novel hydrogen peroxide biosensor based on the coimmobilization of horseradish peroxidase and methylene blue on a carbon nanotube-modified glassy carbon (GC) electrode.26 Li et al. reported thionine-mediated chemistry of carbon nanotubes.27 Some NADH biosensors based on the CNTs and toluidine blue O (TBO) also have been reported.28,29 Azure I, a phenoxiazine dye, has shown very promising properties as a redox mediator, which has been adsorbed on electrode surfaces for the oxidation of NADH.30 The structure of Azure I is similar to other dyes such as thionine, toluidine blue O (TBO), and methylene blue. It can also be adsorbed onto the surface of multi-walled carbon nanotubes (MWNTs) to form a new kind of stable material.31 As is well-known, gold nanoparticles have been extensively used in the field of electroanalytical chemistry, which can provide an environment similar to nature and retain the bioactivity of immobilized biomolecules, offer a high surface to volume ratio, and enhance the electron-transfer kinetics.32 On the basis of the previous reasons, we developed an amperometric enzyme immunoassay for AFP determination based on an Azure I/MWNT composite membrane and gold nanoparticles. In the previous work, we reported that Nafion adsorbed thionine via the ion exchange adsorption to form a stable film.33,34 However, the nearly insulate Nafion blocked the electron propagation, which led to sensitivity problems. In this present work, the Azure I/MWNT composite film with good conductivity, a high electrochemical signal, and biocompatibility can overcome the defect of the Nafion film. Moreover, the obtained Azure I/MWNT composite can provide a remarkable synergistic augmentation to facilitate electron transfer between the active redox center of the enzyme and the electrode surface. At the same time, the MWNT and HRP avoiding the nonspecific absorption instead of BSA significantly amplified the response of the antigen-antibody reaction since both HRP and MWNT exhibited electrocatalytic activity for H2O2 in the strategy. XPS and UV-vis absorption spectroscopy were used to monitor the formation of the Azure I/MWNT composite and the assembly of gold nanoparticles. SEM, TEM, and AFM were employed

to investigate the morphologies or microstructures of the asprepared different modified films. The performance and factors influencing the performance of the resulting immunosensors were studied in detail. The studied immunosensor exhibited a fast analytical time, simple pretreatment procedure, high sensitivity, broader linear range, and lower detection limit. Significantly, the proposed methodology can be readily extended toward the determination of other clinically or environmentally interested biospecies. 2. Materials and Methods 2.1. Reagents. The MWNTs (>95% purity) synthesized by the CVD method were purchased from Chengdu Organic Chemicals Co. Ltd., of the Chinese Academy of Science. Prior to use, MWNTs were treated with concentrated nitric acid to introduce carboxylic acid groups according to ref 35. AFP and anti-AFP were purchased from Biocell Company (Zhengzhou, China). Azure I, HRP, gold chloride (HAuCl4), and sodium citrate were obtained from Sigma Chemical Co. (St. Louis, MO). Sodium dodecyl sulfate (SDS) and hydrogen peroxide (30%, w/v solution) were purchased from Chemical Reagent Company (Chongqing, China). Double distilled water was used throughout this study. Acetate buffer solutions with various pH values were prepared with 0.1 M HAc and 0.1 M NaAc. The supporting electrolyte was 0.1 M NaCl. Gold nanoparticles with a mean size of 16 nm were produced by reducing gold chloride tetrahydrate with citric acid at 100 °C for half an hour.36 2.2. Apparatus. Cyclic voltammetric measurements were carried out with a CHI 600B electrochemistry workstation (Shanghai CH Instruments, Shanghai, China). A three-compartment electrochemical cell containing a platinum wire auxiliary electrode, a saturated calomel reference electrode (SCE), and modified GC (Φ ) 4 mm) as a working electrode were employed for all electrochemical experiments. The UV-vis absorption spectra were recorded in the range of 200-700 nm using a UV-vis spectrometer (UV-vis 8500). The scanning electron micrographs were taken with a scanning electron microscope (SEM, S-4800, Hitachi, Tokyo, Japan). X-ray photoelectron spectroscopy (XPS) measurements were carried out using a VG Scientific ESCALAB 250 spectrometer, using Al KR X-ray (1486.6 eV) as the light source. The atomic force microscopy (AFM) images were obtained with a scanning probe microscope (SPM) (Vecco, Alpharetta, GA). Raman spectra were performed by using a Raman spectrometer (Bruker, RFS 100/S, Karlsruhe, Germany). Transmission electron microscopy (TEM) was carried out on a TECNAI 10 (Philips Fei Co., Hillsboro, OR).

Au Nanoparticles and Azure I C Nanotube Composite Membranes

Figure 1. Cyclic voltammograms of the electrodes at different stages: bare glassy carbon electrode (GC) (a), MWNT/GC (b), Azure I/MWNT/GC (c), nanoAu/Azure I/MWNT/GC (d), anti-AFP/nanoAu/ Azure I/MWNT/GC (e), and HRP/anti-AFP/nanoAu/Azure I/MWNT/ GC (f) in acetate acid buffer (pH ) 5.5). The potential scan rate was 50 mV/s.

2.3. Experimental Measurements. Electrochemical experiments were performed in an unstirred electrochemical cell at room temperature, and the potential was swept from -0.6 to 0.3 (vs SCE) with a sweeping rate of 50 mV/s. After being incubated in a 50 µL incubation solution at 25 °C for 10 min, the immunosensors for AFP were washed carefully with doubly distilled water, and the cyclic voltammetric experiment was performed in 5 mL of pH 5.5 acetate buffer containing 1.4 mM H2O2. 2.4. Fabrication of the Immunosensor. The GC electrode (Φ ) 4 mm) was polished repeatedly with 1.0, 0.3, and 0.05 µm alumina slurry, followed by successive sonication in doubly distilled water and ethanol for 5 min and dried in air. In 10 mL of 0.3 wt % SDS aqueous solution, 10 mg of the acid-treated MWNT was ultrasonically dispersed to give a black suspension of 1.0 mg/mL. The MWNT film was prepared by dropping 15 µL of this black suspension on a cleaned GC electrode surface and then drying slowly in air at room temperature. Subsequently, the Azure I (3 mM) solution was added on the electrode surface and dried at room temperature. During this process, the dye was adsorbed onto the surface of MWNTs. Then, the electrode was immersed into doubly distilled water to remove the non-firmly adsorbed dye molecules until a steady electrochemical response was achieved. Subsequently, the Azure I/MWNT-modified electrode was immersed in a gold nanoparticles solution overnight (about 12 h). Then, the electrode was incubated in anti-AFP solution overnight at 4 °C. Finally, the resulting electrode was incubated in HRP solution for about 4 h at 4 °C to block possible remaining active sites of the gold nanoparticle monolayer and to avoid the nonspecific adsorption instead of BSA and amplify the response of the antigenantibody reaction. The fabricated procedure of the modified electrode was shown in Scheme 1. The finished immunosensor was stored at 4 °C when not in use. 3. Results and Discussion 3.1. Electrochemical Characteristics of the Modified Electrode. Cyclic voltammetry (CV) was an effective and convenient method for probing the feature of the modified electrode surface. In this paper, each assembly step of the modified electrode was followed by an electrochemical characterization. Figure 1 showed cyclic voltammgramms of different modified electrodes in the potential range of -0.6-

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Figure 2. Cyclic voltammograms of the modified electrodes at different scan rates (from inner to outer): 10, 30, 50, 80, 100, 150, 200, 250, 300, and 400 in pH 5.5 acetate acid buffer. The inset shows the dependence of redox peak currents on the potential sweep rates.

0.3 V in a pH 5.5 acetate buffer solution containing 0.1 M NaCl at a scan rate 50 mV/s. No redox peak appeared for the cyclic voltammgramms of the bare GC electrode (curve a, Figure 1). The MWNT-modified electrode showed a very small reductionoxidation peak pair at about 0 V vs SCE (curve b, Figure 1), which was also observed by others MWNT37,38 film electrodes. This contributed to the presence of oxygen-containing moieties on the tube surface. Moreover, the MWNT-modified electrode had larger background current compared with bare GC, probably due to the significant increment of electrode surface-area. In contrast, a pair of well-define redox peak was observed at the Azure I/MWNT-modified electrode (curve c, Figure 1), which was ascribed to the redox reaction of Azure I, showing that Azure I can be easily adsorbed onto the MWNT film. After gold nanoparticles were chemisorbed into the Azure I/MWNT composite via the oppositely charged adsorption technique (curve d, Figure 1), the responses of the peak currents obviously increased as the gold nanoparticles were similar to a conducting wire or an electron-conducting tunnel, which made it easier for the electron transfer to take place. When the anti-AFP molecules were adsorbed onto the gold nanoparticles (curve e, Figure 1), the peak current decreased, and the peak-to-peak separation increased. The reason for this was that anti-AFP acted as an inert electron layer and hindered the electron transfer. At the last step of assembly, HRP was employed to block the possible remaining activity sites of the modified immunosensor, and a further decrease of the peak currents contributed to the adsorption of the protein HRP (curve f, Figure 1). Typical cyclic voltammgramms of the proposed electrode in acetate buffer solution (pH 5.5) at different scan rates were presented in Figure 2. The peak currents of the HRP/anti-AFP/ nanoAu/Azure I/MWNT/GC were directly proportional to the scan rates up to 400 mV/s, indicating a surface confined redox process. 3.2. UV-vis, Raman Spectroscopy, and X-ray Photoelectron Spectroscopy. The modified process of the electrode could also be examined by UV-vis absorption spectra. The UV spectra of MWNT dispersed in an aqueous solution of SDS exhibited a feature absorption at 255 nm (curve d in the inset of Figure 3), which accorded with that of the pristine HiPco MWNT.39,40 Pure Azure I displayed two strong adsorbances at 282 and 586 nm (curve a, Figure 3). The Azure I/MWNT composite also had two characteristic absorption peaks, one in the visible region around 580 nm, and the other located in the


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Figure 5. Raman spectra of MWNT (a) and Azure I/MWNT (b). Figure 3. UV spectra of Azure I (a), Azure I/MWNT (b), and nanoAu/ Azure I/MWNT (c). Inset: MWNT (d).

Figure 4. (A) XPS spectra of MWNT (a) and Azure I/MWNT composite (b). (B) Gold nanoparticles (a) and nanoAu/Azure I/MWNT (b). Inset: narrow scan spectrum of N1s peak.

UV region around 270 nm (curve b, Figure 3). As compared with pure Azure I, the two absorption peaks exhibited a slight deviation, which indicated an interaction between Azure I and MWNT. After gold nanoparticles were adsorbed onto the Azure I/MWNT composite film, the two peaks could also be observed. However, a red shift for the peak at 596 nm, a blue shift for the other peak at 266 nm, and a new absorption peak at 520 nm appeared due to the characteristic peak of the gold nanoparticles (curve c, Figure 3), which provided evidence of the interaction between the oppositely charged Azure I and gold nanoparticles, showing that gold nanoparticles could be preferentially adsorbed onto Azure I. XPS experiments for MWNT, Azure I/MWNT, and nanoAu/ Azure I/MWNT were carried out to obtain key information concerning the chemical state of the film surface. As shown in Figure 4A (curve a), there was an intense C1s peak centered at 286.4 eV due to the C atoms in the MWNTs and a weak O1s peak centered at 532.4 eV due to the oxidation of the MWNTs in the survey. After Azure I was adsorbed on the surface of MWNTs (Figure 4A, curve b), a characteristic peak of N 1s appeared at 400.0 eV, which further confirmed the existence of Azure I on the electrode. Further, we also measured the Au 4f XPS of gold nanoparticles and nanoAu/Azure I/MWNTs. As can be seen in Figure 4B (curve a), the Au 4f XPS from gold nanoparticles was characterized by peaks with binding energies of 83.9 eV for 4f7/2 and 87.6 eV for 4f5/2, both distinctive for Au metal. In contrast, the Au 4f signal was observed at the nanoAu/Azure I/MWNT film (Figure 4B, curve b), indicating that gold nanoparticles were indeed adsorbed on the Azure I/MWNT composite film.

Figure 6. SEM photos of MWNT film (A), Azure I/MWNT (B), nanoAu/Azure I/MWNT (C), and anti-AFP/nanoAu/Azure I/MWNT (D).

To ensure the presence of MWNTs in the composite film, Raman spectroscopy was considered. As shown in Figure 5 (curve a), the MWNT displayed two feature peaks at 1595 and 1285 cm-1. The peak lying at 1595 cm-1 named the G band was due to the splitting of the E2g stretching mode in graphite. The peak at 1285 cm-1, the D band, was related to the defects in the MWNT, which might be attributed to the extent of destruction of carbon nanotubes by strong oxidation.41 As for the Azure I/MWNT composite (Figure 5, curve b), the G and D bands upshifted to 1599 and 1290 cm-1, respectively, which might be due to the interaction between Azure I and MWNTs. In our strategies, the appearance of the G and D bands was the signature of the presence of MWNTs in the composite membrane. On the basis of the results of UV spectroscopy, Raman spectroscopy, and XPS, we concluded that complex reactions on the electrode surface might have occurred successfully as was expected. 3.3. SEM, TEM, and AFM Analysis. The morphologies and microstructures of the as-prepared different modified films were studied by means of SEM and TEM. As shown in the SEM profiles (Figure 6A), the homogeneous dispersion MWNTs could be observed, and most of the MWNTs were in the form of small bundles or single tubes, which would benefit the sensor performance because the well-dispersed MWNTs were electrochemically accessible.42 For comparison, the diameter of MWNTs modified by Azure I (Figure 6B) became larger than


Figure 7. TEM images of MWNT film (A) and nanoAu/Azure I/MWNT (B).

that of MWNT, confirming the formation of Azure I/MWNT composite membranes. Some small bright particles could be observed at the SEM image of nanoAu/Azure I/MWNTs, which were evidence of the presence of gold nanoparticles (Figure 6C). When anti-AFP was adsorbed on nanoAu/Azure I/MWNTs, the corresponding SEM image obviously changed. The MWNTs became blurry due to the presence of anti-AFP (Figure 6D). The TEM images of MWNT films and nanoAu/Azure I/MWNTs are shown in Figure 7. As compared with the TEM image of the MWNT film (Figure 7A), the TEM image of nanoAu/Azure I/MWNTs (Figure 7B) clearly exhibited the presence of numerous small particles, which further confirmed that gold nanoparticles could firmly be adsorbed onto the Azure I/MWNTs composite by electrostatic interactions. AFM studies were further conducted to give insight into the surface topography of the MWNTs, Azure I/MWNTs, nanoAu/ Azure I/MWNTs, and anti-AFP/nanoAu/Azure I/MWNTs. As shown in Figure 8A, it was clearly seen that randomly oriented MWNTs covered the entire surface of the substrate quite homogeneously. However, after adsorbing Azure I (Figure 8B), the diameter of MWNTs coated by Azure I became “fat” as compared with the MWNTs, which was in conformity with the results of SEM. Moreover, the Azure I/MWNT composite was also well-dispersed, implying that the MWNTs would act as a high conductivity nanowire connecting the film domains throughout the composite and directly improve the composite conductivity.43 The AFM images of the nanoAu/Azure I/MWNTs presented many particles due to the coating of gold nanoparticles (Figure 8C). As compared with the nanoAu/Azure I/MWNTs, the AFM image of anti-AFP/nanoAu/Azure I/MWNTs exhibited obvious differences (Figure 8D). The latter showed a smoothing effect, which might be ascribed to anti-AFP molecules filling the interstitial places between nanoparticles and nanoparticles, suggesting that the anti-AFP was successfully immobilized on the surface of gold nanoparticles. On the basis of the previous results, we might conclude that the as-prepared immunosensor could preliminarily be applied for determination of the AFP antigen. 3.4. Cyclic Voltammetric Response of Immunosensor to H2O2. The electrocatalytic reactivity of the HRP layer-modified electrode toward H2O2 was investigated by CV. Figure 9 depicts the electrocatalytic response of the electrode in pH 5.5 acetate acid buffer with and without the presence of H2O2. A pair of oxidation-reduction peaks was observed in the blank acetate acid buffer (Figure 9, curve a), which contributed to the redox reaction of Azure I. Upon the addition of 1.4 mM H2O2 in the substrate solution, the reduction peak current increased obviously from -144.5 to -185.4 µA, while the oxidation current decreased (Figure 9, curve b), suggesting a typical electrocatalytic reduction process of H2O2. Good electrocatalytic activity

J. Phys. Chem. C, Vol. 111, No. 24, 2007 8447 of the immunosensor for H2O2 was attributed to the presence of MWNTs and HRP. This result indicated that the Azure I/MWNT composite could effectively shuttle electrons from the base electrode surface to the redox center of HRP. 3.5. Optimization of Experimental Parameters. 3.5.1. Influence of pH on the Sensor Response. The analytical performance of the immunosensor was related to the pH value of the detection solution. The acidity of the solution greatly affected both the activity of the immobilized protein and the electrochemical behavior of the Azure I mediator. The effect of pH on the CV peak current was tested over a pH range from 4.0 to 6.5 at constant concentrations of AFP and 1.4 mM H2O2. From the results shown in Figure 10, both reduction and oxidation peak potentials shifted negatively with an increase of pH, while the current response decreased with increasing pH. This phenomena was the same as the electrochemical behavior of most dyes.34,44 The formal potential E0', which was estimated as the midpoint of the cathodic and anodic peak potential, showed a linear dependence on pH in the range from 4.0 to 6.5 with a slope of -50.6 mV/pH. The slope value was close to the theoretical value of -59 mV/pH at 25 °C, showing that the proton was transported with an equal number of protons and electrons.45 Considering the activity of the protein and the lifetime of the immunosensor, an acetate acid buffer solution of pH 5.5 was selected for further experiments. 3.5.2. Influence of the Amount of H2O2 on the Sensor Response. The H2O2 concentration in 5 mL of pH 5.5 acetate acid buffer on the steady-state current of the immunosensor was studied because it played an important role in the immunosensor performance. As shown in Figure 11, when the H2O2 concentration was less than 1.4 mM, the reduction peak current increased linearly with increasing H2O2 concentration, then it tended toward a constant value, which corresponded to the saturated station. This showed that the HRP attached to the immunosensor surface had a relatively high catalytic activity. Therefore, the optimal concentration of H2O2 was chosen at 1.4 mM. 3.5.3. Effect of Temperature and Incubation Time on the Immunoreaction. The effect of the immunochemical incubation (i.e., when the antigen-antibody reaction occurs) temperature was examined at the range from 15 to 45 °C. As shown in Figure 12, it was found that the current response increased with increasing temperature up to 35 °C. The lower response at the incubation temperatures higher than 35 °C might be attributed to an irreversible behavior (denaturation of proteins) involved in the process. However, a longer time of high temperatures might decrease the activity of the biomolecule, leading to the deterioration of response signals and lifetime. So, a temperature of 25 °C (room temperature) was selected as a compromise. According to the study of incubation time influence (Figure 13), we found that the amperometric response of the immunosensor to 50 ng/mL AFP in presence of 1.4 mM H2O2 increased with an increment of incubation time and leveled off after 10 min. A longer incubation time did not improve the response. Therefore, 10 min was chosen as the incubation time for the determination of the AFP antigen. 3.6. Amperometric Response of the Immusensor to AFP Concentration. Under optimal immunoassay conditions, the calibration graph for the determination of AFP is shown in Figure 14. The amperometric detection was performed in acetate acid buffer (pH 5.5) at room temperature containing 1.4 mM H2O2 after the immunosensor was incubated with different concentrations of AFP solution for 10 min. The cyclic voltammgramm peak currents of the resulted HRP/anti-AFP/nanoAu/ Azure I/MWNT/GC after the antigen-antibody reaction showed


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Figure 8. AFM images of MWNT membrane (A), Azure I/MWNT composite membrane (B), nanoAu/Azure I/MWNT (C), and anti-AFP/nanoAu/ Azure I/MWNT (D).

Figure 9. Cyclic voltammograms of the immunosensor in pH 5.5 acetate acid buffer without (a) and with 1.4 mM H2O2 (b). Scan rate 50 mV/s.

a decrease with increasing AFP concentration. The reason for this was that the incubation of the immunosensor with AFP solution resulted in the formation of an immunocomplex, which made the block of electrons shuttle between the redox center of HRP and the electrode. As shown in Figure 14 (curve b),

Figure 10. Cyclic voltammograms of the immunosensor in acetate acid buffer at various pH values: (a) 4.0, (b) 4.5, (c) 5.0, (d) 5.5, (e) 6.0, and (f) 6.5 at a scan rate of 50 mV/s.

the percentage decrease of the cyclic voltammgramm peak current was proportional to the AFP concentration in two ranges from 0.1 to 8.0 and 8.0 to 250.0 ng/mL with linear slopes of


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Figure 11. Influence of the amount of H2O2 on the sensor response.

Figure 14. Calibration curves for AFP determination were obtained by using BSA (a) and HRP (b) as blocking agents, respectively. Insets: linear relationship between the amperometric reduction current response and AFP concentration in ranges of (A) 0.1-8.0 ng/mL and (B) 8.0-250.0 ng/mL.

Figure 12. Effect of the temperature on the response of the immunoelectrode.

Figure 15. Lifetime of the immunosensor to 80 ng/mL AFP.

Figure 13. Influence of incubation time on the response of the immnuoelectrode.

2.6068 and 0.1583 µA mL/ng and coefficients of 0.9985 and 0.9994, respectively. The detection limit was 0.04 ng/mL, calculated at 3 times the background noise. As compared with the calibration plot of the HRP blocked electrode, the BSA blocked electrode (Figure 14, curve a), as shown in the inset, exhibited a less steep decline with a narrower linear response from 0.5 to 8.0 and 8.0 to 200 ng/mL. These results indicated that HRP instead of BSA as a blocking agent enhanced the linear range and sensitivity of the electrode to the analyte solution. The reason for this may be that the blocking reagent HRP performed an effective amplification property as expected. 3.7. Selectivity. One of the potential advantages of using biological molecules as recognition elements in biosensors was the selectivity of the biological molecule for its analyte. Possible interfering substances were used to evaluate the selectivity of the immunosensor. The immunosensors were incubated with 160 ng/mL AFP in the incubator containing separately CEA, HBsAg, BSA, l-cysteine, and l-glutamic acid. The imunosensors were separately exposed to 160 ng/mL AFP solution with

interference and without interference. The peak current responses of cyclic voltammgramms in the two solutions show less than 4% difference. The selectivity of the AFP immunosensor based on the highly specific antigen-antibody immunoreaction was satisfactory. 3.8. Repeatability and Stability of the AFP Immunosensor. The AFP solution of 80 ng/mL was repeatedly determined using the proposed immunosensor 5 times, and the current responses, respectively, were -150.8, -149.4, -155.6, -147.6, and -152.9 µA. The relative standard deviation (RSD) was 2.0%. The previous results suggest that the reproducibility of the HRP/ anti-AFP/nanoAu/Azure I/MWNT/GC immunosensor was satisfactory. The proposed immunosensor showed a high stability. The 100 continuous cycle scan was carried out in the potential range from -0.6 to 0.3 with a 50 mV/s scan rate; a 7.9% decrease of the initial response was observed. On the other hand, the longterm storage stability of the enzyme immunosensor was also studied. When not in use, the electrode was suspended above 0.1 M phosphate buffer at 4 °C in a refrigerator. The long-term storage stability of the immunosensor was investigated by measuring the response current to 80 ng/mL AFP every 2-3 days and is shown in Figure 15. During the first 6 days, the response current decreased by about 0.80% of its initial response, in the next 2 weeks by about 2.7%, and by about 4.5% for 1 month. The good stability may be due to the fact that the Azure


Li et al.

TABLE 1: Recovery of Prepared Immunosensor

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sample: R-fetoprotein (ng/mL)

biosensor: found R-fetoprotein (ng/mL)

recovery (%)

3 20 50 80 200

3.2 19.5 51 78.8 204.9

106.7 97.5 102.0 98.5 102.4

I/MWNT composite was stable and that the amount of gold nanoparticles was adsorbed firmly onto the composite monolayer. 3.9. Application. The analytical application of the immunosensor was evaluated by determining the recoveries of 3, 20, 50, 80, and 200 ng/mL AFP by standard addition methods. The experimental results were listed in Table 1. As is seen from it, the recovery was in the range of 97.5-106.7%, which demonstrated that the developed immunoassay might provide a feasible alternative tool for determining the AFP antigen in human serum in a clinical laboratory. 4. Conclusion In this paper, a new strategy based on an Azure I/MWNT composite and a gold nanoparticle-modified GC electrode was proposed for developing an amperometric enzyme immunosensor for AFP. The studied immunosensor possessed three attractive advantages: first, the Azure I/MWNT composite overcame the defect of insulated film and provided a remarkable synergistic augmentation to facilitate the electron-transfer process. Second, gold nanoparticles with larger specific interface areas, desirable biocomapatibility, and a high surface free energy could adsorb more antibodies without the loss of their biological activities. Third, because the MWNTs could also electrocatalyze H2O2, both MWNTs and the HRP used as the blocking reagent significantly amplified the response of the antigen-antibody reaction. With these three advantages, the immunosensor showed a broader linear range, lower detection limit, and high sensitivity. Thus, the proposed method could be extended to the determination of other disease markers. Acknowledgment. This work was supported by the National Natural Science Foundation of China (20675064), the Natural Science Foundation of Chongqing City (CSTC-2004BB4149 and 2005BB4100), the Chinese Education Ministry Foundation for Excellent Young Teachers (2002-40), and the High Technology Project Foundation of Southwest University (XSGX 02), China. References and Notes (1) Wilson, G. S.; Hu, Y. Chem. ReV. 2000, 100, 2693-704. (2) Warsinke, A.; Benkert, A.; Scheller, F. W. Fresen. J. Anal. Chem. 2000, 366, 622-634. (3) Van Emon, J. M.; Lopez-Avila, V. Anal. Chem. 1992, 64, 79-89. (4) Bilitewski, U. Anal. Chem. 2000, 72, 693-701. (5) Gillespie, J. R.; Uversky, V. N. Biochim. Biophys. Acta 2000, 1480, 41-56. (6) Li, T. X. In Assay and Clinical Application of AFP, Modern Clinical Immunoassay, 1st ed.; Military Medical Science Press: Beijing, 2001; p 169.

New Antibody Immobilization Strategy Based on Gold Nanoparticles

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