Electrodeposition of gold–platinum alloy nanoparticles on carbon nanotubes as electrochemical sensing interface for sensitive detection of tumor marker

Electrochimica Acta 56 (2011) 6715–6721

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Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta

Electrodeposition of gold–platinum alloy nanoparticles on carbon nanotubes as electrochemical sensing interface for sensitive detection of tumor marker Ya Li, Ruo Yuan ∗ , Yaqin Chai, Zhongju Song Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China

a r t i c l e

i n f o

Article history: Received 15 January 2011 Received in revised form 19 May 2011 Accepted 19 May 2011 Available online 26 May 2011 Keywords: Amperometric immunosensor Alpha-fetoprotein (AFP) Multi-walled carbon nanotubes (MWCNTs) Gold–platinum alloy nanoparticles (Au–PtNPs) Horseradish peroxidase (HRP)

a b s t r a c t A novel electrochemical sensing interface, electrodeposition of gold–platinum alloy nanoparticles (Au–PtNPs) on carbon nanotubes, was proposed and used to fabricate a label-free amperometric immunosensor. On the one hand, the multiwalled carbon nanotubes (MWCNTs) could increase active area of the electrode and enhance the electron transfer ability between the electrode and redox probe; on the other hand, the Au–PtNPs not only could be used to assemble biomolecules with bioactivity kept well, but also could further facilitate the shuttle of electrons. In the meanwhile, horseradish peroxidase (HRP) instead of bovine serum albumin (BSA) was employed to block the possible remaining active sites and avoid the nonspecific adsorption. With the synergetic catalysis effect of Au–PtNPs and HRP towards the reduction of hydrogen peroxide (H2 O2 ), the signal could be amplified and the sensitivity could be enhanced. Using alpha-fetoprotein (AFP) as model analyte, the fabricated immunosensor exhibited two wide linear ranges in the concentration ranges of 0.5–20 ng mL−1 and 20–200 ng mL−1 with a detection limit of 0.17 ng mL−1 at a signal-to-noise of 3. Moreover, the immunosensor exhibited good selectivity, stability and reproducibility. The developed protocol could be easily extended to other protein detection and provided a promising potential in clinical diagnosis application. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction The elevated concentration of tumor marker in serum may be an early indication of certain cancer. Therefore, it is necessary and important to develop a method with high sensitivity and selectivity for the determination of tumor marker levels in serum. Electrochemical immunosensor, based on highly specific recognition of antibody and antigen, is a very promising technique for the assay of tumor marker and has been applied widely in many fields such as environment analysis [1], food industry [2,3], and clinical applications [4], which was due to their potential utility as specific, simple, low cost, small size and short response time [5]. Various types of electrochemical immunosensors such as amperometric [6,7], potentiometric [8,9], capacitive [10,11] and impedance immunosensors [12,13] have been reported. Among these immunosensors, the amperometric immunosensor is especially promising for its relatively higher sensitivity, low detection limit and wider linear range [14,15]. In the fabrication process of immunosensor, immobilization of biomolecules with bioactivity kept well on the sensing electrode surface has been considered to be one of the most important points, and accordingly there are many literatures about immunosensor

∗ Corresponding author. Tel.: +86 23 68252277; fax: +86 23 68253172. E-mail address: [email protected] (R. Yuan). 0013-4686/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2011.05.066

based on different immobilization matrix and sensing interface [16]. With the rapid development of nanotechnology over the past decade, various nanomaterials have been synthesized and used for the construction of biosensors, especially metallic nanoparticles. Recently, bimetallic alloys have been of considerable interest in the field of catalysis and sensors because of the interaction between two components in bimetallic alloys. They often present many favorable properties in comparison with the corresponding monometallic counterparts, which include high catalytic activity, catalytic selectivity and better resistance to deactivation. Gold–platinum alloy nanoparticles (Au–PtNPs) are very attractive among various bimetallic alloys. Besides large surface-to-volume ratio, good biocompatibility and satisfied conductive capability [17,18], Au–PtNPs possessed excellent catalytic activities towards H2 O2 , methanol and so on, due to the high synergistic action between gold and platinum [19]. So it is significant to develop Au–PtNPs based electrochemical sensors with appropriate characteristics such as high sensitivity, fast response time, wide linear range, better selectivity and reproducibility, which was attribute to the advantages of bimetallic nanoparticles. Based on the above reason, Luo et al. have reported Au–PtNPs for electrocatalytic methanol oxidation reaction and the result was satisfying [20]. On the other hand, carbon nanotubes (CNTs), which could possess unique advantages including enhanced electronic properties, a large edge plane/basal plane ratio, and rapid electrode kinetics, have also been incorporated into electrochemical sensors.

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Fig. 1. Schematic illustration of the stepwise immunosensor fabrication process: (a) adsorption of MWCNTs film, (b) electrodeposition of gold–platinum alloy nanoparticles, (c) anti-AFP loading, (d) HRP blocking, (e) AFP loading.

CNT-based sensors generally have higher sensitivities, lower limits of detection, and faster electron transfer kinetics than traditional carbon electrodes [21]. Based on the above consideration, we utilized the unique properties of MWCNTs and Au–PtNPs to fabricate an amperometric immunosensor. Initially, Au–PtNPs were electrodeposited on the multiwalled carbon nanotubes (MWCNTs) modified glassy carbon electrode by constant potential stripping technique, and then anti-AFP was adsorbed onto the sensing interface of the Au–PtNPs/MWCNTs/GCE. Subsequently, horseradish peroxidase (HRP) instead of bovine serum albumin (BSA) was employed to block possible remaining active sites of the Au–PtNPs to avoid the non-specific adsorption and was further used to amplify response signal. The electron transfer between HRP and electrode surface is the limiting factor in the operation of amperometric signal detection. The hydroquinone (HQ) was used as an electron mediator to shuttle electrons between HRP and the electrode surface. Hydrogen peroxide (H2 O2 ) was added into the working buffer as substrate, and the response signal could be amplified effectively by the synergistic catalysis action of Au–PtNPs and HRP towards the reduction of H2 O2 . Using alpha-fetoprotein (AFP) as model analyte, the proposed biosensor exhibited a wide linear response range for model analyte, good selectivity and sensitivity, which demonstrated the proposed immunosensor possessed potential applications in clinical screening of cancer biomarkers. 2. Experimental 2.1. Reagents and material Anti-AFP and AFP were purchased from Biocell Company (Zhengzhou, China), stored in the frozen state before used. The multi-walled carbon nanotubes (MWCNTs, >95% purity, synthesized by CVD method) were purchased from Chengdu Organic Chemicals Co. Ltd. of the Chinese Academy of Science. Chlorauric acid, chloroplatinic acid, bovine serum albumin (BSA, 96–99%), N, N-dimethylformamide (DMF) and horseradish peroxidase (HRP) were bought from Sigma Chemical Co. (St. Louis, MO, USA). Hydrogen peroxide (H2 O2 , 30%, w/v solution) and hydroquinol (HQ) were obtained from Chemical Reagent Co. (Chongqing, China). All of the chemicals used were of analytical grade and were used as received without further purification. Phosphate-buffered solution

(PBS, 0.1 M) with various pH values was prepared with stock standard solutions Na2 HPO4 and NaH2 PO4 . The supporting electrolyte was 0.1 M KCl. Serum specimens provided by Southwest Hospital of Third Military Medical University (Chongqing, China) were stored at 4 ◦ C in a freezer. Double-distilled water was used throughout this study. 2.2. Apparatus Electrochemical measurements were carried out by CHI 660D electrochemistry workstation (Shanghai CH Instruments Co., China). The scanning electron micrograph was taken with scanning electron microscope (SEM, S-4800, Hitachi). A conventional, threeelectrode cell consisting of the modified glassy carbon electrode (GCE) as the working electrode, a platinum wire as the counter electrode and a saturated calomel electrode (SCE) as the reference electrode was used. 2.3. Fabrication of the amperometric immunosensors The bare GCE were respectively polished with 0.3 and 0.05 ␮m alumina slurry to obtain mirror-like surface and ultrasonically cleaned with ethanol and double distilled water for 5 min to remove the physically adsorbed substance. Then the electrodes were allowed to dry at room temperature. Subsequently, 10 ␮L of black suspension of MWCNTs was dispersed in N, N-dimethylformamide (DMF) was cast on the pretreated bare GC electrode surface and dried in air. After that, the MWCNTs modified GCE was immersed in 2 mL deposition solution (0.2 M Na2 SO4 aqueous solution containing 1 mM HAuCl4 and 1 mM H2 PtCl6 ) [22] and applied a constant potential for 200 s at −0.2 V to obtain Au–PtNPs/MWCNTs modified electrode. Then, it was immersed in anti-AFP solution at 4 ◦ C for 12 h. Finally, the obtained electrode was incubated in HRP solution for 4 h at 4 ◦ C to eliminate non-specific binding effect and block possible remaining active sites. The schematic diagram of the immunosensor was shown in Fig. 1. 2.4. Synthesis of Au–PtNPs Au–PtNPs were synthesized according to the reference [22]. 1 mL 1 mM HAuCl4 and 1 mL 1 mM H2 PtCl6 were mixed with 0.2 M Na2 SO4 aqueous solution. The resulting solution was Au–PtNPs

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7.4 PBS containing 5.0 mM K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ] (1:1) and 0.1 M KCl. Fig. 3A and B displayed the CVs of the fabrication process of the immunosensor with different redox probes, respectively, which both proved the successful fabrication of the sensing interface. 3.2. The influence of MWCNTs on electrochemical sensing interface

Fig. 2. SEM images of gold–platinum alloy nanoparticles.

solution and was stored at 4 ◦ C. Scanning electron micrographs (SEM) were performed to characterize the shape and size of Au–PtNPs (Fig. 2). 2.5. Experimental measurements The characteristic of the stepwise modified electrode were performed by cyclic voltammetry (CV) measurements, with the potential range from −0.3 to +0.4 V (vs SCE) at the rate of 50 mV s−1 in pH 7.4 PBS containing 2.0 mM hydroquinone (HQ). After being incubated with various concentrations of AFP for 12 min at room temperature, the immunosensors capturing AFP were washed carefully with washing buffer and the detection was carried out by differential pulse voltammetry (DPV) in working buffer containing 2.0 mM HQ and 8.0 mM H2 O2 . The immunoassay was based on the change of reduction peak current response (I) before and after the reaction of antibody and antigen. Before the immunoreaction taken place, the current response (I0 ) was recorded. Owing to the formation of bulky immunocomplexes restraining the electron-transfer, the current response of the immunosensor decreased after incubation, which was recorded as I. Therefore, the immunosensor current response was given by: I = I0 − I. 3. Results and discussion 3.1. Electrochemical characterization on electrode surface CV is an effective and convenient tool of showing the changes of electrode behavior after each modification step. Fig. 3A shows CVs of differently modified electrodes in the potential range from −0.3 to +0.4 V in work solution (pH 7.4 PBS containing 2.0 mM HQ). Welldefined CVs, characteristic of diffusion-limited redox processes, were observed at the bare GCE electrode (Fig. 3A, curve a). The peak currents increased after MWCNTs coating on the bare electrode (Fig. 3A, curve b), the reason was that MWCNTs facilitate electron transfer. After Au–PtNPs monolayer was electrodeposited on modified electrode, the current further increased (Fig. 3A, curve c), which was due to the promotion of Au–PtNPs towards the shuttle of electrons. When anti-AFP molecules were immobilized on the electrode surface, a decreased current response signal was obtained (Fig. 3A, curve d). After HRP was immobilized successfully, a further decrease of the peak currents was found with the fact that HRP insulates the conductive support and hinders the transmission of electrons towards the electrode surface (Fig. 3A, curve e). Fig. 3B was a contrast experiment and showed the CVs of the different modified electrodes in the potential range from −0.2 to +0.6 V in pH

In order to investigate the electrochemical properties of MWCNTs monolayer, a comparative study was carried out by calculating electro-active surface areas of different modified electrodes: (a) Au–PtNPs/MWCNTs/GCE, (b) Au–PtNPs/GCE. The CVs of two electrodes were performed in the presence of redox probe Fe(CN)6 3−/4− at a series of scan rates, respectively. An enormous surface areato-volume ratio, which is highly susceptible to heterogeneous redox chemistry with surrounding environment, is pivotal factor. The electro-active surface area of electrode can be estimated for a reversible and diffusion controllable process according to the Randles–Sevcik equation [23]: Ip = 2.69 × 105 A · D1/2 n3/2 v1/2 c where, Ip relates to the redox peak current; n represents the transferring electron number. This constant can be used to estimate n for an electrode. The Fe(CN)6 3−/4− redox system used in this study is one of the most extensively studied redox couples in electrochemistry and exhibits a heterogeneous one-electron transfer (n = 1). c represents the concentration (mol cm−3 ) of the ferricyanide; A is the area of the electrode (cm2 ), D is the diffusion coefficient of the molecule in solution (cm2 s−1 ), which is 6.70 ± 0.02 × 10−6 cm2 s−1 at 25 ◦ C; and v is the scan rate of the potential perturbation (V s−1 ). D, C and n are constants. Ip (as well as the current at any other point on the wave) is proportional to vl/2 . According to the above equation, we can get the approximate value of A. Fig. 4A and B were the CVs of the prepared Au–PtNPs/MWCNTs/GCE and Au–PtNPs/GCE in 5.0 mM Fe(CN)6 3−/4− at different scan rates, respectively. The inset in Fig. 3A and B were respectively linear relations with the anodic and cathodic peak current of Au–PtNPs/MWCNTs/GCE and Au–PtNPs/GCE against the square root of scan rate in the ranges of 10–300 mV s−1 , suggesting a diffusion-controlled redox process. The values of the electro-active surface area were respectively 25.44 mm2 for Au–PtNPs/MWCNTs/GCE and 14.75 mm2 for Au–PtNPs/GCE by the anodic peak current of electrode. In Fig. 4C, it was the CVs of two detected electrodes in the same 5.0 mM Fe(CN)6 3−/4− at scan rate of 90 mV s−1 . Fig. 4C shows that the proposed electrode with MWCNTs (Fig. 4C, curve a) had higher conductivity than contrastive electrode without MWCNTs (Fig. 4C, curve b). This trend is consistent with the electro-active surface area and the peak current. The experimental results suggested that the proposed sensing interface possessed bigger electro-active surface areas and higher conductivity, which could prove the sensing capabilities of the immunosensor. 3.3. The synergistic catalysis effect of HRP and Au–PtNPs In order to make a comparison of catalysis activity of different metal nanoparticles, the electrochemical performance of the HRP/anti-AFP/AuNPs/MWCNTs sensing interface (Fig. 5A): (a) without H2 O2 , (b) with H2 O2 ; HRP/anti-AFP/PtNPs/MWCNTs sensing interface (Fig. 5B): (c) without H2 O2 , (d) with H2 O2 ; and HRP/anti-AFP/Au-PtNPs/MWCNTs sensing interface (Fig. 5C): (e) without H2 O2 , (f) with H2 O2 were evaluated. Compared with their monometallic counterparts of AuNPs and PtNPs, Au–PtNPs exhibited more excellent catalytic property because of the synergistic effect of the Au and Pt.

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I / µA

75

125

0 a b c d e

0

-100

-200

e

-0.25

0.00

B

250

I /µA

I /µA

100

A

150 I / µA

200

200 100 0

a b c d e

0

-125

a db c

a ed b

-250 0.25

0.50

E/V

-0.2

0.0

0.2

c 0.4

0.6

E/V

Fig. 3. The CVs of the modified electrodes in different redox probes: (A) 2.0 mM HQ in PBS of pH 7.4; (B) 0.1 mM K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ] (1:1) PBS (containing 0.1 M KCl, pH 7.4): (a) bare GCE, (b) MWCNTs/GCE, (c) Au–PtNPs/MWCNTs/GCE, (d) anti-AFP/Au–PtNPs/MWCNTs/GCE, (e) HRP/anti-AFP/Au–PtNPs/MWCNTs/GCE. The scan rate was 50 mV s−1 and all potentials are given versus SCE.

The electrocatalytic reactivity of immobilization matrix for the reduction of H2 O2 was investigated by CVs in Fig. 5D. Curves g of Fig. 5D show the CV of the sensing interface with BSA as blocking agent in the presence of H2 O2 . The HRP blocked sensing interface exhibited better electrocatalytic property than BSA blocked sensing interface, owing to the synergistic catalysis effect of HRP and Au–PtNPs. 3.4. Optimization conditions for immunoassay 3.4.1. Influence of pH on the response of immunosensor The pH of the detection solution (0.1 M PBS) had a profound effect on the immunosensor. As shown in Fig. 6A, the influence of pH value on the current responses of the immunosensor was investigated in the range from 6.0 to 8.5. It was found that the current

responses increased with increasing pH value from 6.0 to 7.4 to reach the maximum and decreased thereafter. Thus, the optimal pH 7.4 of working buffer was chosen throughout this study to obtain a high analytical sensitivity. On the other hand, the incubation solution pH was selected as 7.4 (the physiological environment) in order to keep the immunoreaction under optimal condition. 3.4.2. Influence of concentration of hydrogen peroxide The influence of concentration of hydrogen peroxide in working solution for the catalytic activities of the electrode was studied using CV (Fig. 6B). With increasing the concentration of H2 O2 , the reduction peak current increased gradually with the decrease of the oxidation peak current, which indicated that the immobilized Au–PtNPs and HRP on the sensing surface had a typical electrocatalytic reduction process of H2 O2 . The maximum peak current

Fig. 4. The CVs of the different modified electrodes at different scan rates (from inner to outer): 10, 30, 50, 70, 90, 120, 150, 200 and 300 mV s−1 . The inset showed the dependence of redox peak currents on the square root of scan rates. (A) Au–PtNPs/MWCNTs/GCE; (B) Au–PtNPs/GCE; (C) is the CVs of Au–PtNPs/MWCNTs modified electrode at scan rate of 90 mV s−1 : (a) with MWCNTs, (b) without MWCNTs. All experiments were in 5 mM K3 Fe(CN)6 /K4 Fe(CN)6 (1:1) mixture under room temperature and all potentials were given vs. SCE.

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Fig. 5. (A) The CVs of HRP/anti-AFP/AuNPs/MWCNTs modified electrode: (a) without H2 O2 , (b) with H2 O2 ; (B) HRP/anti-AFP/PtNPs/MWCNTs modified electrode: (c) without H2 O2 , (d) with H2 O2 ; (C) HRP/anti-AFP/Au–PtNPs/MWCNTs modified electrode: (e) without H2 O2 , (f) with H2 O2 ; (D) (g) BSA/anti-AFP/Au–PtNPs/MWCNTs/GCE in working solution with H2 O2 . The scan rate was 50 mV s−1 .

Fig. 6. Optimization of experimental parameters: (A) The immunosensor response in 2.0 mM HQ various pH values under room temperature: 6.0, 6.5, 7.0, 7.5, 8.0, 8.5; (B) The current response in the working solution with various concentration of H2 O2 under room temperature; (C) influence of the incubation time on amperometric response of immunosensor when immune-reacted with 100 ng mL−1 AFP. All potentials were given vs. SCE and the scan rate was 50 mV s−1 .

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80

Table 1 Reproducibility assays using four immunosensors prepared in the same conditions.

a

Sample number

Standard value of AFP (ng mL−1 )

Intra-assaya reproducibility (%)

Inter-assayb reproducibility (%)

1 2 3 4

0.5 1 10 20

4.4 2.3 5.7 4.0

5.8 4.7 6.1 3.9

ΔI /µA

60

40

I / µA

-20 -40

a and b are RSD of three measurements.

e

-60

b

20

-80

a -0.1

0.0

0.1

0.2

E/V 0

50

100

150

200

Table 2 The recovery of the proposed immunosensor in human serum. Sample number

Standard value of AFP (ng mL−1 )

Found (ng mL−1 )a

1 2 3 4 5

1 5 10 20 50

1.05 4.86 9.90 21.54 51.25

Recovery (%)

-1

cAFP / (ng mL ) Fig. 7. Calibration plots of the changes of DPV peak current response versus the concentrations of AFP with the different immunosensors under optimal conditions: (a) HRP/anti-AFP/Au–PtNPs/MWCNTs/GCE with H2 O2 in working solution; (b) BSA/anti-AFP/Au–PtNPs/MWCNTs/GCE without H2 O2 in working solution. The inset was the DPV of the proposed immunosensor after incubation in different concentrations of AFP standard solution (from a to e): 20, 50, 100, 150 and 200 ng mL−1 under the optimal conditions.

response occurred in 8.0 mM H2 O2 , which corresponded to the saturated state. Consequently, the optimum concentration of 8.0 mM H2 O2 was employed for the test. 3.4.3. Influence of incubation time on the immunoreactions As shown in Fig. 6C, the influence of immunochemical incubation time on current response was investigated. The immunosensor was incubated in a constant concentration of 100 ng mL−1 AFP for different time. The current response decreased with increasing incubation time and reached a platform at 12 min, indicating the saturated formation of immunocomplex on the modified electrode. Therefore, the incubation time of 12 min was adopted in this work. 3.5. Performance of the immunosensor 3.5.1. DPV response and calibration curve In this study, DPV technique was employed to investigate the response performances of immunosensor under the optimal conditions. The inset of Fig. 7 shows the DPV response of the immunosensor when detecting different concentrations of AFP. Obviously, the DPV peak currents of the immunosensor showed a decrease with the increase of AFP concentration in the incubation solution. The reason was that more antigen molecules could be bound to immobilized antibodies at higher concentrations of antigens, and the immuocomplexes acted as an inert block layer hindering the electron-transfer. As can be seen from Fig. 7a, the DPV peak currents of the electrochemical immunosensor and the concentrations of AFP showed a linear relationship in the concentration range from 0.5 to 20 ng mL−1 and 20 to 200 ng mL−1 with a detection limit of 0.17 ng mL−1 at a signal-to-noise of 3. As comparative study, the immunosensor employed BSA as blocking agent was also prepared in Fig. 7b. The linear range was 5–10 ng mL−1 and 10–100 ng mL−1 with a detection limit of 1.6 ng mL−1 . Compared with the immunosensor employed BSA as blocking agent, the proposed immunosensor displayed higher sensitivity and a wider linear range by the addition of H2 O2 in the working solution. The reason could be contributed to the facts that Au–PtNPs and blocking agent HRP performed an effective amplification properties as expected. 3.5.2. Stability of the immunosensor The stabilities of the immunoassay system were evaluated by successive cycle scan. After 50 cycle successive CV measurements

a

± ± ± ± ±

0.02 0.08 0.16 0.33 0.52

104.6 97.2 99.0 107.6 102.5

Mean value ± SD of three measurements.

under the optimal conditions at 50 mV s−1 , only 2.8% decrease of the initial current was observed. The long-time stability of the immunosensor was investigated with 7 days. The immunosensor was stored in the refrigerator at 4 ◦ C, the relative standard deviation was less than 6%. The excellent performance of the immunosensor owed to the good stability of the prepared Au–PtNPs/MWCNTs sensing interface and the anti-AFP molecules were absorbed firmly on the Au–PtNPs layer which provided a good biocompatible microenvironment. 3.5.3. Reproducibility of the immunosensor The reproducibility of the response of the immunosensor was investigated by intra- and inter-assay coefficients of variation. The intra-assay precision of the analytical method was evaluated by analyzing four concentration levels using the equally prepared immunosnesors. Similarly, the inter-assay precision of the analytical method was also evaluated. The results of intra-assay and inter-assay were shown in Table 1, which suggested that the reproducibility of the proposed immunosensors was satisfying. 3.5.4. Preliminary analysis of real samples To investigate the feasibility of the prepared immunosensor for clinical analysis, the immunosensor was applied to the determination of different concentrations of AFP added into normal blood serum samples. As shown in Table 2, the recovery was in the range of 97.2–107.6%, which indicated that the proposed immunosensor might be preliminarily applied for the direct determination of AFP in clinical diagnosis. 4. Conclusion In this paper, a novel approach for fabrication of immunosensor was described based on HRP/anti-AFP/Au–PtNPs/MWNTs modified GC electrode. The highlights of the prepared immunosensor can be summarized as follows: the proposed electrode showed a large electro-active surface areas and high conductivity. With the synergetic catalysis effect of Au–PtNPs and HRP towards the reduction of hydrogen peroxide (H2 O2 ), the signal could be amplified and the sensitivity could be enhanced. Biomolecules could be immobilized on the Au–PtNPs tightly with the bioactivity kept well. On the basis of the above reasons, the proposed immunosensor showed wide linear range, high stability and reproducibility, superior sensitivity and good catalytic activity. The simple fabrication procedures

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