Biosensors and Bioelectronics 59 (2014) 389–396

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Au nanoparticles/PAMAM dendrimer functionalized wired ethyleneamine–viologen as highly efficient interface for ultra-sensitive α-fetoprotein electrochemical immunosensor Begard Kavosi a, Rahman Hallaj a, Hazhir Teymourian a, Abdollah Salimi a,b,n a b

Department of Chemistry, University of Kurdistan, 66177-15175 Sanandaj, Iran Research Center for Nanotechnology, University of Kurdistan, 66177-15175 Sanandaj, Iran

art ic l e i nf o

a b s t r a c t

Article history: Received 25 December 2013 Received in revised form 7 March 2014 Accepted 24 March 2014 Available online 30 March 2014

In this work, a novel electrochemical immunoassay system is developed for ultrasensitive detection of cancer biomarker, α-fetoprotein (AFP). This immunosensor is constructed by covalent immobilization of polyamidoamine (PAMAM) dendrimer-encapsulated gold nanoparticles (Au–PAMAM) sensing interface on a Au electrode surface, followed by sequential covalent immobilization of ethyleneamineviologen (Vio) electrochemical redox marker and AFP monoclonal antibody (mAb) on the surface of Au–PAMAM. The Au–PAMAM nanocomposite not only led to increase the electrode surface area and accelerate the electron transfer kinetics, but also it could provide a highly stable matrix for the convenient conjugation of biomolecules. Upon immunorecognition of the immobilized AFP to its antibody, the Vio peak current decreased due to the hindered electron transfer reaction on the electrode surface. Through the differential pulse voltammetry (DPV) experiments, it is found that the proposed method could detect AFP antigen at a wide linear range (0.001–45 ng mL  1) and a detection limit down to 130 fg mL  1. The immunosensor exhibited high specificity for AFP detection, extremely short incubation time (5 min), good stability and acceptable reproducibility. Moreover, the fabricated immunosensor could accurately detect AFP concentration in human serum samples demonstrated by excellent correlations with standard ELISA immunoassay. In addition, electrochemical impedance spectroscopy technique was used as an efficient alternative detection system for AFP measurement with detection limit 0.5 ng mL  1 and concentration range up to 40 ng mL  1. The present protocol is shown to be quite promising for clinical screening of cancer biomarkers and point-of-care diagnostics applications. & 2014 Elsevier B.V. All rights reserved.

Keywords: Electrochemical immunosensor Au nanoparticles PAMAM dendrimer Cancer biomarker α-Fetoprotein (AFP)

1. Introduction Highly sensitive and selective detection of protein bio-markers is a vital aspect of medical diagnostics, elucidation of disease vectors, immunology, new drug development and biodefense applications (Kitano, 2002; Figeys, 2003). Specifically, the clinical analysis of cancer biomarker shows great promise for early cancer detection and proteomics research, which will also offers opportunities to understand the fundamental biological processes involved in disease progression and monitoring patient responses to therapy methods (Srinivas et al., 2001; Wilson and Nie, 2006; Du et al., 2010). Therefore, great efforts have been made to achieve highly sensitive detection of tumor markers in recent years.

n Corresponding author at: Department of Chemistry, University of Kurdistan, 66177-15175 Sanandaj, Iran. Tel.: þ98 871 6624 001; fax: þ98 871 662 4008. E-mail addresses: [email protected], [email protected] (A. Salimi). 0956-5663/& 2014 Elsevier B.V. All rights reserved.

The conventional immunoassay methods used for cancer biomarker detection mainly include immune-polymerase chain reaction (PCR) assay (Saito et al., 1999), radioimmunoassay (Goldsmith, 1975), fluorescence immunoassay (Matsuya et al., 2003), chemiluminescence assay (Qin et al., 2012), electrophoretic immunoassay (Schmalzing and Nashabeh, 1997), mass spectrometric immunoassay (Aebersold and Mann, 2003), and enzyme-linked immunosorbent assay (ELISA) (Yates et al., 1999). Although each of these methods offers some advantages, however, they suffer from some common drawbacks such as time-consuming labeling processes, long analysis time and the need for qualified personnel and/or sophisticated instrumentation which limit their wide range use (Ho et al., 2009). Thus, attempts to design and explore novel detection technologies for the earlier and sensitive profiling of cancer biomarkers, especially in the point-of-care applications are highly desirable. Because of the great number of advantages of high sensitivity and specificity, rapid detection, cost efficiency, low manpower requirements, and compact instrumentation that is compatible with portable devices, there has recently been a burgeoning interest in the


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development of electrochemical immunosensors (Hu et al., 2012; Salimi et al., 2013). With the advent of nanoscience and nanotechnology, electrochemical immunosensors that utilize nanomaterials as building blocks are shown to be promising analytical tools (Hu et al., 2013). Up until now, intense amount of research has been conducted toward the development of nanoparticle-based electrochemical immunosensing platforms being able to detect different proteins with good sensitivity and selectivity by combining various electrochemical techniques with functional nanomaterials, such as carbon nanostructures (Ho et al., 2009), graphene nanosheets, (Lin et al., 2012), quantum dots (Ho et al., 2009; Liang et al., 2012) and noble metal nanoparticles (Mani et al., 2009). Most of such immunosensors rely on a multienzyme report probe strategy in which enzyme-functionalized nanoparticles are used as the label to enhance detection sensitivity by increasing the enzyme loading toward a sandwich immunological reaction event (Mani et al., 2009; Jeong et al., 2013). In spite of the promising results reported, however, the practical applications of these report probes are limited that is due to the denaturation and leakage of enzymes (Shoji and Freund, 2001) and their timeconsuming and costly preparation and purification processes (Dai et al., 2009). Therefore, looking for simpler and more costly-effective sensing platforms which on the same time have high sensitivity and specificity is being actively pursued by investigators. Recently, coupling noble metal nanoparticles with organic compounds and biorecognition elements have attracted a major attention. Jia et al. have employed Ag@BSA composite as an effective and versatile platform for the immobilization of retinol-binding protein (RBP) monoclonal antibody and sensitive detection of RBP (Hu et al., 2012). The good performance of Ag@BSA composite has been also demonstrated for the measurement of KB tumor cells (Hu et al., 2013). An electrochemical immunosensor has been reported for detecting osteoproteogerin based on a Au NP-conducting polymer electrode with a linear range from 2.5 to 25 pg mL  1 and detection limit of 2 pg mL  1 (Singh et al., 2008). Among different nanomaterials, Au NPs are the most used metal nanoparticles in the design of electrochemical immunosensors due to their unique abilities of good biocompatibility and easy functionalization with proteins (Lin et al., 2012). In this paper, we report an electrochemical immunosensor for sensitive detection of AFP biomarker based on sequential covalent immobilization of Vio as electrochemical redox indicator and antibody molecules as biorecognition elements on a Au NPs/polyamidoamine dendrimer (Au– PAMAM) modified electrode. AFP, used as a model cancer biomarker, is a major plasma protein produced by the yolk sac and the liver. The AFP expression is often associated with hepatoma and teratoma and has been widely used as a diagnostic biomarker for hepatocellular carcinoma (Du et al., 2010). On the basis of its facile and environmentally friendly preparation, large surface area and abundant outer-shell functional groups available for attachment of biomolecules, prominent electrical conductivity, good stability, and high biocompatibility, the as-prepared 3D Au–PAMAM composite structure showed excellent performance to act as electrochemical sensing interface. Upon the formation of antibody–antigen immunocomplex on the electrode surface, the electron transfer characteristics of the electrochemical probe changes; this can be monitored by both DPV and EIS techniques. Quantitative measurements of AFP in human sera have been demonstrated, showing the potential of practical application of this novel immunosensor for analysis of AFP in clinical samples.

2. Experimental 2.1. Preparation of Au–PAMAM nanocomposite and ethyleneamineviologen (Vio) Some parts of the experimental section including chemicals and apparatus was presented in the supporting information (SI).

The preparation of Au–PAMAM nanocomposite was based on a previously developed procedure (Pan et al., 2005). Thiolterminated PAMAM dendrimer was first synthesized and characterized based on our previous report (Kavosi et al., 2014). In brief, 16 mL of methyl mercaptoacetate was added to 40 mg amineterminated PAMAM dendrimer (G4) dissolved in 80 mL water and stirred at 50 1C for 12 h. The solvent and residual methyl mercapto acetate were removed under vacuum at 75 1C to get thick honey-colored oil. Au nanoparticles were prepared from aqueous solutions of HAuCl4 and trisodium citrate. To 20 mL of distilled water, 40 μL of 0.5 M HAuCl4 solution was added and the mixture was refluxed. Furthermore, to 20 ml of water, 320 mL of trisodium citrate (0.5 M) in water was added and this mixture (20 mL) was added to the boiling HAuCl4 solution. The mixture was refluxed for 20 min and the solution was left to cool at room temperature. The as-prepared Au nanoparticles and 1 wt% thiolterminated PAMAM aqueous solutions were mixed at the ratio of [PAMAM]/[Au]¼ 100:1 (mol/mol) and incubated for 8 h at room temperature to form stable aqueous solutions of Au–PAMAM nanocomposite. The Vio was synthesized according to a previously reported strategy (Wang et al., 2009). In brief, a mixture of 2bromoethanamine (10 Eq) and 4,40 -bipyridine (1 Eq) was heated to reflux in anhydrous acetonitrile under nitrogen for 8 h. After the reaction mixture was cooled down to room temperature, the precipitate was filtered off and purified by recrystallization from ethanol. The as-synthesized Vio was characterized by FTIR spectroscopy technique (Fig. S1). From this figure, the synthesis of Vio can be successfully confirmed by the presence of well-defined bands at 3415 cm  1 assigned to the stretching vibration of amine groups (–NH2) and the bands at 3005 and 1638 cm  1 related to the CH (alkyl) and C ¼N, respectively. 2.2. Fabrication of the immunosensors and AFP immunosensing procedure The 5.0 mm Au disk electrodes were polished with 0.3 μm alumina slurry and washed with water, then cleaned with freshly prepared piranha solution (98% H2SO4, 30% H2O2, 7:3,v/v) for  10 min, followed by washing with copious amounts of distilled water and ethanol. The Au electrode was immersed for 5 h in acetonitrile solution containing 5 mM of4-aminothiophenol. After rinsing, the thiophenol self-assembled electrode was immersed in a saturated Pht solution of toluene for 4 h. Then, the electrode was rinsed with water followed by placing a 10 mL aliquot of Au–PAMAM on the electrode surface for 6 h. Au–PAMAM nanocomposite attached to the electrode through amide bond formation between its outer surface amine groups and –COCl groups of Pht. After that, the Au–PAMAM modified electrode was immersed again in Pht for 4 h. The Vio was coated on the electrode by transferring the electrode into the 2 mM aqueous solution of Vio for 5 h. After washing the electrode and immersing in the Pht for 4 h in the next step, the modified electrode was immersed in 0.1 M PBS containing anti-AFP (20 mg mL  1) and 3% BSA at 4 1C for 12 h. Afterwards, the fabricated electrode (denoted as Au/Au–PAMAM/ mAb) was successively rinsed with water and PBS and finally, it was immersed into antigen AFP solution with different concentrations for 5 min at room temperature. Detection of AFP level was performed by measuring the decrease of catalytic peak current of Vio in DPV or the differences in electron transfer resistance (ΔRet) in EIS induced by incubation with AFP antigen. The Au/mAb and Au/PAMAM/mAb were also prepared to perform control experiments. For preparing the Au/mAb, after forming a thiophenol selfassembled layer on the Au electrode, it was sequentially modified with Pht, Vio, Pht and mAb using the same procedure as above. The Au/PAMAM/mAb electrode was fabricated using a similar procedure to that used for Au/Au–PAMAM/mAb except that

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PAMAM dendrimer was applied instead of Au–PAMAM nanocomposite.

3. Results and discussion 3.1. Sensing principle of Au–PAMAM based AFP immunosensor The schematic representation for the fabrication of electrochemical AFP immunosensor was given in Scheme 1. A layer of 4-aminothiophenol is first self-assembled on Au electrode and then, using Pht, the Au–PAMAM hybrid is coated on the electrode surface. In this step, –NH2 groups present in PAMAM are attached to -COCl groups of Pht, which results in the formation of a steady film on the surface of Au electrode. The Au–PAMAM layer not only could lead to greatly accelerate the electron transfer reactions on the electrode surface, but it could provide a quite suitable interface with good biocompatibilty for the subsequent conjugation of biomolecules. Then, using Pht, Vio molecules carrying –NH2 groups are grafted onto Au–PAMAM surfaces carrying –NH2 groups to form amide bonds. The Pht linking agent is further utilized in the next step to covalently immobilize AFP mAb molecules. Finally, different concentrations of AFP antigen are


incubated with the biosensor for a period of time and by following DPV peak current of Vio as redox indicator to measure the interaction of antigen–antibody immunocomplex, a sensitive and specific electrochemical immunosensor is successfully presented. There are two amplification factors that mainly contribute to the high sensitivity of the presented Au–PAMAM-based immunosensing system; first is the use of Au–PAMAM nanocomposite with high conductivity and remarkably large surface area which can provides a high density of AFP mAb on the electrode surface and the second is the way of signal transduction in which signal produces by molecularly wired Vio redox indicator; since in this way any tiny perturbation on the electrode surface can lead to a large change in the electron transfer characteristics of redox indicator.

3.2. Characterization of Au–PAMAM-based AFP immunosensor It is obvious that the modification of the electrode with Au–PAMAM nanocomposite plays a vital role in preparation of the proposed sensor. In this regard, based on the attachment of Au nanoparticles to internal-SH groups in PAMAM structure, the PAMAM was incorporated by Au nanoparticles to form

Scheme 1. The schematic illustration for fabrication of AFP immunosensor.


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Au–PAMAM nanostructure. We used TEM to reveal the morphology and structure of Au–PAMAM nanocomposite. The low- and high-magnification TEM images of Au–PAMAM hybrid are shown in Fig. S2 (SI). As shown, Au spherical nanoparticles having uniform diameters of ca. 5 nm are encapsulated in a PAMAM dendrimer shell, confirming the successful synthesis of Au– PAMAM nanocomposite. It has been reported that the dendrimers can operate as mediator for the formation of Au nanoparticle selfassembled structure in water (Pan et al., 2005). It has been suggested that the Au nanoparticle was first surrounded by several thiol-terminated dendrimers to form self-assembly monolayer (SAM), which followed by the formation of dendrimer-mediated networks in which each Au nanoparticle may associate with more than one dendrimer molecule and, likewise, each dendrimer molecule may bind to more than one Au nanoparticle. The observed uniform nanostructure of Au–PAMAM may provide a significant increase of the effective electrode surface for loading biomolecules and accelerating electron transfer, and can also enhance the conductivity and remain the excellent biocompatibility for biosensor. The changes in electrode behavior after each immobilization and binding step could be efficiently detected by CV. As shown in Fig. 1, the well-defined redox characteristics of [Fe(CN)6]3  /4  were observed for all investigated electrodes but with variations in the current response and values of peak-to-peak separations (ΔEp). [Fe(CN)6]3  /4  redox probe at bare Au exhibits peaks with ΔEp as  0.14 V. A marked increase in current as well as a decrease in ΔEp (  0.058 V) was noticed at Au/Au–PAMAM electrode in compared to bare Au, which can be ascribed to the large electron transfer capability and high specific surface area of the nanocomposite. This observation also provided additional evidence for the successful immobilization of Au–PAMAM onto the electrode. Upon covalent immobilization of AFP mAb, the current response at the Au/Au–PAMAM/mAb decreased dramatically as compared with that at the Au/Au–PAMAM and ΔEp became 0.061 V. This implies that most of the electrode surface is covered by antibody, with the result of blocking the electron transfer efficiency of [Fe(CN)6]4  /3  at solid/liquid interface and consequently, retarding the interfacial electron-transfer kinetics. The incubation with 10 ng mL  1 AFP for 5 min gave rise to further decrease in peak current as well as an increase in ΔEp to 0.10 V. It means that AFP antigen is adsorbed onto the electrode surface with the result of substantially

Fig. 1. CVs of 2.5 mM [Fe(CN)6]3  /4  in 0.1 M KCl recorded at bare Au, Au/Au– PAMAM, Au/Au–PAMAM/mAb and Au/Au–PAMAM/mAb after incubated with 10 ng mL  1of AFP. Scan rate: 0.1 V s  1.

hindering the electron transfer between the electrode surface and redox couples in solution due to the formation of AFP mAb/ AFP immunocomplex onto the surface of Au–PAMAM modified electrode. In order to show the great signal amplification achieved by Au– PAMAM nanocomposite, a series of control experiments were conducted in PBS (pH 7) by DPV measurements. As shown in Fig. 2A, when AFP was incubated with its mAb directly exposed to the Au electrode, only a small response could be observed. However, a slightly larger response was seen upon the interaction of AFP with Au/PAMAM/mAb electrode which can be ascribed to the larger number of AFP mAb molecules loaded on the electrode surface. The greatest signal amplification was obtained in the case of Au–PAMAM modified Au electrode. As can be seen, the signal response at Au/Au–PAMAM/mAb showed 3- and 10-fold increases compared with Au/PAMAM/mAb and Au/mAb electrodes, respectively. The achieved signal amplification was mainly due to the large specific surface area of the Au–PAMAM nanocomposite which forms a 3D network with abundant surface functional

Fig. 2. (A) DPVs and (B) Nyquist curves recorded at different electrodes of Au/mAb, Au/PAMAM/mAb and Au/Au–PAMAM/mAb before and after incubation with AFP concentrations of 0.05 in (A) and 25 ng mL  1 in (B) as indicated in the figure legend. (A) Was performed in 0.1 M PBS (pH 7) and (B) in 0.1 M KCl containing 2.5 mM [Fe(CN)6]3  /4  at the frequency range of 0.1 Hz–10 kHz.

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groups available to capture much more antibodies at the sensing interface in one hand and on the other hand, it can be attributed to the accelerated electron transfer caused by the presence of Au–PAMAM nanocomposite. The signal amplification was also confirmed by EIS measurements. EIS is a characterization technique that provides electrical information in the frequency domain (Macdonald, 1987). Because of its ability for probing the interfacial properties at the electrode surface and the possibility of performing label-free detections, EIS is increasingly being used for the very sensitive detection of biorecognition events at the electrode surface (Willner and Zayats, 2007; Bonanni and Pumera, 2011). Here, we compare the Nyquist plots obtained with different electrodes of Au/mAb, Au/PAMAM/ mAb and Au/Au–PAMAM/mAb before and after incubated with 15 ng mL  1 AFP in 0.1 M KCl solution containing 5 mM Fe(CN)36  /4  (Fig. 2B). As expected, the Au/Au–PAMAM/mAb electrode provided a much larger response upon the interaction with AFP relative to those when AFP interacted with antibody immobilized on bare Au or Au/PAMAM electrodes. The herein obtained impedance data corroborate the preceding results obtained by DPV, all demonstrate the prominent signal amplification effect achieved by Au–PAMAM nanocomposite. In addition, the EIS measurements were performed in both conditions for two redox probes (i.e., using the [Fe(CN)6]3  /4  as external probe in solution and Vio as internal probe which has been immobilized onto the electrode in one case and the other only using the Vio and in the absence of [Fe(CN)6]3  /4  ). The results are shown in Fig. S3 (SI). It was found that the changes upon AFP addition in the former case (A) are more obvious than those in the latter (B), suggesting that a more sensitive signal response can be obtained using [Fe(CN)6]3  /4  that is due to the fact that [Fe(CN)6]3  /4  with well-known reversible redox properties is a more suitable probe to perform EIS measurements. We also performed a similar measurement by DPV technique in the aforementioned two situations and observed the opposite response (not shown). In this case, the sensitivity using Vio in the absence of [Fe(CN)6]3  /4  was more than that using both probes. This may be explained by the fact that Vio has been covalently immobilized onto the electrode surface and hence, even a very small change occurred in the interface could induce a large change in its redox characteristics and subsequently, a more signal response. 3.3. Optimization of detection conditions The immunoreaction time is an important parameter which greatly affects the analytical performance of the developed immunosensor. In order to investigate the effect of the incubation time on response signal, the fabricated Au/Au–PAMAM/mAb immunosensors were incubated into PBS (pH 7) containing 5 ng mL  1AFP antigen at room temperature for various periods of time. Then, DPV peak currents at the Au/Au–PAMAM/mAb at different incubation times were recorded to choose an optimum. As shown in Fig. S4, the DPV peak current decreased rapidly upon increasing the incubation time to 5 min, but no further significant decreases occurred upon increasing the incubation time any further. Considering the conventional incubation time in other works which is in the range of 30–60 min, the optimal incubation time in our work is very low. Since a long incubation time could result in a large nonspecific signal, this can be a specific advantage of the proposed AFP immunosensor. 3.4. Voltammetric and impedimetric detection of AFP The analytical calibration was performed using various concentrations of AFP antigen to evaluate the sensitivity of the


fabricated immunosensor. As can be seen in Fig. 3A, when the AFP concentration increased, the DPV current signal decreased accordingly which is due to the formation of increasing number of antibody–antigen immunoconjugates, with the result of enhanced hindering of electron transfer reaction of Vio redox probe on the immunosensor surface. The dependence of the DPV current response on the AFP concentration is also presented (inset of Fig. 3A). Each point on the calibration curve corresponds to the mean value obtained from three independent measurements. As can be seen, the calibration curve showed good linear relationship between the current signal and AFP concentration in the range of 0.001–45.0 ng mL  1. The linear relationship for lower concentrations could be represented by the equation, IP(μA) ¼ 12.75 [AFP] þ1.88 (ng mL  1) with a correlation coefficient of 0.9985. The relative standard deviations (RSD) for the measurement of each data point were less than 5.0%. The limit of detection (LOD) (at signal to noise ratio of 3) (Miller and Miller, 2010) was 130 fg mL  1. The obtained LOD surpasses detection limits for AFP by commercial immunoassay methods, as well as most reported AFP immunosensors, such as enzyme-nanoparticle conjugates sandwich immunoassay at oil-water interface (29 pg mL  1) (Su et al., 2012), bienzyme functionalized threelayer composite magnetic nanoparticles (7 pg mL  1) (Zhuo et al., 2009), amperometric enzyme immunosensor based on HRP modified Pt nanoparticles (80 pg mL  1) (Hong et al., 2012), electrodeposited Au–Pt alloy nanoparticles on carbon nanotubes electrochemical immunoassay (170 pg mL  1) (Li et al., 2011), amperometric immunosensor based on amine-functionalized graphene and Au nanoparticles modified CILE (100 pg mL  1) (Huang et al., 2011), label-free electrochemical immunosensor based on graphene and thionine nanocomposite (5.77 pg mL  1) (Wei et al., 2010) and dual signal amplification based on graphene sheets and multienzyme functionalized carbon nanospheres (5.77 pg mL  1) (Du et al., 2010). Also, Table S1 (SI) lists the response characteristics of the proposed immunosensor compared to the some other immunosensors reported in the literature. The results presented in this table obviously show that the proposed immunosensor based on Au–PAMAM nanocomposite displayed better response characteristics compared with the other sensors. The high sensitivity of the presented Au–PAMAM-based immunosensing system relies upon these amplification features: (a) the use of Au–PAMAM nanocomposite with some excellent properties including high conductivity and remarkably large surface area can provide a high density of AFP mAb on the electrode surface; (b) the way of signal transduction in which signal produces by molecularly wired Vio redox indicator may greatly contribute to the observed high sensitivity, since in this way any tiny perturbation on the electrode surface can lead to a large change in the electron transfer characteristics of redox indicator. The reproducibility of the fabricated immunosensor was also studied with intra- and inter-assay precision. The intra-assay precision of the biosensor was evaluated by analyzing the AFP with concentration of 10 ng mL  1 for 5 times. The interassay precision was evaluated by assaying the AFP at the same concentration for 5 electrodes. The intra- and the inter-assay variation coefficients calculated for 10 ng mL  1 were 2.57 and 4.66%, respectively, suggesting that both the intra and inter-assays of the as-designed AFP immunosensor indicated acceptable reproducibility. Due to the high ability of EIS technique for probing the interfacial properties at the electrode surface and the possibility of performing label-free detections, EIS measurement was also applied as an efficient alternative detection system to further confirm the analytical performance of as-proposed system. Fig. 3B displays the Nyquist plots obtained at the Au/Au–PAMAM/mAb electrode after being incubated with different concentrations of


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Fig. 3. DPV and EIS results for immunosensors incubated with different concentrations of target AFP. (A) DPV response of electrochemical immunoassay in 0.1M PBS (pH 7) with increasing AFP concentration from a to p: 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0 and 45.0 ng mL  1; Inset is the corresponding calibration curve between the DPV peak current (corrected for background) and the AFP concentrations. (B) EIS response of electrochemical immunoassay in 0.1 M KCl containing 2.5 mM [Fe(CN)6]3  /4  with increasing AFP concentration from a to i (5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0 and 40.0 ng mL  1) and the inset is the equivalent circuit and calibration curve of the impedance immunosensor for detecting AFP (data obtained were the averages of three measurements).

target AFP in 0.1 M KCl solution containing 2.5 mM Fe(CN)36  /4  .A Randles equivalent circuit R1(CPE[R2W]) was used to fit the experimental data. As shown in this figure, the Ret value increased

with increasing the concentration of AFP that is due to the hindrance of electron transfer process of Fe(CN)36  /4  at electrode surface. The ΔRet was proportional to the AFP concentration in the

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3.6. Analysis of human sera samples In order to demonstrate the potential of the as-proposed immunosensor in clinical application, 4 human serum samples from different people were analyzed by using the Au–PAMAMbased voltammetric immunosensor and were compared to a standard ELISA immunoassay. Table 1 presents the estimated concentrations of AFP in these samples. It could be seen that there is a good agreement between results obtained by two methods, illustrating that the fabricated immunosensor exhibits great promise as a reliable sensor for the detection of AFP in human serum samples.

4. Conclusions

Fig. 4. The DPVs of Au/Au–PAMAM/mAb electrode before (dashed line) and after (solid line) being incubated for 5 min with 1% BSA, 40 ng mL  1 PSA and 20 ng mL  1 AFP in 0.1 M PBS (pH 7).

Table 1 The obtained results for AFP detection in human serum samples by the proposed electrochemical immunosensor and the standard ELISA method. Sample

Proposed method (ng mL  1)

Reference method (ng mL  1)

1 2 3 4

5.2 7 0.1 7.9 7 0.2 9.1 7 0.15 16.5 7 0.2

5 8 9 16


range 5–40 ng mL . The linear equation could be depicted as ΔRet (Ω)¼22.189 [AFP] (ng mL  1) þ8.8071 (R2 ¼0.9966) with a detection limit of 0.5 ng mL  1 (S/N¼ 3) (inset of Fig. 3B), which was higher than the above obtained LOD in DPV measurement but sensitive enough for AFP detection in clinical samples. This clearly indicates that the proposed system can also be used as an efficient ultrasensitive impedimetric immunosensor for AFP detection.

In conclusion, we have developed a novel electrochemical immunosensing platform for the detection of cancer biomarker AFP. The immunosensor was constructed by sequentially covalent immobilizing Vio molecule as redox marker and AFP mAb as recognition element onto the Au–PAMAM nanocomposite modified surface with the help of Pht as linking agent. The presence of Au–PAMAM nanocomposite with excellent properties including high conductivity, prominent biocompatibility and large surface area improved the electrochemical performance of the immunosensor for the diagnosis of AFP. This technique possessed adequate precision and sensitivity, with an LOD of 130 fg mL  1 and an acceptable dynamic range of 5 orders of magnitude at unusual incubation time, 5 min. In addition, the as-proposed immunoassay system showed good specificity and it exhibited satisfactory reproducibility for the analysis of human serum samples. We anticipate that such devices can be readily expanded for detecting other relevant biomarkers, which hold great promise for reliable point-of-care diagnostics of cancer and other diseases, and as tools for intra-operation pathological testing, proteomics, and systems biology.

Acknowledgments This research was supported by the Iranian Nanotechnology Initiative and the Research Office of the University of Kurdistan.

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at References

3.5. Specificity of AFP immunosensor To illustrate whether the strategy is applicable to the detection of AFP in a biological sample, BSA and PSA were used as potential interfering agents to test the selectivity of the immunosensor. In a typical experiment, the immunosensor was incubated with 1% BSA, 40 ng mL  1 PSA and 20 ng mL  1AFP, respectively, and their DPV current response were measured. As shown in Fig. 4, while the signal current is very small after the electrode reacted with BSA or PSA, a significant signal induced by the interaction of the electrode and AFP was observed. This phenomenon indicated that the proposed strategy had good selectivity in AFP detection and could distinguish AFP in complex samples from its analogues. This can be attributed to this fact that the binding event between AFP and antibody is based on the specific recognition between them but not on the other factors, such as nonspecific adsorption.

Aebersold, R., Mann, M., 2003. Nature 422, 198–207. Bonanni, A., Pumera, M., 2011. ACS Nano 5, 2356–2361. Du, D., Zou, Z., Shin, Y., Wang, J., Wu, H., Engelhard, M.H., Liu, J., Aksay, I.A., Lin, Y., 2010. Anal. Chem. 82, 2989–2995. Dai, Z.H., Liu, S.H., Bao, J.C., Ju, H.X., 2009. Chem. Eur. J. 15, 4321–4326. Figeys, D., 2003. Anal. Chem. 75, 2891–2905. Goldsmith, S.J., 1975. Semin. Nucl. Med. 5, 125–152. Ho, Ja-an A., Lin, Y.C., Wang, L.S., Hwang, K.C., Chou, P.T., 2009. Anal. Chem. 81, 1340–1346. Hong, C., Yuan, R., Chai, Y., Zhuo, Y., Yang, X.J., 2012. J. Electroanal. Chem. 664, 20–25. Hu, C., Yang, D.P., Wang, Z., Huang, P., Wang, X., Chen, D., Cui, D., Yang, M., Jia, N., 2013. Biosens. Bioelectron. 41, 656–661. Hu, C., Yang, D.P., Xu, K., Cao, H., Wu, B., Cui, D., Jia, N., 2012. Anal. Chem. 84, 10324–10331. Huang, K.J., Niu, D.J., Sun, J.Y., Zhu, J.J., 2011. J. Electroanal. Chem. 656, 72–77. Jeong, B., Akter, R., Han, O.H., Rhee, C.K., AminurRahman, M., 2013. Anal. Chem. 85, 1784–1791. Kavosi, B., Salimi, A., Hallaj, R., Amani, K., 2014. Biosens. Bioelectron. 52, 20–28. Kitano, H., 2002. Science 295, 1662–1664.


B. Kavosi et al. / Biosensors and Bioelectronics 59 (2014) 389–396

Li, Y., Yuan, R., Chai, Y., Song, Z., 2011. Electrochim. Acta 56, 6715–6721. Liang, G., Liu, S., Zou, G., Zhang, X., 2012. Anal. Chem. 84, 10645–10649. Lin, D., Wu, J., Wang, M., Yan, F., Ju, H., 2012. Anal. Chem. 84, 3662–3668. Macdonald, J.R., 1987. Impedance Spectroscopy. Wiley, New York Mani, V., Chikkaveeraiah, B.V., Patel, V., Gutkind, J.S., Rusling, J.F., 2009. ACS Nano 3, 585–594. Matsuya, T., Tashiro, S., Hoshino, N., Shibata, N., Nagasaki, Y., Kataoka, K., 2003. Anal. Chem. 75, 6124–6132. Miller, J.N., Miller, J.C., 2010. Statics and Chemometrics for Analytical Chemistry, sixth ed. Pearson Education Limited Pan, B., Gao, F., Ao, L., Tian, H., He, R., Cui, D., 2005. Colloids Surf., A 259, 89–94. Qin, G., Zhao, S., Huang, Y., Jiang, J., Ye, F., 2012. Anal. Chem. 84, 2708–2712. Saito, K., Kobayashi, D., Sasaki, M., Araake, H., Kida, T., Yagihashi, A., Yajima, T., Kameshima, H., Watanabe, N., 1999. Clin. Chem. 45, 665–669. Salimi, A., Kavosi, B., Fathi, F., Hallaj, R., 2013. Biosens. Bioelectron. 42, 439–446.

Schmalzing, D., Nashabeh, W., 1997. Electrophoresis 18, 2184–2193. Singh, K., Rahman, A., Son, J.I., Kim, K.C., Shim, Y., 2008. Biosens. Bioelectron. 23, 1595–1601. Shoji, E., Freund, M.S.J., 2001. J. Am. Chem. Soc. 123, 3383–3384. Srinivas, P.R., Kramer, B.S., Srivastava, S., 2001. Lancet Oncol. 2, 698–704. Su, H., Yuan, R., Chai, Y., Zhuo, Y., 2012. Biosens. Bioelectron. 33, 288–292. Wang, D., Crowe, W.E., Strongin, R.M., Sibrian-Vasquez, M., 2009. Chem. Commun. 14, 1876–1878. Wei, Q., Mao, K., Wu, D., Dai, Y., Yang, J., Du, B., Yang, M., Li, H., 2010. Sens. Actuators, A 149, 314–318. Willner, I., Zayats, M., 2007. Angew. Chem. Int. Ed. 46, 6408–6418. Wilson, M.S., Nie, W.Y., 2006. Anal. Chem. 78, 6476–6483. Yates, A.M., Elvin, S.J., Williamson, D.E., 1999. J. Immunoassay 20, 31–44. Zhuo, Y., Yuan, P.X., Yuan, R., Chai, Y.Q., Hong, C.L., 2009. Biomaterials 30, 2284–2290.

PAMAM dendrimer functionalized wired ethyleneamine-viologen as highly efficient interface for ultra-sensitive α-fetoprotein electrochemical immunosensor.

In this work, a novel electrochemical immunoassay system is developed for ultrasensitive detection of cancer biomarker, α-fetoprotein (AFP). This immu...
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