Ultrasensitive electrochemical immunosensor based on Au nanoparticles dotted carbon nanotube–graphene composite and functionalized mesoporous materials

Ultrasensitive electrochemical immunosensor based on Au nanoparticles dotted carbon nanotube–graphene composite and functionalized mesoporous materials

Biosensors and Bioelectronics 33 (2012) 29–35 Contents lists available at SciVerse ScienceDirect Biosensors and Bioelectronics journal homepage: www...

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Biosensors and Bioelectronics 33 (2012) 29–35

Contents lists available at SciVerse ScienceDirect

Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios

Ultrasensitive electrochemical immunosensor based on Au nanoparticles dotted carbon nanotube–graphene composite and functionalized mesoporous materials Juanjuan Lu a , Shiquan Liu b , Shenguang Ge a , Mei Yan a , Jinghua Yu a,∗ , Xiutao Hu a a b

Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, university of Jinan, Jinan 250022, China School of Material Science and Engineering, university of Jinan, Jinan 250022, China

a r t i c l e

i n f o

Article history: Received 10 October 2011 Received in revised form 23 November 2011 Accepted 29 November 2011 Available online 5 January 2012 Keywords: Carbon nanotubes Mesoporous silica nanoparticles Grapheme Electrochemical immunosensor Human chorionic gonadotrophin

a b s t r a c t A facile and sensitive electrochemical immunosensor for detection of human chorionic gonadotrophin (hCG) was designed by using functionalized mesoporous nanoparticles as bionanolabels. To construct high-performance electrochemical immunosensor, Au nanoparticles (AuNPs) dotted carbon nanotubes (MWCNTs)–graphene composite was immobilized on the working electrode, which can increase the surface area to capture a large amount of primary antibodies (Ab1 ) as well as improve the electronic transmission rate. The as-prepared bionanolabels. composed of mesoporous silica nanoparticles (MCM41) coated with AuNPs through thionine linking, showed good adsorption of horseradish peroxidaselabeled secondary anti-hCG antibody. Interlayer thionine was not only a bridging agent between MCM41 and AuNPs but also an excellent electron mediator. The approach provided a good linear response range from 0.005 to 500 mIU mL−1 with a low detection limit of 0.0026 mIU mL−1 . The immunosensor showed good precision, acceptable stability and reproducibility. Satisfactory results were obtained for determination of hCG in human serum samples. The proposed method provides a new promising platform of clinical immunoassay for other biomolecules. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Human chorionic gonadotrophin (hCG) is a secretion of the placenta during pregnancy and gestational trophoblastic diseases. It is increased as a consequence of abnormal placental invasion and placental immaturity. Therefore, it is an important diagnostic marker of pregnancy and one of the most important carbohydrate tumor markers (Handschuh et al., 2007; Vartiainen et al., 2002). Thus, the detection of hCG in serum or urine has been widely employed in clinical situations. At present, conventional methods for determination of hCG are immunoradiometric assay (IRMA) and enzyme-linked immunosorbent assay (ELISA) (Su et al., 2000; Venkatesh and Murthy, 1996). IRMA is extremely sensitive, but it also has the obvious limitations: short shelf life of 125 I-labeled antibody, radiation hazards, complicated wash procedure and expensiveness. And ELISA is less sensitive, time-consuming. Electrochemical immunoassays, based on the high specificity of antigen–antibody interactions with electrochemical transducers, have become important analytical tools

∗ Corresponding author. Tel.: +86 531 82767161. E-mail address: [email protected] (J. Yu). 0956-5663/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2011.11.054

in clinical and biochemical analyses, because they have many advantages including simple instrumentation and operation, fast analysis, high sensitivity and selectivity, wide linear range, and high compatibility with advanced micromachining technologies (Takahashi et al., 2010; Lee et al., 2010; Warsinke et al., 2000). Although the label-free electrochemical measurements make a little requirement for operational steps and reagents, the detectable signal and sensitivity are usually limited. In order to develop a high-performance electrochemical immunosensor, signal amplification and noise reduction are very vital (Bard et al., 2010). In recent years, with the development of nanoscience and nanotechnology, a variety of nanoparticles, such as carbon nanotube, carbon nanosphere and gold nanoparticles, have been applied as the labels in nanoparticle-based amplification platforms which can dramatically enhance the signal intensity of electrochemical immunosensor and lead to ultrasensitive bioassays (Yu et al., 2006; Cui et al., 2008; Wu et al., 2009). Despite extensive efforts launched in this area, it was still a challenge for the fabrication of novel immunosensors using new materials to further achieve sensitive, accurate and facile detection. Carbon nanomaterials, such as carbon nanotubes, carbon nanodots and carbon nanofibers, have been widely used in both analytical and industrial electrochemistry because of chemical

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inertness, low residual current, excellent conductivity, wide potential window, and electrocatalytic activity to a variety of redox reactions (Yanez-Sedeno et al., 2010). Carbon nanotubes (MWCNTs) have extensively been used for electrochemical and optical biosensors due to its unique mechanical, chemical, and electrical properties. The MWCNTs-based biosensors have attracted much attention due to the high surface area-to-weight ratio, excellent mechanical properties, and fast electron-transfer capabilities (Yang et al., 2010a; Kauffman et al., 2010). Graphene nanosheets (GS), a single layer of carbon atoms with a closely packed honeycomb in a two-dimensional lattice, has recently attracted enormous attention in constructing electrochemical biosensors due to its fast electron transportation, high thermal conductivity, excellent mechanical stiffness and good biocompatibility (Xia et al., 2010; Meyer et al., 2007). However, the water solubility of graphene limits their further application in designing biosensors because graphene is hydrophobic and tends to form agglomerates in water (Shan et al., 2009). Recent researches have made efforts to increase the graphene solubility by covalent or non-covalent functionalization method (Wu et al., 2010). Thus, a water-soluble polymers, such as polyvinylpyrrolidone (Bourlinos et al., 2009), chitosan (Kang et al., 2009), and Nafion (Li et al., 2009) were used as disperser to prepare homogeneous GS solution, while the introduction of these polymers could not promote electron transfer well. Herein, we tried to couple the graphene nanosheet with carbon nanotube through the ␲–␲ stacking. This MWCNTs–graphene coupling produces a synergic effect in the electroanalytical performance of the resulting electrode material that can be profited for the development of electrochemical biosensors. Then the AuNPs were dotted on the surface of MWCNTs–graphene composite modified electrode, which can improve the electronic transmission rate as well as increase the surface area to capture a large amount of primary antibodies (Ab1 ). In the sandwich-type immunoassays, enzyme-labeled antibodies are often used for the signal amplification. The enormous signal enhancement associated with the use of nanomaterial labels and the formation of nanomaterial–antibody–antigen assemblies provided the basis for ultrasensitive electrochemical detection of antigens (Liu and Lin, 2007). In this contribution, we use functionalized mesoporous nanoparticles MCM-41 immunocomplex as the signal amplification section. Mesoporous nanoparticles MCM-41 have many unique structural features compared with other solid nanoparticles, including large surface area and high pore volume, ordered porous channels, uniform and tunable pore structure, and great diversity in surface functionalization. These characteristics have the virtue of conjugating more biomolecules, and improving the sensitivity of bioanalysis. The conductivity of porous nanomaterials can be improved by doping electroactive substance into the pores with an in situ synthesized method. Thionine, as an excellent electron mediator was firstly dotted onto/into the MCM-41. The AuNPs decorated on the TH/MCM-41 exhibit many advantages, such as greatly chemical stability, large specific surface area, strong adsorption ability, excellent electrical conductivity and biocompatibility (Park et al., 2010; Parker et al., 2010; Ren et al., 2009). The unique properties of AuNPscodified mesoporous nanoparticles TH/MCM-41 coupled with the conjugation of biological components, provide a promising platform for the development of high-performance electrochemical immunosensors. Herein, we designed a novel electrochemical immunoassay using AuNPs dotted MWCNTs–graphene composite modified electrode, which constructed an effective antibody immobilization matrix and made the immobilized immunocomponents possessed high stability and bioactivity. In addition, the amplified sensitivity was enhanced by using horseradish peroxidase-conjugated hCG (HRP-Ab2 ) linked to the functionalized mesoporous Au/TH/MCM41 nanoparticles.

2. Experimental 2.1. Chemicals Diagnostic kit for human serum hCG, monoclonal hCG capture antibody (Ab1 ) and HRP-labeled hCG signal antibodies (HRP-Ab2 ) were produced by Shanghai Linc-Bio Science Co. Ltd. (Shanghai, China). Bovine serum albumin (BSA, 96–99%), Gold chloride (HAuCl4 ·4H2O), NaBH4 were obtained from Sigma Chemical Co. (St. Louis, MO, USA). hydrogen peroxide (30%, w/v) was obtained from Chemical Reagent Co. (Tianjin, China). The natural graphite powder was obtained from J&K Scientific Ltd. The MWCNTs were purchased from Nanoport Co. Ltd. (Shenzhen, China). Tetraethoxysilane (TEOS) and (1-Hexadecyl) trimethylammonium bromide (CTAB) were purchased from Alfa Aesar. 2.2. Apparatus Electrochemical measurements were carried out with a CHI 660D electrochemistry workstation (Shanghai CH Instruments Co., China). Electrochemical impedance spectroscopy (EIS) was performed on a CHI 604D Electrochemical Workstation (Shanghai CH Instruments Inc., China). A conventional three electrode system was used for all electrochemical measurements: a modified glassy carbon electrode as the working electrode, a Pt and Ag/AgCl electrode were used as the counter electrode and reference electrode, respectively. Scanning electron microscopy (SEM) images were recorded using a JEOL-JSM-6300 scanning electron microscope. Transmission electron microscopy (TEM) investigations were performed using JEOL 4000 EX microscope. Atomic force microscope (AFM) images were achieved by scanning probe microscopy (SPM, Vecco, USA). The nitrogen sorption and pore diameters measurements were performed on Micromeritics ASAP 2020 surface area and porosity analyzer (Quantachrome, United States). 2.3. Bioconjugation of Au/TH/MCM-41 with HRP-Ab2 As shown in Fig. 1, the preparation procedure of the HRP-Ab2 /Au/MCM-41 bioconjugate was as follows: 100 mg Au/TH/MCM-41 (see Supporting information) was initially dispersed into 2 mL Tris buffer (50 mmol L−1 , pH 9.0) and sonicated 10 min to obtain a homogeneous dispersion. 500 ␮L HRP-Ab2 (0.2 mg mL−1 ) was added into the mixture. The reaction is based on the interaction between NH2 or SH groups on the HRPAb2 biomolecules and gold nanoparticles. After incubated for 12 h at 4 ◦ C with gentle stirring, the mixture was washed with Tris buffer, then centrifuged at 5000 rpm for 5 min, repeatedly washed and centrifuged three times. Following that, the precipitation was added into 1% BSA solution to block possible remaining active sites and avoid the nonspecific adsorption. The obtained HRPAb2 /Au/TH/MCM-41 was stored at 4 ◦ C until use. 2.4. Preparation of electrochemical immunosensor The electrochemical immunosensor was prepared as following steps: (i) Prior to surface modification, glassy carbon electrode (GCE) (3 mm diameter) was firstly polished with 1.0, 0.3 and 0.05 ␮m alumina slurry respectively and rinsed thoroughly with absolute alcohol and distilled water in ultrasonic bath, and dried in air at room temperature. (ii) 5 ␮L of graphene nanosheets (Detail synthesis procedures in Supporting information) was initially deposited on the electrode surface, and then dried at room temperature. Then 5 ␮L of acid-treated MWCNTs (2 mg mL−1 , see Supporting information) was cast on electrode and dried under infrared lamp. (iii) AuNPs were electrode-posited on the MWCNTs/GS-modified GCE via multipotential step from 1.055

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Fig. 1. Fabrication process of Au/TH/MCM-41 nanomaterials and measurement protocol of the electrochemical immunosensor.

to 0.045 V for 10 s in 0.50 mol L−1 H2 SO4 solution containing 0.10 mmol L−1 HAuCl4 . (iv) The modified working electrode was incubated with Ab1 (5 ␮L, 50 ␮g mL−1 ) for 1 h, followed by washing with PBS buffer to remove unspecific physically adsorption. (v) The possible remaining active sites on working electrode were blocked by 1% BSA solution. The as-prepared immunosensor (designed as Ab1 /Au/MWCNTs/GS/GCE) was used for detection of hCG analyte. Part of this fabrication process of the electrochemical immunosensor is illustrated in Fig. 1. 2.5. Electrochemical measurements Electrochemical measurements of this immunosensor toward hCG samples or standards were carried out through a sandwichtype immunoassay mode using HRP-Ab2 /Au/TH/MCM-41 as traces and H2 O2 as enzyme substrates. The detection process is depicted as follows. (i) The immunosensor was incubated in PBS with various concentration of hCG for 1 h at 37 ◦ C, and then the electrode was washed extensively to remove unbounded hCG molecules; (ii) the prepared HRP-Ab2 /Au/TH/MCM-41 solution was dropped onto the electrode surface and incubated for another 1 h. (iii) The electrochemical behavior of the immunosensor was recorded in pH 7.0 PBS containing 2.0 mmol L−1 H2 O2 by differential pulse voltammetry (DPV) from 0 to −600 mV with a pulse amplitude of 50 mV and a pulse width of 20 ms. The DPV peak current was collected and registered as the signal of the immunosensor which was relative to the concentration of hCG samples. The detection principle of the stepwise procedure of the sandwich-type immunoassay is illustrated in Fig. 1. 3. Results and discussion 3.1. Characterization of GS, MWCNTs, Au/MWCNTs To developed a high-performance electrochemical immunosensor, the critical issues are to enhance the immobilized amount of the biomolecules on the electrode surface, retain their biologic activity, and provide a good pathway of electron transfer (Hsing et al., 2007; Tang et al., 2008). In this contribution, we tried to construct an improved immunosensing interface using graphene and Au/MWCNTs composite-modified GCE for the immobilization of biomolecules. Graphene with high conductivity and good biocompatibility was expected to improve the electron transfer rate of the electrochemical immunosensor (Soldano et al., 2010). AuNPs, as a three-dimensional structure, were expected to favor a particleenhanced immobilization of antibodies due to the unique physical

and chemical properties of nanoparticles (Zhao and Hsing, 2010). Note that to fully utilize the electrical property and biocompatibility of AuNPs, it is very important to decorate them on the MWCNTs in a dense and well-dispersed way. The graphene was characterized by TEM and atomic force microscopy (AFM). The TEM image (Fig. 2A) displays a view of graphene nanosheets clearly illustrating typical flake-like wrinkled shapes of graphene with irregular size. A detail structure of graphene was shown in Fig. 2A (insert figure) which can be found the crystalline lattice clearly. Fig. 2B is the typical AFM images and height profiles of graphene. The thickness of the graphene sheet obtained is about 1 nm, which are consistent with previously reported thickness of single-layer graphene sheets (Stankovich et al., 2006; Li et al., 2008; Novoselov et al., 2005). A direct evidence for the attachment of AuNPs to the MWCNTs surface is given by the SEM tests. Compared with Fig. 2C, it is observed in Fig. 2D that the AuNPs decorated uniformly on the walls of MWCNTs. Furthermore, this uniform nanostructure provides an efficient electrode surface for loading Ab1 (through the formation of covalent bond between Au and amine groups of the antibody) and accelerating electron transfer. According to the equation of Q = 2nFAcD1/2 −1/2 t1/2 + Qdl + Qads given by Anson (Anson, 1964), the surface area of the Au/MWCNTs/GS/GCE is 0.352 cm2 , which was calculated by the slope of Q versus t1/2 curve obtained by chronocoulometry using 2 mmol L−1 K3 [Fe(CN)6 ] as model. 3.2. Characterization of MCM-41, TH/MCM-41 and Au/TH/MCM-41 As shown in Fig. 3E, the N2 adsorption–desorption curve of MCM-41 mesoporous nanoparticles exhibits a type IV isotherm. The size of the pores estimated by the Kruk–Jaroniec–Sayari (KJS) model is 3.1 nm. The Brunauer–Emmett–Teller (BET) specific surface area and pore volume are 1048 m2 g−1 and 0.588 cm3 g−1 , respectively. The high surface area and quite a number of pores favored for the immobilization of nanoparticles and biomolecules. Fig. 3A and D shows the HRTEM and SEM image of mesoporous silica nanoparticles MCM-41. The particles exhibited a spherical morphology. As shown in Fig. 3A, MCM-41 nanoparticles retained a regular array channels which could be obviously observed. After the immobilization of thionine on the surface of MCM-41 nanoparticles, the mesoporous nanostructures could be also observed (Fig. 3B). However, the pore size decreased and displayed more disorder than unmodified one, which could further confirm the attachment of thionine on MCM-41. The positively charged thionine molecules on the surface displayed strong

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Fig. 2. TEM (A) (HRTEM of graphene in insert figure) and AFM (B) of graphene nanosheets, SEM image of pure MWCNTs (C) and Au/MWCNTs (D).

electrostatic interaction with negative charged AuCl4 − ions, leading to more AuNPs formed on the TH/MCM-41 after reduced by NaBH4 . As shown in Fig. 3C, the further decreasing of the pore size indicated a large number of AuNPs were attached on the TH/MCM41. The appearance of AuNPs provided a biocompatible interface and a good microenvironment for the conjugation of biomolecules. 3.3. Cyclic voltammetry characterization Fig. 4B displays the electrochemical behavior of the nanoparticles-based immunoassay after each step in pH 7.4 PBS. No peak was observed at anti-hCG/Au/MWCNTs/GCE in pH 7.4 PBS (curve a). After the immunosensor was incubated with hCG in the sample solution, a low background current was achieved (curve b), indicated that the formation of the antigen–antibody complex hindered the electron transfer resulted in a great decrease in the background current. After the HRP-Ab2 /Au/TH/MCM-41 was reacted with the immunosensors, however, a couple of well-defined redox peaks at −189 and −252 mV was appeared at the working potential range in pH 7.4 PBS (curve c). The redox waves mainly derived from the immobilized thionine molecules with redox activity on the HRP-Ab2 /Au/TH/MCM-41. The results indicated that the doped thionine, as a good electron mediator, still remained their redox properties in synthesized HRP-Ab2 /Au/TH/MCM-41 immunocomplex and could provide a fast pathway for electron transfer. Moreover, the peak separation was about 60 mV. The good electrochemical behavior might be

owing to the penetrated/coated gold nanoparticles into/onto the mesoporous nanomaterials. When 0.2 mmol L−1 H2 O2 substrate was added into pH 7.4 PBS, an obvious catalytic process with the decrease of anodic peak and the increase of cathodic peak was occurred at the sandwiched immunosensor (curve d). The catalytic current mainly derived from the immobilized HRP toward the reduction of H2 O2 with the aid of the doped thionine as mediator. And it also suggested that the immobilized HRP-Ab2 biomolecules on the HRP-Ab2 /Au/TH/MCM-41 could still maintain their natural bioactivity, and the synthesized Au/TH/MCM-41 could effectively shuttle electrons from the base electrode surface to the redox center of the immobilized biomolecules. Thus, we might quantitatively evaluate the concentration of hCG according to the reduction current. 3.4. EIS characterization of the immunosensor EIS is an effective method to monitor the changes of interfacial properties, allowing the understanding of chemical transformation and processes associated with the conductive electrode surface (Park and Yoo, 2003; Chen et al., 2006; Prodromidis, 2010). We use this method to get information on the impedance changes of the sensor interface in the modification process. Electrochemical impedances of the electrodes were performed in a background solution of 5.0 mmol L−1 K3 Fe(CN)6 containing 0.1 mol L−1 KCl, and the frequency range is at 100 mHz to 10 kHz at 220 mV. Fig. 4C shows the Nyquist plots of EIS corresponding to the stepwise

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Fig. 3. HRTEM image of MCM-41 (A), TH/MCM-41 (B), Au/TH/MCM-41 (C), SEM image of MCM-41 (D), N2 adsorption–desorption isotherm of MCM-41 (E).

Fig. 4. (A) Cyclic voltammograms of (a) bare GCE, (b) GS/GCE, (c) MWCNTs/GS/GCE, (d) Au/MWCNTs/GS/GCE, and (e) BSA/Ab1 /Au/MWCNTs/GS/GCE in 2 mmol L−1 potassium ferricyanide solution (pH 7.0) containing 0.1 mol L−1 KCl at 50 mV/s. (B) Cyclic voltammograms of (a) Ab1 /Au/MWCNTs/GS/GCE, (b) hCG/Ab1 /Au/MWCNTs/GS/GCE, and (c) HRPAb2 /Au/TH/MCM-41 reaction with (b) in pH 7.0 PBS and (d) containing 2 mmol L−1 H2 O2 at 50 mV/s. (C) EIS of (a) bare GCE, (b) GS/GCE, (c) MWCNTs/GCE, (d) MWCNTs/GS/GCE, (e) Au/MWCNTs/GS/GCE, (f) Ab1 /Au/MWCNTs/GS/GCE, (g) hCG/Ab1 /Au/MWCNTs/GS/GCE, (h) HRP-Ab2 /Au/TH/MCM-41 bioconjugate hCG/Ab1 /Au/MWCNTs/GS/GCE in 5.0 mmol L−1 K3 Fe(CN)6 containing 0.1 mol L−1 KCl. (D) Amperometric responses of the immunosensor with the variously secondary-labeled antibody toward different hCG concentrations: (a) HRP-Ab2 /Au/TH/MCM-41, (b) HRP-Ab2 /TH/MCM-41, (c) HRP-Ab2 /Au/MCM-41, (d) HRP-Ab2 /Au in pH 7.0 PBS containing 2.0 mmol L−1 H2 O2 .

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modification processes. The curve (a) shows EIS of the bare GCE. There is a small semicircle (Ret = 143 ) at high frequencies and a linear part at low frequencies. When GS was modified on the GCE surface, a much lower resistance was obtained (curve b, Ret = 85 ), implying that the GS is an excellent electric conducting material and accelerated the electron transfer (as comparison, curve c was EIS of GCE modified with MWCNTs only, Ret = 77 ). IT was observed that the EIS of MWCNTs/GS/GCE (curve d) and Au/MWCNTs/GS/GCE (curve e) displayed an almost straight line in the Nyquist plot, characteristics of a diffusion-limited electrontransfer process. The decrease of impedance in curve d was owing to the synergy of graphene and MWCNTs which can greatly enhance the electron transfer (Li et al., 2010; King et al., 2010; Tang and Gou, 2010). Furthermore, the Au/MWCNTs/GS/GCE showed a much lower resistance than the former, indicating that the introduction of the AuNPs was highly beneficial to the electron transfer. However, after incubation of anti-hCG molecules, the Ret increased to 240  (curve f), suggesting that anti-hCG molecules were successfully immobilized on the surface and blocked the electron exchange between the redox probe and the electrode. Then, the Ret increased again when BSA and hCG were immobilized onto Au/MWCNTs/GS/GCE gradually (curve g, Ret = 680 ), which were caused by the nonconductive properties of biomacromolecule. At last, when HRP-Ab2 /Au/TH/MCM-41 interacted with hCG (curve h), interestingly the resistance decreased to 508 , indicating that the synthesized HRP-Ab2 /Au/TH/MCM-41 possessed high conductivity and good electron transfer efficiency, although the protein adsorption layer acted as barrier to the interfacial electron transfer. So, the EIS results were in accordance with those of cyclic voltammetry (Fig. 4A, detailed descriptions were in Supporting information). 3.5. Comparison of electrochemical responses In this contribution, the assay is based on a sandwich-type immunoassay format using HRP-Ab2 /Au/TH/MCM-41 as traces and H2 O2 as enzyme substrates. So, the label of detection/secondary antibody is crucial. To clarify the advantage of the developed immunoassay using multifunctional mesoporous MCM-41 immunocomplex as signal amplification section, a comparative study of the electrochemical responses of the immunosensor was carried out on the Ab1 /Au/MWCNTs/GS/GCE. We fabricated four kinds of secondary conjugating antibody including HRPAb2 /Au/TH/MCM-41, HRP-Ab2 /TH/MCM-41, HRP-Ab2 /Au/MCM-41 and HRP-Ab2 /Au, which were used for the detection of hCG by using the sandwich-type immunoassay. As shown in Fig. 4D, the immunosensor by using HRP-Ab2 /Au/TH/MCM-41 exhibited higher sensitivity than those of other labeled probes. Some possible explanations might be considered as follows: (i) the mesoporous MCM-41 nanoparticles could display a high surfaceto-volume ratio, which could enhance the immobilized amount of biomolecules; (ii) AuNPs doped on the bionanocomposites with higher capability of electron transfer, which could effectively shuttle electrons from the base electrode surface to the redox center of HRP, meanwhile the nanoparticles can further amplify the specific surface area and enhance the sensitivity of the immunoassay; (iii) thionine could be not only an excellent electron mediator, but also a good cross-linkage reagent between MCM-41 and nanogold, resulting in more HRP-Ab2 loading effectively to amplify the amperometric signal output. Therefore, the proposed immunosensor and immunoassay could display better analytical properties under the same conditions. 3.6. DPV response of immunosensor to hCG concentration To evaluate the sensitivity and dynamic range of the electrochemical immunosensor, routine samples with various

Fig. 5. Calibration curves of the electrochemical immunosensor toward hCG standards in pH 7.0 PBS containing 2 mmol L−1 H2 O2 .

concentrations of hCG were assayed under optimal conditions (optimal conditions see in Supplement). As shown in Fig. 5, the DPV response of the immunosensor increased with the increment of hCG concentrations, and exhibited a good linear relationship with the logarithm of hCG concentration from 0.005 to 500 mIU mL−1 . The linear regression equation was adjusted to I (␮A) = −15.42 − 5.04 × log C [hCG] (mIU mL−1 , R2 = 0.999) with a detection limit (LOD) of 0.0026 mIU mL−1 at a signal-to-noise ratio of 3 (where  is the standard deviation of the blank, n = 11). Compared with other sensors reported previously (Tao et al., 2011; Yang et al., 2009, 2010b, 2011), our proposed immunosensor exhibited a satisfactory detection limit and linear range. The characteristics of other hCG sensors are summarized in Supporting information.

3.7. Reproducibility, stability and selectivity of the immunosensor The reproducibility of the electrochemical immunosensor was evaluated by intra- and interassay coefficients of variation (CVs). The intra-assay precision of the analytical method was evaluated by analyzing four concentration levels five times per run in 5 h. The CVs of intra-assay with this method were 4.2%, 5.0%, 4.5%, and 5.2% at 0.5, 5, 80, and 150 mIU mL−1 of hCG, respectively. Similarly, the interassay CVs on five immunosensors were 5.3%, 4.8%, 5.1%, and 4.3% at 0.5, 5, 80, and 150 mIU mL−1 of hCG, respectively. Thus, the precision and reproducibility of the proposed immunoassay were acceptable. Stability of this immunosensor is a key factor in their application and development. When 100 continuous cyclic scans were carried out in 0.1 mol L−1 PBS (pH 7.0) containing 2 mmol L−1 H2 O2 , only 1.5% decrease of the initial response was observed. When the immunosensor was dried and stored at 4 ◦ , it retained 90% of its initial response after a storage period of 30 days. The slow decrease in response seemed to be related to the gradual deactivation of the immobilized antibody incorporated in the composite. To investigate the differences in response of the immunoassay to interference degree or crossing recognition level, carcinoembryonic antigen (CEA), ␣-fetoprotein (AFP), CA 125, prostate-specific antigen (PSA) and BSA was used in this study. DPV responses of the proposed immunosensor in 80 mIU mL−1 of hCG solutions containing interfering substances of different concentrations were assayed. The results were shown in Table 1A, with the RSD values ranged from 1.12% to 2.45%, so the selectivity of the as-prepared immunosensor was acceptable.

J. Lu et al. / Biosensors and Bioelectronics 33 (2012) 29–35 Table 1A Interference degree/crossing recognition level of the developed immunoassays. Conc. [interfering agents] (ng mL−1 ) or (U mL−1 ); Current (␮A)b a

Crossing reagents

0

20

100

200

RSD (%)

hCG + CEA hCG + AFP hCG + CA 125 hCG + PSA hCG + BSA

−25.11 −25.21 −25.09 −25.13 −25.19

−25.01 −25.15 −24.89 −24.96 −24.72

−24.62 −24.84 −24.28 −24.63 −24.34

−24.18 −24.58 −23.98 −24.07 −23.77

1.71 1.12 2.11 1.89 2.45

a b

Containing 80 mIU mL−1 hCG and various concentrations of interfering agents. The average value of three assays.

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Acknowledgments This work was financially supported by Natural Science Research Foundation of China (21175058), Natural Science Foundation of Shandong Province, China (ZR2011BQ019) and Technology Development Plan of Shandong Province, China (2011GGB01153). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bios.2011.11.054. References

Table 1B Determination of hCG in blood samples. Sample

Proposed immunosensor (mIU mL−1 ) (n = 5)

ELISA (mIU mL−1 )

Relative error (%)

1 2 3 4 5

14.36 25.84 47.57 84.72 102.85

13.79 26.35 48.17 82.32 104.63

4.13 −1.94 −1.25 2.92 −1.70

3.8. Real sample analysis The feasibility of the immunoassay for clinical applications was investigated by analyzing several real samples in comparison with the commercial ELISA method. Five clinical human serum samples were from the Shandong Tumor Hospital. Human serum samples were diluted to different concentrations with a PBS solution of pH 7.0, and each sample was analysed for five times. The results were shown in Table 1B, and the relative error between the two methods ranged from −1.94% to 4.13%. These results indicate no significant difference between the results given by the two methods. Therefore, the proposed immunosensor could be reasonably applied in the clinical determination of hCG. 4. Conclusion In this work, we have designed a novel electrochemical immunosensor based on graphene and MWCNTs/Au modified electrode for the detection of hCG using functionalized mesoporous Au/TH/MCM-41 nanoparticles as signal amplifying probe. The synergy of graphene and MWCNTs can greatly enhance the electron transfer between the electrolyte and electrode. Furthermore, the AuNPs were modified on MWCNTs and TH/MCM-41 simultaneously, as an efficient bio-interface film possessed the biocompatibility, enhanced the conductivity, and enlarged the specific surface area of the sensing interface, which would be very useful for immobilizing antibody more efficiently. More significantly, a novel amplification strategy was introduced to further improve the sensitivity of the immunosensor using Au/TH/MCM-41-labeled secondary antibodies as amplifying probes, which could display low LOD and high sensitivity. Highlight of this work is to open new avenues in the application of AuNPs dotted MWCNTs–graphene composite interface and Au/TH/MCM-41-labeled secondary antibodies as amplifying probes for sensitive electrochemical immunoassay. This strategy can be observed to improve the immunoassay sensitivity, and thus provides a novel promising platform of clinical immunoassay for hCG.

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