Preparation of TiO2 nanosheet-carbon nanotube composite as immobilization platform for both primary and secondary antibodies in electrochemical immunoassay

Analytica Chimica Acta 946 (2016) 40e47

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Preparation of TiO2 nanosheet-carbon nanotube composite as immobilization platform for both primary and secondary antibodies in electrochemical immunoassay Xiaoqiang Liu*, Pepipei Liu, Xiaohe Huo, Xiuhua Liu, Jin Liu Institute of Environmental and Analytical Sciences, College of Chemistry and Chemical Engineering, Henan University, Kaifeng, Henan Province, 475004, PR China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t


TiO2 nanosheet-carbon nanotube composite was synthesized by hydrothermal method.
The composite exhibits improved properties compared to the individuals.
The composite is designed as electrode scaffold for immobilizing primary antibody.
Secondary antibody and horseradish peroxidase are immobilized on the composite as a label.
The tracing label exhibits great amplification effect for immunosensing.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 August 2016 Accepted 16 October 2016 Available online 24 October 2016

TiO2 nanosheets (TNSs) were synthesized and deposited on multi-wall carbon nanotubes (MWCNTs) to form a nano-composite through a hydrothermal method, followed by the characterization with various spectroscopic and microscopic techniques. The TNS-MWCNT composite was then applied as not only an electrode scaffold to immobilize primary antibody, but also as a carrier to load secondary antibody and horseradish peroxidase (HRP). In both cases, bis(sulfosuccinimidyl) suberate sodium salt acted as an amino cross-linker to covalently bind the biomolecules on TNS-MWCNT composite through their surface primary amino groups. After the sandwich-type immunoreaction, HPR was quantitatively captured on the electrode surface via the binding between secondary antibody and antigen, and electrochemical response of the immunosensor was then amplified by a H2O2 mediated HRP catalytic reaction. Using aFetoprotein as a model analyte, a linear range between 0.005 and 320 ng mL1 with a detection limit of 2.0 pg mL1 was achieved by differential pulse voltammetry. The improved immunosensor performance could be attributed to the biocompatibility and high specific surface area of TNS, and excellent electrical conductivity of MWCNTs, which accelerated the electron transfer at the electrode surface. © 2016 Elsevier B.V. All rights reserved.

Keywords: TiO2 nanosheets Multi-wall carbon nanotubes Sandwich-type immunoreaction a-Fetoprotein Nano-composite

1. Introduction * Corresponding author. Tel.: þ86 13781157777. E-mail addresses: [email protected], [email protected] (X. Liu). http://dx.doi.org/10.1016/j.aca.2016.10.020 0003-2670/© 2016 Elsevier B.V. All rights reserved.

Various immunoassay techniques including chemoluminescence immunoassay [1], enzyme-linked immunosorbent

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assay (ELISA) [2], fluorescence immunoassay [3,4] and radioimmunoassay [5] have been extensively applied in the medical field such as cancer screening, early disease diagnosis, therapeutic efficacy and so on [6]. As a powerful supplement to the above techniques, electrochemical immunosensors exhibited certain advantages including operation convenience, simple preparation, rapid analysis, high sensitivity, and uncomplicated instrumentation [7,8]. For example, they are more sensitive than spectrometric or optical immunoassays because the latter necessitate bulky and power-intensive light sources, monochromator and optical detectors. Moreover, turbid and coloured samples may produce false positive signals when the optical methods are used to detect such samples. In recent years, different nanomaterials such as magnetic particles [9e11], noble metal nanomaterials [12,13], carbon nanomaterials [14e16] and oxide nanomaterials [17,18] have been extensively applied for the development of electrochemical sensors to improve their performance including dynamic range, detection of limit, selectivity, stability, precision and etc. Among them, carbon nanomaterials became the most excellent electrode materials due to their outstanding electrical conductivity, good chemical stability, considerable mechanical strength and chemically modifiable surfaces [19]. For example, Malhotra et al. [20] have prepared an electrochemical immunosensor for detecting cancer-related levels of interleukin-6 in squamous cell carcinomas of head and neck cells. The immunosensor was constructed on an electrically conductive, high surface area and densely upright packed single wall carbon nanotube (SWCNT) forest with primary antibody attached to their terminals. The antigen would bind to the primary antibody, followed by the horseradish peroxidase (HRP)-labelled secondary antibody to facilitate a sandwiched assay. High sensitivity was achieved through enzymatic catalysis amplification on SWCNT forest, resulting in a low detection limit of 30 pg mL1 for human interleukin-6 in calf serum. However, the direct formation of the bioconjugate between secondary antibody and signal enzymes involves many complicated steps, which may lead to denaturation of the biomolecules. Accordingly, carbon nanomaterials were also used as the support for immobilizing both signal enzymes and secondary antibody. For example, Zhao et al. [21] fabricated an electrochemical immunosensor involving carbon nanospheres for microcystins-LR (MCLR) detection. In their work, HRP and secondary antibody were coimmobilized on carbon nanospheres to form signal-amplifying labels. Due to the three-dimensional porous structure and high conductivity of carbon nanospheres, this approach yielded a linear range from 0.05 to 15 mg L1 MCLR with a detection limit of 0.016 mg L1. Although carbon nanomaterials behaved well as both the immobilization platform and the support for secondary antibody/ enzymes, they still suffer from certain disadvantages as described below. In the first place, the electrochemical signals resulted from CNT surface carboxyl groups may bring interference to the immunosensor test and affect the detection accuracy. Furthermore, the biocompatibility of carbon materials is not as good as that of semiconductors, therefore, carbon materials have shown weaker capability of retaining bioactivity than semiconductors. In this work, TiO2 nanosheets with high surface area, negligible electrochemical interference and excellent biocompatibility were deposited on highly conductive carbon nanotube surface to form a composite, which retained the advantages of both materials and minimize their shortages [22,23]. For the first time, this composite was applied as both the immobilization scaffold for primary antibody (Ab1) and the carrier for secondary antibody (Ab2) and HRP. This novel strategy will consume less time compared with the traditional electrochemical immunoassay, which has to prepare

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Ab1 scaffold and Ab2 support separately. The obtained immunosensor was applied to detect a model analyte of a-Fetoprotein (AFP) with differential pulse voltammetry (DPV) technique to demonstrate the superiority of TNS-CNT composite over CNT. 2. Experimental 2.1. Materials and reagents Short carboxyl multi-wall carbon nanotubes (MWCNTs) (outside diameter: 20e30 nm, length: 0.5e1 mm) were purchased from Chengdu Organic Chemical Co., Ltd., Chinese Academy of Sciences, China. Titanium (Ⅳ) isopropoxide (TIP; 97%) and diethylenetriamine (DETA; 99%) were purchased from J&K Scientific Ltd., Beijing, China. Mouse monoclonal primary and secondary anti-AFP antibodies (clone no. A14C11 and A46C9) and AFP (96%) (Product no. A0101) were purchased from Shuangliu Zhenglong Biochem. Lab (Chengdu, China) and immediately diluted to the required concentrations with 0.02 M phosphate buffer (pH 7.4) before use. 3aminopropyltriethoxysilane (APTES), suberic acid bis(3-sulfo-Nhydroxysuccinimide ester) sodium salt (BS3), HRP, bovine serum albumin (BSA), 30% H2O2 (v/v) and Nafion were obtained from Sigma-Aldrich (Shanghai, China). Ultrapure water obtained from a Millipore water purification system (18 MU, Milli-Q, Millipore) was used in all the assays. All the other reagents were of analytical grade and used as received. The phosphate buffer solutions (PBS, pH 7.4) were prepared by mixing the stock solutions of 0.05 M KH2PO4 and 0.05 M K2HPO4 containing 0.10 M KCl as the supporting electrolyte. The washing buffer (PBST) consists of PBS (0.05 M, pH 7.4) and 0.05% (w/v) Tween 20 and the blocking solution was PBS (0.05 M, pH 7.4) containing 5% (w/v) BSA. 2.2. Apparatus All electrochemical measurements were conducted at a CHI630C electrochemical workstation (CH Instruments, Shanghai, China) in a conventional three-electrode cell. Glassy carbon electrode (GCE), platinum wire and AgjAgCl (3.0 M KCl) electrode were used as working electrode, counter electrode and reference electrode respectively. All the electrodes were purchased from Gaoshiruilian Co., Ltd., Wuhan, China. Electrochemical impedance spectroscopy was collected at an IM6ex electrochemical station (ZAHNER, Germany). The morphology and chemical composition of the nanocomposite were studied by transmission electron microscope (TEM, Tecnai G2 20, USA), scanning electron microscope (SEM, JSM-7500F, JEOL., Japan), X-ray diffraction (XRD, Bruker D8 Advance, Germany) with Cu Ka radiation and Fourier transform infrared spectra (FT-IR, Nicolet 170, USA). X-ray photoelectron spectra (XPS) were collected on an X-ray photoelectron spectrometer (ESCALAB 250Xi, USA) with a monochromated Al Ka source (hn ¼ 1486.6 eV), 150 W Power and 500 mm beam spot. The spectra were calibrated on the C1s peak (284.8 eV) and analyzed using XPSPEAK41 software. 2.3. Preparation of TNS-MWCNT composite and its amination The TNS-MWCNT composite was prepared as follows: 50 mg of MWCNTs was dispersed in 40 mL isopropyl alcohol by 20 min of ultrasonication, followed by addition of 50 mL DETA. Then 3.6 mL TIP was added to the above dispersion under proper stirring. The mixed solution was subsequently transferred into a Teflon-lined stainless steel autoclave and the autoclave was kept in an electric oven at 200  C for 24 h TiO2 nanosheets are formed through the hydrolysis of TIP catalyzed by DETA, which also aids in assembling TiO2 nanosheets on the surface of carboxyl modified MWCNTs.

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After that, the black precipitate was collected by centrifugation, washed with ethanol and dried at 70  C. To obtain pure TiO2 nanosheets, the as-prepared composite was calcinated at 500  C for 2 h to remove MWCNT from the composite. The amino-functionalized TNS-MWCNT composite were prepared following a protocol with minor modification [24]. In brief, TNS-MWCNTs (120 mg) were dispersed in a solution containing 20 mL ethanol, 1 mL ammonia (28%) and 5 mL APTES. The suspension was mechanically stirred overnight before it was centrifuged. The supernatant was discarded while the precipitate was collected, washed three times with Milli-Q water, and dried in oven at 60  C. 2.4. Preparation of the tracing marker Scheme 1A displays the preparation of the HRPjNH2-TNSMWCNTjAb2 bioconjugate. Initially, BS3 (2 mg) was dissolved in 0.58 mL 0.02 M PBS (pH 7.4) and then 3 mg NH2-TNS-MWCNT was dispersed into BS3 solution with ultrasonication. After that, the dispersion was mixed with 400 mL HRP (2.5 mg mL1) and the mixture was properly stirred for 30 min at room temperature. Afterward, 20 mL Ab2 (1.0 mg mL1) was added into the mixture and incubated at 4  C for 4 h under gentle stirring. The mixture was centrifuged to obtain the bioconjugate, which was then washed with PBS and blocked with 2% BSA. Finally, the bioconjugate was washed again with PBS and suspended in 1.0 mL PBS containing 0.1% BSA. 2.5. Fabrication of the immunosensor and electrochemical immunoassay Scheme 1B illustrates the fabrication process of the single-use immunosensor. Briefly, 6 mg NH2-TNS-MWCNT composite was dispersed in 1 mL 1% Nafion solution after 20 min sonification to obtain a homogeneous dispersion. Next, 5 mL of this suspension was cast on a cleaned GCE and the modified GCE was kept at 4  C overnight. After that, a droplet (10 mL) of 2 mg mL1 BS3 solution

was applied on the modified GCE, which was then left at room temperature for 1 h, followed by washing with distilled water. Subsequently, 5 mL primary antibody (0.5 mg mL1) was incubated on the BS3 modified GCE surface at room temperature for 1 h. The excess antibodies were removed with PBST and PBS. The modified electrode was then exposed to 5 mL of 5% (w/v) BSA solution for 30 min to block possible remaining active sites against nonspecific adsorption before it was washed with PBST and PBS respectively. The resulting modified electrode was stored at 4  C overnight in a 100% moisture-saturated environment. Before the immunoassay, the Ab1 modified GCE was incubated with 5 mL AFP solution for 50 min at room temperature and washed with PBST and PBS successively. Following that, the modified GCE was incubated with 5 mL HRPjNH2-TNS-MWCNTjAb2 bioconjugate for 50 min at 37  C to obtain the immunosensor before it was rinsed with PBST and PBS respectively. The immunosensor, AgjAgCl reference electrode and Pt counter electrode were placed in an electrochemical cell containing 10 mL PBS. Prior to the experiments, the test solution was deoxygenized by high purity nitrogen for 15 min and maintained in nitrogen atmosphere during the detection. Hydroquinone (final concentration 2 mM) and H2O2 (final concentration 1 mM) solutions were injected in the cell and differential pulse voltammetry (DPV) with a pulse amplitude of 50 mV and a pulse width of 50 ms was performed for quantitative detection of AFP. 3. Results and discussion 3.1. TEM, SEM, XPS, XRD, and FT-IR characterization of nanomaterials The microscopic images of MWCNTs and TNS-MWCNT composite are displayed in Fig. 1 to confirm the deposition of TNSs on MWCNT surface. As expected, the TEM image of MWCNTs (Fig. 1(A)) exhibits a hollow and slightly curved tubular structure with an outer diameter ~20e30 nm and length ~0.5e1 mm. After the hydrothermal reaction, the TEM image of the product (Fig. 1(B)) shows that MWCNTs are densely covered by semitransparent nano-

Scheme 1. Schematic illustration of preparation of (A) HRPjTNS-MWCNTjAb2 bioconjugate and (B) immunosensor.

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Fig. 1. TEM images of (A) MWCNTs, (B) and (C) TNS-MWCNT composite; SEM image of (D) TNS-MWCNT composite.

materials. Fig. 1(C) provides a more distinct TEM image of the composite, showing that the single hollow tubular structure of MWCNTs is encapsulated by flocculent nano-materials. However, it is difficult to identify the morphology of the nanostructure on MWCNT surface by the TEM images alone due to the semitransparent character of the nanomaterials. Therefore, the SEM image of the composite (Fig. 1(D)) is provided and it has clearly revealed the curved flake shape of the nanostructure deposited on MWCNT. Both TEM and SEM results have confirmed that the surface contact between the two single components is very intimate, indicating the successful formation of the binary composite. XPS measurement was performed to obtain the chemical composition and surface state information of the samples (Fig. 2). The XPS survey spectrum of the composite (Fig. 2(A)) shows the peaks of C1s, O1s and Ti2p, which are attributed to the main composition elements of the composite. The C1s XPS spectrum of the samples (Fig. 2(B)) displays four peaks centering at 284.1, 285.2, 286.4 and 288.8 eV, which are strongly related to the different carbon-atom bond environments. For example, the peak at ~284.1 eV is ascribed to the graphitic C]C/CeC structure [25]. In addition, the peak at ~285.2 eV is attributed to the C atoms with the defective structure on the carbon nanotube surface, whereas the two peaks at ~286.4 and 288.8 eV correspond to CeO and C]O structure respectively [26]. The XPS spectrum of O1s (Fig. 2(C)) exhibits another two peaks at 532.4 and 530.7 eV attributing to the O atoms in C]O groups and the lattice oxygen [TieO6] respectively [26]. The titanium XPS 2p core level spectrum consists of two sublevels due to the spinorbit splitting [27]. Therefore, two peaks centered at 459.1and 464.6 eV, assigned to Ti2p3/2 and Ti2p1/2 respectively [28] are observed on the XPS high-resolution spectrum of Ti2p (Fig. 2(D)). The above XPS results have clearly indicated that the nanomaterials deposited on MWCNTs belong to titanium oxide. Fig. 3(A) presents the XRD patterns of MWCNTs, TNSs and TNSMWCNT composite as curve a, b and c respectively. As shown on curve a, the (0 0 2) and (1 0 0) reflections of MWCNTs have

generated two typical peaks centered at ~25.9 and 43.6 [29]. The XRD pattern of TNS sample (curve b in Fig. 3(A)) exhibits several typical peaks at 25.3 , 37.9 , 48.0 , 54.1, 54.9, 62.7, 68.8 , 70.2 , 75.0 and 82.8 , which are attributed to the (1 0 1), (0 0 4), (2 0 0), (1 0 5), (2 1 1), (2 0 4), (1 1 6), (2 2 0), (2 1 5) and (2 2 4) crystal faces of anatase TiO2 respectively [30]. Both the reflection peaks ascribed to TiO2 nanosheets and MWCNTs can be observed on curve c in Fig. 3(A), indicating the formation of the TiO2-MWCNT composite. The XRD pattern of TNS-MWCNT has also demonstrated that the titanium oxide formed on MWCNT surface is ascribed to anatase TiO2. However, the peak intensity of the composite is significantly decreased compared with that of the individual components, implying that the formation of the composite has weakened the crystal reflection slightly. FT-IR spectra of (a) MWCNTs, (b) TNSs and (c) TNS-MWCNT composite (Fig. 3(B)) have been investigated to obtain the structural information of these nanomaterials. Fig. 3(B)(a) displays two obvious absorption band centered at ~3440 cm1 and 1630 cm1 respectively, assigning to the hydroxyl stretching and bending vibrations on the surface of MWCNTs [30]. These two absorption bands are also observed on curve b and c, indicating the presence of hydroxyl groups on both TiO2 nanosheets and TNS-MWCNT composite. TiO2 nanosheets have shown a strong adsorption band between 400 and 800 cm1 attributing to the TieO stretching and TieOeTi bridging vibration (Fig. 3(B)(b)) [31], which is also found in the FT-IR spectra of TNS-MWCNT composite (Fig. 3(B)(c)). Therefore, Fig. 3(B) has demonstrated the formation of TNSMWCNT composite. 3.2. EIS characterization of the nanomaterials and the assembly of the immunosensor Electrochemical impedance spectroscopy (EIS) is a powerful technique for investigating the conductivity of the materials and interfacial properties of the modified electrodes [32]. Therefore, EIS

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Fig. 2. XPS analysis of TNS-MWCNT composite: (A) XPS survey scans, high-resolution XPS spectrums of (B) C1s, (C) O1s, and (D) Ti2p.

Fig. 3. (A) XRD patterns of (a) MWCNTs, (b) TNSs and (c) TNS-MWCNT composite; (B) FT-IR spectra of (a) MWCNTs, (b) TNSs and (c) TNS-MWCNT composite.

is used herein to demonstrate the improved conductivity of the TNS-MWCNT composite and the stepwise assembly of the immunosensor. Impedance experiments were performed in 0.1 M KCl solution containing 5 mM K3[Fe(CN)6]jK4[Fe(CN)6] at a constant potential of 0.21 V superimposed by an alternating voltage of 5 mV. All the Nyquist plots in Fig. 4 display a linear portion in the low frequency region and a semicircular portion in the high frequency region over a frequency range from 50 kHz to 100 mHz. It is reported that the semicircular diameter is proportional to the electron transfer resistance (Ret) of redox probe at the modified electrode surface [33]. As expected, the semicircle diameter of TNSjGCE (curve c) is obviously larger than that of TNS-MWCNTjGCE (curve b) and bare GCE (curve a), indicating that the electrical conductivity of TNS-MWCNT has been improved compared to that

of TiO2 alone. With the successive immobilization of primary antibody (Curve d), BSA (Curve e), and AFP/secondary antibody (Curve f) on TNS-MWCNTjGCE, the semicircle diameter in the Nyquist plots increases gradually, indicating successful modification of the biomaterials on the electrode. The nanomaterials and biomolecules assembled on electrode surface have formed the barrier layers to impede the electron transfer, which thus increased Ret. 3.3. Optimization of the immunoassay The immunoreaction time, the amount of TNS-MWCNT, the concentration of primary antibody and the ratio between HRP and secondary antibody in the tracing marker will significantly affect

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Fig. 4. Nyquist plots of (a) bare GCE, (b) TNS-MWCNTjGCE, (c) TNSjGCE, (d) Ab1jTNSMWCNTjGCE, (e) BSAjAb1jTNS-MWCNTjGCE and (f) Ab2jAFPjBSAjAb1jTNSMWCNTjGCE.

the detection signal and therefore should be optimized before the performance evaluation. At room temperature, the incubation time of AFP on Ab1 modified electrode was optimized for the immunoassay. Fig. 5(A) demonstrates that the amperometric response at the immunosensor enhances rapidly with the increase of AFP incubation time until 50 min. After that, the current increase becomes very slow due to the saturated binding between AFP and Ab1 antibody on the electrode surface. To achieve the balance between binding efficiency and immunoreactions time, an incubation time

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of 50 min was used for the later immunoassay. The volume of TNS-MWCNT suspension applied on the electrode surface was also investigated. The amperometric response versus amount of the composite shows a broad peak with the maximum peak value between 5 and 6 mL of TNS-MWCNT suspension (Fig. 5(B)). The excessive of the composite on electrode surface has impeded the charge and electron transfer, which resulted in the signal decrease. Accordingly, 5 mL of TNS-MWCNT suspension was applied on the electrode surface. As shown in Fig. 5(C), the DPV current of the immunosensor has increased dramatically until the concentration of Ab1 reaches 0.5 mg mL1, followed by slight decrease of the current. With the increase of Ab1 concentration, more AFP molecules are bound to Ab1 and more tracing markers are immobilized on electrode surface, leading to larger detection signal. However, the space hinder effect resulted from excessive Ab1 on electrode surface has impeded the binding of the tracing markers and decreased the DPV current. Next, the ratio between HRP and Ab2 concentration on the tracing markers was also optimized because the quantity of HRP significantly affects the detection signal. As shown in Fig. 5(D), the current is increased with the increase of HRP/Ab2 ratio and reaches the current peak at the ratio of 50:1 (W/W). After that, the DPV current drops mildly because the binding between the antigen and Ab2 has been weakened due to the extremely high HRP/Ab2 ratio. That is, the excessive HRP molecules have obstructed Ab2 from binding with the antigen.

3.4. Analytical performance of the immunosensor Under the optimum conditions, the immunosensor was

Fig. 5. Effect of (A) AFP incubation time, (B) amount of TNS-MWCNT, (C) Ab1 concentration and (D) ratio between HRP and Ab2 on DPV currents of the proposed immunosensor.

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Fig. 6. DPV currents obtained at the proposed immunosensor with increasing AFP concentration from curve a (0.005 ng mL1) to I (320 ng mL1); Inset: dynamic curve plotted based on DPV currents versus logarithms of AFP concentration.

assembled and its analytical performance was evaluated. In the presence of H2O2, HRP in the tracing markers converted hydroquinone to benzoquinone, which was then electrochemically reduced to generate a detection signal quantitatively related to AFP concentration. As shown in Fig. 6, the DPV peak currents were enhanced with the increase of AFP concentration and a calibration curve was plotted based on the DPV currents versus the logarithm of AFP concentration (the inset of Fig. 6). A linear range between 0.005 and 320 ng mL1 (r2 ¼ 0.994) was obtained. A detection limit of 2.0 pg mL1 was estimated from the calibration plot equation based on a signal to noise ratio of 3. Specifically, the average detection signal of four blank samples plus three times of their standard deviation was substituted into the calibration plot equation, from which limit of detection was calculated. The analytical performance of the proposed immunosensor is better than most of the AFP immunosensors reported in Table 1, which could be attributed to the excellent biocompatibility, superior electron transfer capability and high surface area of TNS-MWCNT as both the immobilization scaffold for Ab1 and the carrier for HRP and Ab2. 3.5. Selectivity, stability, precision and practical application of the immunosensor The specificity of the proposed immunosensor was tested by introducing several possible interferences including cancer antigen

125, carcinoembryonic antigen, prostate protein antigen, Immunoglobulin G, D-(þ)-glucose, ascorbic acid, and uric acid into the test solutions. In specific, a droplet (5 mL) of mixed solution containing 5 ng mL1 AFP and 100 ng mL1 each of the above interferences was applied on the immunosensor surface, followed by incubation with HRPjNH2-TNS-MWCNTjAb2 bioconjugate. The DPV currents obtained at the above immunosensors were compared with those at the immunosensors in the absence of the interferences and the relative deviation is less than 5.4%. The stability was also studied by measuring the detection signals of the immunosensor every single day for two weeks and the current signal retained 92.7% of the initial value after two weeks storing at 4  C. The reproducibility was investigated by measuring the same sample with six immunosensors prepared in the identical experimental conditions. A relative standard deviation (RSD) of 4.9% was obtained, indicating the good precision of the proposed immunoassay. Three different concentrations (0.2, 5 and 50 ng mL1) of AFP were spiked into blank serum samples respectively to test the analytical reliability and application potential of the proposed immunosensor. A recovery of 98.5%, 96.2% and 104.8% was obtained at three levels of AFP. The detection results were also compared with those obtained by standard enzyme-linked immunosorbent assay and the relative deviation values are less than 2.9%, indicating that the immunosensor can be potentially applied in the clinical detection of AFP. 4. Conclusion In this experiment, a TNS-MWCNT composite was prepared, characterized by various techniques including TEM, SEM, XPS and FT-IR, and applied in the construction of an electrochemical immunosensor. The novelty of the work mainly lies in two aspects. Firstly, the composite remains the merits of both individual components such as high conductivity, low electrochemical interferences, large specific surface area and excellent biocompatibility. In addition, the composite is not only used as an electrode scaffold for immobilizing primary antibody, but also acts as a carrier for secondary antibody and HRP. The assembly of the immunosensor was confirmed by the EIS results. Under the optimum conditions, the proposed immunosensor exhibited a dynamic range from 0.005 to 320 ng mL1 and a detection limit of 2.0 pg mL1, which is much superior over that of many AFP immunosensors based on carbon nanomaterials. This result has demonstrated the strong signal amplification of the tracing marker and the significant property improvement of TNS-MWCNT

Table 1 The analytical performance of the proposed immunosensor and other AFP immunosensors based on carbon nanomaterials. Immunosensors

Linear range (ng mL1)

Limit of detection (pg mL1)

References

HRP-Ab2jAFPjBSAjAb1jTNS-MWCNTjGCE AuNPs/anti-AFP/BSA/AFPeCNTeMnO2 MWCNTs-CDa-Fc-Ab1/BSA/AFP/GOD-CD-Ag-Ab2 MWCNT-PBb-GNPs/anti-AFP/BSA/AFP AuNPs/Ab1/AFP/Cd2þ-Ab2-AuNPs@MWCNTs GCE/rGOc-AuNPs/Ab1/AFP/CNSsd@Thie- AuNPs-Ab2 CNTs/Au NRsf/Ab1/BSA/ HRP-Au NRs-Ab2 MWCNTs/AuePtNPs/anti-AFP/AFP CD-GSg/Ab1/AFP/Pt@CuO-MWCNTs/Ab2 MWCNTs-Ag/Chitosan-MnO2/AuNPs/anti-AFP/AFP

0.005e320 0.2e100 0.001e5 0.01e300 0.01e60 0.01e80 0.1e1 00

2 40 0.2 3 4.5 3.5 30

This work [34] [35] [36] [37] [38] [39]

0.5e20/20-200 0.001e0.5/0.5e20 0.25e12/12-250

170 0.33 80

[40] [41] [42]

a b c d e f g

Cyclodextrins. Prussian blue. Reduced graphene oxide. Carbon nanospheres. Thionine. Gold nanorods. Graphene nanosheet.

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composite relative to other carbon nanomaterials. Author information

[19]

The authors declare no competing financial interest. [20]

Acknowledgments This work was financially supported by National Natural Science Foundation of China (No. U1504215), the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry (Batch 46th) and the grant from College Science and technology innovation team program of Henan Province (No. 141RTSTHN030).

[21]

[22]

[23]

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