• 2019-07
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  • br Preparation and characterization of L


    2.3. Preparation and characterization of L-histidine functionalized rGO
    Graphene oxide was easily synthesized from graphite powder using modified Hummer’s method and then functionalized accord-ing to the reported procedure by Amiri et al. [20]. In brief, 80 mg of DCC was added to GO suspension (5 mg mL−1), stirred mechanically for 30 min at room temperature and sonicated for 30 min. Then, the L-histidine solution (1 mg mL−1, pH = 8.5) was added to the GO–DCC mixture and the reaction mixture was refluxed at 80 ◦ C for 24 h. The obtained product was filtered and washed three times with ace-tonitrile followed by hydrochloric acid and ultrapure water. Finally, the resulting product was dried under an oven at 60 ◦ C for 12 h.
    The representative SEM image of the L-histidine functionalized rGO nanosheets is displayed in Fig. 1A. As seen, morphologi-cal structure of His-rGO consists of a flake-like formation with transparent and regular structure. The functionalization of GO with L-histidine was further supported by the FTIR spectroscopy
    (Fig. 1B). The GO spectrum (solid line) shows the Compound 48/80 bands at 1018 and 1168 cm−1 for C–O stretching vibrations in alkoxy and epoxy groups, respectively. The characteristic peak at 1392 cm−1 is related to the C–O-H deformation vibration. The stretching vibra-tion mode of CO in carboxyl group is appeared at 1745 cm−1. The bands at around 1639 and 3453 cm−1 are assigned to the stretch-ing vibrations CC and O H, respectively. The O-characterized band’s intensities decrease in the case of functionalized graphene oxide (dashed line), suggesting that GO was converted to rGO along with functionalization. The absorption band around 1691 cm−1 and a wide peak in the range of 3580–3600 cm−1 (symmetry and a symmetry vibrations of the primary amine) in His-rGO spectrum indicate the formation of the amide bond with imidazole ring after functionalization of graphene [20].
    2.4. Preparation of MWCNT/His-rGO/thionine/Ab as the biosensing interface
    The surface of GCE was first polished with 1.0, 0.3 and 0.05 m alumina powder, followed by ultrasonic washing with ethanol and distilled water for 5 min. Then, an aliquot of 10.0 L of 1.0 mg mL−1 MWCNT suspension in water was pipeted on the electrode sur-face and dried under an infrared lamp. To further modification of GCE, it was coated by casting of 10.0 L of the synthesized bifunc-tional His-rGO suspension (1.0 mg mL−1) on the electrode surface. To immobilization of redox indicator on the electrode surface, the GCE/MWCNT/bifunctional His-rGO was immersed in a 2.5% GA in PBS for 1 h [21]. After rinsing, 5.0 L of thionine solution (1.0 mM, 0.1 M PBS of pH 7.4) was dropped on the modified electrode surface for 12 h. During this time, the primary amine (-NH2) in thionine was covalently attached to the primary amino group of bifunc-tional rGO via GA linker. Then, the modified electrode was washed with double distilled water to wash away un-immobilized electro-chemical probes. Subsequently, anti-PSA Ab (10.0 L, 1.0 M) was  immobilized on the MWCNT/bifunctional His-rGO/thionine mod-ified electrode surface through amide bond formation between activated carboxyl groups and amine groups of the antibody. For this purpose, 20 L of EDC (20 mM) and NHS (40 mM) solutions were coated on the GCE/MWCNT/bifunctional His-rGO/thionine for 2 h to activate the carboxyl-terminated surface of the rGO, followed by rinsing with PBS of pH 7.4 [14]. After immobilization of Ab, the biosensing interface modified electrode was dipped in a BSA solution (0.25%) for 30 min to decrease the non-specific binding, and dried with N2 again. Finally, the GCE/MWCNT/bifunctional His-rGO/thionine/Ab was rinsed thoroughly with water to wash away the loosely adsorbed antibodies and stored at 4 ◦ C when not in use.
    2.5. Immunosensing process of PSA
    The immunosensing was based on the typical process for signal-off assay. First, the modified electrode was incubated with 10.0 L of PSA standard solutions with different concentrations for 25 min at room temperature, followed by rinsing thoroughly with double distilled water and PBS to remove the unbound analytes. Then, the voltammetric responses of the immunosensor were recorded for the quantitative detection of PSA tumor marker. All the steps of the immunosensor fabrication and the voltammetric detection of PSA are schematically shown in Fig. 2.
    3. Results and discussion
    3.1. Electrochemical characterization of the PSA immunosensor
    Each step of electrode modification has been confirmed with cyclic voltammetry (CV) in 0.1 M PBS (pH 7.4) (Fig. 3A). The CV of MWCNT/His-rGO modified GCE did not show any significant redox peak in the potential window of −0.6–0.2 V. Thus, empha-sizing that the proposed modifier nanofilm is electro inactive in the used potential window. However, when the modified elec-trode was incubated with thionine solution (1 mM, 0.1 M PBS of pH 7.4) for 12 h, a pair of sharp and well-defined reversible redox peaks were observed at the approximate potential of −0.21 V, as a result of thionine reduction. As the figure suggests, the GCE/MWCNT/His-rGO presents no observable peak, but the GCE/MWCNT/His-rGO/thionine modified electrode displays a pair of well-defined redox peaks in the potential range. This can be attributed to the characteristic of the electron transfer process of the thionine/leucothionine redox couple on the modified elec-trode surface. The interaction of anti-PSA antibody with rGO makes the biosensing interface bigger, which blocks the electron trans-fer of thionine and decreases redox signals (Fig. 3B). When PSA molecules are captured on the interface by anti-specific Abs, the signals of thionine further decrease due to the thickening of the electron transfers.