b http www glpbio com simage
Since the first graphene-based biosensor was reprted by Shan et al. , a variety of nanoplatforms have been developed using the graphene nanocomposites for the sensing of biological com-pounds [13–15]. In this work, we reported an acid-base bifunctional rGO by introducing L-histidine, as an essential amino acid, at the surface of nanosheets. Interestingly, L-histidine functionalized rGO (His-rGO) promised covalently binding redox mediator, thionine, to the graphene surface to give detectable electrochemical sig-nals. Notably, the His-GO also exhibited significantly chemical reactivity to the amine monoclonal antibodies, mainly through amidation procedure. In fact, this valuable characteristic provided the covalent attachment of the bioreceptor and the electroac-tive probe on the rGO surface. Meanwhile, His-rGO possessed the carboxyl and amine groups on its basal planes. These rich func-tional groups render rGO as a nice nanoplatform in a signal-off immunosensing strategy through obstructing the electron transfer of thionine probes by antibodies. A further decrease of the sensor response was achieved by incubating the antibodies with the target biomolecules. Because the observed signal suppression is linked to the change of electron transfer rate that occurs upon binding, this protocol allows for detection of a specific target, even within the incredibly complex media existing within the biological system. Inspired by this mechanism, we developed a signal-off immunosen-sor that can provide both the sensitivity and specificity for PSA detection. In the proposed electrochemical immunosensor, multi-walled carbon nanotube (MWCNT)/His-rGO based signal amplified nanoplatform presented both excellent characteristics of CNTs and rGO nanosheets. It should be noted that the carbon nanotubes are promising materials for sensing applications due to several intrigu-ing properties. In particular, their large length-to-diameter aspect ratios provide the high surface-to-volume ratios. Moreover, CNTs have an outstanding ability to mediate electron-transfer kinetics for a wide range of electroactive species . In fact, due to the high surface area and electronic conductivity associated with the well-organized and featuring packed vertical oriented film, the CNTs have been explored in design sensitive assays. Among the various signal amplification strategies employed by nanomaterials, the use of CNTs remained as one of the most effective ones.
2.1. Reagents and chemicals
All chemicals were of analytical grade purity and used as received. Natural graphite powder, hydrochloric 14605-22-2 (HCl, 37%), nitric acid (HNO3, 65%), sulfuric acid (H2SO4, 98%), ethanol
(C2H5OH, ≥99.8%), potassium permanganate (KMnO4, 99.9%) and hydrogen peroxide (H2O2, 30%) were purchased from Merck (Darmstadt, Duitsland, Germany). N,N -dicyclohexylcarbodiimide (DCC, 99%), MWCNT, glutaraldehyde solution (GA, 25% in water), bovine serum albumin (BSA, 98%), human serum albumin (HAS, ≥97%), immunoglobulin G (IgG, ≥95%), thio- ˜ nine acetate salt (90%), L-histidine (C6H9N3O2, ≥99%), N-hydroxysuccinimide (NHS, 98%), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC HCl, ≥98%), potassium hexacyanoferrate(III) (K3Fe(CN)6, ≥99.0%), potassium hexacyano-ferrate(II) trihydrate (K4Fe(CN)6 0.3H2O, ≥99.95%) and potassium chloride (KCl, ≥99.0%) were supplied by Sigma Aldrich (St. Louis, MO, USA). Human PSA and anti-PSA antibody were purchased from CanAg Company (Gothenburg, Sweden). Vascular endothelial growth factor (VEGF165) was obtained from InvitrogenTM (Carlsbad, CA, USA). The solutions were prepared with double-distilled water, with a specific resistance of >18 M cm, and stored at 4 ◦ C. The supporting electrolyte and pH of solution have a great influence on the immunosensor response because of alkaline or acid solutions may break the antigen–antibody linkage and damage antigen and antibody . Therefore, a 0.1 M phosphate buffer solution (PBS) prepared with Na2HPO4 and NaH2PO4 was selected as the support-ing electrolyte in the process of electrochemical measurements and the pH was adjusted at the neutral level (pH = 7.4) according to the previous literature [17–19].
Cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) were per-formed using a PGSTAT 302 N electrochemical workstation (Autolab, Kanaalweg, Utrecht, Netherlands). The working electrode was a modified glassy carbon electrode (GCE, i.d = 3.0 mm, Azar Electrode, Urmia, Iran), and auxiliary and reference electrodes were a Pt wire and Ag/AgCl electrode (KCl, 3 M), respectively. The topography of bifunctional GO nanosheets was investigated by scanning electron microscopy (SEM) by a KYKY instrument, Model EM3200 (Madell Technology Corporation, Ontario, CA, USA). Fourier transform infrared (FTIR) spectra were recorded on the range of 400–4000 cm−1 using a Brucker Vector 22 FT-IR Spectrom-eter (Silberstreifen, Rheinstetten, Germany). A 780 pH Meter was used for measuring pH at 25 ◦ C (Metrohm, Zofingen Switzerland). A sonoreactor UTR200 (Teltow, Hielscher, Germany) with a max-imum output power of 200 W, operating frequency of 24 kHz and ultrasonic intensity of 80 W cm-2 was employed for ultrasound-assisted exfoliation of graphite oxide. Double-distilled water was produced using the Milli-Q system (Millipore, Bedford, MA, USA).