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We describe an electrochemical immunosensor for the simultaneous determination of alpha-fetoprotein (AFP) and prostate specific antigen (PSA) via a modified glassy carbon electrode. Silica nanoparticles (200–300 nm i.d.) with good monodispersity and uniform shape were synthesized, and the following species were then consecutively immobilized on their surface: gold nanoparticles (AuNPs; 5–15 nm i.d.), secondary antibody (Ab2) and the redox-probes Azure A or ferrocenecarboxy acid (Fc). In parallel, two types of primary antibodies (Ab1) were co-immobilized on the surface of the dissolved reduced graphene oxide sheets (rGO) that were also decorated with AuNPs. In the presence of antigens (AFP or PSA), the Ab2/Si@AuNPs carrying Azure A and Fc are attached to the AuNP/rGO conjugate via a sandwich type immunoreaction. Differential pulse voltammetry (DPV) was employed to measure the resulting changes in the signal of Fc or Azure A. Two well-resolved oxidation peaks, one at −0.48 V (corresponding to Azure A) and other at + 0.12 V (corresponding to Fc; both vs. SCE) can be observed in the DPV curves. Under optimal conditions, AFP and PSA can be simultaneously determined in the range from 0.01 to 25 ng mL‾1 for AFP, and from 0.012 to 25 ng mL‾1 for PSA. The detection limits are 3.3 pg mL‾1 for AFP and 4.0 pg mL‾1 for PSA (at a signal-to-noise ratio of 3). The method was applied to (spiked) real sample analysis, and the recoveries are within 96.0 and 107.2 % for PSA, and within 100.9 and 105.8 % for AFP, indicating that this dual immunosensor matches the requirements of clinical analysis.

An electrochemical sensing platform was developed for the amperometric detection of β-nicotinamide adenine dinucleotide (NADH) through the integration of a multi-walled carbon nanotube (MWCNT) into electropolymerized phenothiazine dyes. The composite containing MWCNT and poly(phenothiazine) was prepared by electro-oxidative polymerization of phenothiazine derivatives, Azure B, Azure A and thionine, into an MWCNT/ poly(diallyldimethylammonium chloride) (PDDA) multilayer, which was constructed by electrostatic layer-by-layer assembly on a glassy carbon electrode. The three phenothiazine monomers used in this study exhibited similar electrochemical behaviors. Azure B was used extensively as a model monomer for the investigation. Electrochemical techniques and scanning electron microscopy were used to demonstrate that the porous composite was formed and that the carbon nanotube served as a nano-sized backbone for the loading of polymeric phenothiazine. The electrocatalytic current for NADH oxidation was enhanced as the number of layers increased, implying that the increase of NADH-accessible poly(phenothiazine) and the three-dimensional arrangement of the poly(phenothiazine)-coated MWCNT in the composite facilitated electron and NADH transfer. Under optimal conditions, the detection limit for NADH decreases to 7.0 × 10−8 M at a potential of 0.1 V (versus Ag/AgCl) using a {MWCNT/PDDA}8−poly(Azure B) composite modified glassy carbon electrode, with a response time of about 5 s. This work demonstrates that the electropolymerization of the phenothiazine monomer into a pre-formed multilayer containing MWCNT can be used for the controllable preparation of stable MWCNT/poly(phenothiazine) composites on electrode surfaces, which have the potential to provide a platform for electrochemical biosensors based on NAD+-dependent dehydrogenase enzymes.