We present an electrochemically reduced graphene oxide (ERGO)-based electrochemical immunosensing platform for the ultrasensitive detection of an antigen by the sandwich enzyme-linked immunosorbent assay (ELISA) protocol. Graphene oxide (GO) sheets were initially deposited on the amine-terminated benzenediazonium-modified indiun tin oxide (ITO) surfaces through both electrostatic and π-π interactions between the modified surfaces and GO. This deposition was followed by the electrochemical reduction of graphene oxide (GO) for preparing ERGO-modified ITO surfaces. These surfaces were then coated with an N-acryloxysuccinimide-activated amphiphilic polymer, poly(BMA-r-PEGMA-r-NAS), through π-π stacking interactions between the benzene ring tethered to the polymer and ERGO. After covalent immobilization of a primary antibody on the polymer-modified surfaces, sandwich ELISA was carried out for the detection of an antigen by use of a horseradish peroxidase (HRP)-labeled secondary antibody. Under the optimized experimental conditions, the developed electrochemical immunosensor exhibited a linear response over a wide range of antigen concentrations with a very low limit of detection (ca. 100 fg/mL, which corresponds to ca. 700 aM). The high sensitivity of the electrochemical immunosensor may be attributed not only to the enhanced electrocatalytic activity owing to ERGO but also to the minimized background current owing to the reduced nonspecific binding of proteins.
Simple and sensitive competitive immunosensors for small molecules are difficult to obtain, especially in serum containing numerous interfering species (ISs) with different concentrations. Herein, we report a washing-free and sensitive (competitive) displacement immunosensor for cortisol in human serum, based on electron mediation of Os(bpy)Cl between an electrode and a redox label [oxygen-insensitive diaphorase (DI)] (i.e., electrochemical-enzymatic redox cycling). The anticortisol IgG-DI conjugate bound to a cortisol-immobilized electrode is displaced by competitive binding of cortisol in serum and diffuses away from the electrode during incubation; therefore, the concentration of the displaced conjugate near the electrode becomes very low, even without washing. Electrochemically interfering ascorbic acid is converted to a redox-inactive species by ascorbate oxidase during incubation. The remaining bound conjugate mainly contributes to electrochemical currents. Compared with ferrocene methanol, Fe(CN), and Ru(NH), the electrochemical and redox cycling behaviors of Os(bpy)Cl are influenced significantly less by ISs in serum. Comparative studies reveal that washing-free displacement assay shows better cortisol-induced signal change than three other assays. The surface concentration of cortisol immobilized on the electrode is optimized, because the electrochemical signal is highly dependent on the surface concentration. When the washing-free displacement immunosensor is applied for the detection of cortisol in artificial serum, cortisol is measured with a detection limit of ∼30 pM within 12 min. The cortisol concentrations measured in clinical serum samples agree well with those obtained using a commercial instrument. The new immunosensor is highly promising for the simple, sensitive, and rapid point-of-care detection of small molecules.
Biosensors for ultrasensitive point-of-care testing require dried reagents with long-term stability and a high signal-to-background ratio. Although ortho-substituted diaromatic dihydroxy and aminohydroxy compounds undergo fast redox reactions, they are not used as electrochemical signaling species because they are readily oxidized and polymerized by dissolved oxygen. In this report, stable, solid 1-amino-2-naphthyl phosphate (1A2N-P) and ammonia-borane (HN-BH) are respectively employed as a substrate for alkaline phosphatase (ALP) and a reductant for electrochemical-chemical (EC) redox cycling. ALP converts 1A2N-P to 1-amino-2-naphthol (1A2N), which is then employed in EC redox cycling using HN-BH. The oxidation and polymerization of 1A2N by dissolved oxygen is significantly prevented in the presence of HN-BH. The electrochemical measurement is performed without modification of indium-tin oxide (ITO) electrodes with electrocatalytic materials. For comparison, nine aromatic dihydroxy and aminohydroxy compounds, including 1A2N, are evaluated to achieve fast EC redox cycling, and four strong reductants, including HN-BH, are evaluated to achieve a low background level. The combination of 1A2N and HN-BH allows the achievement of a very high signal-to-background ratio. When the newly developed combination is applied to the detection of creatine kinase-MB (CK-MB), the detection limit for CK-MB is ∼80 fg/mL, indicating that the combination allows ultrasensitive detection. The concentrations of CK-MB in clinical serum samples, determined using the developed system, are in good agreement with the concentrations obtained using a commercial instrument. Thus, the use of stable, solid 1A2N-P and HN-BH along with bare ITO electrodes is highly promising for ultrasensitive and simple point-of-care testing.
We report in situ generation of aldehyde-functionalized benzenediazonium cation (ABD) and its use as a suitable linker molecule for fast and selective immobilization of biomolecules on indium-tin-oxide (ITO) electrode surfaces. We prepared ABD through a new reaction procedure, a simultaneous diazotation of the amine group and deprotection of the aldehyde group from an aniline derivative, 2-(4-aminophenyl)-1,3-dithiane, which was revealed on the ITO electrode surfaces through the electrodeposition of the reaction product and the characterization of the resulting surfaces with cyclic voltammetry, X-ray photoelectron spectroscopy, and protein immobilization. We also showed that successive electrodeposition of ABD and probe molecules on individually addressable microarray electrode surfaces can provide a useful platform for efficient detection of multianalyte. The usage of ABD has been demonstrated by the patterning of three different probe molecules on a single substrate and the simultaneous detection of two target molecules.
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