A conductive multi‐catalyst system consisting of Fe3O4 magnetic nanoparticles (MNPs) and oxidative enzymes co‐entrapped in the pores of mesoporous carbon is developed as an efficient and robust electrochemical biosensing platform. The construction of the nanocomposite begins with the incorporation of MNPs by impregnating Fe(NO3)3 on a wall of mesoporous carbon followed by heat treatment under an Ar/H2 atmosphere, which results in the formation of magnetic mesoporous carbon (MMC). Glucose oxidase (GOx) is subsequently immobilized in the remaining pore spaces of the MMC by using glutaraldehyde crosslinking to prevent enzyme leaching from the matrix. H2O2 generated by the catalytic action of GOx in proportion to the amount of target glucose is subsequently reduced into H2O by the peroxidase mimetic activity of MNPs generating cathodic current, which can be detected through the conductive carbon matrix. To develop a robust and easy‐to‐use electrocatalytic biosensing platform, a carbon paste electrode is prepared by mechanically mixing the nanocomposite or MMCs and mineral oil. Using this strategy, H2O2 and several phenolic compounds are amperometrically determined employing MMCs as peroxidase mimetics, and target glucose was successfully detected over a wide range of 0.5 × 10−3 to 10 × 10−3 M, which covers the actual range of glucose concentration in human blood, with excellent storage stability of over two months at room temperature. Sensitivities of the biosensor (19 to 36 nA mM−1) are about 7–14 times higher than that of the biosensor using immobilized GOx in mesoporous carbon without MNPs under optimized condition. The biosensor is of considerable interest because of its potential for expansion to any oxidases, which will be beneficial for use in practical applications by replacing unstable organic peroxidase with immobilized MNPs in a conductive carbon matrix.

The operation of an electrochemical real-time PCR system, based on intercalative binding of methylene blue (MB) with dsDNA, has been demonstrated. PCR was performed on a fabricated electrode-patterned glass chip containing MB while recording the cathodic current peak by measuring the square wave voltammogram (SWV). The current peak signal was found to decrease with an increase in the PCR cycle number. This phenomenon was found to be mainly a consequence of the lower apparent diffusion rate of the MB-DNA complex (D(b) = 6.82 × 10(-6) cm(2) s(-1) with 612 bp dsDNA) as compared to that of free MB (D(f) = 5.06 × 10(-5) cm(2) s(-1)). Utilizing this signal changing mechanism, we successfully demonstrated the feasibility of an electrochemical real-time PCR system by accurately quantifying initial copy numbers of Chlamydia trachomatis DNA templates on a direct electrode chip. A standard calibration plot of the threshold cycle (C(t)) value versus the log of the input template quantity demonstrated reliable linearity and a good PCR efficiency (106%) that is comparable to that of a conventional TaqMan probe-based real time PCR. Finally, the system developed in this effort can be employed as a key technology for the achievement of point-of-care genetic diagnosis based on the electrochemical real-time PCR.

We report a novel method of electrochemical signaling from antigen-antibody interactions at immunoelectrodes with bioelectrocatalyzed enzymatic signal amplification. For the immunosensing surface construction, a poly(amidoamine) G4-dendrimer was employed not only as a building block for the electrode surface modification but also as a matrix for ligand functionalization. As a model biorecognition reaction, the dinitrophenyl (DNP) antigen-functionalized electrode was fabricated and an anti-DNP antibody was used. Glucose oxidase (GOX) was chosen to amplify electrochemical signal by enzymatic catalysis. The signal amplification strategy introduced in this study is based on the back-filling immobilization of biocatalytic enzyme to the immunosensor surface, circumventing the use of an enzyme-labeled antibody. The non-labeled native antibody was biospecifically bound to the immobilized ligand, and the activated enzyme (periodate-treated GOX) reacted and "back-filled" the remaining surface amine groups on the dendrimer layer by an imine formation reaction. From the bioelectrocatalyzed signal registration with the immobilized GOX, the surface density of biospecifically bound antibody could be estimated. The DNP functionalization reaction was optimized to facilitate the antibody recognition and signaling reactions, and approximately 6% displacement of surface amine to DNP was found to be an optimum. From quartz crystal microbalance measurement, immunosensing reaction timing and the surface inertness to the nonspecific biomolecular binding were tested. By changing the surface functionalization level of DNP in the calibration experiments, immunosensors exhibited different dynamic detection ranges and limits of detection, supporting the capability of parameters modulation for the immunosensors. For the anti-DNP antibody assay, the fabricated immunosensor having 65% functionalization ratio exhibited the linear detection range of 10(-4) to 0.1 g/L protein and a limit of detection around 2 x 10(-5) g/L.

The signal amplification technique of peptide nucleic acid (PNA)-based electrochemical DNA sensor was developed in a label-free and one-step method utilizing enzymatic catalysis. Electrochemical detection of DNA hybridization on a PNA-modified electrode is based on the change of surface charge caused by the hybridization of negatively charged DNA molecules. The negatively charged mediator, ferrocenedicarboxylic acid, cannot diffuse to the DNA hybridized electrode surface due to the charge repulsion with the hybridized DNA molecule while it can easily approach the neutral PNA-modified electrode surface without the hybridization. By employing glucose oxidase catalysis on this PNA-based electrochemical system, the oxidized mediator could be immediately reduced leading to greatly increased electrochemical signals. Using the enzymatic strategy, we successfully demonstrated its clinical utility by detecting one of the mutation sequences of the breast cancer susceptibility gene BRCA1 at a sample concentration lower than 10(-9) M. Furthermore, a single base-mismatched sample could be also discriminated from a perfectly matched sample.

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