Yaojuan Hu

Electrochemical Approach for Detection of Extracellular Oxygen Released from Erythrocytes Based on Graphene Film Integrated with Laccase and 2,2-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)

This work develops a novel electrochemical approach for detection of the extracellular oxygen released from human erythrocytes. The sensing is based on the bioelectrocatalytic system of graphene integrated with laccase (Lac) and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) toward the reduction of oxygen. ABTS and laccase are assembled on the surface of graphene, which is synthesized by a chemistry route, utilizing the pi-pi and electrostatic interactions of these components. Transmission electron microscopy (TEM), atomic force microscopy (AFM), and FT-IR spectroscopy demonstrate that graphene has been successfully synthesized, and ABTS and laccase have been effectively assembled on a graphene surface with the formation of Lac-ABTS-graphene hybrid. The voltammetric results indicate that ABTS can be used as a redox mediator when it is in immobilized form. The hybrid deposited on the glassy carbon (GC) electrode is demonstrated to be a good bioelectrocatalyst for the reduction of oxygen with inherent enzyme activity, accepted stability, high half-wave potential (ca.670 mV vs NHE), and unimpeded electrical communication to the copper redox sites of laccase. Therefore, this study has not only established a novel approach of detection of extracellular oxygen but also provided a general route for fabricating a graphene-based biosensing platform via assembling enzymes/proteins on a graphene surface.

Electrochemical approach for the specific detection of hepatitis C virus based on site-specific DNA cleavage of BamHI endonuclease

This work proposes a new electrochemical approach for specific detection of hepatitis C virus (HCV) based on the site-specific cleavage of BamHI endonuclease. The method was developed by immobilizing a synthetic probe DNA (a short thiol-capped single strand oligonucleotide) on the surface of a gold electrode via the –SH group at the 5′-terminus of the probe, and conjugating the electroactive label of ferroceneacetic acid (FcA) moiety to the 3′-terminus of the probe via formation of a covalent bond between the –NH2 and –COOH groups. The FcA-labeled probe was then hybridized with target cDNA (an oligonucleotide related to HCV) and cleaved by BamHI endonuclease (a site-specific endonuclease recognizing the duplex symmetrical sequence 5′–GGATCC–3′ and catalyzing double-stranded cleavage between the guanines). After digestion by BamHI, the DNA hybrid was cleaved at a specific site, and the FcA label was removed from the gold electrode surface leading to a decrease or disappearance of the electrochemical signal of the label. The extent of decrease was related to the concentration of target cDNA in solution, which forms the basis of quantitative detection of target cDNA. The detection is based on the variation of voltammetric signal (Δi) of FcA label before and after digestion with BamHI. It was demonstrated that the value of Δi has a linear relationship with the concentration of the HCV DNA (cDNA) ranging from 0.05 to 4.0 μM with a detection limit of (0.5 ± 0.2) nM at a signal/noise of 3. Moreover, the developed method has a high selectivity with ability to discriminate the complementary target DNA sequence from single-base mismatched DNA sequence, and can also be used for detection of HCV in real clinical samples. The major advantages of this enzymatic cleavage assay are its good specificity, ease of performance, and the ability to perform real-time monitoring. The proposed protocol can be taken as a general method of DNA detection and is expected to be applicable to other types of DNA analysis.