Amperometric hydrogen peroxide biosensor based on covalently immobilizing thionine as a mediator

A novel amperometric hydrogen peroxide biosensor based on the immobilization of hemoglobin on the 2,6-pyridinedicarboxylic acid (PDC) polymer, thionine and nano-Au was successfully fabricated. In this strategy, PDC polymer acted as the matrices to covalently immobilize the thionine, and then hemoglobin was successfully adsorbed on the nano-Au which was electro-deposited on to thionine modified electrode surface. The preparation process of modified electrode was characterized with electrochemical impedance spectroscopy and atomic force microscope. The analytical performance of proposed biosensor toward H₂O₂ was investigated by cyclic voltammetry and chronoamperometry. The resulted biosensor exhibited fast amperometric response (within 6 s) to H₂O₂, and linear range was from 9.1 μM to 5.0 mM with the detection limit of 2.6 μM (S/N = 3). The apparent Michaelis–Menten constant (K _M^app) was evaluated to be 3.2 mM. Furthermore, the resulted biosensor showed good stability and reproducibility.     

Study on an amperometric immunosensor based on Nafion-cysteine composite membrane for detection of carcinoembryonic antigen

In this work, protonated l-cysteine was entrapped in Nafion (Nf) membrane by cation exchange function, forming Nf-Cys (cysteine) composite membrane, which was more stable, compact, biocompatible, and favorable for mass and electron transfer compared with Nf film solely. Then gold (Au) nanoparticles were adsorbed onto the electrode surface by thiol groups on the composite membrane. After that, nano-Au monolayer was formed, onto which carcinoembryonic antibody was loaded to prepare carcinoembryonic antigen (CEA) immunosensor. The results indicated that the immunosensor had good current response for CEA using potassium ferricyanide as the redox probe. A linear concentration range of 0.01 to 100 ng/ml with a detection limit of 3.3 pg/ml (signal/noise = 3) was observed. Moreover, the morphology of the modified Au substrates was investigated with atomic force microscopy, and the electrochemical properties and performance of modified electrodes were investigated by cyclic voltammograms and electrochemical impendence spectroscopy. The results exhibited that the immunosensor has advantages of simple preparation, high sensitivity, good stability, and long life expectancy. Thus, the method can be used for CEA analysis.     

A sensitive amperometric immunosensor for carcinoembryonic antigen detection with porous nanogold film and nano-Au/chitosan composite as immobilization matrix

A sensitive amperometric immunosensor for carcinoembryonic antigen (CEA) was prepared. Firstly, a porous nano-structure gold (NG) film was formed on glassy carbon electrode (GCE) by electrochemical reduction of HAuCl₄ solution, then nano-Au/Chit composite was immobilized onto the electrode because of its excellent membrane-forming ability, and finally the anti-CEA was adsorbed onto the surface of the bilayer gold nanoparticles to construct an anti-CEA/nano-Au/Chit/NG/GCE immunosensor. The characteristics of the modified electrode at different stages of modification were studied by cyclic voltammetry (CV). The gold colloid, chitosan and nano-Au/Chit were characterized by transmission electron microscopy and UV–vis spectroscopy. In addition, the performances of the immunosensor were studied in detail. The resulting immunosensor offers a high-sensitivity (1310 nA/ng/ml) for the detection of CEA and has good correlation for detection of CEA in the range of 0.2 to 120.0 ng/ml with a detection limit of 0.06 ng/ml estimated at a signal-to-noise ratio of 3. The proposed method can detect the CEA through one-step immunoassay and would be valuable for clinical immunoassay.     

DNA point mutation detection based on DNA ligase reaction and nano-Au amplification: a piezoelectric approach

A novel piezoelectric method for DNA point mutation detection based on DNA ligase reaction and nano-Au-amplified DNA probes is proposed. A capture probe was designed with the potential point mutation site located at the 3' end and a thiol group at the 5' end to be immobilized on the gold electrode surface of quartz crystal microbalance (QCM). Successive hybridization with the target DNA and detection probe of nano-Au-labeled DNA forms a double-strand DNA (dsDNA). After the DNA ligase reaction and denaturing at an elevated temperature, the QCM frequency would revert to the original value for the target with single-base mismatch, whereas a reduced frequency response would be obtained for the case of the perfect match target. In this way, the purpose of point mutation discrimination could be achieved. The current approach is demonstrated with the identification of a single-base mutation in artificial codon CD17 of the beta-thalassemia gene, and the wild type and mutant type were discriminated successfully. The scanning electron microscope (SEM) image showing that plenty of gold nanoparticles remained on the electrode surface demonstrated that the nano-Au label served as an efficient signal amplification agent in QCM assay. A detection limit of 2.6 x 10(-9)mol/L of oligonucleotides was achieved. Owing to its ease of operation and low detection limit, it is expected that the proposed procedure may hold great promise in both research-based and clinical genomic assays.