Effect of Poly-l-Lysine Polycation on the Glucose Oxidase/Ferricyanide Composite-Based Second-Generation Blood Glucose Sensors.

Second-generation glucose biosensors are presently the mainstream commercial solution for blood glucose measurement of diabetic patients. Screen-printed carbon electrodes (SPCEs) are the most-used substrate for glucose testing strips. This study adopted hydrophilic and positively charged α-poly-l-lysine (αPLL) as the entrapment matrix for the immobilization of negatively charged glucose oxidase (GOx) and ferricyanide (FIC) on SPCEs to construct a disposable second-generation glucose biosensor. The αPLL modification is shown to facilitate the redox kinetics of FIC and ferrocyanide on the SPCEs. The SPCEs coated with 0.5 mM GOx, 99.5 mM FIC, and 5 mM αPLL had better sensitivity for glucose detection due to the appreciable effect of protonated αPLL on the promotion of electron transfer between GOx and FIC. Moreover, the SPCEs coated with 0.5 mM GOx, 99.5 mM FIC, and 5 mM αPLL were packaged as blood glucose testing strips for the measurement of glucose-containing human serum samples. The glucose testing strips had good linearity from 2.8 mM to 27.5 mM and a detection limit of 2.3 mM. Moreover, the 5 mM αPLL-based glucose testing strips had good long-term stability to maintain GOx activity in aging tests at 50 °C.

Monitoring extracellular K+ flux with a valinomycin-coated silicon nanowire field-effect transistor.

A silicon nanowire field-effect transistor (SiNW-FET) coated with a polyvinyl chloride (PVC) membrane containing valinomycin (VAL) was employed as a biosensor (referred to as VAL-PVC/SiNW-FET) to detect the K(+)-efflux from live chromaffin cells. The detection sensitivity of K(+) with the VAL-PVC/SiNW-FET covers a broad range of concentrations from 10(-6) to 10(-2) M. The apparent association constants between VAL and Li(+), Na(+), K(+), and Cs(+) in Tris buffer solution were determined to be 67±42, 120±23, 5974±115, and 4121±140 M(-1), respectively. By culturing chromaffin cells on the VAL-PVC/SiNW-FET, the conductance was significantly increased by nicotine stimulation in a bath buffer without Na(+). The K(+) concentration at the cell surface was determined to be ~20 µM under the stimulation of 5 mM nicotine. These results demonstrate that the VAL-PVC/SiNW-FET is sensitive and selective to detect the released K(+) from cells and is suitable for applications in cellular recording investigations.

Novel steroid-sensing model and characterization of protein interactions based on fluorescence anisotropy decay.

Intramolecular binding of a ligand with an alkyl link, (-CH(2))(3), covalently bound to a residue near the active site of the protein forms a novel steroid-sensing model. A genetically engineered big up tri, open(5)-3-ketosteroid isomerase (KSI) was designed to conjugate uniquely with this ligand at its Cys-86 through the formation of a disulfide bond. The steady-state protein-ligand binding, mediated by hydrophobic interactions, was confirmed with fluorescence spectra, and the fluorophore-labeled peptide sequence was identified with tandem mass spectra. A comparison of steady-state fluorescence spectra of various fluorophore-labeled KSI mutants reveals that the emission characteristics vary with environmental factors. An evaluation of the decay of the fluorescence anisotropy of the fluorophore indicates the existence of an intramolecular protein-ligand binding interaction. The measurement of time-resolved fluorescence anisotropy of various protein-ligand complexes yielded values of anisotropy decay representing the degrees of freedom of the fluorophore related to its location, inside or outside the steroid-binding domain. When 19-norandrostenedione (19-NA) was added to this protein-ligand system, competitive binding between the ligand and the steroid was observed; this finding confirms the feasibility of the design of steroid detection with engineered KSI. On integration of this protein-ligand system with a silicon-based nanodevice (a p-type field-effect transistor with an ultrathin body), a noncharged steroid, 19-NA, became detectable at a micromolar level ( Biosens. Bioelectron. 2008 , 23 , 1883 ).