Incorporation of ligand-receptor binding-site models and transistor-based sensors for resolving dissociation constants and number of binding sites.

Ligand-receptor binding site model is used to elucidate the binding affinity between ligands and receptors, with transistor-based sensors. AlGaN/GaN high electron mobility transistors (HEMTs) were immobilised with antibodies and human immunodeficiency virus type 1 reverse transcriptase enzymes to detect peptides and human immunodeficiency virus drugs, respectively. The signals generated by the sensors because of the binding of the ligands to the receptors were fitted into the binding-site models and analysed. The dissociation constants of the ligand-receptor pairs and the number of the binding sites on the receptors were revealed. The results are very consistent with the data reported by the other methods from the literatures. The incorporation of the HEMTs and the binding-site models is demonstrated to be useful for studying the mechanism of the biomolecular interaction and the application for quick and cost-effective drug developments.

Realization of an ultra-sensitive hydrogen peroxide sensor with conductance change of horseradish peroxidase-immobilized polyaniline and investigation of the sensing mechanism.

In this study, we fabricate an ultra-sensitive hydrogen peroxide sensor by using horseradish peroxidase (HRP)-immobilized conducting polymer, polyaniline (PANI). With the proposed detection mechanism, hydrogen peroxide first oxidizes HRP, which then oxidizes polyaniline, thus resulting in decreased conductivity of the polyaniline thin film. The reduced HRP can be further oxidized by hydrogen peroxide and the cycle of the oxidation/reduction would continue until all hydrogen peroxide are reacted, leading to the high sensitivity of the sensor due to the signal contributed from all hydrogen peroxide molecule. The detection limit of this sensor is only 0.7 nM. The detectable concentration of H2O2 is from 0.7 nM to 1 µM. Beyond 1 µM, the sensor gradually saturates and some H2O2 remains, indicating the inhibition of HRP activity at high concentration of H2O2. There is no response to hydrogen peroxide once the PANI is standalone without HRP immobilized, showing the enzymatic reaction is required in the process of hydrogen peroxide detection. The simple process for the sensor fabrication allows the sensor to be cost-effective and disposable. This electronic hydrogen peroxide sensor is promising in applications for low concentration hydrogen peroxide detections, such as the reactive oxygen species (ROS) in oxidative stress studies.

Investigation of C-terminal domain of SARS nucleocapsid protein-Duplex DNA interaction using transistors and binding-site models.

AlGaN/GaN high electron mobility transistors (HEMTs) were used to sense the binding between double stranded DNA (dsDNA) and the severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid protein (N protein). The sensing signals were the drain current change of the HEMTs induced by the protein-dsDNA binding. Binding-site models using surface coverage ratios were utilized to analyze the signals from the HEMT-based sensors to extract the dissociation constants and predict the number of binding sites. Two dissociation constants, K D1 = 0.0955 nM, K D2 = 51.23 nM, were obtained by fitting the experimental results into the two-binding-site model. The result shows that this technique is more competitive than isotope-labeling electrophoretic mobility shift assay (EMSA). We demonstrated that AlGaN/GaN HEMTs were highly potential in constructing a semiconductor-based-sensor binding assay to extract the dissociation constants of nucleotide-protein interaction.