Pin-Zhu Qin

Time-resolved fluorescence aptamer-based sandwich assay for thrombin detection

In the present study, the authors report a novel sensitive method for the detection of thrombin using time-resolved fluorescence sensing platform based on two different thrombin aptamers. The thrombin 15-mer aptamer as a capture probe was covalently attached to the surface of glass slide, and the thrombin 29-mer aptamer was fluorescently labeled as a detection probe. A bifunctional europium complex was used as the fluorescent label. The introduction of thrombin triggers the two different thrombin aptamers and thrombin to form a sandwich structure. The fluorescence intensity is proportional to the thrombin concentration. The present sensing system could provide both a wide linear dynamic range and a low detection limit. The proposed sensing system also presented satisfactory specificity and selectivity. Results showed that thrombin was retained at the aptamer-modified glass surface while nonspecific proteins were removed by rinsing with buffer solution. This approach successfully showed the suitability of aptamers as low molecular weight receptors on glass slides for sensitive and specific protein detection.

Time-resolved fluorescence based DNA detection using novel europium ternary complex doped silica nanoparticles

A two-probe tandem DNA hybridization assay including capture DNA(1), probe DNA(2), and target DNA(3) was prepared. The long-lived luminescent europium complex doped nanoparticles (NPs) were used as the biomarker. The complex included in the particle was Eu(TTA)(3)(5-NH(2)-phen)-IgG (ETN-IgG), the europium complex Eu(TTA)(3)(5-NH(2)-phen) linking an IgG molecule. Silica NPs containing ETN-IgG were prepared by the reverse microemulsion method, and were easy to label oligonucleotide for time-resolved fluorescence assays. The luminophores were well-protected from the environmental interference when they were doped inside the silica network. The sequences of Staphylococcus aureus and Escherichia coli genes were designed using software Primer Premier 5.0. Amino-modified capture DNA(1) was covalently immobilized on the common glass slides surface. The detection was done by monitoring the fluorescence intensity from the glass surface after the hybridization reaction with the NPs labeled probe DNA(2) and complementary target DNA(3). The sensing system presented short hybridization time, satisfactory stability, sensitivity, and selectivity. This approach was successfully employed for preliminary application in the detection of pure cultured E. coli, it might be an effective tool for pathogen DNA monitoring.

A novel bifunctional europium chelate applied in quantitative determination of human immunoglobin G using time-resolved fluoroimmunoassay

The authors demonstrate herein a novel time-resolved fluoroimmunoassay (TRFIA) protocol for quantification of human IgG with the new bifunctional chelate Eu(TTA)₃(5-NH₂-phen) (ETNP) labeling the goat anti-human IgG. The immunoassay was conducted by following the typical procedure for sandwich-type immunoreactions. Goat anti-human IgG was immobilized on aldehyde-modified glass slides. The human IgG analyte was first captured by the primary antibody and then sandwiched by a secondary antibody labeled with the chelate ETNP. The experimental procedure was simple to follow and gave desirable levels of sensitivity and low limits of detection. To the best of our knowledge, this is the first application of the new chelate, ETNP, in an immunoassay. In comparison to typical organic, fluorescent compounds and other lanthanide fluorescent chelates used in immunoassay, the detection sensitivity of our method using ETNP chelate in the solid phase was greatly improved and a concentration of human IgG about 5 μg/L could be detected under optimal conditions. The main result of this work shows that the new chelate ETNP can be applied as a powerful fluorescent labeling material for constructing ultrasensitive TRFIAs. The detection of human IgG, using ETNP as the chelate, is a model example of the effectiveness of this immunoassay. Many other types of antigen-antibody immunoassays should be possible using the protocol described herein.