Highly sensitive fluorescence detection of avidin/streptavidin with an optical interference mirror slide.

This paper presents highly sensitive fluorescence detections of avidin and streptavidin using an optical interference mirror (OIM) slide consisting of a plane mirror covered with an optical interference layer. Compared with a common glass slide, the OIM slide can enhance the fluorescence from a dye by more than 100-fold. We fabricated an OIM slide by depositing an optical interference layer of Al(2)O(3) on an Ag mirror. To enhance the fluorescence maximally, the optimal thickness of the Al(2)O(3) layer was estimated from optical interference theory. For detections of protein, avidin/streptavidin labeled with fluorescein, Cy3, and Cy5 were detected with biotin immobilized on an OIM slide with the optimal Al(2)O(3) thickness. We achieved a sensitivity improvement of more than 50-fold, comparing with a glass slide. Such a high degree of improvement would be a significant contribution to further progress in biomedical research and medical diagnostics.

High-performance biosensing using arrays of plasmonic nanotubes.

We show that aligned gold nanotube arrays capable of supporting plasmonic resonances can be used as high performance refractive index sensors in biomolecular binding reactions. A methodology to examine the sensing ability of the inside and outside walls of the nanotube structures is presented. The sensitivity of the plasmonic nanotubes is found to increase as the nanotube walls are exposed, and the sensing characteristic of the inside and outside walls is shown to be different. Finite element simulations showed good qualitative agreement with the observed behavior. Free standing gold nanotubes displayed bulk sensitivities in the region of 250 nm per refractive index unit and a signal-to-noise ratio better than 1000 upon protein binding which is highly competitive with state-of-the-art label-free sensors.

A fluorescence polarization assay to quantify biotin and biotin-binding proteins in whole plant extracts using Alexa-Fluor 594 biocytin.

A high-throughput fluorescence polarization assay has been developed for the detection of biotin and biotin-binding proteins in whole leaf extracts. Various groups are investigating the insecticidal properties of avidin and other biotin-binding proteins expressed in leaves of transgenic plants. The methods commonly used to quantify biotin and avidin in leaf extracts are enzyme-linked immunosorbent assay (ELISA) and Western blotting. Here we describe a homogeneous fluorescence polarization (FP) method that quantifies transgenic avidin in whole leaf extract by the simple addition of the fluorescent avidin ligand Alexa-Fluor 594 biocytin (AFB). The FP assay exploits the fact that AFB excites and emits in regions of the spectrum that are relatively free of background fluorescence in leaf extract. Transgenic leaf avidin can be quantified within 1-2 h by the FP method, in comparison with 1-2 days for ELISA and Western blotting. The FP method can also measure the amount of biotin in control leaves, not expressing avidin. Functional avidin levels of 1.54 microM (26.1 microg/g leaf tissue) were detected in tobacco leaves expressing vacuole-targeted avidin. Control leaves had biotin levels of around 0.74 microM (approximately 0.18 microg/g leaf tissue). Reagent costs are minimal: typically AFB is used at concentrations of 1-10 nM, avidin is used at 1-100 nM, and sample volumes are 20 microL in 384-well microplates.

Development of drug targeting based on recombinant expression of the chicken avidin gene.

The chemistry required for covalent biotinylation of drugs, radiopharmaceuticals and other ligands is highly developed, and a large number of biotinylated reagents can be readily synthesized. In order to investigate whether expression of avidin cDNA in mammalian cells might be useful as part of a drug targeting strategy, we transiently expressed the avidin gene in two human tumor cell lines (the cervical carcinoma cell line, HeLa, and the liver derived line, Hep G2). Avidin protein as detected by either immunohistochemistry or binding of streptavidin-biotin complexes was present and functional following transient expression. This result indicated that the mechanisms underlying avidin oligomerization which are necessary for proper protein folding are present within mammalian carcinoma cell lines. Next, we generated a producer cell line (derived from psi2) capable of releasing a recombinant retrovirus encoding chicken avidin, and a tumorigenic murine breast cancer cell line (16/C) with stable avidin expression. We show that these cell lines are suitable for conferring functional expression of avidin in vitro. These experiments establish a means by which avidin gene expression can be explored as a mechanism for targeted gene delivery of biotin-derivitized drugs in vitro, and have important implications for utilization of this strategy in vivo.