No abstract

Biotinylated, highly luminescent CdSe-ZnS quantum dot (QD) conjugates were prepared and used in immunofiltration assays. Water-soluble quantum dot surfaces having a homogeneous negative charge density at basic pH were initially coated with a two-domain recombinant maltose-binding protein appended with a positively charged leucine zipper. Biotin functionalization of these electrostatically stabilized QD-protein complexes was then carried out using amine-reactive NHS biotin. These protein-coated biotin-functionalized quantum dot conjugates were incorporated into flow immunofiltration/displacement assays employing Affi-gel agarose resin for antibody immobilization, analyte capture, and immune complex formation with a second biotinylated antibody. A key component of the assay was the use of tetranitromethane-modified NeutrAvidin, used to link the biotinylated QDs to the immune complexes and facilitate their release at basic pH for subsequent quantification. This assay methodology was used to detect as little as 10 ng/mL staphylococcal enterotoxin type-B.

A fluorescence-based multianalyte immunosensor has been developed for simultaneous analysis of multiple samples. While the standard 6 x 6 format of the array sensor has been used to analyze six samples for six different analytes, this same format has the potential to allow a single sample to be tested for 36 different agents. The method described herein demonstrates proof of principle that the number of analytes detectable using a single array can be increased simply by using complementary mixtures of capture and tracer antibodies. Mixtures were optimized to allow detection of closely related analytes without significant cross-reactivity. Following this facile modification of patterning and assay procedures, the following nine targets could be detected in a single 3 x 3 array: Staphylococcal enterotoxin B, ricin, cholera toxin, Bacillus anthracis Sterne, Bacillus globigii, Francisella tularensis LVS, Yersiniapestis F1 antigen, MS2 coliphage, and Salmonella typhimurium. This work maximizes the efficiency and utility of the described array technology, increasing only reagent usage and cost; production and fabrication costs are not affected.