We describe the preparation and characterization of bioinorganic conjugates in which luminescent semiconductor CdSe-ZnS core-shell nanocrystal quantum dots (QDs) were coupled to antibodies through the use of an avidin bridge adsorbed to the nanocrystal surface via electrostatic self-assembly. Avidin, a highly positively charged protein, was found to adsorb tightly to QDs modified with dihydrolipoic acid, which gives their surface a homogeneous negative charge. QD conjugation to biotinylated antibodies subsequently is readily achieved. Fluoroimmunoassays utilizing these antibody conjugated QDs were successful in the detection of protein toxins (staphylococcal enterotoxin B, cholera toxin). QD-antibody conjugates formed in such a facile manner permit their use as a common immuno reagent, and in the development of multianalyte detection.

We describe the preparation and characterization of bioinorganic conjugates made with highly luminescent semiconductor CdSe-ZnS core-shell quantum dots (QDs) and antibodies for use in fluoroimmunoassays. The conjugation strategy employs an engineered molecular adaptor protein, attached to the QDs via electrostatic/hydrophobic self-assembly, to link the inorganic fluorophore with antibodies. In this method, the number of antibodies conjugated to a single QD can be varied. In addition, we have developed a simple purification strategy based on mixed-composition conjugates of the molecular adaptor and a second two-domain protein that allows the use of affinity chromatography. QD-antibody conjugates were successfully used in fluoroimmunoassays for detection of both a protein toxin (staphylococcal enterotoxin B) and a small molecule (2,4,6-trinitrotoluene).

Biomolecules labeled with luminescent colloidal semiconductor quantum dots (QDs) have potential for use in numerous applications, including fluoro-immunoassays and biological imaging. QD labels exhibit size-tunable narrow-band luminescent emission and high resistance to photodegradation. They also exhibit efficient Fö rster energy transfer between neighboring QDs of different sizes and their emission is readily quenched by bound fluorescent dyes. In this paper, we describe preliminary results aimed at defining conditions for the design and preparation of nanoscale QD-bioconjugate sensors based on fluorescence quenching. We envision building sensor assemblies that employ quantum dots linked with dye-labeled biological receptors that utilize donor-acceptor energy transfer between QDs and receptors for conducting recognition-based assays. In particular, we report the effects of varying the concentration of energy acceptors bound to nanocrystal surfaces under both soluble and solid phase conditions on quenching phenomena.Introduction Strong quantum confinement of the charge carriers in semiconductor nanocrystals (quantum dots, QDs) increases their effective band gap energy significantly with decreasing particle size, resulting in size dependence of QD light absorption and luminescence spectra [1-3]. Colloidal QDs are approximately spherical nanocrystals with surfaces that can be derivatized with a variety of functional capping groups (surface ligands), allowing their dispersion in a range of solvents, including aqueous environments. Several groups have shown that surface ligands can be used to attach various biological molecules to form QD-bioconjugates [4][5][6][7]. As an alternative to conventional fluorophores, QDs offer a number of attractive features, including high resistance to photodegradation, relatively narrow and symmetric luminescence bands, and simultaneous excitation of several sizes of QDs (thus several colors) over a broad range of wavelengths. Advantages in using luminescent QDs to synthesize bioconjugates with potential use in biological imaging applications and in developing QD-based nanosensors have been elaborated [4][5][6].Our recent work has focused on the design and preparation of aqueous-compatible dithiol-capped CdSe-ZnS QDs having carboxylic acid terminal groups linked with bifunctional asymmetrically charged fusion proteins for QD-bioconjugate formation where conjugation is driven by electrostatic self-assembly [6]. In previous works, we studied the formation of QD-bioconjugates with two proteins, E. coli maltose binding