Fluoroimmunoassays Using Antibody-Conjugated Quantum Dots

Abstract

Luminescent colloidal semiconductor nanocrystals (quantum dots) are robust inorganic fluoro phores that have the potential to circumvent some of the functional limitations encountered by organic dyes in sensing and biotechnological applications. Quantum dots exhibit size-dependent tunable, narrow fluorescence emission spectra that span the visible spectrum and have broad absorption spectra. This allows simultaneous excitation of several particle sizes at a single wavelength with emission at multiple wavelengths. Quantum dots also provide a high-resistance threshold to chemical degradation and photodegradation. We have developed a conjugation strategy for the attachment of antibodies to quantum dots based on electrostatic interactions between negatively charged dihydrolipoic acid (DHLA)-capped CdSe-ZnS core-shell quantum dots and positively charged proteins (natural or engineered) that serve to bridge the quantum dot and antibody. This chapter details the materials and methods for synthesis of the DHLA-capped CdSe-ZnS core-shell quantum dots, the construction and preparation of recombinant proteins, the conjugation of antibodies to quantum dots, and the use of antibody-coated quantum dots in a fluoroimmunoassay.

In vitro and in vivo documentation of quantum dots labeled Trypanosoma cruzi–Rhodnius prolixus interaction using confocal microscopy

“…This marker could be used to determine the percentage of positive fluorescent parasites or when conjugated to molecules, as antibody, to localize a specific antigen on the parasite surface. Many reports also demonstrate the use of QDs conjugated to antibodies, named bioinorganic conjugates, for use in fluoroimmuno assays (Goldman et al 2002(Goldman et al , 2005Sweeney et al 2008). Tokumasu et al (2004) showed a kind of parasite and vertebrate host cells interaction such the mechanism of innate protection of erythrocytes infected with Plasmodium falciparum using quantum dots technique.…”

Semiconductor Nanomaterials-Based Fluorescence Spectroscopic and Matrix-Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometric Approaches to Proteome Analysis

“…This method was based on the electrostatic noncovalent interactions between bioconjugates of CdSe/ZnS QDs and antibodies. Goldman’s group described a conjugation strategy for the attachment of antibodies to quantum dots based on the electrostatic interactions between the negatively charged dihydrolipoic acid (DHLA)-capped CdSe-ZnS core-shell QDs and positively charged proteins (natural or engineered) that serve to bridge the quantum dot and antibody [49]. The same group prepared bioinorganic conjugates luminescent semiconductor NCs (CdSe–ZnS core-shell QDs) with antibodies and used as probes in multiplexed fluoroimmunoassays [50].…”

Semiconductor Quantum Dots for Biomedicial Applications

“…In one common approach, the original hydrophobic coatings are replaced by water-soluble functional molecules (e.g., dithiothreitol [38–40], mercaptocarbonic acids [41–44], 2-aminoethanethiol [33,45], dihydrolipoic acid [34–36,46,47], oligomeric phosphines [37,48], peptides [49–57], and cross-linked dendrons [58–61]) through the ligand exchange reactions. Because the optical properties of the inorganic core are often very sensitive to the surface, the ligand exchange process may result in poorer performance, particularly in the case of quantum dots [62].…”