Conjugating antibodies with quantum dots

Quantum (q) dots have a number of advantages over conventional organic fluorophores, and their application may prove fruitful to constructing input-output models for different neuron types. The dots can be as small as 5-8 nanometers, although their hydrophobic region has been reported to be at least 16 nm. Their small size allows researchers to combine them to individual protein molecules and thus visualize intracellular processes.

Pathak et al (2007) considered the actual binding potential of 605 nm q dots to the most common human antibody, immunoglobulin G, which has two light chains and two heavy chains linked by disulfide bonds--see a picture of the molecule here. It's important to validate that the quantum dots not only bind to the antibody but that they bind in such a way that the antibody can still bind to its typical ligand and have normal biological function. Since the light chain is the part of the antibody that binds to other proteins, it needs to be oriented outward, and moreover the antibody molecule itself should not be cleaved by the q dot binding.

Of their two techniques, direct conjugation and biotin-streptavidin based conjugation, the latter yielded significantly more q dots bound correctly per antibody molecule. In this latter technique, the researchers coated their q dot with the protein streptavidin and added biotin groups to the immunoglobins, likely at some of the antibody's primary amine groups (see biotinylation). The noncovalent interaction between streptavidin and biotin has one of the lowest known dissociation constants, ~ 10^-15 mol/L, and thus it leads to a strong interaction between the q dot and the antibody.

Ultimately, the highest ratio they were able to produce was 1.3 +/- 0.35 of antibodies bound per q dot with a 2:1 antibody to q dot molar ratio. Since not all of these bound antibodies will be functional, in part because some will have the light chain of the immoglobulin molecule blocked, that number represents an upper bound on functional antibodies. The researchers note that since there is currently no way to control the binding orientation of immoglobulin molecules, Brownian motion means there is no guaranteed way to ensure functionally bound antibodies. More on q dots and neuro to come--this will be an important tech in the years to come, no doubt.

Reference

Pathak S, et al. 2007 Characterization of the Functional Binding Properties of Antibody Conjugated Quantum Dots. doi:10.1021/nl062706i, pdf.