Biomedical Engineering Reference
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sensitivity for reliable specifi c labeling of target proteins. In contrast, the number of
antibodies bound to quantum dots via the strepavidin-biotin system resulted in a
more biologically reasonable 0.60 + 0.14 IgG molecules per quantum dot for a 1:1
IgG:quantum dot molar ratio and, as would be expected, 1.3 + 0.35 IgG molecules
per quantum dot for a 2:1 ratio. This is equivalent to a functional volume of 0.943 mL
of antibody for a 2:1 molar ratio or 0.53 mL for 1:1 molar ratio starting from 4 mL
of biotinylated antibody conjugated to streptavidin quantum dots from a 0.5 mg/mL
stock concentration. We acknowledge that these numbers are an approximation,
since light chains near the quantum dot surface attached to a heavy chain bound to
the quantum dot as part of a partial fragment would be sterically unavailable for
antigen binding but could still dissociate following DTT reduction. However, this
may represent a small source of error because it may be sterically diffi cult for bound
heavy-light chain domains to bind to the quantum dot, therefore thermodynami-
cally favoring the functional partial fragment orientation (see Fig. 3b ). In any case,
this error would contribute to an overestimation of the number of functional anti-
bodies conjugated to a quantum dot, and therefore represent an upper bound on the
number of putative functional antibodies, further emphasizing the signifi cance of
the results we present here.
These results are signifi cantly less than the suggested estimates of about two to
ten antibodies conjugated per quantum dot. To the best of our knowledge, no conju-
gation reaction can control the binding orientation of IgG molecules. Consequently,
due to Brownian motion, the number of bound functional antibodies is almost cer-
tainly less than the number of total bound IgG. This is not considered by TEM
imaging approaches that measure the size (i.e., diameter) of antibody-quantum dot
complexes in order to estimate the number of bound antibodies. An important ques-
tion is: Why did covalent conjugations result in lower numbers of functional anti-
bodies compared to streptavidin-biotin conjugations? One possible explanation is
that DTT-reduced antibody fragments attaching to the surface of quantum dots leave
few opportunities for light chain fragments to be properly oriented outward and
available for protein binding, since of the three reduced fragment types only partial
fragments result in functional antibodies and even then the orientation of the partial
fragment binding to the quantum dot surface must be correct to allow the light chain
fragment to point outward in order to interact with its ligand. In biotin-streptavidin
conjugations, the antibody is never cleaved, leaving the whole molecule bound to
the quantum dot surface and structurally offering more opportunities for light chain
fragments to bind their targets. It is plausible that other covalent, conjugation chem-
istries result in higher yields of functional antibodies, comparable to those we report
for streptavidin-biotin conjugates or even higher, but it cannot simply be assumed
so since, as we show here, at least one well-established and commonly used cova-
lent conjugation reaction results in very low numbers of functional antibody on
quantum dots. We propose that functionalized quantum dot labeling of biological
preparations need to be preceded by the experimental determination of the number
of functionalized antibodies per quantum dot, especially given the variability in
conjugation methods between different labs. These considerations have a direct
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