Nanoparticles as fluorescence labels: is size all that matters?

Abstract

Fluorescent labels are often used in bioassays as a means to detect and characterize ligand-receptor binding. This is due in part to the inherently high sensitivity of fluorescence-based technology and the relative accessibility of the technique. There is often little concern raised as to whether or not the fluorescent label itself affects the ligand-receptor binding dynamics and equilibrium. This may be particularly important when considering nanoparticle labels. In this study, we examine the affects of nanoparticle (quantum dots and polymer nanospheres) fluorescent labels on the streptavidin-biotin binding system. Since the nanoparticle labels are larger than the species they tag, one could anticipate significant perturbation of the binding equilibrium. We demonstrate, using fluorescence cross-correlation spectroscopy, that although the binding equilibria do change, the relative changes are largely predictable. We suggest that the nanoparticles' mesoscopic size and surface tension effects can be used to explain changes in streptavidin-biotin binding.

FIGURE 1

Schematic diagram of relative sizes. (A) Biotin. (B) Streptavidin. (C) 525 streptavidin-QD (hydrodynamic radius, 4 nm). (D) 605 biotinylated-QD (hydrodynamic radius, 10 nm). (E) Green streptavidin-fluosphere (hydrodynamic radius, 20 nm). (F) Green biotinylated fluosphere (hydrodynamic radius, 100 nm).

FIGURE 2

(A) Cross-correlation decays for the titration of 1 nM green emitting streptavidin-coated fluospheres with increasing volumes of 605 nm emission biotinylated QDs (from a 10 nM stock QD solution). (B) Representative data set and fit using Eq. 4.

FIGURE 3

Hill plot (Eq. 8 represents fitted line) of a titration of 1 nM green emitting streptavidin-coated fluospheres with increasing volumes of 605 nm emission biotinylated QDs (C_ligand). This plot gives a binding ratio of 1:1 and K_d of 2.3 × 10⁻⁹ M.

FIGURE 4

Unbinding kinetics plot for a 1 nM QD_B-FS_S (1:1) solution into which 100 μM free biotin has been added. The time zero normalized fractional occupancy is plotted such that k_off can be determined directly from a fit using Eq. 12 (solid line).

FIGURE 5

Plots of the streptavidin-biotin dissociation constant (K_d, crossed circles), off-rate constant (k_off, triangles), and normalized on-rate constants (k_on, crossed squares, normalized to remove dependence of the number of reactive sites per particle surface) versus sum of the radii of the interacting pair. Data are grouped according to the biotinylated species with a red QD (red oval around data), green 40-nm-diameter fluosphere (green oval around data) and green 200-nm-diameter fluosphere (black oval around data). Within the plots the streptavidin binding partners are given by a green 40 nm fluosphere or red or green QDs. The smallest binding partner is streptavidin labeled with AF dyes. The details of the data are found in Tables 1 and 2.

FIGURE 6

Comparison of the equilibrium unbinding constants (K_d) for (A) QD_S-QD_B (black), (B) QD_S-FS_B 40 nm (blue), and QD_S-FS_B 200 nm (red) at various ionic strengths.

FIGURE 7

Comparison of dissociation rate constants (k_off) for QD_S-QD_B (black), QD_S-FS_B 40 nm (blue), and QD_S-FS_B 200 nm (red) for different ionic strengths. Inset is an expanded version of the lowest plot.

FIGURE 8

Comparison of calculated association rate constants (k_on) for QD_S-QD_B (black), QD_S-FS_B 40 nm (blue), and QD_S-FS_B 200 nm (red) at various ionic strengths.