Optimizing Quantum Dot Probe Size for Single-Receptor Imaging.

ACS nano (2020-06-12)
Phuong Le, Rohit Vaidya, Lucas D Smith, Zhiyuan Han, Mohammad U Zahid, Jackson Winter, Suresh Sarkar, Hee Jung Chung, Pablo Perez-Pinera, Paul R Selvin, Andrew M Smith
ABSTRACT

Quantum dots (QDs) are nanocrystals with bright fluorescence and long-term photostability, attributes particularly beneficial for single-molecule imaging and molecular counting in the life sciences. The size of a QD nanocrystal determines its physicochemical and photophysical properties, both of which dictate the success of imaging applications. Larger nanocrystals typically have better optical properties, with higher brightness, red-shifted emission, reduced blinking, and greater stability. However, larger nanocrystals introduce molecular-labeling biases due to steric hindrance and nonspecific binding. Here, we systematically analyze the impact of nanocrystal size on receptor labeling in live and fixed cells. We designed three (core)shell QDs with red emission (600-700 nm) and crystalline sizes of 3.2, 5.5, and 8.3 nm. After coating with the same multidentate polymer, hydrodynamic sizes were 9.2 nm (QD9.2), 13.3 nm (QD13.3), and 17.4 nm (QD17.4), respectively. The QDs were conjugated to streptavidin and applied as probes for biotinylated neurotransmitter receptors. QD9.2 exhibited the highest labeling specificity for receptors in the narrow synaptic cleft (∼20-30 nm) in living neurons. However, for dense receptor labeling for molecular counting in live and fixed HeLa cells, QD13.3 yielded the highest counts. Nonspecific binding rose sharply for hydrodynamic sizes larger than 13.3 nm, with QD17.4 exhibiting particularly diminished specificity. Our comparisons further highlight needs to continue engineering the smallest QDs to increase single-molecule intensity, suppress blinking frequency, and inhibit nonspecific labeling in fixed and permeabilized cells. These results lay a foundation for designing QD probes with further reduced sizes to achieve unbiased labeling for quantitative and single-molecule imaging.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
1-Octadecene, technical grade, 90%
Sigma-Aldrich
Selenium dioxide, ≥99.9% trace metals basis
Sigma-Aldrich
Behenic acid, 99%
Sigma-Aldrich
1,2-Hexadecanediol, technical grade, 90%
Sigma-Aldrich
Cadmium acetate hydrate, ≥99.99% trace metals basis