Optimizing Quantum Dot Probe Size for Single-Receptor Imaging
- 15 July 2020
- journal article
- research article
- Published by American Chemical Society (ACS) in ACS Nano
- Vol. 14 (7), 8343-8358
- https://doi.org/10.1021/acsnano.0c02390
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 (QD(9.2)), 13.3 nm (QD(13.3)), and 17.4 nm (QD(17)(.4)) respectively. The QDs were conjugated to streptavidin and applied as probes for biotinylated neurotransmitter receptors. QD(9.2) exhibited the highest labeling specificity for receptors in the narrow synaptic cleft (similar to 20-30 nm) in living neurons. However, for dense receptor labeling for molecular counting in live and fixed HeLa cells, QD(13.3) yielded the highest counts. Nonspecific binding rose sharply for hydrodynamic sizes larger than 13.3 nm, with QD(17.4) 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.Funding Information
- National Institute of Neurological Disorders and Stroke (R01NS097610, R01NS100019)
- Division of Graduate Education (0965918)
- National Institute of General Medical Sciences (R01GM127497, R01GM131272)
This publication has 84 references indexed in Scilit:
- Multi-Color Quantum Dot Tracking Using a High-Speed Hyperspectral Line-Scanning MicroscopePLOS ONE, 2013
- Quantum dot imaging platform for single-cell molecular profilingNature Communications, 2013
- Two-Photon 3D FIONA of Individual Quantum Dots in an Aqueous EnvironmentNano Letters, 2011
- Non-specific binding of antibodies in immunohistochemistry: fallacies and factsScientific Reports, 2011
- Bright and Compact Alloyed Quantum Dots with Broadly Tunable Near-Infrared Absorption and Fluorescence Spectra through Mercury Cation ExchangeJournal of the American Chemical Society, 2010
- Compact Biocompatible Quantum Dots via RAFT-Mediated Synthesis of Imidazole-Based Random Copolymer LigandJournal of the American Chemical Society, 2009
- Design considerations for tumour-targeted nanoparticlesNature Nanotechnology, 2009
- Renal clearance of quantum dotsNature Biotechnology, 2007
- Elucidating the Mechanism of Cellular Uptake and Removal of Protein-Coated Gold Nanoparticles of Different Sizes and ShapesNano Letters, 2007
- VMD: Visual molecular dynamicsJournal of Molecular Graphics, 1996