There are three issues, all of which are related to blinking, in quantum dot-based particle/molecule tracking. The recently reported "non-blinking quantum dots" address only one of the three issues. Here we report a new class of quantum dot-based composite nanoparticles that emit fluorescence with continuous intensity and alternating color, effectively addressing all three blinking-related issues, as well as providing greatly enhanced brightness compared with single quantum dots.
There is little doubt that quantum dots (semiconductor nanocrystals, or QDs) have revolutionized single molecule tracking [1, 2]. Molecules in question can be easily tagged with these nanometer-sized tiny particles, which serve as fluorescent tracers under a fluorescent microscope. QDs are much brighter than their competitors (organic fluorophores and fluorescent proteins). Another competitive edge of QDs is their photostability: their fluorescence doesn‘t fade over time [1, 2].
However, QD-based tracking is not without problems . First, QDs "blink". In other words, every now and then a QD disappears ("blinks off"), and quickly re-appears ("blinks on"). This means that a QD trajectory can't last indefinitely, since an off-state of blinking kills the trajectory instantly. The blinking dynamics of a QD is essentially random, although on average a QD blinks off every 0.5 seconds. Second, because QDs have large surface area, they are prone to aggregation. This is especially true in complex biological environments, in which a wide variety of molecules attack the QD surface constantly. Even a few defects on the QD surface can cause significant aggregation. Although many methods have been developed to reduce QD aggregation, this problem is difficult to address in environments as complex as biological systems. Here blinking is actually very helpful because it offers a "warning system" to particle aggregation: when serious aggregation occurs, the QD aggregates do not blink any more. Third, when a QD leaves the focal plane of the microscope objective, it disappears as well. Unfortunately this disappearance looks just like a QD blinking off. Knowing that a particle has moved out-of-focus would be helpful for understanding particle motion in three dimensions.
The above three problems are all related to blinking of QDs. After nearly a decade's research since the first successful QD-based single molecule tracking , scientists have finally solved the first problem. Several types of "non-blinking QDs" have been developed, successfully eliminating blinking [4-8]. The designs of these newer nanoparticles are based on the physical origin of QD blinking, i.e. additional charges on QD surface . Thus the following approaches have been used: applying a charge mediator/compensator, coating QDs with a very thick shell, and synthesizing QDs with a gradually changing potential energy function [4-8]. Although these efforts have been very effective in eliminating blinking, the other two blinking-related problems are completely untouched. For example, after blinking is eliminated in the "non-blinking QDs", the precious single (or near single) particle signature used as an "aggregation warning system" is also lost. Although there are several other possible ways to confirm single (or near single) particle status, such as transmission electron microscopy, atomic force microscopy and single photon counting, these methods generally do not meet the needs of actual single molecule tracking studies. If dramatic improvements can be made in the spatial resolution of single photon counting, one day this technique might be able to be used to confirm single (or near single) particle status, but even then it would still be an expensive option compared with simply using the blinking feature of single (or near single) QDs.
Here, we look at the three blinking-related problems as a whole for the first time, and describe a new class of QD-based composite nanoparticles (CNPs) to address these problems simultaneously. Unlike the "non-blinking QDs" developed previously in which the chemical structure of QDs is changed, in a CNP QDs are kept intact. Conceptually, each CNP is essentially a composite of a few QDs of different fluorescent colors (e.g. green and red in figure 2). Because blinking dynamics of QDs are random, a CNP depicted in figure 2 emits nearly continuous fluorescence, with the color alternating between green, red, and yellow (composite color of green and red). Thus, the first and second blinking-related problems are solved by a CNP because it not only is free of blinking-caused interruptions, but also offers a single (or near single) particle signature in the color-alternating feature.
Experimentally, a CNP is made by micelle-templated self assembly (fig. 3). After an oil phase, which is composed of QDs with a hydrophobic surface and amphiphilic polymers dissolved in a non-polar solvent, is added to water, hydrophobic interaction drives the system to form QDs-encapsulated micelles spontaneously. This process is facile and requires no external energy input since it is based on self-assembly. Particle sizes of CNPs range from 25 nm to 40 nm, which are only slightly larger than those of single water-soluble QDs (typically 15 nm).
The continuous fluorescence and alternating-color features of CNPs have been confirmed by fluorescent microscopy (fig. 4). Control samples examined include a large CNP aggregate, which exhibited constant fluorescent color, and single QDs, which showed blinking behavior (fig. 4). Furthermore, a CNP is several times brighter than a QD (fig. 4c and fig. 4d) since each CNP contains several QDs. This remarkable increase in nanoparticle brightness is expected to greatly enhance signal-to-noise ratio in single molecule tracking or detection.
CNPs offer a solution to the third blinking-related problem, too (fig. 5). When a QD blinks, the change in fluorescence cannot be distinguished from the particle moving out of focus (fig. 5a). In contrast, when a constitute QD in a CNP blinks, the CNP undergoes a color-changing event, which is easily distinguishable from CNP moving out of focus (fig. 5b).
Taken together, this new class of QD-based nanoparticles offer solutions to all three blinking-related problems in QD-based particle/molecule tracking simultaneously. Furthermore, a CNP is several times brighter than a QD, possesses relatively small particle size, and can be readily conjugated with various types of biomolecules by well-established bioconjugation methods. Applications of CNPs can include not only single molecule tracking in biological systems, but also some less obvious ones: 1) studying flow mechanics in microfluidic systems, using CNPs as particle tracers, 2) detecting molecules in various settings, taking advantage of the superior brightness of a CNP compared with a QD. With increasing emphasis on single molecule analysis and miniaturization in many fields, CNPs would potentially have far-reaching impact in science, technology and medicine.
The funding of this work is provided by the National Science Foundation CBET-0707969, CMMI-0900377, endowment of the William G. Lowrie family, the Center of Emergent Materials at the Ohio State University, and the Ohio State University.
 Dahan M. et al.: Science 302, 442-445 (2003)
 Reck-Peterson S. L. et al.: Cell 126, 335-348 (2006)
 Smith A. M. et al.: Nature Biotechnology 27, 732-733 (2009).
 Hohng S. et al.: Journal of the American Chemical Society 126, 1324-1325 (2004)
 He H. et al.: Angewandte Chemie-International Edition 45, 7588-7591 (2006)
 Mahler B. et al.: Nature Materials 7, 659-664 (2008)
 Che Y. et al.: Journal of the American Chemical Society 130, 5026-+ (2008)
 Wang X. Y. et al.: Nature 459, 686-689 (2009)
 Nirmal M. et al.: Nature 383, 802-804 (1996)
Related Articles :
Keywords: CNP Fluorescent Microscopy live cell imaging molecular imaging nanoparticles Nanotechnology QD QD-based Composite Nanoparticles Quantum Dot Tracking Semiconductor Nanocrystals Single MoleculeEmail requestCompany Homepage
The Ohio State University
140 West 19th Avenue
43210 Columbus, OH