Strongly emissive individual DNA-encapsulated Ag nanoclusters as single-molecule fluorophores

Abstract

The water-soluble, near-IR-emitting DNA-encapsulated silver nanocluster presented herein exhibits extremely bright and photostable emission on the single-molecule and bulk levels. The photophysics have been elucidated by intensity-dependent correlation analysis and suggest a heavy atom effect of silver that rapidly depopulates an excited dark level before quenching by oxygen, thereby conferring great photostability, very high singlemolecule emission rates, and essentially no blinking on experimentally relevant time scales (0.1 to >1,000 ms). Strong antibunching is observed from these biocompatible species, which emit >10 9 photons before photobleaching. The significant dark-state quantum yield even enables bunching from the emissive state to be observed as a dip in the autocorrelation curve with only a single detector as the dark state precludes emission from the emissive level. These species represent significant improvements over existing dyes, and the nonpower law blinking kinetics suggest that these very small species may be alternatives to much larger and strongly intermittent semiconductor quantum dots.correlation ͉ photophysics ͉ silver nanoclusters ͉ single-molecule spectroscopy ͉ fluorescence intermittency W hile myriad dyes exist with varying photophysical properties (1, 2), organic dye-based single-molecule and even bulk in vivo imaging dynamics studies suffer from low probe brightness, poor photostability (3), and oxygen sensitivity (4). Advances in nanotechnology such as the use of quantum dots (5, 6) have ameliorated some of these issues but at the cost of toxicity (7), broad excitation (8, 9), power-law blinking (10-12), and large probe size (13,14). While quantum dots are readily excited with low-intensity sources, their fluorescence exhibits intermittency on all time scales (10-12), thereby causing problems when used for tracking or imaging studies. Arising from Auger processes (15), these photophysical dynamics are apparent at all excitation intensities and appear without characteristic times. While functionalization, large size (Ϸ10-20 nm in diameter), and cellular uptake are potential problems, the strong nonmolecular power-law fluorescence intermittency is a major drawback of these materials as single-molecule reporters (10-12). Recently, Ϸ35-nm-sized fluorescent nanodiamonds have also been reported as single-molecule emitters, but these also raise concerns about label size (16). Consequently, for both in vitro and in vivo single-molecule studies, fluorophores with high emission rates and excellent photostability must be identified that are completely devoid of blinking on all relevant time scales, while maintaining small overall sizes.By combining the virtues of chemistry and nanotechnology, we have developed few-atom, molecular-scale noble metal nanoclusters as a class of emitters that simultaneously exhibit bright, highly polarizable discrete transitions, good photostability, and small size, all within biocompatible scaffolds (17-20). Recent observations that DNA encapsulates Ag nanoclust...

Single‐Molecule Characterization of Near‐Infrared‐Emitting Silver Nanoclusters

“…A histogram of fl uorescence decay times, built using the average decay time from each C24-AgNC, is shown in Figure 1 the average decay times of the 285 C24-AgNCs is broad and centered around 2.7 ns, slightly shorter than the dominant decay component of 3.09 ns found in the bulk experiments, but still similar to other reports. [ 4,6 ] The broad Gaussian-like distribution of the decay times suggests that the sample does not consist of several distinct species with well-defi ned decay times, but points to an inherent inhomogeneity of the polymer matrix embedded C24-AgNCs. The small difference in the decay time between the bulk sample and the isolated single molecules could be due to changes in the conformation of the DNA in the polymer fi lm compared to in solution.…”

“…This is consistent with previous polycytosine samples that have shown good stability in PVA fi lm. [ 6 ] Not all AgNCs have such excellent chemical and photostability, especially in solution. [ 20,38 ] The fl uorescence decay times are calculated every 10 000 photons and plotted alongside the fl uorescence trajectory (Figure 1 a).…”

“…[ 21,22,34 ] as well as the spectra of AgNCs stabilized in other polycytosine sequences. [ 6 ] Time-correlated single photon counting (TC-SPC) experiments exciting at 635 nm and monitoring the emission at 692 nm were performed. The decay curve could be best fi tted with a four exponential decay model.…”

“…In this case, we focus on the nearinfrared emitters using 633 nm as the excitation wavelength. Although a number of single molecule studies have been performed on AgNC to investigate specifi c photophysical parameters, [ 5,6,8,10,14,25,[27][28][29][30][31][32][33] we simultaneously measure several photophysical properties for each individual cluster, including decay times, emission spectra, and antibunching. This allows us to compare and correlate these photophysical properties within a sample to better understand the photophysical behavior and heterogeneity of these complex as-synthesized AgNC samples.…”

All-thiol-stabilized Ag44 and Au12Ag32 nanoparticles with single-crystal structures

“…[1][2][3][4][5][6][7][8][9][10] Size, composition and surface structure are important factors determining the physical, chemical and biological properties of monolayerprotected noble metal nanoparticles. 4,11,12 To achieve chemical fine-tuning, understanding of their structures at the molecular level is crucial.…”