Small silver clusters that form with short oligonucleotides are distinguished by their strong fluorescence. Previous work showed that red and blue/green emitting species form with the cytosine oligonucleotide dC12. To understand how the bases and base sequence influence cluster formation, the blue/green emitting clusters that form with the thymine-containing oligonucleotides dT12, dT4C4T4, and dC4T4C4 are discussed. With dT12 and dT4C4T4, variations in the solution pH establish that the clusters associate with the N3 of thymine. The small clusters are bound to the larger DNA template, as demonstrated by fluorescence anisotropy, circular dichroism, and fluorescence correlation spectroscopy (FCS) studies. For dT4C4T4, FCS studies showed that approximately 50% of the strands are labeled with the fluorescent clusters. Absorption spectra and the gas dependence of the fluorescence show that nonfluorescent clusters also form following the reduction of the silver cation – oligonucleotide conjugates. Fluorescent cluster formation is favored by oxygen, thus indicating that the DNA-bound clusters are partially oxidized. To elaborate the sequence dependence of cluster formation, dC4T4C4 was studied. Cluster formation depends on the oligonucleotide concentration, and higher concentrations favor a red emitting species. A blue/green emissive species dominates at lower concentrations of dC4T4C4, and it has spectroscopic, physical, and chemical properties that are similar to those of the clusters that form with dT12 and dT4C4T4. These results suggest that cytosine- and thymine-containing oligonucleotides stabilize a preferred emissive silver cluster.

Photostability, inherent fluorescence brightness, and optical modulation of fluorescence are key attributes distinguishing silver nanoclusters as fluorophores. DNA plays a central role both by protecting the clusters in aqueous environments and by directing their formation. Herein, we characterize a new near infrared-emitting cluster with excitation and emission maxima at 750 and 810 nm, respectively that is stabilized within C 3 AC 3 AC 3 TC 3 A. Following chromatographic resolution of the near infrared species, a stoichiometry of 10 Ag/oligonucleotide was determined. Combined with excellent photostability, the cluster's 30% fluorescence quantum yield and 180,000 M −1 cm −1 extinction coefficient give it a fluorescence brightness that significantly improves on that of the organic dye Cy7. Fluorescence correlation analysis shows an optically accessible dark state that can be directly depopulated with longer wavelength co-illumination. The coupled increase in total fluorescence demonstrates that enhanced sensitivity can be realized through Synchronously Amplified Fluorescence Image Recovery (SAFIRe), which further differentiates this new fluorophore. Keywordsnear infrared fluorescence; few-atom silver clusters; DNA templates; Ag nanodot; optical modulation Improved fluorescence sensitivity, largely through background reduction, continues to motivate the development of fluorescence contrast agents in the near infrared spectral region. 1 From 700-1000 nm, not only is scattering diminished relative to shorter wavelengths, but light absorption by hemoglobin, lipids, and water is also minimized. [2][3] Furthermore, endogenous chromophores typically have electronic transitions in the ultraviolet and visible spectral regions, so background autofluorescence is also drastically reduced using near infrared excitation. 1 These spectroscopic features in conjunction with cost-effective instrumentation suggest the great promise of near-infrared based molecular diagnostics. 4 However, the true potential of near infrared contrast agents is restricted by fluorophores with low sustained emission rates at low excitation (brightness), small numbers of emitted photons (photostability), and/or limited compatibility with biological environments. 5 jeff.petty@furman.edu and dickson@chemistry.gatech.edu. Supporting Information Available: Detailed experimental procedures and supplemental figures. This material is available free of charge via the Internet at http://pubs.acs.org. Due to their small size (~1 kDa), organic, transition metal, and lanthanide fluorophores both minimize perturbation of biomolecular interactions and enable high labeling densities to increase detection sensitivity. 6 Also contributing to their prevalence is amenability to synthetic modifications, thereby permitting covalent attachment to specific biomolecules, enhanced aqueous solubility, and modified spectral properties. 5,7-9 Genetically expressed fluorescent proteins are also attractive fluorophores that enable direct and specific correlations of fluores...

When compared with silver nanoparticles, silver clusters comprised of ∼101 atoms are distinguished by their strong fluorescence, and DNA directs and stabilizes particular types of clusters via base-specific interactions. Two main observations considered in this paper are the pH dependence of the fluorescence and the folded conformation of the oligonucleotide−cluster conjugates. Two i-motif forming oligonucleotides (dTA2C4)4 and (dC4A2)3C4 coordinate red and green emissive species, and these fluorescent species are favored in slightly acidic and basic solutions, respectively. The red emission is highest at pH 6, at which the i-motif forms of the oligonucleotides are also stable. When assessed by size exclusion chromatography, the oligonucleotide and cluster conjugate have similar global structures, which indicate that the DNA strands are similarly organized at this pH. The green emission is highest at pH 8−9. In these basic solutions, the oligonucleotide alone is unfolded, yet the green and red cluster−oligonucleotide conjugates have similar shapes. The pH-dependent fluorescence and the compact shapes of the cluster−oligonucleotide conjugates suggest that protons dominate DNA folding for the red emissive species, while the green emissive clusters themselves determine the shape of their DNA matrix. These studies provide the basis for understanding how specific base arrangements and environmental factors influence the formation of this new class of fluorescent nanomaterials.