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.

Few-atom silver clusters harbored by DNA are promising fluorophores due to their high molecular brightness along with their long- and short-term photostability. Furthermore, their emission rate can be enhanced when co-illuminated with low-energy light that optically depopulates the fluorescence-limiting dark state. The photophysical basis for this effect is evaluated for two near infrared-emitting clusters. Clusters emitting at ~800 nm form with C3AC3AC3TC3A and C3AC3AC3GC3A and both exhibit a trap state with λmax ~ 840 nm and an absorption cross section of 5–6 × 10−16 cm2/molec that can be optically depopulated. Transient absorption spectra, complemented by fluorescence correlation spectroscopy studies, show that the dark state has an inherent lifetime of 3–4 μs and that absorption from this state is accompanied by photoinduced crossover back to the emissive manifold of states with an action cross section of ~2 × 10−18 cm2/molec. Relative to C3AC3AC3TC3A, C3AC3AC3GC3A produces a longer-lived trap state and permits more facile passage back to the emissive manifold. With the C3AC3AC3AC3G template, a spectrally distinct cluster forms having emission at ~900 nm and its trap state has a ~four-fold shorter lifetime. These studies of optically-gated fluorescence bolster the critical role of the nucleobases on both the formation and excited state dynamics of these highly emissive metallic clusters.

Conductive and plasmon-supporting noble metals exhibit an especially wide range of sizedependent properties, with discrete electronic levels, strong optical absorption, and efficient radiative relaxation dominating optical behavior at the ~10-atom cluster scale. In this Perspective, we describe the formation and stabilization of silver clusters using DNA templates and highlight the distinct spectroscopic and photophysical properties of the resulting hybrid fluorophores. Strong visible to near-IR emission from DNA-encapsulated silver clusters ranging in size from 5-11 atoms has been produced and characterized. Importantly, this strong Ag cluster fluorescence can be directly modulated and selectively recovered by optically controlling the dark state residence, even when faced with an overwhelming background. The strength and sequence sensitivity of the oligonucleotide-Ag interaction suggests strategies for fine tuning and stabilizing cluster-based emitters in a host of sensing and biolabeling applications that would benefit from brighter, more photostable, and quantifiable emitters in high background environments. KeywordsNoble metals; clusters; modulation; fluorescence; biolabel; imaging; photophysics Few-atom metal clusters exhibit distinctive, size-dependent behaviors along the transition from bulk to molecular scales. For example, as electron mean free path and Fermi screening length scales are approached, the excellent conductivity and optical reflectivity characterizing bulk metals morph into shape-dependent plasmon excitations, size-dependent redox potentials and chemical/biological reactivities, with discrete optical transitions emerging at even smaller sizes. [1][2][3][4] Resulting from their relative inertness and highly polarizable electronic transitions, molecular-scale noble metals have emerged as a promising class of fluorophores for materials and biological imaging. Characterized by excellent brightness and photostability, gold and silver clusters encapsulated by coordinating ligands feature strong optical transitions that vary not only with stoichiometry, charge, and geometry, but can also be strongly influenced by interactions with their encapsulating matrix. [5][6][7][8] As the molecular size scale is approached, few-atom metallic cluster sizes exhibit an insufficient density of states to close the "band gap" as occurs in bulk or nanoparticulate metals, leading to strong size-dependent optical and catalytic properties that in many cases are well-explained by the nuclear shell model for free electron energies and magic cluster sizes. [9][10][11] This approach has led to the extended atom description of metallic clusters and an effective expansion of the periodic table with size-dependent cluster behaviors when metalmetal interactions dominate cluster stability. 12, 13 * dickson@chemistry.gatech.edu. Although the protoplasmonic electronic structure of metal clusters is best observed in bare, unsolvated and isolated clusters, 9, 10, 14 ligands are essential to constrain growth, stabilize, an...

Molecular silver clusters conjugated with DNA act as analyte sensors. Our studies evaluate a type of cluster-laden DNA strand whose structure and silver stoichiometry changes with hybridization. The sensor strand integrates two functions: the 3′ region binds target DNA strands through base recognition while the 5′ sequence C3AC3AC3TC3A favors formation of a near infrared absorbing and emitting cluster. This precursor form exclusively harbors an ~11 silver atom cluster that absorbs at 400 nm and that condenses its single-stranded host. The 3′ recognition site associates with a complementary target strand, thereby effecting a 330 nm red-shift in cluster absorption and a background-limited recovery of cluster emission at 790 nm. One factor underlying these changes is sensor unfolding and aggregation. Variations in salt and oligonucleotide concentrations control cluster development by influencing DNA association. Structural studies using fluorescence anisotropy, fluorescence correlation spectroscopy, and size exclusion chromatography show that the sensor-cluster conjugate opens and subsequently dimerizes with hybridization. A second factor contributing to the spectral and photophysical changes is cluster transformation. Empirical silver stoichiometries are preserved through hybridization, so hybridized, dimeric near infrared conjugates host twice the amount of silver in relation to their violet absorbing predecessors. These DNA structure and net silver stoichiometry alterations provide insight into how DNA-silver conjugates recognize analytes.