TUNING THE MOBILITY OF FLUORESCENT, DNA-TEMPLATED, SILVER NANOCLUSTERS FOR ELECTROPHORETIC SEPARATIONS IN MICROCHANNELS J.T. Del Bonis-O’Donnell, D. Fygenson, S. Pennathur University of California Santa Barbara, USA ABSTRACT We demonstrate that the electrophoretic mobility of DNA-templated fluorescent nanoclusters (AgDNAs) can be finely tuned via DNA sequence without altering the fluorescence spectra, allowing these novel fluorophores to be used as fluorescent mobility markers for electrokinetic-based separation assays using lab on chip devices. Starting with hairpin DNA templates containing varying lengths of poly-dT overhangs, we show that spectrally identical clusters form that can easily be separated electrokinetically. Using short overhangs produces a poly-disperse pair of clusters. We use Alexafluor488 tagged DNA to look in detail at the polydispersity of initial single stranded DNA and compare to the resulting fluorescent clusters. KEYWORDS: microfluidics, separation, fluorophores, silver DNA nanoclusters INTRODUCTION In the last 20 years, electrokinetic-based microfluidic separation devices have emerged as powerful analytical tools for the separation and identification of various analytes [1][2], many of which rely on fluorescence detection owing to its sensitivity and straightforward detection. A particularly powerful separation assay relies on the ability to detect untagged analytes using intermediate fluorescent markers that vary in their electrophoretic mobility [3]. However, fluorescence de- tection is limited to commercially available fluorophores, where little flexibility or control is provided over their electro- kinetic properties. Silver-DNA nanoclusters (AgDNA), a new class of novel fluorophores, are hybrid macromolecules in which a silver superatom is stabilized in aqueous solution by segments of single stranded DNA [4]. AgDNA fluoresce at wavelengths that can be tuned across the visible spectrum and into the infrared by varying the DNA sequence. Their small size, spectral tuning and biocompatibility have lead to a variety of novel uses in biological imaging, genetic analy- sis and chemical detection [5]. However, systematically tuning the physical properties of AgDNA has remained a chal- lenge due to the unpredictable sensitivity of sequence on cluster formation. In this work, we develop an approach for cre- ating a set of spectrally identical AgDNA fluorophores with tunable electrophoretic mobilities for future separation assays. We also use microchip CE to study the conformational heterogeneity of the AgDNA and the single stranded DNA used to make them. EXPERIMENTAL Starting with a hairpin DNA, modifications were made to its sequence that both preserved and altered the secondary structure. While some changes produced clusters that differed both in mobility and spectra, we were able to systematically alter the mobility of one red cluster by appending poly-dT repeats to the 3' end of the hairpin sequence to create overhangs in the stem (Figure 1a). AgDNA nanoclusters were prepared by mixing DNA oligos (IDT DNA, Inc) in 3’ C C C G C A A G G C T A T A G C C T T T T T G T G C ... 5’ C C C G C A A G G C T A T A G C C T G T G C 5’ 3’ 12C-hp10 12C-hp10-T# 500 600 700 800 0 0.2 0.4 0.6 0.8 1 1.2 &íKS &íKSí7 &íKSí7 &íKSí7 SRO\íG7 (PLVVLRQ ,QWHQVLW\ (a) (b) Figure 1: (a) Schematic depicting the DNA sequences and predicted secondary structures used to synthesize fluo- rescent AgDNA nanoclusters. (b) Normalized fluorescence emission under 260 nm excitation for the hairpin 12C- hp10, the overhang poly-dT variants, and a DNA sequence containing only 10 thymine after addition of silver and reduction. 978-0-9798064-6-9/μTAS 2013/$20©13CBMS-0001 1863 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences 27-31 October 2013, Freiburg, Germany