br Acknowledgements The strain of E coli K was generously

Acknowledgements The strain of E. coli (K12) was generously donated by Ms. Dorothy Agnew, Department of Biomedical and Molecular Sciences, Queen’s University. Funding provided by the Natural Sciences and Engineering Research Council (NSERC) – Canada Discovery program and SARC (Queen’s U.) is gratefully acknowledged.
Introduction Optical DNA-chips are widely used to study genome, gene expression, genetic diseases [1] and microbial detection [2]. We can divide the DNA-chip into three basic components: the sensing element or probe specific for target gene (single strand DNA); the labeling molecule, i.e. the conventional fluorophore CY5 (indodicarbocyanine) [3]; the optical detector, i.e. imagers or scanners. The final goal of our work is to integrate the whole biosensor system in a single portable device easy to design and fabricate. To this purpose, the first issue to address is the fluorescent labeling of the target. For DNA labeling, the cyanine dye CY5 is conventionally used. However, it suffers of self-absorption of its fluorescence [4], caused by the proximity of ddr1 and emission’s peaks, at 650nm and 670nm, respectively. At the same time, it is photobleached after prolonged exposure to laser beam (see Fig. 4B). Moreover, its short lifetime (1–3ns) [5], would imply a quite sophisticated electronic and optical systems. Therefore, we studied an alternative fluorophore to be used in DNA-chip application, the tris(2,2′-bipyridyl)ruthenium(II) (Ru(bpy)32+). It is an octahedral metal transition complex composed by the transition metal ruthenium bounded to three heteroaromatic bypiridine units. Its optical properties would allow one to overpass some issues, related to the use of CY5, as already described. The fluorophore has two absorption peaks at 290nm and 450nm, ligand-center (LC) and metal–ligand (MLCT) electronic transitions respectively, and a quantum yield of 0.042±0.002 (compared to 0.2 of Cy5). They are far away from the emission peak at 630nm [6,7], 100nm the closest absorption peak, thus avoiding the fluorescence self-absorption. Therefore, the incident radiation may be shielded using a simple and inexpensive band-pass filter. Moreover, Ru(bpy)32+ fluorescence exhibits a very long lifetime (τ=350ns) [8], allowing one the use of pulsed LED for excitation. Ru(bpy)32+ has been already used in bio-sensing applications: optical environmental sensors [9]; electrical sensors, based on the electro-chemo-luminescence of Ru(bpy)32+[10]; lysozyme based optical aptasensors [11]; light switching experiments, based on direct linkage to DNA [12,13]. In this last case, the fluorophore structure was modified through the substitution of one of the bipyridyl groups with a dipyridophenazine. Thereby, fluorophore emission switched on once intercalated among base pairs of DNA double helix (especially inserted into AA mismatches). However, there are few evidence about Ru(bpy)32+ used in DNA optical sensing applications. For this reason, we studied extensively Ru(bpy)32+ absorption, emission and fluorescence lifetime, focusing on its suitability to realize a portable and easy to use biosensor device for biomedical applications (namely the DNA-chip technology). The investigation has highlighted the fluorophore sensitivity to the environment, as supported by morphological Transmission Electron Microscopy (TEM) analysis.
Materials and methods Powder of tris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrate was purchased from Sigma–Aldrich, while Lumiprobe provided powder of sulfo-Cyanine5 NHS ester. Phosphate buffer saline 10× solution were from Fisher Bioreagents™. The cuvettes were UV-transparent disposable cuvettes Ultra-Micro, 2×3.5mm2 with 10mm optical path; the slides were coverslip glass slide 24×60mm2 with 0.18mm thickness. We dissolved uranyl acetate (EMS) to give a 4% (w/v) final concentration and filtered before the use. For TEM analysis, we used formvar carbon coated nickel grids (300 mesh). The aqueous solution of Ru(bpy)32+ for TEM analysis was prepared using Milli-Q ultrapure water, 18MΩ.