Abstract
The binding state dependent quenching behaviour of functionalized dyes opens perspectives on directly monitoring binding and dissociation events in a catalytic cycle at the single molecule level.
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Asymmetrically amine-substituted perylene diimide dyes were synthesized via the monoanhydride salt (3) (Scheme 1), prepared as in ref. 7, and further functionalized as in ref. 18. First, 3,4,9,10-perylenetetracarboxylic acid dianhydride (0.392 g) was hydrolyzed by adding it to a solution of 0.5 g KOH in 10 mL of water. The reaction mixture was stirred at 90 °C for 1 h, resulting in a yellow fluorescent solution of (2). Precipitation of the monopotassium salt was induced by adding a 10 wt% H3PO4 solution until a pH of ∼4 was reached. After cooling down, this precipitate was separated by filtration, washed with water, and dried at 80 °C. This resulted in compound (3) (0.444 g), which was subsequently suspended in 10 mL of water and cooled in an ice bath. Next, N,N-diethylethylenediamine was added in a molar ratio of 3:1. This reaction mixture was stirred for 3 h, while the ice bath was removed after 30 min. Afterwards, the suspension was acidified with 8 mL of a 0.5 M H2SO4 solution to yield (4) which was separated by filtration and washed with 200 mL of a 0.1 M H2SO4 solution. This compound was immobilized on aminosilane functionalized glass coverslips to yield (5) (vide infra). Compound (6) was readily prepared by reacting 15 mg of (4) with 2.3 mL of methylamine solution (40 wt% in water) overnight at room temperature and subsequently separating and washing the precipitate with water. The formation of (6) was confirmed by NMR spectroscopy. 1H NMR (CDCl3, 300 MHz): δ 8.43–8.60 (m, 8H, aromatic), 4.32 (t, 2H, J = 7.3 Hz), 3.59 (s, 3H), 2.85 (t, 2H, J = 7.3 Hz), 2.70 (q, 4H, J = 6.9 Hz), 1.12 (t, 6H, J = 6.8 Hz); MS-ESI+: m/z 504 (MH+).
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Glass coverslips were cleaned by sonicating them once in acetone, twice in NaOH solution and twice in MQ-water, followed by a UV/ozone treatment. Cleaned coverslips were submerged in CHCl3 in a Teflon container to which 30 μL of a silane mixture was added containing 3-aminopropyltrimethoxysilane and dichlorodimethylsilane in a ratio of 1:100000. Afterwards the functionalized coverslips were again subjected to a short UV/ozone treatment to eliminate fluorescent impurities. Next, the freshly prepared amine functionalized glass surfaces were reacted for 5 min with a drop of very dilute solution of (4) in water to yield (5). Subsequently samples were washed thoroughly with water and dried in an Ar gas stream.
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The wide field microscope consists of an inverted microscope (IX-71, Olympus) with a 100 ×, 1.3 NA oil immersion objective lens and a cooled Electron Multiplying-CCD (cascade 512B, Princeton Instruments Inc.). The wide field illumination was achieved by focusing the expanded circular polarized 488 nm light from an Ar+ laser (Stabilite 2017, Spectra-Physics) onto the back focal plane of the objective. Emission is collected by the same objective and imaged by the CCD after passing through a dichroic mirror and spectral filters removing the excitation light. The image was expanded 3.3 × before the CCD, resulting in a field of view of 24.2 × 24.2 μm. The excitation power was ± 20 kW cm−2.
Starting conditions were: water with a pH of 10–11 to ensure quenched fluorescence through PET. After adding the first drop of acetic acid, further addition didn’t result in a further increase of fluorescence thus proving that the detected spots didn’t originate from fluorescent impurities in the acid. Moreover, observed spots remained localized until bleaching in one step, indicating an effective immobilization.
Confocal measurements (in dioxane) started from acidic conditions to allow localization of single molecules. To perform measurements in basic conditions, an excess of methylamine was subsequently added. The sample was illuminated with 543 nm light. Further details of the experimental setup can be found in ref. 8.
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Dark states were also observed when measuring immobilized perylene diimide molecules without amine functionalization, thus unable to undergo PET quenching. Therefore, it seems that the occurrence of these long lived dark states are not inherent to the system proposed herein but rather to either the way of measuring or the fluorophore, which can both be adjusted without altering the proposed concept.
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Electronic supplementary information (ESI) available: Schematic illustration of the reporter system in a base catalyzed reaction; fluorescence emission spectra of (6) in acidic and basic conditions. See DOI: 10.1039/b821657f
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Ameloot, R., Roeffaers, M., Baruah, M. et al. Towards direct monitoring of discrete events in a catalytic cycle at the single molecule level. Photochem Photobiol Sci 8, 453–456 (2009). https://doi.org/10.1039/b821657f
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DOI: https://doi.org/10.1039/b821657f