Obtenção de marcadores fluorescentes a partir de fontes alternativas

Abstract

Fluorescent stains have a broad application in biological analysis, mainly by labelling cellular components, allowing their visualization. Some pen inks can have fluorescent substances and a variety of other compounds, such as lubricants, antioxidants and surfactants. Purification allows unknown substances to be tested at biological analysis with less influence of impurities. Thereby, new fluorescent markers can be found from alternative sources, like pens, bryophytes, seaweeds and vegetable roots. Likewise, this present work aims to purify fluorescent compounds present at the red pen Pilot BP-1 INOX 1.0 (Brazil), in order to apply it/them in experiments using biological samples. Hereby, it was used a solution of 10% of Pilot pen ink at ethanol. The purification was done with column chromatography, using polarity gradients of the mobile phase of hexane, ethyl acetate and ethanol. Thereby, it was possible to visualize the separation of two main fluorescent compounds inside the column. The purified fractions were analyzed by TLC, evaporated and joined with each other. To identify the chromatographic profile, the compounds were injected in CLAE, using as mobile phase an isocratic solution of methanol and water (65:35) with flow rate of 1,1 ml/min at 35ºC. This experiment revealed that the purified compounds was different of the crude extract. The rhodamines B and 123 was used as controls, however the CLAE analysis did not detect them in the pen extract or in the purified substances. Furthermore, it was made the determination of optical characteristics of this compounds in a spectrofluorometer. The analysis showed that there were differences in the optical behavior between purified substances and the rhodamines used as control. The emission and absorption peaks of the purified fluorophores was close to the values of rhodamine 6G found in literature. The obtained fluorescent substances also showed positive charge by electrophorese analysis, what can be potentially used for interaction with negatively charged biomolecules, like cells and nucleic acids.