Fluorimetric determination of fluorescein at the femtomole level with a self-ordered ring of a sessile droplet on glass slide support

A method for fluorimetric microscopic determination of trace amounts of fluorescein at the femtomole level has been developed. Since the solvent evaporates from the droplet on a hydrophobic-treated glass slide, an outward capillary flow of the solvent from the interior of the droplet occurs. The resulting outward capillary flow then carries the solute to the perimeter of the droplet spot, where the solute accumulates to form a fluorescent self-ordered ring (SOR). Depending on the volume of the droplet of fluorescein solution, SORs of different sizes with an outer diameter less than 1.2 mm and a ring belt width less than 24 μm can be obtained. Data analysis for a digitally imaged SOR using a charge-coupled device (CCD) camera showed that the fluorescein molecules across the fluorescent SOR belt section follow a Gaussian distribution; the maximum fluorescent intensity at central ring belt (I _max) was found to be proportional to fluorescein content. When a 0.1-μL droplet was spotted on the solid support, fluorescein in a range of 0.6–120 femtomol (or 6.2–1200.0 nM) can be detected, and the limit of detection can reach 62 attomol (or 0.6 nM). With the present method, the contents of fluorescein in fluorescein sodium injections were satisfactorily detected with recoveries of 95–105% and RSD of 3.5 and 4.2%, respectively. The text was submitted by the authors in English.