SLAS

Mp27:karoliny:rapidprototypingELISA

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SLAS2012 Posters(1 C)
The Market Place for Lab Automation & Screening  The Market Place

Author(s) name and affiliation: Karoliny Almeida Oliveira, Cristina R. de Oliveira, Lucimeire A. da Silveira and Wendell Karlos Tomazelli Coltro.



This report describes the quick and simple fabrication of toner-based 96-microzone plates by a direct-printing technology for bioanalytical assays. The design of the microplates was drawn in Corel Draw software and laser printed on a polyester film surface. The microplates configuration consisted of 96 wells arranged into 12 columns with 8 wells each. The printer deposits a toner layer (ca. 5 µm thick) on the polyester surface which acts as a hydrophobic barrier to confine small volumes of sample on test zones (wells). The microplates were prepared in two formats, namely of (i) positive microplate (in which a toner line delimiting the microzone area) and (ii) negative microplate (where assay zones are delimited by a toner layer printed over the entire area of the plate). The effects of the sample volume, microplate printing format, zone diameter as well as the toner barrier width on the analytical performance have been investigated. Positive and negative printed microplates have provide a very good inter-zone repeatability (relative standard deviation lower than 7%) allowing us concluding that both configurations can be successfully used on analytical and bioanalytical assays. It has been found that the absorbance response was proportional to the droplet height of sample added to each zone. According to a spherical cap model, the path length in typical toner microzones with diameter between 9 and 4 mm have been estimated to be ca. 0.200 and 1.420 mm, respectively. Using an absorbance microplate reader, a limit of detection (LD) of 77 pmol of methylene blue (MB) dye per zone has been found. Microzones with 7-mm diameter were surrounded by toner barriers designed with width values between 0.5 and 1.0 mm. According to the data obtained, no significative difference was observed. A 96-microzone toner plate was explored to successfully demonstrate its capability of performing enzyme-linked immunosorbent assay (ELISA). For this purpose, human and mouse IgG were immobilized on polyester surface to detect mouse IgG using a rabbit anti-mouse IgG conjugated to peroxidase. ELISA was performed on both standard plastic and printed microplates. It was achieved a LD of 14 fmol/zone for plastic and 6 fmol/zone for printed microplates. In addition, it was demonstrated that ELISA can be performed on toner-based zones using shorter incubation time. This work has been supported by CNPq and INCTBio.