Mp27:karoliny:rapidprototypingELISA
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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.
