The application of laser printing on a transparent poly(ethylene terephthalate) (PET) foil for toner-assisted immobilization of antibodies is employed for the first time in the preparation of two-dimensional arrays for high throughput ELISA. Two protocols for rapid microfabrication of single- and multi-layer immunoassay substrates with spatially controlled wettability are demonstrated, which include essential steps of laser Printing, UV-ozone Irradiation, and ultrasonically assisted toner Delamination (PID). This study demonstrates the potential of laser toner as an effective and easily removable protectant of PET against permanent hydrophilization by UV irradiation. To prove the feasibility and analytical performance of this novel immunosensing platform, an indirect sandwich immunoassay for human C-reactive protein (hCRP) was selected as a model. Dynamic range, covering CRP levels between 10 ng∙mL−1 and 5.62 µg∙mL−1, with a detection limit of 0.656 ng∙mL−1, as well as excellent signal recovery in human serum (97.3 - 104.5%), confirms clinical suitability of the assay in the analysis of real samples. Thanks to the design and fabrication simplicity, use of commonly available materials and up-scalability, presented methodologies can be successfully applied in the production of substrates for high-throughput immunodiagnostics and flexible microfluidics in Lab-on-a-Foil technology.
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