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A moisture barrier layer with high density and uniformity is essential forincreasing the lifespan of optoelectronic devices such as flexible organic solar cells, LEDs,and photodetectors. In this study, various surface pre-treatments (O2 plasma, UVOtreatment, Al seed layer, and thermal annealing process) were performed on a polyethylenenaphthalate (PEN) film to form a functional group of chemical adsorption with the atomiclayer deposition (ALD) precursor. We investigated the effect of the surface pre-treatment ofa PEN substrate for a deposition of Al2O3 thin film in terms of the deposition uniformity andwater vapor transmittance rate (WVTR). For example, the root mean square roughness ofthe bare PEN film/Al2O3 was RRMS=3.26 nm, whereas the O2 plasma treated PEN film/Al2O3had RRMS=0.99 nm because of the presence of a functional group. As a result, WVTR of barePEN film/Al2O3 was 0.83 g/m²/day, whereas that of O2 plasma treated PEN film/Al2O3decreased to 0.38 g/m²/day.