Oleic acid

For the EO NCs preparation, zein
was dissolved in propylene glycol
under magnetic agitation. Then surfactant (Tween 20, Panreac, Madrid,
Spain) and cosurfactants (propanediol, Dupont Tate & Lyle Bioproducts,
TN, USA; denatured alcohol, Valencia, Spain; and oleic acid, Panreac,
Madrid, Spain) were added under agitation until a clear, transparent
solution was obtained. Oregano EO was then added to that mixture to
achieve a homogeneous organic phase. The resultant surfactant–EO–zein
mixture was added under continuous agitation into water at a ratio
of 3:7 (organic phase/water) to form EO NCs. Afterward, 22.5 mL of
the EO NCs (negatively charged) were mixed with 5 mL of 10 mg mL^–1 AC aqueous solution, at pH 3.3, and incubated for
1 h at RT. After that, the sample was centrifuged at 29 500g for 50 min, resuspended in 3.75 mL of Milli-Q water, and
sonicated to disaggregate the NCs for 20 min at 20 °C. The NCs
were functionalized with rabbit protein A antibody using 50/20 mM/mM
of EDAC/sulfo-NHS in 100 mM phosphate buffer, pH 6.5. The reaction
was performed at RT for 24 h with shaking. Then the NCs were centrifuged
for 40 min at 25 000g, resuspended in 500
μL of Milli-Q water, and subjected to further analysis.