Colloids and Surfaces A: Physicochemical and Engineering Aspects 609 (2021) 125786 Available online 22 October 2020 0927-7757/© 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Plasmin determination based on enzymatic digestion of a β-casein layer at the air/water interface Lor´ and Rom´ anszki a, *, Tibor Hianik b , Zs´ ofa Keresztes a a Functional Interfaces Research Group, Institute of Materials and Environmental Chemistry, Research Centre for Natural Sciences address: Magyar tud´ osok k¨ orútja 2., 1117, Budapest, Hungary b Department of Nuclear Physics and Biophysics, Faculty of Mathematics, Physics and Informatics, Comenius University in Bratislava address: Mlynsk´ a dolina F1, 84248, Bratislava, Slovakia HIGHLIGHTS G R A P H I C A L ABSTRACT • Tensiometry-based label-free determi- nation of proteolytic enzyme activity was developed. • Change in surface pressure of interfacial protein layers upon reaction was determined. • Assay linearity of plasmin enzyme against β-casein substrate of 0.7 nM was achieved. • Sensitivity of the method can be enhanced by increasing the concentra- tion of β-casein. A R T I C L E INFO Keywords: Langmuir flm Gibbs monolayer β-casein Proteolytic enzyme Tensiometry Surface pressure ABSTRACT The possibility of enzyme activity determination based on natural substrate digestion at the air/water interface has been investigated through the interaction of a β-casein layer with the protease plasmin. The protein layers were spread from aqueous solutions of β-casein in a 0.1–0.4 g/L concentration range to the air/water interface. 200 μL plasmin was introduced into the water subphase (15 mL) in various initial concentrations (25–1000 nM), corresponding to 0.33–13.3 nM fnal concentration. The decrease of the surface pressure due to the enzymatic degradation of the β-casein monolayers was followed in time, and calibration curves were obtained by plotting either the total surface pressure change, or the maximum rate of the surface pressure change against the plasmin concentration. The method is suitable for label-free determination of plasmin, with assay linearity down to 0.7 nM as the fnal concentration of plasmin in the system, corresponding to 50 nM for the initial, undiluted plasmin sample in the given experimental setup. * Corresponding author. E-mail addresses: romanszki.lorand@ttk.hu (L. Rom´ anszki), tibor.hianik@fmph.uniba.sk (T. Hianik), keresztes.zsofa@ttk.hu (Z. Keresztes). Contents lists available at ScienceDirect Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa https://doi.org/10.1016/j.colsurfa.2020.125786 Received 4 September 2020; Received in revised form 8 October 2020; Accepted 17 October 2020