**4. Conclusions**

We have shown that β-casein forms a stable monolayer via an 11-mercaptoundecanoic acid (MUA) cross-linker at the gold surface of a piezoelectric transducer. The TSM sensor based on a β-casein layer enabled a detection limit of 0.48 ± 0.08 nM for trypsin. The cleavage of β-casein resulted in an increase of resonant frequency and a decrease of motional resistance. Furthermore, we compared the results obtained by the TSM method with a colorimetric assay for quantifying trypsin activity in a volume. This assay was based on AuNPs modified by β-casein and MCH and on the phenomena of surface plasmon resonance (SPR) and yielded a detection limit of 0.42 ± 0.03 nM, which is comparable with the LOD obtained from TSM experiments. We also analyzed the Michaelis–Menten constants, KM, for reverse enzymatic reaction and showed that the KM value for the colorimetric assay (0.56 ± 0.10 nM) is lower in comparison with that obtained in the case of the TSM method (0.92 ± 0.44 nM). This has been explained by better access of trypsin to the β-casein in a volume. The TSM method is useful for the study of the kinetics of the protease's activity, which is not possible via conventional ELISA or HPLC methods. The obtained results can be considered as a first step toward the application of a TSM sensor and colorimetric assays based on β-casein for the label-free detection of trypsin activity. For practical application in medical diagnostics, both acoustic and optical methods need additional validation in complex biological fluids such as blood or blood plasma. In addition, the sensitivity of the TSM method can be improved by the application of hydrophobic substrates for casein immobilization. We anticipate in this case that the detection limit can be improved at least five times. The improved sensitivity of detection is important for working with diluted bilogical samples in order to minimize the matrix effect.

**Author Contributions:** Investigation, validation, formal analysis, writing—original draft preparation, I.P.; investigation, validation, formal analysis, writing—original draft preparation, S.M.; investigation and formal analysis, writing—review and editing, M.T. (Marek Tatarko); conceptualization, writing— original draft preparation, writing—review and editing, supervision, project administration, funding acquisition, T.H.; methodology, project administration, writing—review and editing, M.T. (Michael Thompson). All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie gran<sup>t</sup> agreemen<sup>t</sup> No. 690,898 and by Science Grant Agency VEGA, project No. 1/0419/20.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.
