*2.1. Reagents*

Ultrapure water obtained by reverse osmosis (Thermo Scientific, Waltham, MA, USA, ρ = 18.2 MΩ cm) was used for the preparation of all aqueous solutions. As a medium, 10 mM, pH 7.4 phosphate-buffered saline (PBS) was used (10 mM Na2HPO4, 2 mM KH2PO4, 2.7 mM KCl and 137 mM NaCl), prepared from tablets (Sigma-Aldrich, Darmstadt, Germany, Cat. No. P4417). In the experiments, trypsin from bovine pancreas (≥90%, ≥7500 BAEE units/mg solid, Sigma-Aldrich, Darmstadt, Germany, Cat. No. T9201) served as a model protease. The concentration of stock bovine β-casein (≥98%, Sigma-Aldrich, Darmstadt, Germany, Cat. No. C6905) solutions, prepared in PBS, was 0.5 mg/mL. 11-Mercaptoundecanoic acid (MUA, Sigma-Aldrich, Cat. No. 450561), *N*- (3-dimethylaminopropyl)-*N*-ethylcarbodiimide (EDC, ≥98%, Sigma-Aldrich, Cat. No. E6383), and N–Hydroxysuccinimide (NHS, Sigma-Aldrich, Darmstadt, Germany, Cat. No. 130672) were employed for β-casein immobilization. The chemicals needed to prepare the gold nanoparticles, such as auric acid (HAuCl4), sodium citrate, and 6-mercapto-1-hexanol (MCH), were purchased from Sigma-Aldrich (Darmstadt, Germany). All experiments were carried out at 20 ◦C.

#### *2.2. Cleaning and Modification of Gold Electrode-Coated Quartz Crystals*

Symmetric gold electrode-coated quartz discs (Total Frequency Control, Storrington, UK, working area, 0.2 cm2) with a fundamental frequency of 8 MHz were cleaned in a basic Piranha solution (29% NH3, 30% H2O and H2O2 with volumetric 1:5:1 ratio, respectively) for 25 min. After this treatment, the crystals were washed three times with deionized water and stored in ethanol. After drying in a flow of nitrogen, the TSM crystals were immersed in 2 mM MUA and were incubated for 16 h to form a self-assembled monolayer. After this step, the crystals were rinsed several times with deionized water and dried under nitrogen, followed by incubation for 20 min in a 20 mM EDC and 50 mM NHS mixture in order to activate the carboxylic groups of MUA for the further immobilization of bovine β-casein on the gold electrode of the quartz sensor. The scheme of modification of the TSM transducer as well as the cleavage of β-casein by trypsin is shown in Figure 1.

**Figure 1.** The scheme for modification of the gold layer on a TSM transducer and the cleavage of β-casein by trypsin.

### *2.3. TSM Measurements*

AT-cut 8.0 MHz gold electrode-coated quartz crystals, modified on one side by MUA with activated carboxylic groups by NHS/EDC as described above, were incorporated into a home-built flow-through thickness shearing mode (TSM) acoustic wave device sensor system. The setup and general configuration of the flow-through system is described in reference [19]. One side of the crystal was exposed to liquid, the other one was exposed to air. The liquid was introduced using a syringe pump (Genie Plus, Torrington, CT, USA). Runs were performed with the crystals in the vertical position and at ambient temperature (approximately 20 ◦C). The modified crystal was secured in the holder using two O-rings. The gold electrodes were kept in contact with the gold leads in the holder. Resonance frequency, f, and motional resistance, R m, were determined based on the Butterworth–van Dyke (BVD) model of a quartz crystal resonator [19]. The resonant frequency represents the energy storage and reflects the mass changes of the oscillating layer, while R m is related to the dissipation of energy and provides evidence of changes in the shearing viscosity of the layer [22]. The measuring procedure was as follows. Each slide was flushed through with PBS at a flow rate of 50 μL/min until a stable baseline was achieved (45 min), using the flow-through injection system. This step was necessary to remove any weakly adsorbed molecules at the surface of the TSM transducer. Next, the pump was momentarily stopped. The β-casein solution (0.5 mg/mL in PBS) was slowly introduced to the sample, while the PBS was exchanged out in order to minimize pressure effects to the system. β-casein was introduced at a rate of 50 μL/min for approximately 45 min. Once again, the pump was momentarily stopped, and the sample input tube was slowly placed back into the PBS solution. The PBS was re-introduced at a rate of 50 μL/min to remove any loosely bound casein until a stable baseline was achieved. Changes of the resonant frequency and motional resistance were recorded. For proteolysis measurements, solutions with various concentrations of trypsin in PBS (0.1, 0.5, 1, 5, 10, and 20 nM) were flowed over TSM crystals with an immobilized β-casein layer at a flow rate of 50 μL/min. Trypsin and β-casein solutions were freshly prepared before each experiment.

#### *2.4. Synthesis and Modification of AuNPs*

AuNPs were prepared using a modified citrate method described in reference [28]. Briefly, 100 mL of HAuCl4 (0.01%) was heated to boiling under vigorous stirring, which was followed by the addition of 5 mL of sodium tris-citrate solution (1%). The solution was left boiling while stirring until it turned a deep red. Then, we let the AuNPs solution cool down and stored it in the dark. In order to modify the gold nanoparticles with casein, we added 2 mL of 0.1 mg/mL β-casein to 18 mL of the AuNPs solution. After 2 h of incubation at room temperature without stirring, the gold nanoparticles were further incubated with 200 μL of 1 mM MCH overnight for approximately 18 h. The scheme of modification of AuNPs is showed in Figure 2.

### *2.5. Sprectrophotometric Assay*

For the colorimetric assay, we prepared 0.95 mL of AuNPs. Trypsin was dissolved in deionized water, and 0.05 mL of trypsin from the stock solution was added to each cuvette. The concentration of trypsin in cuvettes was 0.1, 0.5, 1, 5, and 10 nM at 1 mL total volume of solution. We also used a reference cuvette where only 0.05 mL of protease-free water was added to the AuNPs solution. We measured the spectra of the AuNPs before trypsin addition (0 min), just after trypsin addition (approximately 1 s), and then every 15 min up to 60 min. The measurement was repeated 3 times. We multiplied the value of absorbance at time t = 0 by the dilution factor to correct the changes in absorbance intensity caused by the initial protease addition. Absorbance was measured by UV-1700 spectrophotometer at a temperature of around 20 ◦C and in the wavelength range of 220–800 nm (Shimadzu, Kyoto, Japan).

**Figure 2.** The scheme for modification of gold nanoparticles (AuNPs) by β-casein and by 6-mercapto-1-hexanol (MCH) as well as the cleavage of β-casein by trypsin. Before enzymatic digestion, functionalized AuNPs were stable due to steric stabilization. After the AuNPs were subjected to protease cleavage, the casein was removed from the surface of AuNPs/MCH/β-casein. This caused the destabilization of the NPs, followed by their aggregation.

#### *2.6. Analysis of Casein Adsorption and Hydrolysis Processes*

The surface concentration (Γ*QCM*, ng/cm2) of the adsorbed β-casein layer on the TSM transducer was determined by a modified Sauerbrey Equation (1) as follows:

$$
\Gamma\_{\rm QCD} = \frac{-\mathcal{A}\sqrt{\mu\rho}\Delta f}{2f\_0^2},
\tag{1}
$$

where A is the area of the electrode, *ρ* = 2.648 g/cm<sup>3</sup> is the density of quartz, μ = 2.947 × 10<sup>11</sup> g/cms<sup>2</sup> is the shear modulus of AT-cut crystal, and *f* 0 is fundamental resonant frequency [29]. The Sauerbrey equation is strongly valid for thin rigid films at the surface of quartz crystal in vacuum. However, in a liquid, the viscoelastic contribution can affect the frequency changes. Through analysis of the motional resistance, Rm, it is possible to estimate whether the mass or viscosity is dominant in frequency changes. It has been shown that the slope of |Δf/ΔRm| can be used for quantitative estimation whether the changes in frequency can be attributed to mass or to viscosity effects. For ideal rigid films, the ΔRm values are practically zero. This means that |Δf/ΔRm| parameters higher than a certain critical value can be assigned to the mass effect [30]. According to the calculations made in ref. [30] for the AT cut quartz crystal with fundamental frequency *f* 0 = 8 MHz, |Δf/ΔRm| = 10.37 Hz/Ω.

The frequency changes following the addition of the trypsin were normalized to the changes of the resonant frequency caused by adsorption of the β-casein at the surface of TSM crystal. This allowed consideration of a possible variation in the properties of the β-casein layers that were subsequently cleaved by trypsin. The normalized frequency changes were expressed as ΔfN = (ΔfTRY/Δfcasein) × 100(%), where ΔfTRY are changes in frequency following the addition of trypsin at certain concentration of the protease and Δfcasein are changes in frequency caused by the formation of a β-casein layer.

An inverse Michaelis–Menten (MM) model [31] was used to describe the dependence of the normalized frequency changes vs. concentration of trypsin at fixed concentration of the β-casein at the surface of TSM transducer:

$$
\Delta f\_N = \left(\Delta f\_N\right)\_{max} \frac{C\_{TRY}}{K\_M + C\_{TRY}} \tag{2}
$$

where (Δ*fN*)*max* is the maximal change of the frequency that corresponds to the maximum rate of enzyme reaction achieved by the system happening at saturating enzyme concentration, *CTRY* is the concentration of trypsin, *KM* is the reverse Michalis–Menten constant that is equal to the trypsin concentration that achieves half of maximum rate. The hydrolysis of β-casein in a volume was modeled with an inverse MM Equation (3) as well

$$\frac{A\_0 - A\_{15}}{A\_0} 100 = v\_{\text{max}} \frac{C\_{TRY}}{K\_M + C\_{TRY}} \,\text{}\tag{3}$$

where *A*0 is the absorbance of AuNPs before exposure to trypsin, *A*15 is the absorbance after 15 min of exposure to trypsin, and *vmax* = [100 × (*A*0 − *A*15)/ *A*0]*max* represents the maximum rate achieved by the system.
