**1. Introduction**

Peptidases, more frequently referred to as proteases, are a group of enzymes that irreversibly hydrolyze a peptide bond in an amino acid sequence through the nucleophilic attack and subsequent hydrolysis of a tetrahedral intermediate. They play critical roles in biological and physiological processes such as blood clotting, digestion, and a variety of cellular activities [1,2]. Proteases are highly involved in the dairy industry as well, where their activity is directly linked to the shelf life of dairy products [3]. Owing to their specificity, protease activity-based biosensors are used in various diseases diagnostics [4–6]. For example, pancreatic diseases such as cystic fibrosis, acute pancreatitis, or the acute phase of chronic pancreatitis are associated with the increased trypsin level of 2.1–71.42 nM in the serum of patients [7,8]. In the healthy physiological condition, the concentration of trypsin varies in magnitude. Additionally, levels of trypsin differ between serum and intestinal levels. For serum levels for fasting individuals, the concentration of trypsin was measured from 4 to 20 nM [9,10]. The intestinal level of trypsin depends on the location in theintestineandrangesfrom4to30μM,whichismuchhigherthaninserum[11].

Moreover, the inhibitors of these proteases are successfully employed as therapeutic agents [2,12,13].

**Citation:** Piovarci, I.; Melikishvili, S.;Tatarko, M.; Hianik, T.; Thompson, M.Detection of Sub-Nanomolar Concentration of Trypsin by Thickness-Shear Mode Acoustic Biosensor and Spectrophotometry. *Biosensors* **2021**, *11*, 117. https:// doi.org/10.3390/bios11040117

Received: 11 February 2021 Accepted: 6 April 2021 Published: 11 April 2021

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Trypsin is an extremely important serine protease of the chymotrypsin family. It is produced in the pancreas and it plays crucial roles in the small intestine. Trypsin catalyzes the hydrolysis of consumed proteins and activates protease proenzymes as part of the digestive system. It is highly specific toward the cleavage of peptide bonds at the carboxyl side of lysine or arginine. Trypsin is often used as a model protease because it is inexpensive and readily available [14–16]. Standard assays for the detection of proteases such as trypsin usually utilize fluorogenic and chromogenic substrates. Those assays are useful, practical, and highly sensitive. However, spectroscopic assays are incapable of measuring protease activity in highly colored and turbid samples such as cells, tissue lysates, or milk. Therefore, the development of a new label-free method for detecting protease activity without interruption from impurity inclusions is needed [1,15,17].

The thickness-shear mode (TSM) acoustic wave biosensor may present an attractive platform for the development of cost-effective and highly sensitive techniques for trypsin detection. The use of TSM devices is a well-known method for the detection of mass changes due to depositions or chemical/biochemical reactions on its surface. It is also an established method for detecting changes in the viscoelastic properties of the contacting material. Therefore, the TSM biosensor is a sensitive tool for the study of molecular interactions on surfaces [18]. Moreover, the coupling of a flow injection analysis (FIA) system to a TSM sensor device permits the monitoring of kinetic processes that take place at the surface of the sensor [19]. The TSM device applies a high-frequency AC voltage across an AT-cut quartz crystal on which, due to the piezoelectric effect, an acoustic shear wave is generated and propagated through the sensing layer perpendicular to the surface of the crystal [20]. It has a low noise level and higher Q-factor in clinical liquids such as tissue fluids and serum. Compared to other common biosensing technologies, TSM electroacoustic resonators have the combined advantages of high sensitivity and low cost, label-free detection of analyte, and simple operation without the requirement of bulky detection systems [21]. Moreover, in contrast with traditional quartz crystal microbalance (QCM) techniques, the analysis of complex impedance spectra allows for the receipt of information about changes in the properties of layers even with the adsorption of relatively small molecules that do not contribute to the mass but only to the viscoelastic properties of the layer [22]. The multi-harmonic QCM method has previously been applied for the detection of plasmin and trypsin at the surface of β-casein layers [23]. This method allows the detection of these proteases at the sub-nM level. However, the possible contribution of viscoelastic effects has not been analyzed.

In addition to the acoustic methods also the colorimetric assay based on gold nanoparticles (AuNPs) for protease detection is of increased interest. AuNPs have attracted tremendous interest because of their optical and electronic properties, which are tunable by changing the size, shape, surface chemistry, or aggregation state. Colloidal AuNPs have a distinctive red color, which arises from the tiny dimensions of the AuNPs. The changes in the UV–vis spectra of the resultant colloids are measured to investigate the size effect of AuNPs on the surface plasmon resonance (SPR). Interestingly, the red color of citratestabilized AuNPs turns to blue when they are aggregated [24]. This approach has been widely applied to various methods for colorimetric detection of analytes via the aggregation of AuNPs [25], including those of trypsin detection [26].

In this work, we designed an analytical method based on the TSM biosensor for the real-time and label-free detection of trypsin. Using TSM frequency responses, we studied the assembly and stability of self-assembled β-casein layers on a quartz crystal electrode and measured the dynamics of TSM response and changes in motional resistance during casein cleaving by the protease.

Additionally, we compared the sensitivity of the TSM method with another label-free assay of the protease activity by employing AuNPs coated by β-casein and 6-mercaptohexanol (MCH). Unlike the surface-sensitive TSM biosensor, the suggested approach was volume-sensitive, thus allowing us to monitor tryptic activity in the reaction mixture. We used an approach developed by Chuang et al. [26]. However, instead of gelatin, β-casein

was used as a substrate for trypsin. β-casein adsorbed on AuNPs kept the modified nanoparticles stably suspended in solution.

Considering the results obtained, we believe that the proposed approaches constitute rapid, cost-efficient, sensitive and useful tools for protease analysis. This paper is an extension of a conference paper published in the 1st International Electronic Conference on Biosensors [27].

#### **2. Materials and Methods**
