**1. Introduction**

Proteases represent a very wide and important group of enzymes found in a broad range of biological systems [1]. Proteases play an important role in the digestion process and participate in various pathological processes [2,3]. Chymotrypsin is a serine protease present in the human digestive system that participates in protein cleavage in the intestines [4]. Together with trypsin, chymotrypsinogen is ejected into the duodenum, where trypsin cleaves it into the active form [5]. Chymotrypsin activity is closely related to the activity of trypsin, which, along with plasmin, is an important enzyme in milk. Activity of plasmin is correlated to the quality of milk where the protease cleaves the proteins, mainly casein micelles affecting the milk flavor, shelf-life or cheese yield [6]. In pathology and medicine, chymotrypsin also has anti-inflammatory effects and has been successfully used to reduce post-operation complications after cataract surgery [7]. Measuring chymotrypsin activity can also be used for differential diagnosis [8].

Thus, development of sensitive, inexpensive, fast, and easy to use methods for detection of chymotrypsin or other milk proteases would be beneficial to disease diagnostics and control of dairy quality. However, there are no simple and effective assays that can be used for these purposes ye<sup>t</sup> available. Protease detection is currently based on the detection of α-amino groups cleaved from the protein substrate using optical or high-performance liquid chromatography (HPLC) methods. The method that can be used for fast analysis of the protease concentration is based on enzyme-linked immunosorbent assay (ELISA) with a limit of detection (LOD) of about 0.5 nM for chymotrypsin [9,10]. However, the above-mentioned methods do not allow study of the kinetics of substrate digestion.

In this paper we test three methods for chymotrypsin detection: QCM, spectrophotometric, and DLS.

The QCM method is based on measurement of the resonant frequency, f, of shearing oscillations of AT-cut quartz crystal, as well as motional resistance, Rm, and is also known

**Citation:** Piovarci, I.; Hianik, T.; Ivanov, I.N. Detection of Chymotrypsin by Optical and Acoustic Methods. *Biosensors* **2021**, *11*,63. https://doi.org/10.3390/ bios11030063

Received: 30 January 2021 Accepted: 23 February 2021 Published: 26 February 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

as thickness shear mode method (TSM). The protease substrates, such as β-casein or short specific peptides, are immobilized on thin gold layers sputtered at a QCM transducer. High frequency voltage, typically in the range of 5–20 MHz, induces shearing oscillations of the crystal. The fundamental resonance frequency of the crystal, f0, depends on the physical properties of the quartz viscosity of the medium to which the crystal surface is exposed, as well as on the molecular interactions at the surface. The R m value is sensitive to shearing viscosity, which is due to the molecular slip between the protein layer and surrounding water environment. Using Sauerbrey Equation (1) [11], one can link the change in resonant frequency to the mass bound to the surface of the electrode.

$$
\Delta \mathbf{f} = -2 \mathbf{f}\_0 \, ^2 \Delta \mathbf{m} / \mathcal{A} (\mu\_\mathbf{q} \rho\_\mathbf{q})^{1/2} \, ^\prime \prime \, \tag{1}
$$

where fo is the fundamental resonant frequency (Hz), A is the active crystal area (in our case: 0.2 cm2), ρq is quartz density (2.648 g cm<sup>−</sup>3), Δm is the mass change (g), ρq is the shear modulus of the crystal (2.947 × 10<sup>11</sup> g cm<sup>−</sup><sup>1</sup> s<sup>−</sup>2). This Equation is valid only for a rigid layer in vacuum. In a liquid environment and for relatively soft layers, the viscosity contribution can be estimated by measurements of R m.

We modified the surface of the QCM crystal with a layer of β-casein. The resulting mass added to the sensor leads to the decrease of the resonant frequency, f, and increase of motional resistance, R m. Chymotrypsin will cleave β-casein, which results in an increase in f and decrease in R m values. The mass sensitive QCM method was used for the detection of trypsin activity using synthesized peptide chains [12]. Poturnayova et al. used βcasein layers to detect activity of plasmin and trypsin with LOD around 0.65 nM [13]. Incorporation of machine learning algorithm for analysis of multiharmonic QCM response allowed detection of trypsin and plasmin with LOD of 0.2 nM and 0.5 nM, respectively. The applied algorithm in the work of Tatarko et al. allowed us to distinguish these two proteases within 2 min [14].

We also used the spectrophotometric method based on measurement of absorbance of the dispersion of gold nanoparticles (AuNPs) coated by 6-mercapto-1-hexanol (MCH) and β-casein. AuNPs demonstrate a surface plasmon resonance (SPR) effect, which arises from the oscillating electromagnetic field of light rays getting into contact with the free electrons in metallic nanoparticles and induces their coherent oscillation, which have strong optical absorption in the UV-vis part of the spectrum. The SPR absorbance of AuNPs depends on the surrounding medium and on the distance between nanoparticles [15]. In the work by Diouani, AuNPs modified with casein were used to detect Leishmania infantum using amperometric methods [16]. Chen et al. modified AuNPs with a trypsin-specific peptide sequence [17]. After the trypsin cleavage, the gold nanoparticles aggregated, which was detected by monitoring changes in the UV-vis spectrum. The detection limit of this method was estimated to be around 5 nM. Svard et al. modified gold nanoparticles with casein or IgG antibodies for detection trypsin or gingipain activity, by measuring SPR peak shift (blue shift for trypsin and red shift for gingipain) and reporting LOD of less than 4.3 nM for trypsin and gingipain [18]. Goyal et al. developed method of immobilization of gold nanoparticles on a paper membrane [19]. The protease activity then led to aggregation of the gold nanoparticles on the membrane and resulted in a colorimetric response in a visible part of the spectrum detectable by the naked eye. AuNPs modified by gelatin that served as a substrate for proteinase digestion have also been used for detection of other proteases such as trypsin and matrix metalloproteinase-2 [20]. In our work, we modified the gold nanoparticles with β-casein and MCH using protocol from Ref. [20]. The β-casein protects the AuNPs from aggregation. Addition of the chymotrypsin and subsequent cleavage of the β-casein caused nanoparticles aggregation due to loss of the protective shell. This effect was observed by measuring UV-vis spectra of nanoparticle dispersion.

We also used dynamic light scattering (DLS) method which uses Brownian motion and the Rayleigh scattering of the light from particles to assess their size [21]. The intensity of the scattered light (which depends on particle concentration) changes over time because of particle aggregation. The auto-correlation function that correlates the intensity of

scattered light with its intensity after an arbitrary time is used to discern the size of the particles. The auto-correlation function also depends on diffusion coefficient of the nanoparticles [22]. In DLS experiments we used AuNPs modified with β-casein. After addition of the chymotrypsin, we were able to observe the cleavage of the casein layer without AuNPs aggregation that resulted in a decrease of the size of nanoparticles.

This report is an extension of a manuscript published in proceeding of the 1st International Electronic Conference on Biosensors [23].

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