Detection of Chymotrypsin by Optical and Acoustic Methods

biosensors

Article Detection of Chymotrypsin by Optical and Acoustic Methods

Ivan Piovarci 1, Tibor Hianik 1,* and Ilia N. Ivanov 2

1 Department of Nuclear Physics and Biophysics, Faculty of Mathematics, Physics and Informatics, Comenius University, Mlynska Dolina F1, 842 48 Bratislava, Slovakia; [email protected] 2 Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA; [email protected] * Correspondence: [email protected]

Abstract: Chymotrypsin is an important proteolytic enzyme in the human digestive system that cleaves milk proteins through the hydrolysis reaction, making it an interesting subject to study the activity of milk proteases. In this work, we compared detection of chymotrypsin by spectrophotometric dynamic light scattering (DLS) and quartz crystal microbalance (QCM) methods and determined the limit of chymotrypsin detection (LOD), 0.15 ± 0.01 nM for spectrophotometric, 0.67 ± 0.05 nM for DLS and 1.40 ± 0.30 nM for QCM methods, respectively. The sensors are relatively cheap and are able to detect chymotrypsin in 3035 min. While the optical detection methods are simple to implement, the QCM method is more robust for sample preparation, and allows detection of chymotrypsin in non-transparent samples. We give an overview on methods and instruments for detection of chymotrypsin and other milk proteases.

Keywords: chymotrypsin; β-casein; nanoparticles; UV-vis spectroscopy; dynamic light scattering; quartz crystal microbalance

1. Introduction Citation: Piovarci, I.; Hianik, T.; Proteases represent a very wide and important group of enzymes found in a broad Ivanov, I.N. Detection of range of biological systems [1]. Proteases play an important role in the digestion process Chymotrypsin by Optical and and participate in various pathological processes [2,3]. Chymotrypsin is a serine protease Acoustic Methods. Biosensors 2021, 11, present in the human digestive system that participates in protein cleavage in the in- 63. https://doi.org/10.3390/ testines [4]. Together with trypsin, chymotrypsinogen is ejected into the duodenum, where bios11030063 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 Received: 30 January 2021 plasmin is correlated to the quality of milk where the protease cleaves the proteins, mainly Accepted: 23 February 2021 Published: 26 February 2021 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

Publisher’s Note: MDPI stays neutral to reduce post-operation complications after cataract surgery [7]. Measuring chymotrypsin with regard to jurisdictional claims in activity can also be used for differential diagnosis [8]. published maps and institutional afﬁl- Thus, development of sensitive, inexpensive, fast, and easy to use methods for detec- iations. tion 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 yet 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 Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. of the protease concentration is based on enzyme-linked immunosorbent assay (ELISA) This article is an open access article with a limit of detection (LOD) of about 0.5 nM for chymotrypsin [9,10]. However, the distributed under the terms and above-mentioned methods do not allow study of the kinetics of substrate digestion. conditions of the Creative Commons In this paper we test three methods for chymotrypsin detection: QCM, spectrophoto- Attribution (CC BY) license (https:// metric, and DLS. creativecommons.org/licenses/by/ The QCM method is based on measurement of the resonant frequency, f, of shearing 4.0/). oscillations of AT-cut quartz crystal, as well as motional resistance, Rm, and is also known

Biosensors 2021, 11, 63. https://doi.org/10.3390/bios11030063 https://www.mdpi.com/journal/biosensors Biosensors 2021, 11, 63 2 of 14

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 Rm 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.

2 1/2 ∆f = −2fo ∆m/A(µqρq) , (1)

where fo is the fundamental resonant frequency (Hz), A is the active crystal area (in our 2 −3 case: 0.2 cm ), ρq is quartz density (2.648 g cm ), ∆m is the mass change (g), ρq is the shear modulus of the crystal (2.947 × 1011 g cm−1 s−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 Rm. 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, Rm. Chymotrypsin will cleave β-casein, which results in an increase in f and decrease in Rm values. The mass sensitive QCM method was used for the detection of trypsin activity using synthesized peptide chains [12]. Poturnayova et al. used β- casein 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 Biosensors 2021, 11, 63 3 of 14

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 Interna- tional Electronic Conference on Biosensors [23].

2. Materials and Methods 2.1. Chemicals

Auric acid (HAuCl4), sodium citrate, β-casein (Cat. No. C6905), 6-mercapto-1-hexanol (MCH, Cat. No. 725226), phosphate buffered saline (PBS) tablets (Cat. No. P4417), 11-mercaptoundecanoic acid (MUA, Cat. No. 450561), N-(3-Dimetylaminopropyl)-N0- etylcarbodiimid (EDC, Cat. No. E6383), N-Hydroxisuccinimid (NHS, Cat. No. 130672) and α-chymotrypsin (Cat. No. C3142) were of highest purity and purchased from Sigma- Aldrich (Darmstadt, Germany). Standard chemicals (p.a. grade), NaOH, HCl NaOH, NH3, and H2O2 were from Slavus (Bratislava, Slovakia). Deionized water was prepared by Purelab Classic UV (Elga, High Wycombe, UK).

2.2. Spectrophotometric UV-vis Method Gold nanoparticles (AuNPs) were prepared by modified citrate method [24]. In short, ◦ 100 mL of 0.01% chloroauric acid (HAuCl4) was heated at around 98 C and then 5 mL of 1% sodium tris-citrate was added. This solution was maintained at the temperature 98 ◦C and stirred by magnetic stirrer until it turned deep red (for about 15 min). Then the solution of AuNPs was cooled down and stored in the dark. To modify the gold nanoparticles with β-casein, we added 2 mL of 0.1 mg/mL aqueous β-casein into 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. MCH removes the surface charge of nanoparticles and thus facilitates their aggregation [20]. This is reflected by a color change to violet. However, nanoparticles (NPs) are protected from full aggregation due to the presence of a β-casein layer. Addition of chymotrypsin caused cleavage of β-casein, and as a result, the NPs aggregate. This was reflected by changes of the color of the solution to blue and then it became colorless. For the experiments, we prepared 0.95 mL of NPs. Chymotrypsin was dissolved in deionized water and 0.05 mL of chymotrypsin from the stock solution (concentration 100 nM) was added to each cuvette (1 mL standard cuvette, type UV transparent, Sarstedt, Nümbrecht, Germany). The concentration of chymotrypsin in cuvettes was 0.1, 0.3, 0.5, 0.7, 1; 5, and 10 nM at 1 mL of the 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 (total volume 1 mL). The spectra of the AuNPs were measured before protease addition (t = 0 min), just after protease addition (approximately 30 s) and then every 15 min up to 60 min. The measurements were repeated 3 times. The value of absorbance at time t = 0 has been multiplied by the dilution factor to correct the changes in absorbance intensity caused by the initial protease addition. Absorbance was measured by UV-1700 spectrophotometer (Shimadzu, Kyoto, Japan). The scheme of AuNPs modification and chymotrypsin cleavage is presented in Figure1. BiosensorsBiosensors2021 2021, ,11 11,, 63 x FOR PEER REVIEW 44 of 1414

Figure 1. The scheme of gold nanoparticles (AuNPs) modification by 6-mercapto-1-hexanol (MCH) andFigureβ-casein 1. The and scheme cleavage of gold of β nanoparticles-casein by chymotrypsin. (AuNPs) modification by 6-mercapto-1-hexanol 2.3.(MCH) DLS and Method β-casein and cleavage of β-casein by chymotrypsin. 2.3. DLSThe AuNPsMethod prepared as described in Section 2.2. were incubated with 0.1 mg/mL of aqueous β-casein solution overnight in a volume ratio of 1:9 of β-casein to AuNPs (MCH The AuNPs prepared as described in Section 2.2. were incubated with 0.1 mg/mL of was not used in this case). Addition of the chymotrypsin to the solution of AuNPs modified aqueous β-casein solution overnight in a volume ratio of 1:9 of β-casein to AuNPs (MCH by β-casein did not lead to any discernible color change; however, it was possible to detect thewas decreased not used sizein this of thecase). AuNPs Addition using of ZetaSizer the chymotrypsin Nano ZS (Malvernto the solution Instruments, of AuNPs Malvern, modi- UK).fied by First, β-casein the size did of not AuNPs lead to was any measured discernible in color 1 mL change; standard however, cuvette it (1 was mL possible standard to cuvette,detect the type decreased UV transparent, size of the Sarstedt, AuNPs Nümbrecht, using ZetaSizer Germany). Nano ZS Then (Malvern the 0.1 mLInstruments, of water solutionMalvern, containing UK). First, various the size chymotrypsin of AuNPs was concentrations measured in was 1 mL added standard to each cuvette cuvette. (1 ThemL finalstandard concentration cuvette, type of chymotrypsin UV transparent, in Sars cuvettestedt, Nümbrecht, was 0.1, 0.3, Germany). 0.5, 0.7, 1; 5Then and the 10 0.1 nM mL at 1.1of water mL total solution volume containing of solution. various The size chymotrypsin of nanoparticles concentrations was measured was beforeadded additionto each cu- of chymotrypsinvette. The final (t concentration= 0) right after of the chymotrypsin addition (approximately in cuvettes was 1 min) 0.1, 0.3, and 0.5, after 0.7, 30 1; min. 5 and 10 nM at 1.1 mL total volume of solution. The size of nanoparticles was measured before 2.4.addition Quartz of Crystal chymotrypsin Microbalance (t = 0) (QCM) right after Method the addition (approximately 1 min) and after 30 min.The acoustic QCM sensor was prepared using an AT-cut quartz piezocrystals (fo = 8 MHz, ICM, Oklahoma, OK, USA) with sputtered thin gold layers of an area A2.4. = 0.2Quartz cm2 Crystal, that served Microbalance as electrodes. (QCM) First,Method the crystal was carefully cleaned as follows. It wasThe exposed acoustic to QCM a basic sensor Piranha was solutionprepared (H using2O2 :NHan AT-cut3:H2O quartz = 1:1:5 piezocrystals mL). The crystals (fo = 8 wereMHz, immersed ICM, Oklahoma, for 25 min OK, in USA) this solution,with sputtered in beakers thin ingold a water layers bath of an (temperature area A = 0.2 wascm2, approximatelythat served as electrodes. 75 ◦C). Subsequently, First, the crystal the crystalswas carefully were withdrawn,cleaned as follows. rinsed It with was distilled exposed water,to a basic and Piranha returned solution to thebeaker (H2O2:NH with3:H a2 newO = 1:1:5 dose mL). of Piranha The crystals solution were on immersed the reverse for side 25 ofmin the in crystal. this solution, This was in beakers repeated in threea water times. bath On (temperature the last extraction, was approximately the crystals 75 were °C). washedSubsequently, three timesthe crystals with distilled were withdrawn, water and ri thennsed with ethanoldistilled andwater, placed and returned in a bottle to containingthe beaker with ethanol a new for dose storage of Piranha at room solution temperature. on the reverse The clean side crystalof the crystal. was incubated This was overnightrepeated three for 16–18 times. h atOn room the last temperature extraction with, the 2 crystals mM MUA were dissolved washedin three ethanol. times MUA with isdistilled a carboxylic water acidand withthen with a sulfide ethanol group and (SH). placed The in sulfide a bottle moiety containing interacts ethanol with for the storage gold onat room the crystal temperature. to form The a self-assembled clean crystal was layer. incubated After incubation, overnight thefor 16–18 crystal h wasat room washed tem- withperature ethanol, with distilled 2 mM MUA water, dissolved and 20 mM in ethanol. EDC and MUA 50 mM is a NHS carboxylic were appliedacid with for a 25sulfide min. Thesegroup substances(SH). The sulfide react withmoiety the interacts carboxyl with moiety the gold of MUA on the and crystal activate to form them a self-assem- to form a covalentbled layer. bond After with incubation, amino acids. the crystal Subsequently, was wa theshed crystal with ethanol, was washed distilled by distilled water, and water, 20 driedmM EDC with and nitrogen, 50 mM and NHS placed were inapplied an acrylic for 25 flow min. cell These (JKU substances Linz, Austria). react Thewith cell the was car- filledboxyl withmoiety PBS of buffer MUA using and activate a Genie them plus 2011to form step a pumpcovalent (Kent bond Scientific, with amino Torrington, acids. Sub- CT, USA)sequently, at a flow the crystal rate of 200wasµ washedL/min. by After distill fillinged water, the cell, dried we switchedwith nitrogen, the flow and to placed the rate in µ β ofan 50 acrylicL/min. flow Then,cell (JKU 1 mg/mL Linz, Austria). of -casein The dissolved cell was filled in PBS with was PBS allowed buffer to using flow a under Genie theplus crystal 2011 step modified pump by(Kent MUA Scientific, layer. AfterTorrington, 35 min, CT, only USA) pure at PBSa flow was rate flowed of 200 in μL/min. order β β toAfter remove filling the the unbound cell, we switched-casein. the All flow steps to ofthe the rate preparation of 50 μL/min. of Then,-casein 1 mg/mL layer were of β- casein dissolved in PBS was allowed to flow under the crystal modified by MUA layer. Biosensors 2021, 11, x FOR PEER REVIEW 5 of 14

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After 35 min, only pure PBS was flowed in order to remove the unbound β-casein. All steps of the preparation of β-casein layer were recorded using a research quartz crystal recordedmicrobalance using (RQCM) a research instrument quartz crystal (Maxtek, microbalance East Syracuse, (RQCM) NY, instrumentUSA). (Maxtek, East Syracuse,After NY,binding USA). of β-casein to the electrode surface and stabilizing the resonant fre- quencyAfter (washing binding out of βall-casein unbound to the residues), electrode we surface applied and chymotrypsin stabilizing the to resonantthe crystal fre- at quencyconcentrations (washing of out1 pM, all 10 unbound pM, 100 residues),pM, 1 nM, we 10 appliednM and chymotrypsin20 nM. After 35 to min the of crystal chymo- at concentrationstrypsin application, of 1 pM,the PBS 10 pM, was 100let to pM, flow 1 nM,into 10the nM cell and until 20 the nM. resonant After 35 frequency min of chy- sta- motrypsinbilized. The application, change in casein the PBS coated was QCM let to re flowsonant into frequency the cell until from the application resonant frequencyof chymo- stabilized.trypsin to stabilization The change in in PBS casein corresponds coated QCM to the resonant amount of frequency casein cleaved from applicationfrom the layer. of chymotrypsinAfter lower concentrations to stabilization (1 in pM, PBS 10 corresponds pM, 100 pM), to the we amountapplied of a caseinhigher cleavedconcentration from the of layer.chymotrypsin After lower (at least concentrations at a concentration (1 pM, 102 or pM,ders 100 of pM),magnitude we applied higher a). higher In such concentra- measure- tionments, of chymotrypsinwe analyzed the (at degree least at of a cleavage concentration as the change 2 orders in of frequency magnitude from higher). the initial In such state measurements,to a steady-state we value. analyzed All measuremen the degreets of were cleavage performed as the changeat PBS, inpH frequency 7.4. from the initialFor state optical to a steady-state and gravimetric value. methods, All measurements the limit of were detection performed (LOD) at PBS,was determined pH 7.4. usingFor following optical andEquation: gravimetric methods, the limit of detection (LOD) was determined using following Equation: LODLOD = 3.3 = 3.3× (SD)/S,× (SD)/S, (2)(2) wherewhere SDSD isis standardstandard deviationdeviation ofof thethe samplesample withwith lowestlowest concentrationconcentration andand SS isis slopeslope determineddetermined fromfrom fitfit ofof linearlinear partpart ofof thethe calibrationcalibration curve.curve. TheThe sequencesequence ofof QCMQCM operationoperation includingincluding surfacesurface modificationmodification andand sensingsensing cleavagecleavage ofofβ β-casein-casein byby chymotrypsinchymotrypsin usingusing QCMQCM piezocrystalpiezocrystal andand isis presentedpresented inin FigureFigure2 .2.

Figure 2. The scheme of modification of the piezocrystal and the cleavage of β-casein byFigure chymotrypsin. 2. The scheme of modification of the piezocrystal and the cleavage of β-casein by chymotrypsin. 3. Results and Discussion 3.1.3. Results Detection and of Discussion Chymotrypsin by Optical Method 3.1. DetectionIn the first of seriesChymotrypsin of experiments, by Optical we Method studied the cleavage of the β-casein at the surface of theIn AuNPs the first by series UV-vis of experiments, and DLS methods. we studied AuNPs the modified cleavage byof theβ-casein β-casein and at MCH the surface were usedof the in AuNPs optical by detection UV-vis method. and DLS Figure methods.3 shows AuNPs the changemodified in theby β absorption-casein and spectra MCH afterwere eachused stepin optical of AuNPs detection modification. method. TheFigure modification 3 shows the of AuNPschange within theβ -caseinabsorption resulted spectra in aafter shift each of the step maximum of AuNPs of modification. absorbance byThe around modification 5 nm toward of AuNPs higher with wavelengths β-casein resulted and inin aa slightshift of increase the maximum in absorbance. of absorbance After by addition around of 5 nm MCH toward which higher replaces wavelengths the β-casein and protectivein a slight layerincrease leads in toabsorbance. broadening After of absorption addition of peak, MCH and which shifts replaces by 60 nm the toward β-casein higher pro- wavelengths,tective layer leads indicating to broadening increase in of size absorption due to aggregation peak, and ofshifts AuNP. by The60 nm results toward agree higher well withwavelengths, Ref. [20] forindicating AuNPs modifiedincrease in by size gelatin due andto aggregation MCH. of AuNP. The results agree well with Ref. [20] for AuNPs modified by gelatin and MCH. BiosensorsBiosensors 2021 ,2021 11, x, 11FOR, x FORPEER PEER REVIEW REVIEW 6 of 614 of 14

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1.6 unmodified gold nanoparticles 1.6 unmodified gold nanoparticles β−casein 1.4 β−casein 1.4 MCH + β−casein MCH + β−casein 1.2 1.2 525 525 520 1.0 1.0 520 584 584 0.8 0.8

0.6 0.6 Absorbance Absorbance 0.4 0.4

0.2 0.2

0.0 0.0 300300 400 400 500 500 600 600 700 700 800 800 Wavelength (nm) Wavelength (nm) Figure 3. Absorption spectra of unmodified gold nanoparticles (AuNP) (black) and those modified FigureFigure 3. Absorption 3. Absorption spectra spectra of unmodified of unmodified gold gold nano nanoparticlesparticles (AuNP) (AuNP) (black) (black) and and those those modified modified by β-casein (red) and by β-casein + MCH (blue). The numbers at upper part of absorption peaks byby β-caseinβ-casein (red) (red) and and by by β-caseinβ-casein + MCH + MCH (blue). (blue). The The numbers numbers at atupper upper part part of ofabsorption absorption peaks peaks are are wavelengths in nm. arewavelengths wavelengths in in nm. nm. The changes of absorbance spectra of AuNPs suspension have been measured during TheThe changes changes of ofabsorbance absorbance spectra spectra of ofAuNP AuNPss suspension suspension have have been been measured measured during during thethe chymotrypsinthe chymotrypsin chymotrypsin cleavage cleavage cleavage at at 0 at 0min, min,0 min, 0.5 0.5 min,0.5 min,15 min,15 min,15 min, min, 30 30 min min30 min and and and 60 60 min. min.60 min. Changes Changes Changes in in spec- spectra in spec- traover overtra over time time time for for two twofor differenttwo different different concentration concentration concentration of of chymotrypsin chymotrypsin of chymotrypsin are are presented arepresented presented in in Figure Figure in Figure4. 4. At 4. Ata aAt relativelyrelatively a relatively lowlow low concentration concentration of chymotryps chymotrypsin of chymotrypsin (0.1 (0.1in (0.1nM), nM), nM), we we didwe did didnot not observenot observe observe significant significant significant changeschangeschanges of ofthe of the theabsorbance absorbance absorbance (Figure (Figure (Figure 2a).4a). 2a).However, However, However, at at higher at higher higher chymotrypsin chymotrypsin chymotrypsin concentration, concentration, concentration, aroundaroundaround 10 10nM, 10 nM, nM, a substantial a a substantial substantial red red redshift shiftshift of the ofof thesptheectra spectraspectra was was observed observed (up (upto (up 615 to to 615nm). 615 nm).nm). It can It It canbe can be alsobealso seen also seen that seen that after that after maximum after maximum maximum shifting shifting shifting substa substa substantialntialntial decrease decrease decrease of the of theabsorbance of theabsorbance absorbance with with time with time occurred.timeoccurred. occurred.

1.4 0 min 1.4 0 min 1.4 0 min 1.4 0 min 0.5 min 0.5 min 0.5 min 0.5 min 1.2 15 min 1.2 15 min 1.2 15 min 1.2 15 min 30 min 30 min 30 min 30 min 1.0 45 min 1.0 45 min 1.0 45 min 1.0 45 min 60 min 60 min 60 min 60 min 0.8 0.8 0.8 0.8 0.6 0.6 0.6 0.6 Absorbance Absorbance Absorbance Absorbance 0.4 0.4 0.4 0.4 0.2 0.2 0.2 0.2 (a) (a) (b) (b) 0.0 0.0 0.0 0.0 300 400 500 600 700 800 300300 400 400 500 500 600 600 700 700 800 800 300 400 500 600 700 800 Wavelength (nm) WavelengthWavelength (nm) (nm) Wavelength (nm) FigureFigureFigure 4. 4.Changes 4.Changes Changes of ofabsorbance of absorbance absorbance spectra spectra spectra of of the theof suspensionthe suspension suspension of of AuNPs AuNPs of AuNPs modiﬁedmodified modified byby ββ -caseinby-casein β-casein andand MCHandMCH MCH in in time time in fortime for (a ()fora 0.1) 0.1 (a nM) 0.1 nMchymotrypsin chymotrypsinnM chymotrypsin and and (b and()b 10) 10 ( nMb nM) 10 chymotrypsin. chymotrypsin.nM chymotrypsin.

FigureFigureFigure 5 5shows shows5 shows the the absorbancethe absorbance absorbance and and and change change change of of maximum of maximum maximum position position position of of absorbance of absorbance absorbance peak peak peak in intimein time time for for allfor all concentrations all concentrations concentrations of ofchymotrypsin of chymotrypsin chymotrypsin studied. studied. studied. The rate of decrease in AuNP absorbance is higher for concentration of chymotrypsin 5 and 10 nM, and at lower concentration of chymotrypsin the rate of change is much slower (Figure5a). The maximum position of absorbance peak shifted with time substantially at higher chymotrypsin concentrations (5 and 10 nM). For 5 and 10 nM chymotrypsin the maximum position of absorption peak was stabilized at around 615 nm, while for lower concentrations it increased with time almost linearly (Figure5b). In order to prepare the calibration curve, we ﬁt the change of absorbance vs. time with linear curve and then differentiated this model numerically to obtain the values of dA/dt. The calibration curve is presented on Figure6a. Biosensors 2021, 11, x FOR PEER REVIEW 7 of 14

630 0.90 0 nM (a) 0.1 nM (b) 620 0.85 0.3 nM 0.5 nM 0.80 610 0.7 nM 1 nM 0.75 5 nM 0 nM 600 10 nM 0.1 nM Biosensors 20210.70, 11, x FOR 0.3 nM PEER REVIEW 7 of 14 Biosensors 2021, 11, 63 0.5 nM 590 7 of 14 Absorbance 0.65 0.7 nM 1 nM 0.60 5 nM 580 10 nM 0.55 (nm) position peak Abs. 570 0 102030405060 630 0 102030405060 0.90 0 nM (a) Time (min) 0.1 nM Time (min) (b) 620 0.85 0.3 nM Figure 5. Changes in the absorbance (a) and in maximum position of absorbance 0.5 peak nM (b) vs. time for different chymo- 0.80 610 0.7 nM trypsin concentrations in a suspension of AuNPs modified by β-casein and MCH. 1 nM The results represent mean ± SD obtained 0.75from 3 independent measurements at each concentration of chymotrypsin. 5 nM 0 nM 600 10 nM 0.1 nM 0.70 0.3 nM 0.5 nM The rate of decrease in AuNP absorbance590 is higher for concentration of chymotrypsin

Absorbance 0.65 0.7 nM 5 and 10 nM, and at lower concentration of chymotrypsin the rate of change is much 1 nM 580 0.60 5 nM slower (Figure 5a). The maximum position of absorbance peak shifted with time substan- 10 nM 0.55 tially at higher chymotrypsin concentrations(nm) position peak Abs. 570 (5 and 10 nM). For 5 and 10 nM chymotryp- 0 102030405060 sin the maximum position of absorption peak 0was 102030405060stabilized at around 615 nm, while for Time (min) Time (min) lower concentrations it increased with time almost linearly (Figure 5b). In order to prepare FigureFigure 5. Changes5. Changes in the in absorbancethe absorbancethe calibration (a) and (a) inand maximumcurve, in maximum we position fit the position change of absorbance of ofabsorbance ab peaksorbance (b peak) vs. vs. time(b time) vs. for timewith different for linear different chymotrypsin curve chymo- and then concentrationstrypsin concentrations in a suspension in adifferentiated ofsuspension AuNPs modiﬁed of thisAuNPs model by βmodified-casein numerically and by MCH.β-casein to The obtain and results MCH. the represent values The results of mean dA/dt. represent± SD The obtained meancalibration from± SD 3ob- curve independenttained from measurements 3 independent atis measurements eachpresented concentration on at Figure each of chymotrypsin.concentration 6a. of chymotrypsin.

The rate of decrease in AuNP absorbance18 is higher for concentration of chymotrypsin 18 (a) 5 and 10 nM, and at lower concentration16 (b)of chymotrypsin the rate of change is much 16 slower (Figure 5a). The maximum position14 of absorbance peak shifted with time substan-

) 14 ) 1 - 12 tially at higher chymotrypsin concentrations-1 12 (5 and 10 nM). For 5 and 10 nM chymotryp- 10 sin the maximum position of absorption10 peak was stabilized at around 615 nm, while for 8 lower concentrations it increased with time8 almost linearly (Figure 5b). In order to prepare 6 the calibration curve, we fit the change of6 absorbance vs. time with linear curve and then 4 dA/dt (min differentiated this model numerically dA/dt (min to4 obtain the values of dA/dt. The calibration curve − 2 is presented on Figure 6a. − 2 0 0 −2 −182 18 (a)0246810 16 (b)012345 16 Chymotrypsin [nM] 14 Chymotrypsin [nM]

) 14 ) 1 - -1 12 FigureFigure 6. 126.(a ()a Calibration) Calibration curve curve for for chymotrypsin chymotrypsin fitted fitted by reverseby reverse Michaelis—Menten Michaelis—Menten model. model. dA/dt dA/dt is numerical is numerical derivation deriva- 10 oftion linear of 10 modellinear model of absorbance of absorbance change change at time att = time 0 and t = corresponds 0 and corresponds to the rate to ofthe enzyme rate of reaction.enzyme reaction. (b) Linear (b part) Linear of calibra- part of 8 8 tioncalibration curve − dA/dtcurve − vs.dA/dt concentration vs. concentration of chymotrypsin of chymotrypsin for calculation for calculation of limit of of detection limit of (LOD).detection−dA/dt (LOD). = (6.30−dA/dt± 0.23)= (6.30× ± 6−4 −1 −1 −4 −1 2 6 100.23)−4 min × 10−1 nM min−1 nM+ (1.9 +± (1.90.4) ± ×0.4)10 ×− 104 min min−1. (R. (R2 == 0.993),0.993), LODLOD == 0.150.15 ±± 0.010.01 nM. nM. 4 dA/dt (min dA/dt (min 4 − − 2 WeWe were were able able to to use use reverse reverse Michaelis—Menten Michaelis—Menten2 model model to to analyze analyze the the obtained obtained data. data. 0 However,However, instead instead of substrateof substrate concentration, concentration,0 the concentrationthe concentration of chymotrypsin, of chymotrypsin, c, has beenc, has −2 usedbeen in used this model:in this model: v = vmax v [c/(K= vmaxM [c/(K+ c)],M + where −c)],2 where v and v v andmax arevmax the are rate the and rate maximum and maximum rate 0246810 012345 ofrate enzyme of enzyme reaction, reaction, respectively, respectively, and K andM = K 3.89M = ±3.891.24 ± 1.24 nM nM is reverse is reverse Michaelis—Menten Michaelis—Men- Chymotrypsin [nM] Chymotrypsin [nM]2 2 constantten constant obtained obtained from from the fit the using fit using the Michaelis—Menten the Michaelis—Menten model model (R = 0.96)(R = 0.96) (Figure (Figure6b). −3 −1 max ± × −3 −1 Figure 6. (a) Calibration curveIn6b). our Infor case, our chymotrypsin case, v = dA/dt v = dA/dt fitted and andby v maxreverse v = (5.3= Michaelis—Menten(5.3 ±0.9) 0.9) × 1010 minmin model.. However,. dA/dt However, is thisnumerical this model model deriva- was was used tion of linear model of absorbanceusedonly onlyformally change formally because at time because t of= 0 different and of corresponds different restraints restraints. to .the The rate main Theof enzyme assumptions main assumptionsreaction. of ( bthe) Linear excess of the part enzyme excess of calibration curve −dA/dt enzymevs.and concentration limited and limitedsubstrate of chymotrypsin substrate concentration concentration for calculation was reversed wasof limitreversed in ofthis detection case in thisand (LOD). caseinstead, − anddA/dt the instead, = concentra- (6.30 ± the 0.23) × 10−4 min−1 nM−1 + (1.9concentration ± 0.4) × 10−4 min of− the1. (R enzyme2 = 0.993), changedLOD = 0.15 while ± 0.01 the nM. substrate was presented in excess. This implies different meaning of KM (compared to the Michaelis—Menten model) which now representsWe thewere concentration able to use reverse of enzyme Michaelis—Menten at which the rate ofmodel reaction to analyze is half ofthe the obtained maximum data. insteadHowever, of concentration instead of substrate of substrate. concentration, A limitation the of concentration this approach of is chymotrypsin, the assumption c, of has substratebeen used excess; in this nevertheless, model: v = v itmax can [c/(K beM used + c)], for where good v approximationand vmax are the [rate25]. and To calculate maximum LOD,rate weof enzyme used only reaction, part of respectively, the calibration and curve KM = from 3.89 0± 1.24 to 5 nM,nM is where reverse the Michaelis—Men- plot of dA/dt vs.ten c was constant almost obtained linear. The from results the fit are using shown the inMichaelis—Menten Figure6b. model (R2 = 0.96) (Figure 6b).The In our LOD case, of thev = dA/dt optical and method vmax = (5.3 of chymotrypsin ± 0.9) × 10−3 min detection,−1. However, 0.15 this± 0.01model nM, was was used −1 −1 −1 calculatedonly formally from because the Equation of different (2) using restraints SD = 0.29. The min main assumptionsand S = 6.3 minof the excessnM . enzyme This valueand islimited 3.3 times substrate lower thanconcentration that of the was ELISA reversed method in this reported case and in the instead, literature, the concentra- around 0.5 nM [10]. However, in contrast with ELISA which requires specific antibodies, the Biosensors 2021, 11, x FOR PEER REVIEW 8 of 14

tion of the enzyme changed while the substrate was presented in excess. This implies different meaning of KM (compared to the Michaelis—Menten model) which now represents the concentration of enzyme at which the rate of reaction is half of the maximum instead of concentration of substrate. A limitation of this approach is the assumption of substrate excess; nevertheless, it can be used for good approximation [25]. To calculate LOD, we used only part of the calibration curve from 0 to 5 nM, where the plot of dA/dt vs. c was almost linear. The results are shown in Figure 6b. The LOD of the optical method of chymotrypsin detection, 0.15 ± 0.01 nM, was calculated from the Equation (2) using SD = 0.29 min−1 and S = 6.3 min−1 nM−1. This value is 3.3 times lower than that of the ELISA method reported in the literature, around 0.5 nM Biosensors 2021, 11, 63 8 of 14 [10]. However, in contrast with ELISA which requires specific antibodies, the method based on AuNPs is much easier and faster. Detection time of chymotrypsin using the optical method is about 30 min. The detection of the chymotrypsin with β-casein and MCH modifiedmethod AuNPs based on can AuNPs be done is much in one easier step. and The faster. disadvantage Detection of time this of method chymotrypsin is that only using transparentthe optical samples method can is about be used 30 min. for detection. The detection This restriction of the chymotrypsin can be lifted with usingβ-casein the sur- and faceMCH sensitive modified gravimetric AuNPs canmethod be done (see inSection one step. 3.3).The disadvantage of this method is that only transparent samples can be used for detection. This restriction can be lifted using the 3.2.surface Detection sensitive of Chymotrypsin gravimetric by method DLS Method (see Section 3.3). We measured the Z-average size of the non-modified AuNPs, which was found to be 3.2. Detection of Chymotrypsin by DLS Method around 20 nm. The size is bigger than the assumed size of the prepared AuNPs of around 15 nm We[22]. measured This is explained the Z-average by the size fact ofthat the DLS non-modified technique tends AuNPs, to whichoverestimate was found the size to be ofaround the gold 20 nanoparticles nm. The size isdue bigger to the than hydrat theion assumed sphere size around of the the prepared AuNPs. AuNPs The Z-average of around size15 of nm the [22 AuNPs]. This ismodified explained with by β the-casein fact that was DLS around technique 35 nm. tends Figure to 7. overestimate shows the plot the sizeof theof Z-average the gold nanoparticles size of AuNPs due modified to the hydration by β-casein sphere at time around 0 and the 30 AuNPs. min at The presence Z-average of β varioussize of concentrations the AuNPs modified of chymotrypsin. with -casein Incubation was around of AuNPs 35 nm. with Figure chymotrypsin7. shows the plotre- of the Z-average size of AuNPs modified by β-casein at time 0 and 30 min at presence of sulted in decrease of Z-average size, which is more remarkable at presence of 5 nM and various concentrations of chymotrypsin. Incubation of AuNPs with chymotrypsin resulted 10 nM protease concentrations. The variation in AuNP size at time 0 is related to the orig- in decrease of Z-average size, which is more remarkable at presence of 5 nM and 10 nM inal size of nanoparticles, as well as rather fast cleavage of casein by protease, especially protease concentrations. The variation in AuNP size at time 0 is related to the original size at its higher concentrations. However, even at relatively high chymotrypsin concentra- of nanoparticles, as well as rather fast cleavage of casein by protease, especially at its higher tions (10 nM) the average size did not reach those of naked AuNPs. This is evidence that concentrations. However, even at relatively high chymotrypsin concentrations (10 nM) the cleavage was not complete and there is still a residual β-casein layer around AuNPs. This average size did not reach those of naked AuNPs. This is evidence that cleavage was not also explains why incomplete aggregation was observed in UV-vis experiments. complete and there is still a residual β-casein layer around AuNPs. This also explains why incomplete aggregation was observed in UV-vis experiments.

60 0 nM 55 0.1 nM 50 0.3 nM 0.5 nM 45 0.7 nM 1 nM 40 5 nM 10 nM size (nm) 35 30

average 25 Z 20 15 01 2830

Time (min) FigureFigure 7. 7.ChangeChange in inZ-average Z-average size size of of AuNPs AuNPs modified modiﬁed by by ββ-casein-casein at at time time 0 0and and 30 30 min min at at pres- presence enceof variousof various concentrations concentrations of chymotrypsin of chymotrypsin (see (s theee insert).the insert). The The results results represent represent mean mean±SD ±SD obtained obtainedfrom 3 independentfrom 3 independent measurements measurements at each concentrationat each concentration of chymotrypsin. of chymotrypsin.

We also constructed a calibration curve based on the percentual change of Z-average size in 30 min (Figure8a) and ﬁtted this by reverse Michaelis—Menten model. About 25% of the Z-average size was observed in 30 min. However, we can also see that the data has quite large (5 nm) standard deviation (obtained from 3 independent experiments at each concentration of chymotrypsin) affecting accuracy of concentration measurements, and the LOD of the sensor. Nevertheless, it is still a useful method to detect presence of chymotrypsin in the sample. The standard deviation could be improved by increasing number of measurements of the sample. It is important to note that the enzyme reaction was used without buffering the solution, which could also lead to a large value of standard deviation. The recommended buffer for chymotrypsin is 100 mM Tris-HCl at pH 7.8 (optimum pH) containing 10 mM CaCl2 for stability. However, since we observed AuNPs aggregation in buffer, the experiments were carried in un-buffered solution. The calibration curve seems to be saturated near the 10 nM chymotrypsin. Biosensors 2021, 11, x FOR PEER REVIEW 9 of 14

We also constructed a calibration curve based on the percentual change of Z-average size in 30 min (Figure 8a) and fitted this by reverse Michaelis—Menten model. About 25% of the Z-average size was observed in 30 min. However, we can also see that the data has quite large (5 nm) standard deviation (obtained from 3 independent experiments at each concentration of chymotrypsin) affecting accuracy of concentration measurements, and the LOD of the sensor. Nevertheless, it is still a useful method to detect presence of chymotrypsin in the sample. The standard deviation could be improved by increasing number of measurements of the sample. It is important to note that the enzyme reaction was used without buffering the solution, which could also lead to a large value of standard deviation. The recommended buffer for chymotrypsin is 100 mM Tris-HCl at pH 7.8 (op-

Biosensors 2021, 11, 63 timum pH) containing 10 mM CaCl2 for stability. However, since we observed AuNPs9 of 14 aggregation in buffer, the experiments were carried in un-buffered solution. The calibration curve seems to be saturated near the 10 nM chymotrypsin.

35 30 (a) 30 25 (b)

25 20 20 15 15 10 10 size (%) size (%)

Δ Δ 5 5 0 0

−5 −5 0246810 0.0 0.2 0.4 0.6 0.8 1.0 Chymotrypsin [nM] Chymotrypsin [nM]

FigureFigure 8. 8.(a ()a The) The dependence dependence of of the the changes changes of of Z-average Z-average size size (∆ (size)Δsize) vs. vs. concentration concentration of of chymotrypsin chymotrypsin for for suspension suspension of of AuNPsAuNPs modified modified by byβ -caseinβ-casein measured measured by by DLS DLS method. method. The The curve curve represents represents fit fit according according to to reverse reverse Michaelis—Menten Michaelis—Menten model (see above). Δsize = [Zaverage(30 min)−Zaverage(0 min)]/Zaverage(0 min). (b) The linear part of calibration curve (red) used model (see above). ∆size = [Zaverage(30 min)−Zaverage(0 min)]/Zaverage(0 min). (b) The linear part of calibration curve (red) for calculation of LOD (Δsize = 12.47% nM−1 c + 2.71%, R2 = 0.87, where c is the concentration of chymotrypsin). For com- used for calculation of LOD (∆size = 12.47% nM−1 c + 2.71%, R2 = 0.87, where c is the concentration of chymotrypsin). For parison we show first order reaction fit (blue color) (Δsize = 15.09%−13.83%e−(c−0.006 nM)/0.45nM, R2 = 0.998, where c is the con- comparison we show first order reaction fit (blue color) (∆size = 15.09%−13.83%e−(c−0.006 nM)/0.45nM,R2 = 0.998, where c is centration of chymotrypsin). The results represent mean ± SD obtained from 3 independent measurements at each con- thecentration concentration of chymotrypsin. of chymotrypsin). The results represent mean ± SD obtained from 3 independent measurements at each concentration of chymotrypsin. Using a Michaelis—Menten reverse model from the fit of the results presented on Using a Michaelis—Menten reverse model from the fit of the results presented on Figure 8a we obtained for KM = 1.03 ± 0.26 nM (R2 = 0.998). This value is almost four times Figure8a we obtained for K = 1.03 ± 0.26 nM (R2 = 0.998). This value is almost four less than that obtained via spectrophotometricM methods. This can be explained by addition times less than that obtained via spectrophotometric methods. This can be explained by of MCH in the spectrophotometric method, which can interfere with the cleavage of β- addition of MCH in the spectrophotometric method, which can interfere with the cleavage casein and decelerate the reaction. Both optical methods, however, should be able to de- of β-casein and decelerate the reaction. Both optical methods, however, should be able to tect the protease activity with similar precision. We took the linear part of the calibration detect the protease activity with similar precision. We took the linear part of the calibration curve (Figure 8b) and calculated LOD = 0.67 ± 0.05 nM. LOD value was calculated from curve (Figure8b) and calculated LOD = 0.67 ± 0.05 nM. LOD value was calculated from the Equation (2) using SD = 2.54 (%) and S = 12.47 (%) nM−1. This value is 4.5 times higher the Equation (2) using SD = 2.54 (%) and S = 12.47 (%) nM−1. This value is 4.5 times higherin comparison in comparison with those with obtained those obtained by spectrophotometric by spectrophotometric method. method. The possible The possible reason is less reproducible data in the case of Zaverage measurement in comparison with the absorb- reason is less reproducible data in the case of Zaverage measurement in comparison with theance absorbance method. method.The time Theof measurement time of measurement is practically is practically the same the for same both foroptical both methods. optical methods.In the case In theof the case DLS of the method, DLS method, the preparatio the preparationn of the of AuNPs the AuNPs is by is one by onestep step easier easier and andsince since there there is isno no MCH MCH in in the the sample sample the the AuNP AuNPss are are more stablestable thanthan thosethose used used in in the the spectrophotometricspectrophotometric method. method.

3.3.3.3. Detection Detection of of Chymotrypsin Chymotrypsin by by Gravimetric Gravimetric Method Method ForFor the the gravimetric gravimetric method, method, we we first first modified modified the the surface surface of of the the QCM QCM piezocrystal piezocrystal withwith MUA MUA and and then then byby ββ-casein.-casein. By By monitoring monitoring resonant resonant frequency, frequency, f, f,and and motional motional re- resistance,sistance, R Rmm, ,it it was was possible possible to studystudy allall stepssteps ofof preparationpreparation of of the the sensing sensing surface. surface. The The valuevalue of of motional motional resistance resistance reflects reflects the the viscosity viscosity contribution contribution caused caused by by non-ideal non-ideal slip slip between β-casein-layer and the surrounding water environment [26]. This is presented in Figure9. The activation of carboxylic groups of MUA with EDC/NHS lead to only a small shift in resonance frequency. The addition of β-casein resulted in a fast drop of resonant frequency of about 170 Hz. After changing the flow with buffer, the frequency increased due to the removal of nonspecifically bound β-casein. The resulting frequency shift after washing of the surface corresponded to 120 Hz. From the Sauerbrey equation, we can calculate the change in mass on QCM biosensor, which corresponded to about 165 ng of mass added. With the knowledge of the molecular weight of β-casein Mw = 24 kDa, we could calculate the surface density of β-casein: Г = 34.5 pM/cm2. From the changes in motional resistance Rm, we could estimate the contribution of surface viscosity into resonant frequency. Since the Rm value decreases and increases proportionally to the change of frequency on a rather small value, the change in motional resistance is caused mainly by added weight. Therefore, one can assume that the β-casein layer was relatively rigid, which justifies application of the Sauerbrey equation. Biosensors 2021, 11, x FOR PEER REVIEW 10 of 14

between β-casein-layer and the surrounding water environment [26]. This is presented in Figure 9. The activation of carboxylic groups of MUA with EDC/NHS lead to only a small shift in resonance frequency. The addition of β-casein resulted in a fast drop of resonant frequency of about 170 Hz. After changing the flow with buffer, the frequency increased due to the removal of nonspecifically bound β-casein. The resulting frequency shift after washing of the surface corresponded to 120 Hz. From the Sauerbrey equation, we can calculate the change in mass on QCM biosensor, which corresponded to about 165 ng of mass added. With the knowledge of the molecular weight of β-casein Mw = 24 kDa, we could calculate the surface density of β-casein: Г = 34.5 pM/cm2. From the changes in motional resistance Rm, we could estimate the contribution of surface viscosity into resonant frequency. Since the Rm value decreases and increases proportionally to the change of frequency on a rather small value, the change in motional resistance is caused mainly by added weight. Therefore, one can assume that the β-casein layer was relatively rigid, Biosensors 2021, 11, 63 which justifies application of the Sauerbrey equation. 10 of 14

50 4 EDC/NHS 1 mg/mL β-casein 0 2

−50 R

0 )

−100 Ω ( −2 m

f (Hz) −150 R Δ

−4 Δ −200 f −250 −6 H2O PBS PBS −300 −8 0 20 40 60 80 100 120 140 Time (min)

Figure 9. Kinetics of resonant frequency, f (blue), and motional resistance, Rm (black), changes dur- Figure 9. Kinetics of resonant frequency, f (blue), and motional resistance, Rm (black), changes during ingmodiﬁcation modification of of piezocrystal piezocrystal by byβ -casein.β-casein. The The carboxylic carboxylic groups groups of of MUA MUA that that were were chemisorbed chemi- at sorbed at the crystal were first activated by EDC/NHS. The moments of addition of various com- the crystal were ﬁrst activated by EDC/NHS. The moments of addition of various compounds as pounds as well as washing the surface by water and PBS are shown by arrows. well as washing the surface by water and PBS are shown by arrows.

AfterAfter ββ-casein-casein was was bound bound to to the surface,surface, wewe could could study study its its cleavage cleavage by by different different con- concentrationscentrations of chymotrypsinof chymotrypsin under under ﬂow flow condition condition for 35 min.for 35 An min. example An example of the changes of the of changesresonant of frequency resonant frequency and motional and resistance motional following resistance the following addition the of 10addition nM chymotrypsin of 10 nM chymotrypsin are shown in Figure 10. In the presence of chymotrypsin, the resonant fre- Biosensors 2021, 11, x FOR PEER REVIEWare shown in Figure 10. In the presence of chymotrypsin, the resonant frequency increased11 of 14 quencyby 35 Hz,increased but motional by 35 Hz, resistance but motional decreased resistance by 1.6 Ω decreased. This is clear by 1.6 evidence Ω. This of is the clear cleavage evi- denceof β-casein of the cleavage by chymotrypsin. of β-casein Decrease by chymotrypsin. of motional Decrease resistance of motional can be due resistance to an increase can be of due to an increase of molecular slip, which can be caused by weaker viscosity contribu- · molecular slip, which can be caused by weaker viscosity contribution. tion. 6 1 mg/mL β−casein 0 5 −30 4 −60 ) 3 Ω ( − 90 m f (Hz) 2 R

Δ 10 nM chymotrypsin −120 Δ 1 PBS −150 0 −180 PBS −1 0 20406080100120140

Time (min)

FigureFigure 10. 10. KineticsKinetics of ofthe the changes changes of of resonant resonant frequency, frequency, f f(blue), (blue), and and motional resistance, RRmm (black), (black),following following modiﬁcation modifica oftion piezocrystal of piezocrystal by β-casein by β-casein and addition and addition of 10 nM of of10 chymotrypsin.nM of chymotrypsin. Addition Additionof various of various compounds compounds as well asas washingwell as washin of theg surface of the bysurface PBS isby shown PBS is byshown arrows. by arrows.

BasedBased on on the the changes changes of of the the frequency, frequency, we we constructed constructed a calibration curve forfor differentdiffer- entconcentrations concentrations of of chymotrypsin chymotrypsin (Figure (Figure 11 a).11a). For For determination determination of LOD,of LOD, we we also also prepared pre- paredcalibration calibration curve curve at a lowat a concentrationlow concentration range range where where the dependence the dependence was almostwas almost linear linear(Figure (Figure 11b). 11b). The The LOD LOD for gravimetricfor gravimetric detection detection of chymotrypsin, of chymotrypsin, 1.40 1.40± 0.30 ± 0.30 nM nM was wascalculated calculated from from the the Equation Equation (2) (2) using using SD SD = = 2.20 2.20 (%) (%) and and S S == 5.175.17 (%) nM−1.1 .This This value value waswas 2.8 2.8 times times higher higher than than that that reported reported by by ELISA ELISA methods methods and and 9.3 9.3 times times higher higher than than for for opticaloptical method method of ofdetection detection reported reported here. here. We We should should mention mention that that gravimetric gravimetric detection detection requires careful handling of the cell because even small changes in liquid pressure can affect the measurements. This method is, however, more tolerant to the presence of different components in the sample, such as added fat present in natural milk. The gravimetric method of protease detection requires time similar to optical methods. It is also important to mention the uniformity of the modified materials. The changes of frequency in gravimetric methods and absorbance maximum position in optical methods did not differ significantly from sample to sample, suggesting that it is not a source of significant error (large standard deviation).

5 (a) 18 (b) 16 4 14 3 12

2 (%)f Δ 10

8 df/dt (Hz/min) 1 6 0 4 0 5 10 15 20 0.0 0.2 0.4 0.6 0.8 1.0

Chymotrypsin [nM] Chymotrypsin [nM] Figure 11. (a) Calibration curve for chymotrypsin fitted by reverse Michaelis—Menten model, v = df/dt was the first derivation of frequency obtained from kinetic curve. (b) Calibration curve: changes in frequency Δf = (Δfchymo)/(Δfcasein) × 100 where Δfchymo = f − f0 (where f is steady state frequency following addition of chymotrypsin and washing the surface by PBS and f0 those prior addition of chymotrypsin) is change in frequency after chymotrypsin cleavage and Δfcasein is frequency change after formation of β-casein layer. vs. lower chymotrypsin concentration range for determination of LOD. Biosensors 2021, 11, x FOR PEER REVIEW 11 of 14

6 1 mg/mL β−casein 0 5 −30 4 −60 ) 3 Ω ( − 90 m f (Hz) 2 R

Δ 10 nM chymotrypsin −120 Δ 1 PBS −150 0 −180 PBS −1 0 20406080100120140

Time (min)

Figure 10. Kinetics of the changes of resonant frequency, f (blue), and motional resistance, Rm (black), following modification of piezocrystal by β-casein and addition of 10 nM of chymotrypsin. Addition of various compounds as well as washing of the surface by PBS is shown by arrows.

Based on the changes of the frequency, we constructed a calibration curve for different concentrations of chymotrypsin (Figure 11a). For determination of LOD, we also prepared calibration curve at a low concentration range where the dependence was almost linear (Figure 11b). The LOD for gravimetric detection of chymotrypsin, 1.40 ± 0.30 nM Biosensors 2021, 11, 63 11 of 14 was calculated from the Equation (2) using SD = 2.20 (%) and S = 5.17 (%) nM−1. This value was 2.8 times higher than that reported by ELISA methods and 9.3 times higher than for optical method of detection reported here. We should mention that gravimetric detection requiresrequires carefulcareful handlinghandling ofof thethe cellcell becausebecause eveneven smallsmall changeschanges inin liquidliquid pressurepressure cancan affectaffect the measurements. This This method method is, is, howe however,ver, more more tolerant tolerant to the to thepresence presence of dif- of differentferent components components in the in sample, the sample, such suchas added as added fat present fat present in natural in milk. natural The milk. gravimet- The gravimetricric method methodof protease of protease detectio detectionn requires requires time similar time similar to opti tocal optical methods. methods. It is Italso is also im- importantportant to tomention mention the the uniformity uniformity of ofthe the modified modified materials. materials. The The changes changes of offrequency frequency in ingravimetric gravimetric methods methods and and absorbance absorbance maximum maximum position position in optical in optical methods methods did not did differ not differsignificantly significantly from sample from sample to sample, to sample, suggesting suggesting that it that is not it is a notsource a source of significant of significant error error(large (large standard standard deviation). deviation).

5 (a) 18 (b) 16 4 14 3 12

2 (%)f Δ 10

8 df/dt (Hz/min) 1 6 0 4 0 5 10 15 20 0.0 0.2 0.4 0.6 0.8 1.0

Chymotrypsin [nM] Chymotrypsin [nM] FigureFigure 11.11.( (aa)) CalibrationCalibration curve curve for for chymotrypsin chymotrypsin ﬁtted fitted by by reverse reverse Michaelis—Menten Michaelis—Menten model, model, v = v df/dt = df/dt was was the the ﬁrst first deriva- deri- vation of frequency obtained from kinetic curve. (b) Calibration curve: changes in frequency Δf = (Δfchymo)/(Δfcasein) × 100 tion of frequency obtained from kinetic curve. (b) Calibration curve: changes in frequency ∆f = (∆fchymo)/(∆fcasein) × 100 where Δfchymo = f − f0 (where f is steady state frequency following addition of chymotrypsin and washing the surface by where ∆fchymo = f − f0 (where f is steady state frequency following addition of chymotrypsin and washing the surface PBS and f0 those prior addition of chymotrypsin) is change in frequency after chymotrypsin cleavage and Δfcasein is fre- by PBS and f those prior addition of chymotrypsin) is change in frequency after chymotrypsin cleavage and ∆f is quency change0 after formation of β-casein layer. vs. lower chymotrypsin concentration range for determination ofcasein LOD. frequency change after formation of β-casein layer. vs. lower chymotrypsin concentration range for determination of LOD. (∆f = 5.17%nM−1c + 9.94%, R2 = 0.84, where c is the concentration of chymotrypsin). The results represent mean ± SD obtained from 3 independent measurements at each concentration of chymotrypsin.

It is also interesting to compare the KM values determined in optical and gravimetric experiments. From the results presented above, it can be seen that KM value in the case of AuNPs-based optical assay (3.89 nM and 1.03 nM for spectrophotometric and DLS methods respectively) were lower in comparison with those based on gravimetric measurements 2 (KM = 8.6 ± 3.6 nM, R = 0.999). This can be evidence of better access of β-casein substrate for chymotrypsin in AuNPs in comparison with those immobilized at the surface of piezo- electric transducer. This effect can probably explain the lower LOD for spectrophotometric method of chymotrypsin detection with those based on gravimetric method. In Table1 we present comparison of other published methods for detection of chymotrypsin. Results obtained in our work have either comparable or higher LOD, but in most cases are faster in comparison to other methods.

Table 1. The comparison of LOD and detection time of chymotrypsin detection.

Method LOD Detection Time Reference ELISA 0.5 nM 3.5 h [10] Liquid crystals protease assay 4 pM 3 h [27] Electrophoresis 20 pM 1 h [28] NIR ﬂuorescent probe 0.5 nM 35 min [29] Ratiometric ﬂuorescence probe 0.34 nM 30 min [30] UV-Vis, AuNPs 0.15 ± 0.01 nM 30 min This work DLS, AuMPs 0.67 ± 0.05 nM 30 min This work TSM 1.40 ± 0.30 nM 35 min This work Biosensors 2021, 11, 63 12 of 14

4. Conclusions Wedetermined LOD for detection of chymotrypsin by gravimetric (LOD = 1.40 ± 0.30 nM) spectrophotometric (LOD = 0.15 ± 0.01 nM) and DLS method (LOD = 0.67 ± 0.05 nM). Spec- trophotometric method showed the best value of LOD, even when compared to commercial ELISA (LOD = 0.5 nM). We also determined a steady-state constant KM for the different methods with reverse Michaelis—Menten equation. The largest KM value was found for gravimetric method of chymotrypsin detection (KM = 8.6 ± 3.6 nM), followed by spectrophotometric method (KM = 3.89 ± 1.24 nM),) and then DLS method (KM = 1.03 ± 0.26 nM). We can explain observed differences in KM values by difference in α-chymotrypsin activity, which is highest and least impeded on gold nanoparticles modified with β-casein. Addition of MCH decelerates the reaction and immobilization of β-casein on the gold surface slows the α-chymotrypsin ability to cleave β-casein. The detection time for methods that we tested was comparable and takes around 30 min for chymotrypsin determination. All methods required preparation of the sensing layers or modification of AuNPs overnight. The AuNPs or gravimetric sensors could be stored for a long time (more than one month) at 4 ◦C. In terms of difficulty in operation, the optical methods offered the easier way to measure chymotrypsin. With prepared AuNPs modified by β-casein and MCH, the spectrophotometric method required only one step of protease detection based on measurement of absorbance changes after 30 min, which was simpler in comparison with ELISA. DLS method based on AuNPs requires also only one step of measurement of the Z-average. The spectrophotometric method required only 50 µL of sample, the DLS method used 100 µL, while the gravimetric method used around 2 mL. One of the advantages of the gravimetric method is that it is more robust to “impurities” in the sample. The gravimetric method can be used with natural, no-transparent samples containing fat, minerals, or other proteins, just like in milk. Optical assays require a transparent sample; however, DLS method is a little less sensitive to changes in chymotrypsin concentrations. In terms of cost of analysis, the production of gold nanoparticles is relatively inexpensive and can be scaled to industrial amounts. For optical detection of chymotrypsin, gold nanoparticles should be surface-modified using inexpensive chemicals (β-casein and MCH). While gravimetric methods also use inexpensive chemicals for modification, but the cost of quartz crystal would raise the overall cost of the sensor. This cost offset can be reduced by multiple use of the same crystal when the sensing layer is regenerated. All methods have a distinct advantage and disadvantage compared to the currently used ELISA. In contrast with ELISA, the optical and gravimetric assay are not specific to the protease. Non-specificity of response can be addressed by using chymotrypsin-specific peptide substrate [13] or by integration of advanced machine learning algorithms [14]. In conclusion, we demonstrated advantages and disadvantages of spectrophotometric, DSL and gravimetric methods in detecting chymotrypsin. These methods can be applied also for detection of other proteases and can be useful for further application in the food industry and in medicine for real-time monitoring of the protease activity. In future work we plan to explore application of the presented techniques for analysis of natural milk samples (paying particular attention to gravimetric methods). Many new analytical methods use fluorometric or colorimetric molecules for detection of protease activity [31]. Gold nanoparticles seem to be good alternative component for colorimetric detection or for amplification of existing signal (for example increase of Raman signal from a sample using gold nanoparticles). It is clear that efforts furthering the development of new low-cost methods, easily implementable in practice, which would be sensitive, and exhibiting long-term stability, still need to be developed [32].

Author Contributions: Conceptualization, T.H. and I.N.I.; methodology, I.P.; formal analysis, I.P.; investigation, I.P.; resources, T.H. and I.N.I.; writing—original draft preparation, I.P.; writing—review and editing, T.H. and I.N.I.; visualization, I.P.; supervision, T.H. and I.N.I.; project administration, T.H.; funding acquisition, T.H. and I.N.I. All authors have read and agreed to the published version of the manuscript. Funding: A portion of this research was conducted at the Center for Nanophase Materials Sciences, which is a DOE Ofﬁce of Science User Facility, project No. CNMS2018-293. This work was funded Biosensors 2021, 11, 63 13 of 14

under the European Union’s Horizon 2020 research and innovation program through the Marie Skłodowska-Curie grant agreement No 690898 and by Science 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. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

References 1. Bond, J.S. Proteases: History, discovery, and roles in health and disease. J. Biol. Chem. 2019, 294, 1643–1651. [CrossRef][PubMed] 2. Lojda, Z. The importance of protease histochemistry in pathology. Histochem. J. 1985, 17, 1063–1089. [CrossRef][PubMed] 3. Antalis, T.M.; Shea-Donohue, T.; Vogel, S.N.; Sears, C.; Fasano, A. Mechanisms of disease: Protease functions in intestinal mucosal pathobiology. Nat. Clin. Pract. Gastroenterol. Hepatol. 2007, 4, 393–402. [CrossRef] 4. Appel, W. Chymotrypsin: Molecular and catalytic properties. Clin. Biochem. 1986, 19, 317–322. [CrossRef] 5. Tomlinson, G.; Shaw, M.C.; Viswanatha, T. Chymotrypsin(s). Methods Enzymol. 1974, 34, 415–420. [CrossRef][PubMed] 6. Ismail, B.; Nielsen, S.S. Invited review: Plasmin protease in milk: Current knowledge and relevance to dairy industry. J. Dairy Sci. 2010, 93, 4999–5009. [CrossRef][PubMed] 7. Harris, G.S. Alpha-chymotrypsin in cataract surgery. Can. Med. Assoc. J. 1961, 85, 186–188. [CrossRef][PubMed] 8. Skacel, M.; Ormsby, A.H.; Petras, R.E.; McMahon, J.T.; Henricks, W.H. Immunohistochemistry in the differential diagnosis of acinar and endocrine pancreatic neoplasms. Appl. Immunohistochem. Mol. Morphol. 2000, 8, 203–209. [CrossRef][PubMed] 9. Chen, L.; Daniel, R.M.; Coolbear, T. Detection and impact of protease and lipase activities in milk and milk powders. Int. Dairy J. 2003, 13, 255–275. [CrossRef] 10. Chymotrypsin ELISA Kit. Available online: http://img2.creative-diagnostics.com/pdf/DEIA10041,Chymotrypsin.pdf (accessed on 7 October 2020). 11. Kankare, J. Sauerbrey equation of quartz crystal microbalance in liquid medium. Langmuir 2002, 18, 7092–7094. [CrossRef] 12. Dong, Z.-M.; Cheng, L.; Zhang, P.; Zhao, G.-C. Label-free analytical performances of peptide-based QCM biosensor for trypsin. Analyst 2020, 145, 3329–3338. [CrossRef][PubMed] 13. Poturnayova, A.; Castillo, G.; Subjakova, V.; Tatarko, M.; Snejdarkova, M.; Hianik, T. Optimization of the cytochrome c detection by acoustic and electrochemical methods based on aptamer sensors. Sens. Actuators B Chem. 2017, 228, 817–827. [CrossRef] 14. Tatarko, M.; Muckley, E.; Subjakova, V.; Goswami, M.; Sumpter, B.; Hianik, T.; Ivanov, I. Machine learning enabled acoustic detection of sub-nanomolar concentration of trypsin and plasmin in solution. Sens. Actuators B Chem. 2018, 272, 282–288. [CrossRef] 15. Huang, X.; El-Sayed, M.A. Gold nanoparticles: Optical properties and implementations in cancer diagnosis and photothermal therapy. J. Adv. Res. 2010, 1, 13–28. [CrossRef] 16. Diouani, M.F.; Ouerghi, O.; Belgacem, K.; Sayhi, M.; Laouini, D. Casein-conjugated gold nanoparticles for amperometric detection of leishmania infantum. Biosensors 2019, 9, 68. [CrossRef][PubMed] 17. Chen, G.; Yusheng, X.; Zhang, H.; Wang, P.; Cheung, H.; Yang, M.; Sun, H. A general colorimetric method for detecting protease activity based on peptide-induced gold nanoparticle aggregation. RSC Adv. 2014, 4, 6560–6563. [CrossRef] 18. Svard, A.; Neilands, J.; Palm, E.; Svensater, G.; Bengtsson, T.; Aili, D. Protein-Functionalized Gold Nanoparticles as Refractometric Nanoplasmonic Sensors for the Detection of Proteolytic Activity of Porphyromonas gingivalis. ACS Appl. Nano Mater. Am. Chem. Soc. 2020, 3, 9822–9830. [CrossRef] 19. Goyal, G.; Alagappan, P.; Liedberg, B. Protease Functional Assay on Membrane. Sens. Actuators B Chem. 2019, 305, 127442. [CrossRef] 20. Chuang, Y.-C.; Li, J.-C.; Chen, S.-H.; Liu, T.-Y.; Kuo, C.-H.; Huang, W.-T.; Lin, C.-S. An optical biosensing platform for proteinase activity using gold nanoparticles. Biomaterials 2010, 31, 6087–6095. [CrossRef] 21. Goldburg, W.I. Dynamic light scattering. Am. J. Phys. 1999, 67, 1152–1160. [CrossRef] 22. Naiim, M.; Boualem, A.; Ferre, C.; Jabloun, M.; Jalocha, A.; Ravier, P. Multiangle dynamic light scattering for the improvement of multimodal particle size distribution measurements. Soft Matter 2015, 11, 28–32. [CrossRef][PubMed] 23. Piovarci, I.; Hianik, T.; Ivanov, I.N. Comparison of optical and gravimetric methods for detection of chymotrypsin. Proceedings 2020, 60, 42. [CrossRef] 24. Kimling, J.; Maier, M.; Okenve, B.; Kotaidis, V.; Ballot, H.; Plech, A. Turkevich method for gold nanoparticle synthesis revisited. J. Phys. Chem. B 2006, 110, 15700–15707. [CrossRef] 25. Románszki, L.; Tatarko, M.; Jiao, M.; Keresztes, S.; Hianik, T.; Thompson, M. Casein probe-based fast plasmin determination in the picomolar range by an ultra-high frequency acoustic wave biosensor. Sens. Actuators B Chem. 2018, 275, 206–214. [CrossRef] 26. Martin, S.J.; Granstaff, V.E.; Frye, G.C. Characterization of a quartz crystal microbalance with simultaneous mass and liquid loading. Anal. Chem. 1991, 63, 2272–2281. [CrossRef] Biosensors 2021, 11, 63 14 of 14

27. Chen, C.-H.; Yang, K.-L. Oligopeptide immobilization strategy for improving stability and sensitivity of liquid-crystal protease assays. Sens. Actuators B Chem. 2014, 204, 734–740. [CrossRef] 28. Lefkowitz, R.B.; Schmid-Schönbein, G.W.; Heller, M.J. Whole blood assay for elastase, chymotrypsin, matrix metalloproteinase-2, and matrix metalloproteinase-9 activity. Anal. Chem. 2010, 82, 8251–8258. [CrossRef][PubMed] 29. Mu, S.; Xu, Y.; Zhang, Y.; Guo, X.; Li, J.; Wang, Y.; Liu, X.; Zhang, H. A non-peptide NIR ﬂuorescent probe for detection of chymotrypsin and its imaging application. J. Mater. Chem. B. 2019, 7, 2974–2980. [CrossRef] 30. Chen, Y.; Cao, J.; Jiang, X.; Pan, Z.; Fu, N. A sensitive ratiometric ﬂuorescence probe for chymotrypsin activity and inhibitor screening. Sens. Actuators B Chem. 2018, 273, 204–210. [CrossRef] 31. Magdalena, W.; Lesner, A. Future of Protase Activity Assays. Curr. Pharm. Des. 2012, 19, 1062–1067. [CrossRef] 32. Shi, H.; Liu, C.; Cui, J.; Cheng, J.; Lin, Y.; Luo, R. Perspective on chymotrypsin detection. New J. Chem. R. Soc. Chem. 2020, 44, 20921–20929. [CrossRef]