This review is focused on the application of surface and volume-sensitive acoustic methods for the detection of milk proteases such as trypsin and plasmin. While trypsin is an important protein of human milk, plasmin is a protease that plays an important role in the quality of bovine, sheep and goat milks. The increased activity of plasmin can cause an extensive cleavage of β-casein and, thus, affect the milk gelation and taste. The basic principles of surface-sensitive acoustic methods, as well as high-resolution ultrasonic spectroscopy (HR-US), are presented. The current state-of-the-art examples of the application of acoustic sensors for protease detection in real time are discussed. The application of the HR-US method for studying the kinetics of the enzyme reaction is demonstrated. The sensitivity of the acoustics biosensors and HR-US methods for protease detection are compared.

The determination of protease activity is very important for disease diagnosis, drug development, and quality and safety assurance for dairy products. Therefore, the development of low-cost and sensitive methods for assessing protease activity is crucial. We report two approaches for monitoring protease activity: in a volume and at surface, via colorimetric and acoustic wave-based biosensors operated in the thickness-shear mode (TSM), respectively. The TSM sensor was based on a β-casein substrate immobilized on a piezoelectric quartz crystal transducer. After an enzymatic reaction with trypsin, it cleaved the surface-bound β-casein, which increased the resonant frequency of the crystal. The limit of detection (LOD) was 0.48 ± 0.08 nM. A label-free colorimetric assay for trypsin detection has also been performed using β-casein and 6-mercaptohexanol (MCH) functionalized gold nanoparticles (AuNPs/MCH-β-casein). Due to the trypsin cleavage of β-casein, the gold nanoparticles lost shelter, and MCH increased the attractive force between the modified AuNPs. Consequently, AuNPs aggregated, and the red shift of the absorption spectra was observed. Spectrophotometric assay enabled an LOD of 0.42 ± 0.03 nM. The Michaelis–Menten constant, K_M, for reverse enzyme reaction has also been estimated by both methods. This value for the colorimetric assay (0.56 ± 0.10 nM) is lower in comparison with those for the TSM sensor (0.92 ± 0.44 nM). This is likely due to the better access of the trypsin to the β-casein substrate at the surface of AuNPs in comparison with those at the TSM transducer.

A multiharmonic quartz crystal microbalance (QCM) has been applied to study the viscoelastic properties of the aptamer-based sensing layers at the surface of a QCM transducer covered by neutravidin following interaction with bacteria Listeria innocua. Addition of bacteria in the concentration range 5 × 10³–10⁶ CFU/mL resulted in a decrease of resonant frequency and in an increase of dissipation. The frequency decrease has been lower than one would expect considering the dimension of the bacteria. This can be caused by lower penetration depth of the acoustics wave (approximately 120 nm) in comparison with the thickness of the bacterial layer (approximately 500 nm). Addition of E. coli at the surface of neutravidin as well as aptamer layers did not result in significant changes in frequency and dissipation. Using the Kelvin–Voight model the analysis of the viscoelastic properties of the sensing layers was performed and several parameters such as penetration depth, Γ, viscosity coefficient, η, and shear modulus, μ, were determined following various modifications of QCM transducer. The penetration depth decreased following adsorption of the neutravidin layer, which is evidence of the formation of a rigid protein structure. This value did not change significantly following adsorption of aptamers and Listeria innocua. Viscosity coefficient was higher for the neutravidin layer in comparison with the naked QCM transducer in a buffer. However, a further increase of viscosity coefficient took place following attachment of aptamers suggesting their softer structure. The interaction of Listeria innocua with the aptamer layer resulted in slight decrease of viscosity coefficient. The shearing modulus increased for the neutravidin layer and decreased following aptamer adsorption, while a slight increase of µ was observed after the addition of Listeria innocua.

We analyzed the possibility of the detection of cytochrome c (cyt c) being physically adsorbed on lipid films or covalently bounded to 11-mercapto-1-undecanoic acid (MUA) chemisorbed on the gold layer using quartz crystal microbalance with dissipation monitoring (QCM-D). The negatively charged lipid film composed of a mixture of zwitterionic DMPC and negatively charged DMPG phospholipids at a molar ratio of 1:1 allowed the formation of a stable cyt c layer. Addition of DNA aptamers specific to cyt c, however, resulted in removal of cyt c from the surface. The interaction of cyt c with the lipid film and its removal by DNA aptamers were accompanied by changes in viscoelastic properties evaluated using the Kelvin–Voigt model. Cyt c covalently bound to MUA also provided a stable protein layer already at its relatively low concentrations (0.5 μM). A decrease in the resonant frequency following the addition of gold nanowires (AuNWs) modified by DNA aptamers was observed. The interaction of aptamers with cyt c on the surface can be a combination of specific and non-specific interactions due to electrostatic forces between negatively charged DNA aptamers and positively charged cyt c.

Early detection of foodborne pathogens is significant for ensuring food safety. Nowadays, the detection of pathogens found in food can take up to 72 h and it might take a week to confirm a positive sample. While standardized methods give test results in a shorter period, the reoccurring costs for each measurement are high. Therefore, it is necessary to develop technology that will be low-cost, fast, simple and accurate enough. Biosensors in combination with nucleic acid aptamers offer such possibilities. This work is focused on the development and testing of a biosensor based on DNA aptamers for detection of pathogenic bacteria Listeria innocua using the method of multi-harmonic quartz crystal microbalances (QCM). The aptasensor was prepared on the surface of a piezo crystal, whose frequency was affected by deposited mass. An aptamer specific to the genus Listeria spp. was used for the detection of this pathogen, which includes 16 subspecies, out of which 3 are excluded as their antigen structure differs from other species (L. murrayi, L. grayi, L. ivanovii). We found that addition of the pathogens at the surface of QCM transducer modified by aptamers resulted in the decrease of the resonant frequency in concentration depending manner. We also confirmed the specificity of the aptamer used for Listeria innocua, as neglected response of the sensor took place for E. coli for which Listeria spp. has some partial antigens identical and thus can cause cross-reactions in serological tests. The developed aptasensor showed promising sensitivity and specificity for real-time detection of Listeria innocua, with a detection time of 30 min. The achieved limit of detection was approximately 1.6 × 10³ CFU/mL.

We used the research quartz crystal microbalance (RQCM) to monitor regulatory effects of plasmin and trypsin in the presence of their inhibitor α₂-antiplasmin. The gold surface of quartz crystals was modified with a β-casein layer that served as a substrate for protease digestion. The addition of plasmin or trypsin as well as their mixtures with α₂-antiplasmin resulted in an increase of resonant frequency, f, and in a decrease of motional resistance, R_m, depending on the molar ratio of protease: antiplasmin. At equimolar concentrations of protease and α₂-antiplasmin (5 nM:5 nM) full inhibition of protease activity took place. Monitoring of plasmin activity on an hourly and daily basis revealed a prominent effect of autolysis and decrease of plasmin activity in freshly activated samples. The degree of inhibition as well as plasmin half-life (t_1/2 = 2.48 ± 0.28 days) connected with its degradation was determined.