Detection of Sub-Nanomolar Concentration of Trypsin by Thickness-Shear Mode Acoustic Biosensor and Spectrophotometry

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, KM, 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.

Radiation-Activated Pre-Differentiated Retinal Tissue Monitored by Acoustic Wave Biosensor

A thickness-shear mode acoustic wave biosensor operated within a flow-through system was used to examine the response of mouse retinal tissue to radiation. Control experiments conducted with respect to exposure of the bare gold electrodes of the device under various conditions of light intensity and bathing solution yielded reversible changes in resonant frequency (Fs) and motional resistance (Rm). The magnitude of transient changes was proportional to light intensity, but independent of solution type. These alterations in acoustic parameters were ascribed to acoustic coupling phenomena at the electrode-to-liquid interface. Pre-differentiated retina from mouse samples deposited on the thickness shear mode (TSM) electrode exposed to a high light intensity condition also exhibited reversible changes in both Fs and Rm, compared to control experiments involving a coating used to attach the tissue to the electrode. In this case, the radiation-instigated reversible responses for both acoustic parameters exhibited a reduction in magnitude. The changes are ascribed to the alteration in viscoelasticity of the retinal matrix on the TSM electrode surface. The precise biophysical mechanism responsible for the changes in Fs and Rm remains a challenge, given the complex make up of retinal tissue.