Casein probe–based fast plasmin determination in the picomolar range by an ultra-high frequency acoustic wave biosensor

Abstract

Detection of residual plasmin activity in milk represents a difficult challenge for the dairy industry. Conventional methods are either too expensive or incapable of providing enough data from UHT treated milk. Acoustic wave–based biosensors operated in the thickness shear mode (TSM) showed potential for the detection of proteolysis of β-casein, a milk protein by protease plasmin. An ultra-high frequency device, the electromagnetic piezoelectric acoustic sensor (EMPAS), designed to enhance the sensitivity of TSM, was tested for detection of plasmin at low concentrations. β-casein layers immobilised on the hydrophilic or hydrophobized surfaces of EMPAS quartz discs served as substrate for the enzyme. In contrast with conventional TSM devices, the shearing oscillations in EMPAS are induced contactless, by a magnetic coil located 30 μm below the quartz crystal. This configuration allows the registration of unusually high harmonics (up to the 49th-53rd), thus enhancing the sensitivity of detection. On both surface types, the adsorbed β-casein mass and the stability of the layer was compared, with the result that hydrophobic surfaces provide superior conditions for immobilisation than the hydrophilic case. Consequent proteolysis measurements of these substrate layers were carried out in a broad plasmin concentration range (32 pM–10 nM) in flow mode. Initial reaction rates measured at different enzyme concentrations have been used to construct a calibration curve based on an inverse Michaelis–Menten type equation. The sensitivity of the EMPAS allowed measurements of as low as 32 pM concentration of plasmin, reaching (and often exceeding) levels comparable to state of the art techniques like ELISA. The presented method however, unlike ELISA, is effective on a timescale of minutes.