A Dual-Readout Photonic Sensor for Simultaneous Measurement of Enzyme Activity and Concentration

Enzyme assays are a cornerstone of basic biology and clinical diagnosis. Typically, enzyme activity is measured, but concentration of the enzyme is also of interest, as are comparisons between concentration and activity. In these situations, separate concentration (i.e., ELISA) and activity (i.e., absorbance) assays are required to fully quantify. Here, we report a multiplex disposable photonic biosensor for simultaneous measurement of enzyme activity and concentration. Capture of the enzyme by a ring resonator-bound antibody produces a red shift in resonance, which can be referenced to a nonspecific binding control. At the same time, enzyme-mediated degradation of a ring-bound substrate produces a resonance blue shift, which can be referenced to a peptide inert to enzymatic cleavage. We tested the dual assay with human Cathepsin-L, dysfunction of which is a hallmark of several diseases, including COVID-19, kidney failure, and cancer. Both assays were found to be well-behaved analytically, with lower limits of detection of 2.0 ng·mL^-1 (concentration) and 1.8 ng·mL^-1 (activity), well within the range clinically relevant concentrations. Further assessment with a panel of 25 single-donor human serum samples confirmed utility of the assay in a complex, biologically relevant matrix. This approach therefore serves as a useful method for Cathepsin-L detection, and a prototype for other dual-mode photonic enzyme assays.