Switchable Peroxidase-mimicking Nanosensor for the Determination of Enzyme Biomarkers in Clinical and Veterinary Samples

Detection of enzyme biomarkers originating from either bio-fluids or contaminating microorganisms is of utmost importance in clinical diagnostics and food safety. Herein, we present a simple, low-cost and easy-to-use nanosensor based on the switchable peroxidase-mimicking activity of plasmonic gold nanoparticles (AuNPs) that can catalyse for the oxidation of 3,3’,5’5-tetramethylbenzidine (TMB) for the determination of protease and lipase biomarkers. The surface of AuNPs is modified with casein or Tween 20, showing dual functionalities (Figure 1). The first function of the coating molecules is to suppress the intrinsic peroxidase-mimicking activity of AuNPs by up to 87.4%, due to surface shielding effects. Secondly, casein and Tween 20 also function as recognition sites for enzyme biomarkers. In the presence of protease or lipase, the enzymes bind and catalyse the degradation of the coating layers on the AuNP surface, resulting in the recovery of peroxidase-mimicking activity. This is shown visually in the development of a blue coloured product (oxidised TMB) or spectroscopically as an increase in absorbance at 370 nm and 650 nm. This mechanism allows the detection of protease and lipase at 44 and 218.3 ng/mL, respectively, using their corresponding substrate-coated AuNPs in 90 min. The nanosensor circumvents issues associated with current methods of detection in terms of ease of use, compatibility with point-of-care testing, low-cost production and short analysis time. The sensing approach has also been applied to detect protease spiked in ultra-heat treated (UHT) milk and synthetic human urine samples at a limit of detection of 490 and 176 ng/mL, respectively, showing great potential in clinical diagnostics, food safety and quality control.