Automatic chemiluminescence detection of reactive oxygen species in seawater and saliva using novel 3D-printed flow-cell geometries

Abstract

[eng] Reactive oxygen species (ROS) are highly reactive molecules which might be divided in two types: free oxygen radicals (e.g. O2 ·−, ·OH) and non-radical ROS (e.g. H2O2, 1O2). ROS are naturally generated in both biological and marine systems, but the homeostatic levels may be altered. In fact, the alteration of ROS levels in biological systems can trigger oxidative stress, associated with the damage of biomolecules like proteins, lipids, or nucleic acids. This work aims at developing a suitable automatic analytical methodology able to reliably quantify ROS at the concentration levels expected in seawater and biological samples, such as saliva. For this purpose and taking into account the unsteadiness, and low concentrations of ROS in the studied matrices, a flow-through system with flash chemiluminescence (CL) detection based on the luminol chemistry has been resorted. In order to improve the conventional spirally-shaped CL detection flow-cell with cylindrical cross-section, 3D printing technology has been chosen for rapid and simple prototyping and production of complex designs at low cost. Hence, four 3D printed flow-cells with novel cross sections geometries: semicylindrical, pyramidal shape, cubic and 5-side prism, were evaluated and compared with the cylindrical one with the aim of enhancing the capture of the elicited light from the flash CL reaction by maximizing the liquid volume close to the detection system. In addition, three different reagent/sample confluence types were included in the 3D printed cell design: T, Y and a modified Y types. Computer flow dynamic simulations were also performed to investigate the flow field for the distinct designs and get knowledge on the mixing performance and the magnitude of the flow rates versus position at the inlet of the flow cell. Other chemical and hydrodynamic parameters of the flow-through system were also studied (reagent pH, in-situ preparation of the chemilumogenic reagents, reagent/sample flow-rates, blank signal minimization, injection volume and dispersion effect throughout the flow conduits). Finally, the optimal 3D printed flow-cell (5-side prism cross-section with Y confluence type) in combination with the optimized methodology was applied to the determination of hydrogen peroxide, as a model analyte, in saliva and seawater matrices.