Iron protoporphyrin IX-hyaluronan hydrogel-supported luminol chemiluminescence for the detection of nitric oxide in physiological solutions

Abstract

Due to its role as a free radical signal-transducing agent with a short lifespan, precise measurement of nitric oxide (^●NO) levels presents significant challenges. Various analytical techniques offer distinct advantages and disadvantages for ^●NO detection. This research aims to simplify the detection process by developing a hydrogel system using iron(III)-protoporphyrin IX (hemin)-loaded hyaluronan for the detection of ^●NO in solution. Various hydrogel formulations were created, and the effects of their components on hydrogel-supported luminol chemiluminescence (CL) kinetics, radical scavenging, and physicochemical properties were analysed through factorial analysis. The candidate formulations were then evaluated using two ^●NO donors. An increase in the degree of crosslinking in unloaded formulations enhanced interactions with the CL reaction components, hydrogen peroxide (H₂O₂) and luminol, thereby affecting light generation. However, hemin loading negated these effects, resulting in more prominent luminescence kinetics in formulations with lower crosslinking degrees. Similarly, ^●NO influenced the kinetics differently, interacting with both the CL reaction and hydrogel components. Hemin-loaded formulations exhibited enhanced signal propagation when exposed to ^●NO, followed by H₂O₂ and luminol, whereas reversing the order of addition inhibited this propagation. The magnitude of these luminescence changes depended on the type and concentration of the ^●NO donor, demonstrating greater sensitivity to ^●NO levels compared to amperometric sensing. These findings suggest that the studied hydrogel platform has potential for the facile and accurate detection of ^●NO in solution, requiring minimal sample sizes.