Enzymes-encapsulated hydrogels for Electrochemiluminescence (ECL) sensing applications
Authors:   , Ferraraccio Lucia S. 
Published: October 2022

Nowadays, the development of innovative biosensors based on non-invasive techniques for a reliable and precise real time detection of vital parameters has become the new frontier for the scientific research. In this thesis, a novel prototype of ECL-based biosensor involving different enzymes is proposed. The primary aim of this project was to determine the possibility to apply an ECL approach to encapsulated and immobilised enzymes for the detection of their activity. Particular attention has been placed on the encapsulation of enzymes to maintain their catalytic properties unaffected. Alginate hydrogels and cryogels have been used as matrices for the encapsulation and the development of an amperometric ECL-based biosensor. Both the entrapment processes have been deeply described and their characterisation is reported. The first ECL-based biosensor examined regards the oxidase enzymes and luminol system. It is well established that the oxidase activity can be determined by measuring the amount of H2O2 produced by the redox reaction between the encapsulated enzyme and its corresponding substrate. The utilisation of luminol is determinant for the ECL emission by reacting with the produced hydrogen peroxide, hence for the investigation of the enzymatic reaction. Different aspects of the oxidase-luminol system have been deepened, including the statistical analysis, Michaelis-Menten study, limits of detection and quantification, interferences, and real sample analysis. The second system disclosed in this thesis regards the ECL-based biosensor concerning the employment of dehydrogenase enzymes, [Ru(bpy)3]2+ and NAD+. These enzymes have been successfully encapsulated into alginate hydrogel matrices following the same procedure adopted for the encapsulation on the oxidase group. Likewise, the electrochemical analysis is reported showing the cyclic voltammetry and ECL results, the statistical analysis obtained from the study of mean and standard deviation of three repetitions for each enzyme leading to the final considerations regarding Michaelis-Menten parameters, limits of detection and quantification. The last section of this work is dedicated to the improvement of the proposed biosensors by examining the electro-polymerisation of the two luminophores. Firstly, the electro-polymerisation of the luminol for the generation of polyluminol is proposed with particular attention on the development of a bilayer system composed of a first layer of ECL-active polymer on the GCE surface and a second layer obtained with the deposition of the encapsulated enzyme to be analysed. Different essential aspects for a successful electro-polymerisation have been discussed namely different scan rates, number of scans, supporting electrolytes and electrolytic solution. The electrochemical analysis is also reported showing a very efficient detection of the different enzymatic activities (glucose oxidase, lactate oxidase, Horseradish peroxide and cholesterol oxidase) through the generation of H2O2 and the consequent reaction with the polyluminol film. The statistical analysis of the bilayer systems is brought showing the promising results of this novel ECL-biosensor. Finally, preliminary results are mentioned regarding the development of a new ECL-active film, not yet reported in literature, gained by applying the electro-polymerisation to [Ru(bpy)3]2+. In like manner, the main features have been investigated to achieve a highly performant ECL-active film on the GCE surface such as different scan rates effect, number of scans and very determinant the supporting electrolyte and solvent used to form the ideal electrolytic solution for the generation of the here named Poly-[Ru(bpy)3]2+. The UV and FTIR characterisation are proposed leading to the discovery of the oligomeric nature of the electro-polymerised film. Preliminary electrochemical tests have been performed by building a bilayer system with the Poly-[Ru(bpy)3]2+ layer and the superimposed alginate hydrogels containing dehydrogenase enzymes. Multiple aspects of this newfound material still need to be investigated and further explored.