Co-encapsulation of enzymes and antibodies for chemical deactivation of pathogens on paper

Bioactive paper is obtained through the modification of paper substrate with biomolecules and reagents. It is used in the development of novel biosensors that are portable, disposable and inexpensive, aimed at capturing, detecting and in some cases deactivating pathogens. Generally bioactive papers are made by incorporating biomolecules such as enzymes and/or antibodies on to paper. The immobilization of such biomolecules on solid surfaces is widely used for different diagnostic applications such as in immunosensors and immunoassays but due to the sensitive nature of enzymes, their large scale incorporation into paper has faced several difficulties especially under industrial papermaking conditions. The functionalization of paper at large scale is possible because paper can be easily coated with a layer of microcapsules, which have proven to be an efficient immobilization platform for enzymes and to allow. In this study, we developed a generic alginate-based platform incorporating microcapsules that can be applied to current paper production processes to prepare antibacterial bioactive paper with the ability to capture pathogens on its surface and to deactivate them by producing an anti-pathogenic agent. The design of the antibacterial platform is based on constant production of hydrogen peroxide as the antibacterial agent inside the alginate microcapsules. Hydrogen peroxide production is achieved through oxidation of glucose, catalyzed by the enzyme glucose oxidase encapsulated inside the alginate beads. The different steps of development included the entrapment of glucose oxidase inside alginate microcapsules, the reinforcement and surface activation of microcapsules by adding an additional layer of chitosan, investigating the possibility of immobilization of antibodies (human immunoglobulin G as a model antibody) on the surface of microcapsules and, finally, verifying the antibacterial properties of the system against Escherichia coli K-12 (E. coli K-12) as a representative pathogen. During development, certain measurements and observations were made using various analytical methods and techniques such as Bradford protein assay, oxygen electroanalysis, optical and confocal laser canning microscopy (CLSM), matrix assisted laser desorption/ionization- time of flight mass spectrometry (MALDI-TOF-MS), etc. Appropriate tests were performed to validate the successful modification of microcapsules and to ensure that glucose oxidase is still active after each modification. It was found that the encapsulated glucose oxidase maintained the specific enzymatic activity of 120±30 U/g. Subsequent efforts were made to immobilize glucose oxidase on gold NPs of two different diameters (10.9 nm and 50 nm) to enhance the enzymatic activity and increase the encapsulation efficiency.   The results obtained during this study demonstrate successful modifications on alginate microcapsules and also a successful response of such antibacterial platform regarding deactivation of the pathogen representative, E. coli K-12. The threshold for the enzymatic activity was found to be 1.3×10-2 U/ml for E. coli K-12 growth inhibition of 6.7×108 cells/ml. Further studies are needed to assess the efficiency of immobilized antibody in the capture of pathogens and also to incorporate the platform onto paper and to validate the efficiency of the system once it is coated on paper.

Keyphrases: Antibacterial paper, Glucose oxidase encapsulation, Alginate microcapsules, E. coli growth inhibition