EUROPEAN UNION EUROPEAN SOCIAL FUND OPERATIONAL PROGRAMME SCIENCE AND EDUCATION FOR SMART GROW Green Technologies Innovative Materials Research network GreTInMat Four-day working meeting Starosel, Bulgaria, June 18 – 21, 2018 LAYER-BY-LAYER IMMOBILIZATION OF ENZYMES AND NANOPARTICLES ONTO CELLULOSE SUPPORT Vera Semerdzhieva, Dessislava Marinkova, Raya Raykova University of Chemical Technology and Metallurgy, Department of Biotechnology 8, St. Kliment Ohridski, Blvd., 1756 Sofia, Bulgaria vera.semerdzhieva.91@gmail.com Our Team: Dessislava Antonova Marinkova-Kaloyanova, Associated Professor affiliation University of Chemical Technology and Metallurgy (UCTM), Department of Biotechnology, Sofia, Bulgaria e-mail: dmarinkova@yahoo.com Scopus ID: 24367128100 Raya Nikolova Raykova, Assistant Professor affiliation University of Chemical Technology and Metallurgy (UCTM), Department of Biotechnology, Sofia, Bulgaria e-mail: raianikolova@gmail.com Scopus ID: 55390822200 Vera Angelova Semerdzhieva, PhD student affiliation University of Chemical Technology and Metallurgy (UCTM), Department of Biotechnology, Sofia, Bulgaria e-mail: vera.semerdzhieva.91@gmail.com

Motivation and Background EUROPEAN UNION EUROPEAN SOCIAL FUND OPERATIONAL PROGRAMME SCIENCE AND EDUCATION FOR SMART GROW LAYER-BY-LAYER IMMOBILIZATION OF ENZYMES AND NANOPARTICLES ONTO CELLULOSE SUPPORT Motivation and Background Biosensors [1] Biofuel cells [2] [1] Shruthi GS, Amitha CV, Blessy Baby Mathew, Biosensors: A Modern Day Achievement, Department of Biotechnology, Sapthagiri College of Engineering, Bangalore, Karnataka, India [2] Michelle Rasmussen, Sofiene Abdellaoui, Shelley D. Minteer, Enzymatic biofuel cells: 30 years of critical advancements, Biosensors and Bioelectronics Volume 76, 15 February 2016, Pages 91-102 2 Acknowledgment: „The research networkhas been financially supported by the Operational Programme "Science and education for smart growth" 2014-2020 of the European Union cofounded by the European Social Fund through the project BG05M2ОP001-2.009-0015 “Support for the development of capacity of doctoral students and young researchers in the field of engineering, natural and mathematical sciences”

EUROPEAN UNION EUROPEAN SOCIAL FUND OPERATIONAL PROGRAMME SCIENCE AND EDUCATION FOR SMART GROW LAYER-BY-LAYER IMMOBILIZATION OF ENZYMES AND NANOPARTICLES ONTO CELLULOSE SUPPORT Figure 3. SEM image of cellulose surface [3] Figure 1. Schematic presentation of layer-by-layer assembly of enzymes with poly(ethyleneimine) (PEI) Figure 2. Schematic presentation of layer-by-layer assembly of enzymes with PEI and crosslinking with glutaraldehyde (GA) [3] M. Almostasim Mahmud, Eric J. M. Blondeel, Moufeed Kaddoura and Brendan D. MacDonald, Creating compact and microscale features in paper-based devices by laser cutting, The Royal Society of Chemistry 2016, DOI: 10.1039/c6an02208a; Acknowledgment: „The research networkhas been financially supported by the Operational Programme "Science and education for smart growth" 2014-2020 of the European Union cofounded by the European Social Fund through the project BG05M2ОP001-2.009-0015 “Support for the development of capacity of doctoral students and young researchers in the field of engineering, natural and mathematical sciences”

EUROPEAN UNION EUROPEAN SOCIAL FUND OPERATIONAL PROGRAMME SCIENCE AND EDUCATION FOR SMART GROW LAYER-BY-LAYER IMMOBILIZATION OF ENZYMES AND NANOPARTICLES ONTO CELLULOSE SUPPORT Figure 4. SEM image of Cellulose/ Nanofibrillated cellulose (NFC’s) composite Table 1. Kinetic parameters of free glucose oxidase and glucose oxidase assembled with nanoparticles on the surface of cellulose Table 2. Kinetic parameters of free laccase and laccase assembled with nanoparticles on the surface of cellulose Figure 5. SEM image of Cellulose / Multiwalled carbon nanotubes (MWCNT’s) composite Free enzyme Cellulose / NFC’s Cellulose / MWCNT’s Cellulose / NFC’s / MWCNT’s KM, M 3,06.10-3 3,4.10-3 4,7.10-3 4,9.10-3 vmax, M.min-1 3,83 0,45 0,47 0,48 kcat, s-1 5,98.104 2,9.104 3.104 2,8.104 kcat/KM, M-1.s-1 1,95.107 8,41.106 6,4.106 5,9.106 Free enzyme Cellulose / NFC’s Cellulose / MWCNT’s Cellulose / NFC’s / MWCNT’s KM, M 1,01.10-4 2,8.10-4 4,04.10-4 2,7.10-4 vmax, M.min-1 0,67 0,023 0,020 0,017 kcat, s-1 5,71.103 2,54.102 1,83.102 1,45.102 kcat/KM, M-1.s-1 1,42.107 9,16.105 4,52.105 5,43.105 Figure 6. SEM image of Cellulose / NFC’s / MWCNT’s composite Acknowledgment: „The research networkhas been financially supported by the Operational Programme "Science and education for smart growth" 2014-2020 of the European Union cofounded by the European Social Fund through the project BG05M2ОP001-2.009-0015 “Support for the development of capacity of doctoral students and young researchers in the field of engineering, natural and mathematical sciences”

Figure 8. Chemical structure of Aflatoxin B1 EUROPEAN UNION EUROPEAN SOCIAL FUND OPERATIONAL PROGRAMME SCIENCE AND EDUCATION FOR SMART GROW Other recent projects: Detection of Mycotoxins in Food Samples with QCM Self-assembled monolayers of 3-mercaptopropionic acid were prepared on the gold quarz surface followed by activation of the monolayers with EDC (1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide). NHS (N-hydroxysulfosuccinimide) was connected to the activated groups before the process of immobilization of antibodies. Different concentrations of Aflatoxin B1-BSA conjugate in milk samples in the range of μg.L-1 were investigated in real time antigen-antibody reaction. Figure 7. Chemical structure of Ochratoxin A The current work investigates kinetic reaction of milk gels acidification in the presence in the presence of different concentrations of Ochratoxin A. A thickness shear mode quarz resonator technique was applied for the measurements in the MHz frequency range. Yoghurt samples contaminated with different concentrations of Ochratoxin A and Ochratoxin A-BSA conjugate were investigated. Figure 8. Chemical structure of Aflatoxin B1 5 5 Acknowledgment: „The research networkhas been financially supported by the Operational Programme "Science and education for smart growth" 2014-2020 of the European Union cofounded by the European Social Fund through the project BG05M2ОP001-2.009-0015 “Support for the development of capacity of doctoral students and young researchers in the field of engineering, natural and mathematical sciences”

Other recent projects: Bio-products for Diabetes Treatment EUROPEAN UNION EUROPEAN SOCIAL FUND OPERATIONAL PROGRAMME SCIENCE AND EDUCATION FOR SMART GROW Other recent projects: Bio-products for Diabetes Treatment Chamomile Sumac [4] Table 3. Plant extracts with high inhibitory activity Plants IC₅₀ Erythroxylum laurifolium Lam. (Erythroxylaceae) 7.5 mg/mL Elaeodendron orientale Jacq. 1.7 mg/mL Antidesma madagascariensis Lam. 2.2 mg / mL [4] <https://ghr.nlm.nih.gov/condition/type-2-diabetes> 6 6 Acknowledgment: „The research networkhas been financially supported by the Operational Programme "Science and education for smart growth" 2014-2020 of the European Union cofounded by the European Social Fund through the project BG05M2ОP001-2.009-0015 “Support for the development of capacity of doctoral students and young researchers in the field of engineering, natural and mathematical sciences”

Thank you for your attention! EUROPEAN UNION EUROPEAN SOCIAL FUND OPERATIONAL PROGRAMME SCIENCE AND EDUCATION FOR SMART GROW Green Technologies Innovative Materials Research network GreTInMat IInd working meeting Starosel, Bulgaria, June 18 – 21, 2018 Research network GreTInMAT Green Technologies & Innovative Materials Coordinated by the University of Chemical Technology and Metallurgy, Bulgaria Thank you for your attention! Acknowledgment: „The research network establishment and development is financially supported by the Operational Programme "Science and education for smart growth" 2014-2020 of the European Union cofounded by the European Social Fund through the project BG05M2ОP001-2.009-0015 “Support for the development of capacity of doctoral students and young researchers in the field of engineering, natural and mathematical sciences”

Thank you for your attention! EUROPEAN UNION EUROPEAN SOCIAL FUND OPERATIONAL PROGRAMME SCIENCE AND EDUCATION FOR SMART GROW Thank you for your attention! Acknowledgment: „The research networkhas been financially supported by the Operational Programme "Science and education for smart growth" 2014-2020 of the European Union cofounded by the European Social Fund through the project BG05M2ОP001-2.009-0015 “Support for the development of capacity of doctoral students and young researchers in the field of engineering, natural and mathematical sciences”