1. 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
Our Team:
Dessislava Antonova Marinkova-Kaloyanova, Associated Professor
affiliation University of Chemical Technology and Metallurgy (UCTM), Department of Biotechnology, Sofia, Bulgaria
Scopus ID:
Raya Nikolova Raykova, Assistant Professor
affiliation University of Chemical Technology and Metallurgy (UCTM), Department of Biotechnology, Sofia, Bulgaria
Scopus ID:
Vera Angelova Semerdzhieva, PhD student
affiliation University of Chemical Technology and Metallurgy (UCTM), Department of Biotechnology, Sofia, Bulgaria

2. Motivation and Background
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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 2
Acknowledgment:
„The research networkhas been financially supported by the Operational Programme "Science and education for smart growth" of the European Union cofounded by the European Social Fund through the project BG05M2ОP “Support for the development of capacity of doctoral students and young researchers in the field of engineering, natural and mathematical sciences”

3. 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: /c6an02208a;
Acknowledgment:
„The research networkhas been financially supported by the Operational Programme "Science and education for smart growth" of the European Union cofounded by the European Social Fund through the project BG05M2ОP “Support for the development of capacity of doctoral students and young researchers in the field of engineering, natural and mathematical sciences”

4. 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,
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,
2,8.10-4 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" of the European Union cofounded by the European Social Fund through the project BG05M2ОP “Support for the development of capacity of doctoral students and young researchers in the field of engineering, natural and mathematical sciences”

5. Figure 8. Chemical structure of Aflatoxin B1
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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" of the European Union cofounded by the European Social Fund through the project BG05M2ОP “Support for the development of capacity of doctoral students and young researchers in the field of engineering, natural and mathematical sciences”

6. 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] < 6 6
Acknowledgment:
„The research networkhas been financially supported by the Operational Programme "Science and education for smart growth" of the European Union cofounded by the European Social Fund through the project BG05M2ОP “Support for the development of capacity of doctoral students and young researchers in the field of engineering, natural and mathematical sciences”

7. 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" of the European Union cofounded by the European Social Fund through the project BG05M2ОP “Support for the development of capacity of doctoral students and young researchers in the field of engineering, natural and mathematical sciences”

8. 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" of the European Union cofounded by the European Social Fund through the project BG05M2ОP “Support for the development of capacity of doctoral students and young researchers in the field of engineering, natural and mathematical sciences”