Investigation of the Polyphenol Oxidase Enzyme Thin Film as a Copper Sulfate (CuSO4) Sensor

Öz

In this study, thin films were produced with the polyphenol oxidase (PPO) enzyme on chitosan-gelatine bio-composite via the spin coating technique. Bio-composite material was used for the PPO enzyme layer and for stable thin film transfer on the substrate. In the thin films produced, the PPO enzyme layer forms the active site and is the part where the thin film is in direct contact with the external environment. In this way, any external environmental change to the thin film directly affects the PPO enzyme layer. A copper sulfate (CuSO4) solution in pure water solvent was used to observe the external changes in the environment of the PPO enzyme layer. The copper sulfate in this solution is a pollutant and is the harmful chemical to be detected. Therefore, its usability in sensor applications was investigated by measuring the response of the PPO enzyme to copper sulfate. Production stages of thin films were followed by electrical and Quartz crystal microbalance (QCM) methods. In addition, electrical measurements and the QCM system have been used for liquid media sensor measurements of thin films. By recording the time-dependent contaminant response of the PPO enzyme thin film, the usability of enzymes as an active layer in sensor applications was studied.

Anahtar Kelimeler

• Polyphenol oxidase enzyme
• bio-sensor
• quartz crystal microbalance

Polifenol Oksidaz Enzim İnce Filminin Bakır Sülfat (CuSO4) Sensörü Olarak Kullanılabilirliğinin İncelenmesi

Öz

Bu çalışmada spin kaplama tekniği kullanılarak çitosan-jelatin biyokompozit alt-tabakası üzerine polifenol oksidaz (PPO) enzimi ile ince filmler üretilmiştir. Biyo-kompozit malzeme, PPO enzim tabakasının alttaş üzerine kararlı ince film transferi amacıyla kullanılmıştır. Üretilen ince filmlerde PPO enzim tabakası aktif bölgeyi oluşturarak, ince filmin dış ortam ile direkt temas halinde olduğu kısımdır. Bu sayede ince filmin herhangi bir dış ortam değişimi, PPO enzim tabakası üzerine doğrudan etki etmektedir. PPO enzim tabakasının dış ortam değişimlerini duyarlılığını gözlemlemek amacıyla saf su çözücüsünde bakır sülfat (CuSO4) çözeltisi kullanılmıştır. Bu çözeltideki bakır sülfat kirletici olup tespit edilmek istenilen zararlı kimyasal görevindedir. Dolayısıyla, PPO enziminin bakır sülfata karşı tepkisi ölçülerek sensör uygulamalarındaki kullanılabilirliği araştırılmıştır. İnce filmlerin üretim aşamaları elektriksel ve Kuartz kristal mikrobalans (QCM) yöntemleriyle takip edilmiştir. Ayrıca elektriksel ölçümler ve QCM sistemi, ince filmlerin sıvı ortam sensör ölçümleri için kullanılmıştır. PPO enzim ince filminin zamana bağlı kirletici tepkisi kaydedilerek enzimlerin sensör uygulamalarında aktif tabaka olarak kullanılabilirlikleri araştırılmıştır.

Anahtar Kelimeler

• Polifenol oksidaz enzimi
• biyo-sensör
• kuartz kristal mikrobalans

Kaynakça

1. [1] Silva L. F., Mastelaro V. R., Catto A. C., Escanhoela C. A., Bernardini S., Zilio S. C., Longo E., Aguir K. 2015. Ozone and nitrogen dioxide gas sensor based on a nanostructured SrTi0.85Fe0.15O3 thin film. J. Alloys and Compounds, 638, 374-379.
2. [2] Zhai Z., Sun Y., Hao X., Li C. 2023. Capacitive Gas Sensors Based on a ZIF-67/PAN Nanofiber Membrane to Detect Volatile Organic Compounds. Applied Surface Sci., 621, 156833
3. [3 Kılınc N., Ozturk S., Atilla D., Gurek A., Ahsen V., Ozturk Z. Z., 2012. Electrical and NO2 sensing properties of liquid crystalline phthalocyanine thin films. Sensors and Actuators B, 173, 203-210.
4. [4] Banimuslem H., Hassan A., Basova T., Esenpınar A. A, Tuncel S., Durmus M., Gürek A. G., Ahsen V., 2015. Dye-modified carbon nanotubes for the optical detection of amines vapors. Sensors Actuators B, 207, 224-234.
5. [5] Wang B., Zhang L., Li B., Li Y., Shi Y., Shi T., 2014. Synthesis, characterization, and oxygen sensing properties of functionalized mesoporous silica SBA-15 and MCM-41 with a Pt(II)–porphyrin complex. Sensors Actuators B, 190, 93-100.
6. [6] Hyodo T., Ishibashi C., Matsuo K., Kaneyasu K., Yanagi H., Shimizu Y., 2012. CO and CO2 sensing properties of electrochemical gas sensors using an anion-conducting polymer as an electrolyte. Electrochimica Acta, 82, 19-25.
7. [7] Koshets I. A., Kazantseva Z. I., Shirshov Y. M., Cherenok S. A., Kalchenko V. I., 2005. Calixarene films as sensitive coatings for QCM-based gas sensors. Sensors Actuators B, 106, 177–181.
8. [8] Uzunoglu T., Evyapan M., Beyaztas S., Yildirim G., Kusku T., 2020. Immobilization xanthine oxidase on interdigitated electrode for biosensor investigation. Fresenius Environmental Buletin, 29(8), 7122-7129.

9. [9] Rai U. N., Tripathi R. D., Vajpayee P. 2002. Bioacumulation of toxic metals ( Cr, Cd, Pb and Cu) by seeds of euryale ferox salisb. (makhana). Chemosphere, 46, 267- 272.
10. [10] Gu Y. G., Ning J. J., Ke C. L., Huang H. H., 2018. Bioaccessibility and human health implications of heavy metals in different trophic level marine organisms: A case study of the South China Sea. Ecotoxicol Environ Saf, 163, 551-557.
11. [11] Pandey N., Sharma C. P., 2002 Effect of Heavy metals Co+2, Ni+2 and Cd+2 on growth and metabolism of cabbage. Plant Sci, 163, 753-758.
12. [12] Dinçer A., Becerik S., Aydemir T. 2012. Immobilization of tyrosinase on chitosan-clay composite beads. International Journal of Biological Macromolecules, 50, 815-820.
13. [13] Agarwal P., Dubey S., Singh M., Singh R. P. 2016. Aspergillus niger PA2 tyrosinase covalently ımmobilized on a novel eco-friendly bio-composite of chitosan-gelatin and its evaluation for L-Dopal. Frontiers in Microbiology, 7, https://doi.org/10.3389/fmicb.2016.02088.
14. [14] Uzunoğlu S. B., Uzunoğlu T., Koçsuz S., Evyapan M., Arslan O. 2021. Metal ion effects Polyphenol Oxidase Covalently immobilized on a Bio-Composite. Cellular and Molecular Biology, 67, 50-55.
15. [15] Sauerbrey G. 1959. The use of quartz oscillators for weighting thin layers and for microweighting, Z. Phys., 155, 206.
16. [16] Jundale D., Pawar S., Chougule M., Godse P., Patil S., Raut B., Sen S., Patil V. 2011. Nanocrystalline CuO thin films for H2S monitoring: microstructural and optoelectronic characterization. Journal of Sensor Technology, 1, 36-46.
17. [17] Mecea V. M. 2005. From quartz crystal microbalance to fundamental principles of mass measurements. Analytical Letters, 38, 753-767.
18. [18] Koli P. B, Kapadnis K. H, Deshpande U. G, Tupe U. J, Shinde S. G, Ingale R. S. 2021. Fabrication of thin film sensors by spin coating using sol-gel LaCrO 3 Perovskite material modified with transition metals for sensing environmental pollutants, greenhouse gases and relative humidity. Environmental Challenges, 3, 100043.
19. [19] Tyona M. D., 2013. A theoritical study on spin coating technique. Advances in Materials Research, 2(4), 195-208.
20. [20] Pattananuwat P., Tagaya M., Kobayashi T. 2015. A novel highly sensitive humidity sensor based onpoly(pyrrole-co-formyl pyrrole) copolymer film: AC and DC impedance analysis. Sensors and Actuators B, 209, 186-193.
21. [21] Zhang F., Di C., Berdunov N., Hu Y., Hu Y., Gao X., Meng Q., Sirringhaus H., Zhu D. 2013. Ultrathin Film Organic Transistors: Precise Control of Semiconductor Thickness via Spin-Coating. Advanced Materials, 25, 1401-1407.
22. [22] Evyapan M., Hanoosh W. S., Hassan A. K. 2017. Determination of Volatile Organic Compounds by a Novel Polymer Spin-Coated Thin Film and Surface Plasmon Resonance. Analytical Letters, 50(16), 2579-2594.
23. [23] Moiz S.A., Ahmed M. M., Karimov K. S., Mehmood M. 2007. Temperature-dependent current–voltage characteristics of poly-N-epoxypropylcarbazole complex. Thin Solid Films, 516, 72-77.