O-Fenilendiamin ve Benzokinon Bazlı Polimer Filmle Oluşturulan Kolesterol Biyosensörünün Elektrokimyasal Karakterizasyonu

Öz

Amaç: Kandaki kolesterol konsantrasyonunun tespiti, kardiyovasküler, hepatobiliyer hastalıklar dahil olmak üzere birçok hastalığın tanı ve izleminde önemli bir parametredir. Günümüzde rutin olarak kolesterol analizi için spektrofotometrik, HPLC ve amperometrik yöntemler gibi çeşitli yöntemler kullanılmaktadır. Biyosensörlerin yüksek hassasiyet, hızlı yanıt, düşük maliyet, küçük boyut, sürekli çevrimiçi algılama ve tekrarlanabilir sonuçlar gibi avantajları olduğu için kolesterol tayinine yönelik bir biyosensör yöntemi geliştirilmesi amaçlanmıştır. Yöntem: Altın elektrot yüzeyine elektrokimyasal olarak o-fenilendiamin ve benzokinon içeren asetonitril-su ortamından polimer film immobilizasyonu yapıldı. Polimer film immobilize elektrot yüzeyine kendiliğinden oluşan tek tabaka yöntemiyle kolesterol oksidaz (ChOx,E.C 1.1.3.6) enzimi immobilize edildi. Hazırlanan elektrotlarla kolesterol tayini için optimizasyon ve karakterizasyon çalışmaları yapıldı. Bulgular: Kolesterol tayini, bienzimatik olarak 0.70 V Ag/AgCl'de enzimatik olarak üretilen H2O2'nin oksidasyon akımının izlenmesi yoluyla gerçekleştirilmiştir. Au/oPD-BQ/ChOx enzim elektrotu için fosfat tamponu, 0.1M, pH 7.0 ve 30°C, optimum biyosensör koşulları olarak belirlendi. Doğrusal çalışma aralığı 9.8×10-6-1.1×10-2 mM'dir. Kolesterol tespit limiti 9.8×10-6 mM'dir. Sonuç: Hazırlanan elektrot yüzeyine polimer film aracılığıyla kolesterol oksidaz immobilize edilmesiyle kolesterol tespiti için uygun, basit, tekrarlanabilir ve stabil bir yöntem geliştirilmiştir. Tasarladığımız sensör klinikte birçok analitin tespitinde geliştirilmek üzere yaygınlaştırılabilir.

Anahtar Kelimeler

• Benzokinon
• biyosensör
• kolesterol
• o-fenilendiamin
• polimer film

Electrochemical Characterization of Cholesterol Biosensor Formed by Polymer Film Based on o-Phenylenediamine and Benzoquinone

Öz

Aim: The detection of cholesterol concentration in the blood is an important parameter in the diagnosis and follow-up of many diseases, including cardiovascular and hepatobiliary diseases. Nowadays, various methods have been used for the routine analysis of cholesterol, including spectrophotometric, high-performance liquid chromatography (HPLC), and amperometric methods. Since biosensors have advantages such as high sensitivity, fast response, low cost, small size, continuous online detection, and reproducible results, it is aimed to develop a biosensor method for cholesterol determination. Methods: Polymer film immobilization was performed on the gold electrode surface electrochemically in an acetonitrile-water medium containing o-phenylenediamine and benzoquinone. Cholesterol oxidase (ChOx, E.C. 1.1.3.6) enzyme was immobilized on the polymer film-immobilized electrode surface by the self-forming monolayer method. Optimization and characterization studies were carried out for the determination of cholesterol with the prepared electrodes. Results: The determination of cholesterol was performed via monitoring the oxidation current of enzymatically produced H2O2 at 0.70 V Ag/AgCl. Optimum biosensor conditions were determined for phosphate buffer at 0.1M, pH 7.0, and 30°C for the Au/oPD-BQ/ChOx enzyme electrode. The linear working range is 9.8×10-6-1.1×10-2 mM, and the cholesterol detection limit is 9.8×10-6 mM. The analysis of total cholesterol in solutions was performed using the proposed Au/oPD-BQ/ChOx electrode. Conclusion: A convenient, simple, reproducible, and stable method was developed for the determination of cholesterol by immobilizing cholesterol oxidase on the prepared electrode surface through a polymer film. The sensor we designed can be expanded to improve the detection of many analytes in the clinic.

Anahtar Kelimeler

• Benzoquinone
• cholesterol
• biosensor
• o-phenylenediamine

Kaynakça

1. Ahmad R, Tripathy N, Park JH, Hahn YB. A comprehensive biosensor integrated with a ZnO nanorod FET array for selective detection of glucose, cholesterol and urea. Chemical Communications. 2015;51(60):11968-11971.
2. Ahmadalinezhad A, Chen A. High-performance electrochemical biosensor for the detection of total cholesterol. Biosensors & Bioelectronics. 2011;26(11):4508-4513.
3. Arya SK, Datta M, Malhotra BD. Recent advances in cholesterol biosensor. Biosensors & Bioelectronics. 2008;23(7):1083-1100.
4. Albuquerque T, Oliveira MB, Sanches-Silva A, Costa HS. Cholesterol determination in foods: Comparison between high performance and ultra-high performance liquid chromatography. Food Chemistry. 2016;193:18-25.
5. Mungroo NA, Suresh N. Biosensors for the detection of antibiotics in poultry industry—a review. Biosensors. 2014;4:472-493.
6. Kumar S, Singh R, Kaushik BK, Chen NK, Yang QS, Zhang X. LSPR-based cholesterol biosensor using hollow core fiber structure. IEEE Sensors Journal. 2019;19:7399-7406.
7. Hammond JL, Formisano N, Estrela P, Carrara S, Tkac J. Electrochemical biosensors and nanobiosensors. Essays in Biochemistry. 2016;60(1):69-80.
8. Mansuriya BD, Altintas Z. Applications of graphene quantum dots in biomedical sensors. Sensors. 2020;20(4):1072-1075.

9. Teles FR, Fonseca LP. Applications of polymers for biomolecule immobilization in electrochemical biosensors. Materials Science and Engineering. 2008;28(8):1530-1543.
10. Gooding JJ, Mearns F, Yang W, Liu J. Self‐assembled monolayers into the 21st century: recent advances and applications. Electroanalysis: An International Journal Devoted to Fundamental and Practical Aspects of Electroanalysis. 2003;15(2):81-96.
11. Parnianchi F, Kashanian S, Nazari M, Santoro C, Bollella P, Varmira K. Highly selective and sensitive molecularly imprinting electrochemical sensing platform for bilirubin detection in saliva. Microchemical Journal. 2021;168:106367.
12. Putzbach W, Ronkainen NJ. Immobilization techniques in the fabrication of nanomaterial-based electrochemical biosensors: A review. Sensors. 2013;13(4):4811-4840.
13. Iqbal S, Ahmad S. Recent development in hybrid conducting polymers: Synthesis, applications and future prospects. Journal of Industrial and Engineering Chemistry. 2018;60:53-84.
14. Ghosh S, Ahmad R, Khare SK. Immobilization of cholesterol oxidase: An overview. The Open Biotechnology Journal. 2018;12(1):15-21.
15. Vidal JC, Espuelas J, Castillo JR. Amperometric cholesterol biosensor based on in situ reconstituted cholesterol oxidase on an immobilized monolayer of flavin adenine dinucleotide cofactor. Analytical Biochemistry. 2004;333(1):88-98.
16. Qin HM, Zhu Z, Ma Z, et ai. Rational design of cholesterol oxidase for efficient bioresolution of cholestane skeleton substrates. Scientific Reports. 2017;7(1):163-175.
17. Yamada K, Koroleva A, Laughlin M, Oksanen N, Akhgari A, Safronova V. Characterization and overproduction of cell-associated cholesterol oxidase ChoD from streptomyces lavendulae YAKB-15. Scientific Reports. 2019;9(1):1-8.
18. Fischer LM, Tenje M, Heiskanen AR, et al. Gold cleaning methods for electrochemical detection applications. Microelectronic Engineering. 2009;86(4):1282-1285.
19. Carrington AM, Manuel DG, Fieguth PW, et al. Deep ROC analysis and AUC as balanced average accuracy, for improved classifier selection, audit and explanation. IEEE Transactions on Pattern Analysis and Machine Intelligence. 2022;45(1):329-341.
20. Matharu Z, Sumana G, Arya SK, Singh SP, Gupta V, Malhotra BD. Polyaniline Langmuir− Blodgett film based cholesterol biosensor. Langmuir. 2007;23:13188-13192.
21. Arya SK, Solanki PR, Datta M, Malhotra BD. Recent advances in self-assembled monolayers based biomolecular electronic devices. Biosensors and Bioelectronics. 2009;24:2810-2817.
22. Rappolt M, Vidal M, Kriechbaum M, et al. Structural, dynamic and mechanical properties of POPC at low cholesterol concentration studied in pressure/temperature space. European Biophysics Journal. 2003;31:575-585.
23. Thakur N, Mandal D, Nagaiah TC. A novel NiVP/Pi-based flexible sensor for direct electrochemical ultrasensitive detection of cholesterol. Chemical Communications. 2022;58:2706-2709.
24. Ou X, Tan X, Wei S, Chen S, Zhang J, Liu X. Electrochemiluminescence biosensor for cholesterol detection based on AuNPs/l-cys–C 60 nanocomposites. Analytical Methods. 2014;6:3804-3810.