Use of L-Lactate Dehydrogenase Immobilized on Carboxylated Multiwalled Carbon Nanotubes/Polyaniline/Pencil Graphite Electrode as a Lactate Biosensor

Abstract

In this study, L-Lactate dehydrogenase (L-LDH) was covalently immobilized on carboxylated multiwalled carbon nanotubes (cMWCNT)/polyaniline (PANI)/pencil graphite electrode (PGE) and used as a lactate biosensor. Electrochemical polymerization of PANI was carried out using a three-electrode cell technique via cyclic voltammretry (CV). The characterization of LDH/cMWCNT/PANI/PGE electrode was achieved using electrochemical and scanning electron microscopy (SEM) techniques. The effect of applied potential, pH, lactate concentration, and NAD+ (with and without) on sensor response was assessed. The optimal pH was determined as 7.0. Biosensor response increased with increasing of lactate concentration. The current values were obtained as 0.026 and 0.038 mA cm-2 for 0.166 and 1.331 mM lactate solution with NAD+ as a cofactor, respectively at +0.2 V. The results indicate that LDH/cMWCNT/PANI/PGE biosensor was more sensitive than the biosensor without carbon nanotubes.

Keywords

• Biosensor
• Lactate Dehydrogenase
• Pencil Graphite Electrode
• Carbon Nanotubes

Use of L-Lactate Dehydrogenase Immobilized on Carboxylated Multiwalled Carbon Nanotubes/Polyaniline/Pencil Graphite Electrode as a Lactate Biosensor

Abstract

Bu çalışmada, L-Laktat dehidrojenaz (L-LDH), karboksillenmiş çok duvarlı karbon nanotüpler (cMWCNT)/polianilin (PANI)/kalem grafit elektrot (PGE) üzerinde kovalent olarak immobilize edilmiş ve laktat biyosensörü olarak kullanılmıştır. PANI’ nin elektrokimyasal polimerizasyonu, dönüşümlü voltametri (CV) ile üç elektrot tekniği kullanılarak gerçekleştirildi. LDH/cMWCNT/PANI/PGE elektrotunun karakterizasyonu, taramalı elektron mikroskobu (SEM) ve elektrokimyasal deneylerle yapılmıştır. Uygulanan potansiyel, pH ve laktat derişiminin NAD+ (ile ve olmadan) sensör yanıtı üzerine etkileri araştırılmıştır. Optimum pH 7,0 olarak belirlendi. Biyosensör yanıtının, laktat derişimi ile birlikte arttığı belirlendi. Akım değerleri, kofaktör olarak NAD+ ile +0,2 V’ ta 0,166 ve 1,331 mM laktat çözeltisi için sırasıyla 0,026 ve 0,038 mA cm-2 olarak belirlendi. Sonuçlar, LDH/cMWCNT/PANI/PGE biyosensörün, karbon nanotüp kullanılmayan biyosensöre göre daha duyarlı olduğunu göstermiştir.

Keywords

• Biyosensör
• Laktat Dehidrojenaz
• Kalem Grafit Elektrot
• Karbon Nanotüpler

References

1. Kemp G. Lactate accumulation, proton buffering, and pH change in is chemically exercising muscle. Am J Physiol Regul Integr Comp Physiol. 2005;289(3):895-901.
2. Bravo I, Revenga-Parra, M, Pariente, F, Lorenzo, E. Reagent-Less and Robust Biosensor for Direct Determination of Lactate in Food Samples. 2017;Sensors:17(1), 144.
3. Kucherenko IS, Topolnikova YV, Soldatkin OO. Advances in the biosensors for lactate and pyruvate detection for medical applications: A review. Trac-Trends Anal. Chem.2019;110: 160-172.
4. Bakker J, Gris P, Coffernils M, Kahn RJ, Vincent JL.Serial blood lactate levels can predict the development of multiple organ failure following septic shock. Am. J. Surg.1996;171: 221-226.
5. Artiss JD, Karcher RE, Cavanagh, KT, Collins SL, Peterson VJ, Varma, S, et al. A liquid-stable reagent for lactic acid levels. Am. J. Clin. Pathol.2000;114:139-143.
6. Kuşbaz A, Göcek İ, Baysal G, Kök FN, Trabzon L, Kizil H, K et al. Lactate detection by colorimetric measurement in real human sweat by microfluidic based biosensor on flexible substrate. J. Text. Inst. 2019;110(12):1725-1732.
7. Basavaiah K, Nagegowda P, Somashekar BC, Ramakrishna V. Spectrophotometric and titrimetric determination of ciprofloxacin based on reaction with cerium (IV) sulphate. Sci. Asia. 2006;32:403-409.
8. Yang L, Overdorf G, Kissinger P. Determination of Lactate with liquid chromatography/electrochemistry coupled with a lactate oxidase imer. Curr. Sep. 1997; 16:15-18.

9. Suzuki M, Akaguma H. Chemical cross-talk in flow-type integrated enzyme sensors. Sens. Actuat. B. 2000;64:136–141.
10. Schmitt RE, Molitor HR, Wu T. Voltammetric method for the determination of lactic acid using a carbon paste electrode modified with cobalt phthalocyanine. Int. J. Electrochem. Sci. 2012;7:10835-10841.
11. Nishijima T, Nishina M, Fujiwara K. Measurement of lactate levels in serum and bile using proton nuclear magnetic resonance in patients with hepatobiliary diseases: Its utility in detection of malignancies. Jpn. J. Clin. Oncol. 1997;27:13-17.
12. Rathee K, Dhull V, Dhull R, Singh S. Biosensors based on electrochemical lactate detection: A comprehensive review. Biochem. Biophys. Rep. 2016;5:35-54.
13. Salimi A, Noorbakhsh A, Mamkhezri H. Ghavam R. Electrocatalytic Reduction of H2O2 and Oxygen on the Surface of Thionin Incorporated onto MWCNTs Modified Glassy Carbon Electrode: Application to Glucose Detection. Electroanalysis. 2007;19:1100-1108.
14. He XR, Yu JH, Ge SG, Zhang XM, Lin Q, Zhu H, at al. Amperometric l-lactate biosensor based on sol–gel film and multi-walled carbon nanotubes/platinum nanoparticles enhancement. Chin. J. Anal. Chem. 2010;38:57-61.
15. McCreery RL. Advanced Carbon Electrode Materials for Molecular Electrochemistry. Chem. Rev. 2008;108:2646-2687.
16. Simon E, Halliwell CM, Toh CS, Cass AEG, Bartlett PN. Oxidation of NADH produced by a lactate dehydrogenase immobilised on poly(aniline)–poly(anion) composite films. J. Electroanal. Chem. 2002;538-539:253-259.
17. Syedmoradi L, Daneshpour M, Alvandipour M, Gomez FA, Hajghassem H, Omidfar K. Point of care testing: The impact of nanotechnology. Biosens Bioelectron. 2017;87:373-387.
18. Lai J, Yi Y, Zhu P, Shen J, Wu K, Zhang L. et al. Polyaniline-based glucose biosensor: A review. J Electroanal Chem. 2016; 782:138-153.
19. Yazdanparast S, Benvidi A, Banaei M, Nikukar H, Tezerjani MD, Azimzadeh M. Dual-aptamer based electrochemical sandwich biosensor for MCF-7 human breast cancer cells using silver nanoparticle labels and a poly(glutamic acid)/MWNT nanocomposite. Mikrochim Acta 2018;185:405.
20. Hadian NS, Faridnouri Hassan, Zare EN. Glucose biosensing based on glucose oxidase immobilization on carboxymethyl chitosan/polyaniline/multi-walled carbon nanotubes nanocomposite. Diamond and Related Materials. 2024;148:111423.
21. Özdemir I, Tülek A, Karaaslan B, Yildirim D. Evaluation of multi-walled carbon nanotubes bearing aldehyde groups of different lengths for the immobilization of Geobacillus kaustophilus l-asparaginase. Molecular Catalysis. 2024;555:113903.
22. Varan N, Alagöz D, Toprak A, Korkmaz H, Yildirim D. Immobilization of pullulanase from Bacillus licheniformis on magnetic multi-walled carbon nanotubes for maltooligosaccharide production. Chemical Papers. 2024;7878:9529–9542.
23. Musameh M, Wang J, Merkoci A, Lin Y. Low-potential stable NADH detection at carbon-nanotube-modified glassy carbon electrodes. Electrochem Commun. 2002;4(10): 743-746.
24. Goran JM, Lyon JL, Stevenson KJ. Amperometric detection of L-lactate using nitrogen-doped carbon nanotubes modified with lactate oxidase. Anal Chem. 2011;83(21):8123-8129.
25. Agui L, Eguilaz M, Pena-Farfal C, Yanez-Sedeno P, Pingarron JM. Lactate dehydrogenase biosensor based on an hybrid carbon nanotube-conducting polymer modified electrode. Electroanalysis. 2009; 21(3-5):386-391.
26. Jaryal VB, Kumar S, Singh D, Gupta N. Thiourea‐Modified Multiwalled Carbon Nanotubes as Electrochemical Biosensor for Ultra‐Precise Detection of Dopamine. ChemNanoMat. 2024;10(6): e202300637.
27. Silva W, Guedes EAB, Faustino LC, Goulart MOF, Gerôncio ETS. Tailored Electrochemical Biosensor with poly-diallydimethylammonium chloride-functionalised multiwalled carbon nanotubes/gold nanoparticles/manganese dioxide, and Haemoglobin for Sensitive Hydrogen Peroxide Detection. Talanta. 2024:276;126290.
28. Yulianti ES, Rahman S, Rizkinia M, Zakiyuddin A. Low-cost electrochemical biosensor based on a multi-walled carbon nanotube-doped molecularly imprinted polymer for uric acid detection. Arabian Journal of Chemistry. 2024;17(4):105692.
29. Salimi A, Noorbakhsh A, Mamkhezri H, Ghavami R. Electrocatalytic reduction of H2O2 and oxygenon the surface of thionin incorporated onto MWCNTs modified glassy carbon electrode: application to glucose detection. Electroanalysis. 2007; 19(10):1100-1108.
30. Alagoz D, Toprak A, Yildirim D, Tükel SS, Fernandez-Lafuente R. Modified silicates and carbon nanotubes for immobilization of lipase from Rhizomucor miehei: Effect of support and immobilization technique on the catalytic performance of the immobilized biocatalysts. Enzyme Microb. Technol. 2021;144:109739.
31. Purushothama HT, Arthoba Nayaka Y, Vinay MM, Manjunatha P, Yathisha RO, Basavarajappa KV. Pencil graphite electrode as an electrochemical sensor for the voltammetric determination of chlorpromazine. Journal of Science: Advanced Materials and Devices. 2018;3:161-166.
32. Batra B, Narwal V, Pundir CS. An amperometric lactate biosensor based on lactate dehydrogenase immobilized onto graphene oxide nanoparticles-modified pencil graphite electrode. Eng. Life Sci. 2016;16:786-794.
33. Tamborelli A, Mujica ML, Amaranto M, Barra JL, Rivas G, Godino A, Dalmasso P. L-Lactate Electrochemical Biosensor Based on an Integrated Supramolecular Architecture of Multiwalled Carbon Nanotubes Functionalized with Avidin and a Recombinant Biotinylated Lactate Oxidase. Biosensors (Basel). 2024;14:196.
34. Xia Z, Zuo W, Li H, Qiu L, Mu R,Wang Q, et al. Wearable cellulose textile matrix self-powered biosensor sensing lactate in human sweat. J Appl Electrochem. 2024;54:1137–1152.
35. Darshna, Nandi I, Srivastava P, Chandra P. Clinically Deployable Electro-Immunosensing Device Comprising Bioactive Glass-MWCNT for Alkaline Phosphatase Detection in Human Serum Samples. ACS Appl Bio Mater. 2025;20;8(1):741-753.
36. Zhang M, Gorski W. Electrochemical sensing based on redox mediation at carbon nanotubes. Anal. Chem. 2002;77:3960-3965.
37. Musameh M, Wang, J, Merkoci A, Lin Y. Low-potential stable NADH detection at carbon-nanotube-modified glassy carbon electrodes. Electrochem. Commun. 2002;4:743-746.
38. Istrate OM. Rotariu L, Bala C. Amperometric L-Lactate Biosensor Based upon a Gold Nanoparticles/Reduced Graphene Oxide/Polyallylamine Hydrochloride Modified Screen-Printed Graphite Electrode. Chemosensors. 2021; 9:74.
39. Rahman MM, Shiddiky MJA, Rahman MA, Shim YB. Lactate biosensor based on lactate dehydrogenase/nictotinamide adenine dinucleotide (oxidized form) immobilized on a conducting polymer/multiwall carbon nanotube composite film. Analytical Biochemistry. 2009;384:159-165.
40. Wang J, Musameh M. Carbon nanotube/Teflon composite electrochemical sensors and biosensors. Anal. Chem. 2003;75:2075-2079.
41. Chen J, Bao J, Cai C, Lu T. Electrocatalytic oxidation of NADH at an ordered carbon nanotubes modified glassy carbon electrode, Anal. Chim. Acta. 2004;516:29-34.
42. Banks CE, Moore RR, Davies TJ, Compton RG. Investigation of modified basal plane pyrolytic graphite electrodes: definitive evidence for the electrocatalytic properties of the ends of carbon nanotubes. Chem. Commun. 2004;16:1804-1805.
43. Tian L, Cai L, Ding Z, Zhou Y, Zhang Y, Liu Q, et al. Sweat lactate biosensor based on lactate oxidase immobilized with flower-like NiCo2O4 and carbon nanotubes, Microchemical Journal. 2024;200:11041.
44. Luo X, Killard AJ, Morrin A, Smyth MR. Enhancement of a conducting polymer-based biosensor using carbon nanotube-doped polyaniline. Anal Chim Acta. 2006;575:39-44.
45. Granot E, Basnar B, Cheglakov Z, Katz E, Willner I. Enhanced bioelectrocatalysis using single-walled carbon nanotubes (SWCNTs)/polyaniline hybrid systems in thin-film and microrod structures associated with electrodes. Electroanalysis. 2006;18:26-34.
46. Tsai YC, Chen SY, Liaw HW. Immobilization of lactate dehydrogenase within multiwalled carbon nanotube-chitosan nanocomposite for application to lactate biosensors. Sens. Actuators B Chem. 2007;125(2):474-481.
47. Rassaei L, Olthuis W, Tsujimura S, Sudhölter EJ, Berg AVD. Lactate biosensors: current status and outlook. Anal Bioanal Chem. 2014;406(1);123-37.