Facile immobilization of cholesterol oxidase on Pt,Ru-C nanocomposite and ionic liquid-modified carbon paste electrode for an efficient amperometric free cholesterol biosensing

Sanja Mutić; Dalibor Stanković; Zoltán Kónya; Jasmina Anojčić

doi:10.1007/s00216-023-04847-9

Facile immobilization of cholesterol oxidase on Pt,Ru-C nanocomposite and ionic liquid-modified carbon paste electrode for an efficient amperometric free cholesterol biosensing

Anal Bioanal Chem. 2023 Sep;415(23):5709-5722. doi: 10.1007/s00216-023-04847-9. Epub 2023 Jul 16.

Authors

Sanja Mutić¹, Dalibor Stanković^{2

3}, Zoltán Kónya^{4

5}, Jasmina Anojčić⁶

Affiliations

¹ Department of Chemistry, Biochemistry and Environmental Protection, Faculty of Sciences, University of Novi Sad, Trg D. Obradovića 3, 21000, Novi Sad, Serbia.
² Faculty of Chemistry, University of Belgrade, Studentski Trg 12-16, 11000, Belgrade, Serbia.
³ Department of Radioisotopes, "Vinča" Institute of Nuclear Sciences - National Institute of the Republic of Serbia, University of Belgrade, Mike Petrovića Alasa 12-14, 11000, Belgrade, Serbia.
⁴ Department of Applied and Environmental Chemistry, University of Szeged, Rerrich Béla Tér 1, 6720, Szeged, Hungary.
⁵ ELKH-SZTE Reaction Kinetics and Surface Chemistry Research Group, Rerrich Béla Tér 1, 6720, Szeged, Hungary.
⁶ Department of Chemistry, Biochemistry and Environmental Protection, Faculty of Sciences, University of Novi Sad, Trg D. Obradovića 3, 21000, Novi Sad, Serbia. jasmina.anojcic@dh.uns.ac.rs.

PMID: 37453937
DOI: 10.1007/s00216-023-04847-9

Abstract

In present work, the enzyme cholesterol oxidase (ChOx) was immobilized by Nafion® (Naf) on Pt,Ru-C nanocomposite and an ionic liquid (IL)-modified carbon paste electrode (CPE) in order to create cholesterol biosensor (Naf/ChOx/Pt,Ru-C/IL-CPE). The prepared working electrodes were characterized using scanning electron microscopy-energy-dispersive spectrometry, while their electrochemical performance was evaluated using electrochemical impedance spectroscopic, cyclic voltammetric, and amperometric techniques. Excellent synergism between IL 1-allyl-3-methylimidazolium dicyanamide ([AMIM][DCA]), Pt,Ru-C, and ChOx, as modifiers of CPE, offers the most pronounced analytical performance for improved cholesterol amperometric determination in phosphate buffer solution pH 7.50 at a working potential of 0.60 V. Under optimized experimental conditions, a linear relationship between oxidation current and cholesterol concentration was found for the range from 0.31 to 2.46 µM, with an estimated detection limit of 0.13 µM and relative standard deviation (RSD) below 5.5%. The optimized amperometric method in combination with the developed Naf/ChOx/Pt,Ru-C/IL-CPE biosensor showed good repeatability and high selectivity towards cholesterol biosensing. The proposed biosensor was successfully applied to determine free cholesterol in a human blood serum sample via its enzymatic reaction product hydrogen peroxide despite the presence of possible interferences. The percentage recovery ranged from 99.08 to 102.81%, while RSD was below 2.0% for the unspiked as well as the spiked human blood serum sample. The obtained results indicated excellent accuracy and precision of the method, concluding that the developed biosensor can be a promising alternative to existing commercial cholesterol tests used in medical practice.

Keywords: Cholesterol; Cholesterol oxidase; Electrochemical biosensor; Human blood serum sample; Ionic liquids; Pt,Ru–C nanocomposite.

MeSH terms

Biosensing Techniques* / methods
Carbon / chemistry
Cholesterol / analysis
Cholesterol Oxidase / chemistry
Electrodes
Enzymes, Immobilized / chemistry
Humans
Ionic Liquids* / chemistry
Nanocomposites* / chemistry

Substances

Carbon
Cholesterol Oxidase
Ionic Liquids
Cholesterol
Enzymes, Immobilized

Grants and funding

451-03-47/2023-01/200125/Ministarstvo Prosvete, Nauke i Tehnološkog Razvoja