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Amperometric biosensors based on alcohol oxidase and peroxidase–like nanozymes for ethanol determination

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Abstract

The aim of the current research is to design alcohol oxidase-based amperometric biosensors (ABSs) using hybrid metallic nanoparticles as artificial peroxidases (PO) or PO-like nanozymes (NZs). A lot of metallic PO-like NZs were synthesized and tested with respect to their ability to substitute natural PO in solution and on amperometric electrode. The most effective PO mimetics were coupled with alcohol oxidase (AOX) on graphite electrodes (GEs) and characterized. Two types of modified GEs, namely, the AOX/nAuCePt/GE and the AOX/nFePtAu/GE show the highest sensitivities to ethanol (2600 A⋅M−1⋅m−2 and 1250 A⋅M−1⋅m−2, respectively), low limits of detection (1.5 µM and 2.2 µM), broad linear ranges (5 – 100 µM and 12 – 120 µM), as well as satisfactory storage stabilities. The most sensitive bioelectrode AOX/nAuCePt/GE was used as ABS for ethanol determination in real samples. The practical feasibility of the constructed ABS was demonstrated by determination of ethanol in beverages, human blood and saliva.

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References

  1. Bollella P (2022) Enzyme-based amperometric biosensors: 60 years later … Quo Vadis? Anal Chim Acta 340517. https://doi.org/10.1016/j.aca.2022.340517

  2. Verma D, Singh KRB, Yadav AK et al (2022) Internet of things (IoT) in nano-integrated wearable biosensor devices for healthcare applications. Biosens Bioelectron: X 11:100153. :https://doi.org/10.1016/j.biosx.2022.100153

  3. Matzeua G, Florea L, Diamonda D (2015) Advances in wearable chemical sensor design for monitoring biological fluids. Sens Actuat B: Chem 211:403–418. https://doi.org/10.1016/j.snb.2015.01.077

    Article  CAS  Google Scholar 

  4. Somboon T, Phatchana R, Tongpoothorn W, Sansuk S (2022) A simple and green method for determination of ethanol in liquors by the conductivity measurement of the uncatalyzed esterification reaction. LWT 154:112593. https://doi.org/10.1016/j.lwt.2021.112593

    Article  CAS  Google Scholar 

  5. Pinheiro ACN, Ferreira VS, Lucca BG (2022) Stamping method based on 3D printing and disposable napkin: cheap production of paper analytical devices for alcohol determination in beverages aiming forensics and food control. Microchem J 180:107604. https://doi.org/10.1016/j.microc.2022.107604

    Article  CAS  Google Scholar 

  6. Mansur AR, Oh J, Lee HS et al (2022) Determination of ethanol in foods and beverages by magnetic stirring-assisted aqueous extraction coupled with GC-FID: a validated method for halal verification. Food Chem 366:130526. https://doi.org/10.1016/j.foodchem.2021.130526

    Article  CAS  Google Scholar 

  7. Debebe A, Redi-Abshiro M, Chandravanshi BS (2017) Non-destructive determination of ethanol levels in fermented alcoholic beverages using Fourier transform mid-infrared spectroscopy. Chem Cent J 11:27. https://doi.org/10.1186/s13065-017-0257-5

    Article  CAS  Google Scholar 

  8. Sriariyanun M, Mutrakulcharoen P, Tepaamorndech S et al (2019) Rapid spectrophotometric method for quantitative determination of ethanol in fermentation products. Orient J Chem 35(2):744–750. https://doi.org/10.13005/ojc/350234

    Article  CAS  Google Scholar 

  9. Duangdeewong C, Choengchan N, Wattanasin P, Teerasong S (2022) Direct determination of ethanol in alcoholic beverages based on its anti-aggregation of melamine-silver nanoparticle assembly. Talanta 250:123751. https://doi.org/10.1016/j.talanta.2022.123751

    Article  CAS  Google Scholar 

  10. Bevanda AM, Ivankovic A, Talic S et al (2021) Determination of ethanol in wine and distilled alcoholic beverages using a smartphone as a detector. Int J Comput Theoretical Chem 9(2):43–47. https://doi.org/10.11648/j.ijctc.20210902.13

    Article  CAS  Google Scholar 

  11. Boroujerdi R, Abdelkader A, Paul R (2022) Highly selective detection of ethanol in biological fluids and alcoholic drinks using indium ethylenediamine functionalized graphene. Sens Diagn 1:566–578. https://doi.org/10.1039/D2SD00011C

    Article  CAS  Google Scholar 

  12. Wozniak MK, Wiergowski M, Namiesnik J, Biziuk M (2019) Biomarkers of alcohol consumption in body fluids - possibilities and limitations of application in toxicological analysis. Curr Med Chem 26(1):177–196. https://doi.org/10.2174/0929867324666171005111911

    Article  CAS  Google Scholar 

  13. Thepchuay Y, Sonsa-ard T, Ratanawimarnwong N et al (2020) Paper-based colorimetric biosensor of blood alcohol with in-situ headspace separation of ethanol from whole blood. Anal Chim Acta 1103:115–121. https://doi.org/10.1016/j.aca.2019.12.043

    Article  CAS  Google Scholar 

  14. Taylor L, Remeškevičius V, Saskoy L et al (2021) Determination of ethanol in micro-volumes of blood by headspace gas chromatography: statistical comparison between capillary and venous sampling sites. Med Sci Law 61(2):86–96. https://doi.org/10.1177/0025802420928632

    Article  Google Scholar 

  15. Hernandez J, Kaun KR (2022) Alcohol, neuronal plasticity, and mitochondrial trafficking. Proc Natl Acad Sci USA 119(29):e2208744119. https://doi.org/10.1073/pnas.2208744119

    Article  CAS  Google Scholar 

  16. Knabbe J, Protzmann J, Schneider N et al (2022) Single-dose ethanol intoxication causes acute and lasting neuronal changes in the brain. Proc Natl Acad Sci USA 119(25):e2122477119. https://doi.org/10.1073/pnas.2122477119

    Article  CAS  Google Scholar 

  17. Alaouie AM, Alawieh MB (2020) Critical evaluation of single point measurement-based quantitative analyses in forensic breath alcohol. Forensic Sci Int 2:100101. https://doi.org/10.1016/j.fsir.2020.100101

    Article  Google Scholar 

  18. Jones AW, Cowan JM (2020) Reflections on variability in the blood–breath ratio of ethanol and its importance when evidential breath-alcohol instruments are used in law enforcement. Forensic Sci Res 5(4):300–308. https://doi.org/10.1080/20961790.2020.1780720

    Article  Google Scholar 

  19. Laposat M (1999) Assessment of ethanol intake current tests and new assays on the Horizon. Am J CIin Pathol 112:443–450. https://doi.org/10.1093/ajcp/112.4.443

    Article  Google Scholar 

  20. Keim ME, Bartfield JM, Raccio-Robak N et al (1999) Accuracy of an enzymatic assay device for serum ethanol measurement. J Toxicol Clin Toxicol 37(1):75–81. https://doi.org/10.1081/clt-100102411

    Article  CAS  Google Scholar 

  21. Bendtsen P, Hultberg L, Carlsson M, Jones AW (1999) Monitoring ethanol exposure in a clinical setting by analysis of blood, breath, saliva, and urine. Alcohol Clin Exp Res 23:1446–1451. https://doi.org/10.1111/j.1530-0277.1999.tb04665.x

    Article  CAS  Google Scholar 

  22. Jones AW (1979) Distribution of ethanol between saliva and blood in man. Clin Exp Pharmacol Physiol 6:53–59. https://doi.org/10.1111/j.1440-1681.1979.tb00007.x

    Article  CAS  Google Scholar 

  23. Costa NG, Antunes JC, Paleo AJ, Rocha AM (2022) A review on flexible electrochemical biosensors to monitor alcohol in sweat. Biosensors 12(4):252. https://doi.org/10.3390/bios12040252

    Article  CAS  Google Scholar 

  24. Rao TM, Reddy PDS, Ramani P (2015) Detection of alcohol in saliva for blood alcohol concentration using alcohol saliva strip test: a forensic aid. J Dr NTR Univ Health Sci 4(1):24–29. https://doi.org/10.4103/2277-8632.153310

    Article  Google Scholar 

  25. Mojumder S, Das T, Das S et al (2022) Y and Al co-doped ZnO-nano powder based ultrasensitive trace ethanol sensor: a potential breath analyzer for fatty liver disease and drunken driving detection. Sens Actuat B: Chem 372:132611. https://doi.org/10.1016/j.snb.2022.132611

    Article  CAS  Google Scholar 

  26. Kock FVC, Rocha TC, Araújo GM et al (2019) Time-domain NMR: a novel analytical method to quantify adulteration of ethanol fuel with methanol. Fuel 258:116158. https://doi.org/10.1016/j.fuel.2019.116158

    Article  CAS  Google Scholar 

  27. Avila LM, Franco dos Santos AP, Mançano de Mattos DI et al (2018) Determination of ethanol in gasoline by high-performance liquid chromatography. Fuel 212:236–239. https://doi.org/10.1016/j.fuel.2017.10.039

    Article  CAS  Google Scholar 

  28. Oliver JD, Gaborieau M, Castignolles P (2014) Ethanol determination using pressure mobilization and free solution capillary electrophoresis by photo-oxidation assisted ultraviolet detection. J Chromatogr A 1348:150–157. https://doi.org/10.1016/j.chroma.2014.04.076

    Article  CAS  Google Scholar 

  29. Hu X, Cao H, Dong W, Tang J (2021) Ratiometric fluorescent sensing of ethanol based on copper nanoclusters with tunable dual emission. Talanta 233:122480. https://doi.org/10.1016/j.talanta.2021.122480

    Article  CAS  Google Scholar 

  30. Ratanawimarnwong N, Sinpun M, Chankaw P et al (2020) Simple flow system with in-line gas-diffusion unit for determination of ethanol employing hypsochromic shift of visible absorbance band of methyl orange. Talanta 206:120234. https://doi.org/10.1016/j.talanta.2019.120234

    Article  CAS  Google Scholar 

  31. Vázquez-Guardado A, Ramirez-Flores JA, Lopez-Galmiche G et al (2019) Detection of ethanol concentration using a generic optical sensor platform. Comput Sist 23(1):27–31. https://doi.org/10.13053/CyS-23-1-3138

    Article  Google Scholar 

  32. Li B, Chen Y, Peng A et al (2019) Improved photoelectrochemical properties of tungsten oxide by modification with plasmonic gold nanoparticles for the non-enzymatic sensing of ethanol. J Colloid Interface Sci 537:528–535. https://doi.org/10.1016/j.jcis.2018.11.061

    Article  CAS  Google Scholar 

  33. Gayda G, Stasyuk N, Klepach H et al (2019). Promising bioanalytical approaches to wine analysis. Ch. 12, In: The Science of Beverages, Grumezescu AM, Holban AM (eds): pp. 419–458. Woodhead Publishing Inc. https://doi.org/10.1016/B978-0-12-816681-9.00012-6

  34. Azevedo AM, Miguel D, Prazeres F et al (2005) Ethanol biosensors based on alcohol oxidase. Biosen Bioelectron 21(2):235–247. https://doi.org/10.1016/j.bios.2004.09.030

    Article  CAS  Google Scholar 

  35. Smutok O, Ngounou B, Pavlishko H et al (2006) A reagentless bienzyme amperometric biosensor based on alcohol oxidase/peroxidase and an Os-complex modified electrodeposition paint. Sens Actuat B Chem 113:590–598. https://doi.org/10.1016/j.snb.2005.07.055

    Article  CAS  Google Scholar 

  36. Stasyuk N, Gayda G, Zakalskiy A et al (2019) Amperometric biosensors based on oxidases and PtRu nanoparticles as artificial peroxidase. Food Chem 285:213–220. https://doi.org/10.1016/j.foodchem.2019.01.117

    Article  CAS  Google Scholar 

  37. Smutok O, Kavetskyy T, Prokopiv T (2021) New micro/nanocomposite with peroxidase-like activity in construction of oxidases-based amperometric biosensors for ethanol and glucose analysis. Anal Chim Acta 1143:201–209. https://doi.org/10.1016/j.aca.2020.11.052

    Article  CAS  Google Scholar 

  38. Stasyuk N, Demkiv O, Gayda G et al (2022) Highly porous 3D gold enhances sensitivity of amperometric biosensors based on oxidases and CuCe nanoparticles. Biosensors 12:472. https://doi.org/10.3390/bios12070472

    Article  CAS  Google Scholar 

  39. Gayda G, Demkiv O, Klepach H et al (2019) Effective technologies for isolating yeast oxido-reductases of analytical importance. Ch. 5, In: Non-conventional Yeasts: from Basic Research to Application, Sibirny A. (ed.): pp. 119–151. Springer Nature Switzerland AG, Cham, Switzerland. https://doi.org/10.1007/978-3-030-21110-3

  40. Klepach HM, Zakalskiy AE, Zakalska OM et al (2021) Alcohol oxidase from the methylotrophic yeast Ogataea polymorpha: isolation, purification, and bioanalytical application In: Barile M (ed) Flavins and Flavoproteins, E-Publishing Inc, pp. 231–248. https://doi.org/10.1007/978-1-0716-1286-6_15

  41. Camacho C, Matías JC, Chico B et al (2007) Amperometric biosensor for hydrogen peroxide, using supramolecularly immobilized horseradish peroxidase on the β-cyclodextrin-coated gold electrode. Electroanal 19(24):2538–2542. https://doi.org/10.1002/elan.200703993

    Article  CAS  Google Scholar 

  42. Asif M, Liu H, Aziz A et al (2017) Core-shell iron oxide-layered double hydroxide: High electrochemical sensing performance of H2O2 biomarker in live cancer cells with plasma therapeutics. Biosens Bioelectron 97:352–359. https://doi.org/10.1016/j.bios.2017.05.057

    Article  CAS  Google Scholar 

  43. Stasyuk N, Smutok O, Demkiv O et al (2020) Synthesis, catalytic properties and application in biosensorics of nanozymes and electronanocatalysts: a review. Sensors 20(16):4509. https://doi.org/10.3390/s20164509

    Article  CAS  Google Scholar 

  44. Stasyuk N, Demkiv O, Gayda G (2022) Reusable alcohol oxidase-nPtCu/alginate beads for highly sensitive ethanol assay in beverages. RSC Adv 12(33):21309–21317. https://doi.org/10.1039/D2RA02106D

    Article  CAS  Google Scholar 

  45. Sivashankar S, Kumar NS, Mazumder KRN (2020) Amperometric biosensors as an analytical tool in food, dairy and fermentation industries. Int J Pharm Sci Res 65(1):68–82. https://doi.org/10.47583/ijpsrr.2020.v65i01.010

    Article  CAS  Google Scholar 

  46. Stasyuk N, Gayda G, Demkiv O, Darmohray L, Gonchar M, Nisnevitch M (2021) Amperometric biosensors for L-arginine determination based on L-arginine oxidase and peroxidase-like nanozymes. Appl Sci 11(15):7024. https://doi.org/10.3390/app11157024.5]

    Article  CAS  Google Scholar 

  47. Gonchar MV, Maidan MM, Pavlishko HM, Sibirny AA (2001) A new oxidase-peroxidase kit for ethanol assays in alcoholic beverages. FTB 39:37–42. http://www.ftb.com.hr/archives/103-volume-39-issue-no-1/743-a-new-oxidase-peroxidase-kit-for-ethanol-assays-in-alcoholic-beverages

  48. Alpeeva IS, Vilkanauskyte A, Ngounou B et al (2005) Bi-enzyme alcohol biosensors based on genetically engineered alcohol oxidase and different peroxidases. Microchim Acta 152:21. https://doi.org/10.1007/s00604-005-0407-7

    Article  CAS  Google Scholar 

  49. AOAC (2004) Definitions and calculations of horrat values from intralaboratory data. Available online: http://www.readbag.com/aoac-dietsupp6-dietary-supplement-web-site-horrat-slv. Accessed 1 Oct 2017

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Acknowledgements

The authors would like to thank the Polish Academy of Sciences for financial support of exchange visits in 2022.

Funding

This research was partially supported by the National Research Foundation of Ukraine (project Nos.: 2020.02/0100 “Development of new nanozymes as catalytic elements for enzymatic kits and chemo/biosensors”), National Academy of Sciences of Ukraine: the programs: “Smart sensor devices of a new generation based on modern materials and technologies” and “Double-acting anticancer drugs to overcome the acquired resistance of malignant cells to chemotherapy” (0122U200211), Grant “New dual casein kinase 2 inhibitors and histone deacetylase for targeted tumor chemotherapy” for research laboratories/groups of young scientists in priority areas of science and technology in 2021–2022.

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Authors and Affiliations

  1. Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, 79005, Ukraine

    Nataliya Stasyuk, Olha Demkiv, Galina Gayda, Oksana Zakalska & Mykhailo Gonchar

  2. Institute of Physical Chemistry, Polish Academy of Sciences, 01-224, Warsaw, Poland

    Wojciech Nogala

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Stasyuk, N., Demkiv, O., Gayda, G. et al. Amperometric biosensors based on alcohol oxidase and peroxidase–like nanozymes for ethanol determination. Microchim Acta 189, 474 (2022). https://doi.org/10.1007/s00604-022-05568-z

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