Electrochemical determination of glutathione in hemolyzed erythrocytes

Document Type : Article


1 Department of Chemistry, Faculty of Science, Yazd University, Yazd, 89195-741, Iran

2 Department of Chemistry, Payame Noor University, Tehran, 19395-4697, Iran


The physiological significance of determining glutathione (GSH) and its oxide form is obvious from their applications in clinical practices such as diagnostic experiments for diabetes, Parkinson’s disease, and cancers. Such an important detemination still needs the development of certain experimental procedures that are easy, fast, and cheap enough to implement. These procedural advantages can be provided through electrochemical methods. Therefore, in this study, at the surface of a glassy carbon electrode (GCE), a composite of functionalized multi-walled carbon nanotubes (MWCNTs) and formazon was used as a mediator to determine GSH electrochemically. The results indicated that this modified GCE is electrocatalytically very active for glutathione oxidation. Several techniques including cyclic voltammetry (CV), scanning electron microscopy (SEM), and differential pulse voltammetry (DPV) were used to characterize the electrode. Also, such kinetic parameters as the charge transfer rate constant and the transfer coefficient were calculated. In optimized conditions, there was a linear relationship between the DPV peak current of GSH oxidation and GSH concentration in the ranges of 1.0-100.0 and 100.0-800.0 µM at pH 7.0. As for the detection limit, it was found to be 0.73 µM.


1. Lagman, M., Ly, J., Saing, T., Singh, M. K., Tudela, E. V., Morris, D., Chi, P. T., Ochoa, C., Sathananthan, A., and Venketaraman, V., “Investigating the causes for decreased levels of glutathione in individuals with type II diabetes”, PLoS One, 10(3), pp. 1–19 (2015).
2.     Mazzetti, A. P., Fiorile, M. C., Primavera, A., and Lo Bello, M., “Glutathione transferases and neurodegenerative diseases”, Neurochem Int, 82, pp. 10–18 (2015).
3.     Pinnen, F., Sozio, P., Cacciatore, I., Cornacchia, C., Mollica, A., Iannitelli, A., Dâaurizio, E., Cataldi, A., Zara, S., Nasuti, C., and Di Stefano, A., “Ibuprofen and glutathione conjugate as a potential therapeutic agent for treating alzheimer’s disease”, Arch Pharm (Weinheim), 344(3), pp. 139–148 (2011).
4.     Vidyasagar, M. S., Kodali, M., Prakash Saxena, P., Upadhya, D., Murali Krishna, C., Vadhiraja, B. M., Fernandes, D. J., and Bola Sadashiva, S. R., “Predictive and prognostic significance of glutathione levels and DNA damage in cervix cancer patients undergoing radiotherapy”, Int J Radiat Oncol Biol Phys, 78(2), pp. 343–349 (2010).
5.     Feng, J., Huang, P., Shi, S., Deng, K. Y., and Wu, F. Y., “Colorimetric detection of glutathione in cells based on peroxidase-like activity of gold nanoclusters: A promising powerful tool for identifying cancer cells”, Anal Chim Acta, 967, pp. 64–69 (2017).
6.     Allocati, N., Masulli, M., Di Ilio, C., and Federici, L., “Glutathione transferases: Substrates, inihibitors and pro-drugs in cancer and neurodegenerative diseases”, Oncogenesis, 7(1) (2018).
7.     Kowalska, K., Zalewska, M., and Milnerowicz, H., “The Application of Capillary Electrophoresis in the Determination of Glutathione in Healthy Women ’ s Blood”, J Chromatogr Sci, 53(21), pp. 353–359 (2014).
8.     Meister, A., “Glutathione metabolism and its selective modification”, JournalofBiologicalChemistry, 263(33), pp. 17205–17208 (1988).
9.     Knapen, M. F. C. M., Zusterzeel, P. L. M., Peters, W. H. M., and Steegers, E. A. P., “Glutathione and glutathione-related enzymes in reproduction: A review”, Eur J Obstet Gynecol Reprod Biol, 82(2), pp. 171–184 (1999).
10.     Mills, B. J. and Lang, C. A., “Differential distribution of free and bound glutathione and cyst(e)ine in human blood”, Biochem Pharmacol, 52(3), pp. 401–406 (1996).
11.     Kand’ár, R., Žáková, P., Lotková, H., Kučera, O., and Červinková, Z., “Determination of reduced and oxidized glutathione in biological samples using liquid chromatography with fluorimetric detection”, J Pharm Biomed Anal, 43(4), pp. 1382–1387 (2007).
12.     Šegan, S., Opsenica, I., Zlatović, M., Milojković-Opsenica, D., and Šolaja, B., “Quantitative structure retention/activity relationships of biologically relevant 4-amino-7-chloroquinoline based compounds”, J Chromatogr B Anal Technol Biomed Life Sci, 1012–1013, pp. 144–152 (2016).
13.     Ensafi, A. A., Khayamian, T., and Hasanpour, F., “Determination of glutathione in hemolysed erythrocyte by flow injection analysis with chemiluminescence detection”, J Pharm Biomed Anal, 48(1), pp. 140–144 (2008).
14.     Bergel, A., Souppe, J., and Comtat, M., “Enzymatic amplification for spectrophotometric and electrochemical assays of NAD+ and NADH”, Anal Biochem, 179(2), pp. 382–388 (1989).
15.     Wang, Z., Han, P., Mao, X., Yin, Y., and Cao, Y., “Sensitive detection of glutathione by using DNA-templated copper nanoparticles as electrochemical reporters”, Sensors Actuators, B Chem, 238, pp. 325–330 (2017).
16.     Majumder, M. K., Kaushik, B. K., and Manhas, S. K., “Novel spatially arranged mixed carbon nanotube bundle interconnects - Impact on delay and power”, Sci Iran, 20(6), pp. 2341–2347 (2013).
17.     Mazloum-Ardakani, M., Dehghani-Firouzabadi, A., Sheikh-Mohseni, M. A., Benvidi, A., Mirjalili, B. B. F., and Zare, R., “A self-assembled monolayer on gold nanoparticles modified electrode for simultaneous determination of isoproterenol and uric acid”, Meas J Int Meas Confed, 62, pp. 88–96 (2015).
18.     Gorgin Karaji, Z., Houshmand, B., Abbasi, S., and Faghihi, S., “Electrochemical anodic oxidation process of porous titanium granules for biomedical applications”, Sci Iran, 22(6), pp. 2745–2751 (2015).
19.     Mazloum-Ardakani, M., Farbod, F., and Hosseinzadeh, L., “An Electrochemical Sensor Based on Nickel Oxides Nanoparticle/ Graphene Composites for Electrochemical Detection of Epinephrine”, J Nanostruct, 6(4), pp. 293–300 (2016).
20.     Mazloum-Ardakani, M., Ahmadi, S. H., Safaei Mahmoudabadi, Z., and Khoshroo, A., “Nano composite system based on fullerene-functionalized carbon nanotubes for simultaneous determination of levodopa and acetaminophen”, Meas J Int Meas Confed, 91, pp. 162–167 (2016).
21.     Faramarzi, V., Ahmadi, V., Ghane Golmohamadi, F., and Fotouhi, B., “A biosensor based on plasmonic wave excitation with diffractive grating structure”, Sci Iran, 0(0), pp. 0–0 (2017).
22.     AfzaliTabar, M., Alaei, M., Ranjineh khojasteh, R., Motiee, F., and Rashidi, A. M., “Preference of Nano porous graphene to Single-Walled Carbon Nanotube (SWCNT) for preparing Silica Nano hybrid Pickering Emulsion for potential Chemical Enhanced Oil Recovery (C-EOR)”, Sci Iran, 0(0), pp. 0–0 (2017).
23.     Bagheri, Z., Ranjbar, B., Azizi, A., Latifi, H., Zibaii, M. I., and Tohidi Moghadam, T., “Plasmonic Circular Dichroism Study of Gold Nanorod-Quadruplex Nanobioconjugates”, Sci Iran, 0(0), pp. 0–0 (2017).
24.     Shahmiri, M. R., Bahari, A., Karimi-Maleh, H., Hosseinzadeh, R., and Mirnia, N., “Ethynylferrocene-NiO/MWCNT nanocomposite modified carbon paste electrode as a novel voltammetric sensor for simultaneous determination of glutathione and acetaminophen”, Sensors Actuators, B Chem, 177, pp. 70–77 (2013).
25.     Yuan, B., Zeng, X., Xu, C., Liu, L., Ma, Y., Zhang, D., and Fan, Y., “Electrochemical modification of graphene oxide bearing different types of oxygen functional species for the electro-catalytic oxidation of reduced glutathione”, Sensors Actuators, B Chem, 184, pp. 15–20 (2013).
26.     Munteanu, G., Dempsey, E., and McCormac, T., “Novel ultrasensitive and ultrafast voltammetric determination of biological aminochromes on the copper nanodoped mercury monolayer carbon fiber electrode”, J Electroanal Chem, 650(1), pp. 105–115 (2010).
27.     Wendland, T. R., Muntean, B. S., Kaur, J., Mukherjee, J., Chen, J., Tan, X., Attygalle, D., Collins, R. W., Kirchhoff, J. R., and Tillekeratne, L. M. V., “In Situ Self Assembly of Thiolated ortho-Quinone Capped Electrocatalysts for Bioanalytical Applications”, Electroanalysis, 23(10), pp. 2275–2279 (2011).
28.     Calvo-Marzal, P., Chumbimuni-Torres, K. Y., Höehr, N. F., and Kubota, L. T., “Determination of glutathione in hemolysed erythrocyte with amperometric sensor based on TTF-TCNQ”, Clin Chim Acta, 371(1–2), pp. 152–158 (2006).
29.     Ndamanisha, J. C., Bai, J., Qi, B., and Guo, L., “Application of electrochemical properties of ordered mesoporous carbon to the determination of glutathione and cysteine”, Anal Biochem, 386(1), pp. 79–84 (2009).
30.     Tang, H., Chen, J., Nie, L., Yao, S., and Kuang, Y., “Electrochemical oxidation of glutathione at well-aligned carbon nanotube array electrode”, Electrochim Acta, 51(15), pp. 3046–3051 (2006).
31.     Abiman, P., Wildgoose, G. G., and Compton, R. G., “Electroanalytical exploitation of nitroso phenyl modified carbon-thiol interactions: Application to the low voltage determination of thiols”, Electroanalysis, 19(4), pp. 437–444 (2007).
32.     Osorio, A. G., Silveira, I. C. L., Bueno, V. L., and Bergmann, C. P., “H 2 SO 4 /HNO 3 /HCl-Functionalization and its effect on dispersion of carbon nanotubes in aqueous media”, Appl Surf Sci, 255(5 PART 1), pp. 2485–2489 (2008).
33.     Datsyuk, V., Kalyva, M., Papagelis, K., Parthenios, J., Tasis, D., Siokou, A., Kallitsis, I., and Galiotis, C., “Chemical oxidation of multiwalled carbon nanotubes”, Carbon N Y, 46(6), pp. 833–840 (2008).
34.     Nicholson, R. S., “Theory and Application of Cyclic Voltammetry f m Measurement of Electrode Reaction Kinetics”, Anal Chem, 37(11), pp. 1351–1355 (1965).
35.     Bard, A. J. and Faulkner, L. R., ELECTROCHEMICAL METHODS Fundamentals and Applications (1944).
36.     Anderson, M. E., “Determination of Glutathione and Glutathione Disulphide in Biological Samples”, Annu Rev Biochem, 113(1983), pp. 548–555 (1985).
37.     Raoof, J.-B., Ojani, R., and Baghayeri, M., “Simultaneous electrochemical determination of glutathione and tryptophan on a nano-TiO2/ferrocene carboxylic acid modified carbon paste electrode”, Sensors Actuators B Chem, 143(1), pp. 261–269 (2009).
38.     Hassanvand, Z. and Jalali, F., “Electrocatalytic determination of glutathione using transition metal hexacyanoferrates (MHCFs) of copper and cobalt electrode posited on graphene oxide nanosheets”, Anal Bioanal Chem Res, 5(1), pp. 115–129 (2018).
39.     Lowinsohn, D., Lee, P. T., and Compton, R. G., “Towards Detection of Total Antioxidant Concentrations of Glutathione, Cysteine, Homocysteine and Ascorbic Acid Using a Nanocarbon Paste Electrode”, Int J Electrochem Sci, 9(7), pp. 3458–3472 (2014).
40.     Olmos Moya, P. M., Martínez Alfaro, M., Kazemi, R., Alpuche-Avilés, M. A., Griveau, S., Bedioui, F., and Gutiérrez Granados, S., “Simultaneous Electrochemical Speciation of Oxidized and Reduced Glutathione. Redox Profiling of Oxidative Stress in Biological Fluids with a Modified Carbon Electrode”, Anal Chem, 89(20), pp. 10726–10733 (2017).
41.     Areias, M. C. C., Shimizu, K., and Compton, R. G., “Voltammetric detection of glutathione: An adsorptive stripping voltammetry approach”, Analyst, 141(10), pp. 2904–2910 (2016).
42.     Benvidi, A., Dehghan, P., Dehghani-Firouzabadi, A., Emtiazi, H., Zare, H. R., and Mazloum-Ardakania, M., “Construction of a nanocomposite sensor by modification of carbon paste electrode with reduced graphene oxide and a hydroquinone derivative: Simultaneous determination of glutathione and penicillamine”, Anal Methods, 7(1), pp. 5538–5544 (2015).
43.     Yuan, B., Zhang, R., Jiao, X., Li, J., Shi, H., and Zhang, D., “Amperometric determination of reduced glutathione with a new Co-based metal-organic coordination polymer modified electrode”, Electrochem commun, 40, pp. 92–95 (2014).
44.     Tian, H., Wang, T., Fu, Y., Yu, Y., Guo, C., and Hu, J., “Electrochemical determination of glutathione using an annealed nickel ion implanted-modified electrode”, J Electrochem Soc, 161(9), pp. 191–195 (2014).
45.     Dong, Y., Sheng, Q., Zheng, J., and Tang, H., “A nonenzymatic reduced glutathione sensor based on Ni-Al LDHs/MWCNTs composites”, Anal Methods, 6(21), pp. 8598–8603 (2014).
46.     Lee, P. T., Goncalves, L. M., and Compton, R. G., “Electrochemical determination of free and total glutathione in human saliva samples”, Sensors Actuators, B Chem, 221, pp. 962–968 (2015).
47.     Liu, B., Ma, C., Li, Y., Kou, Y., Lu, J., Jiang, X., and Tan, L., “Voltammetric determination of reduced glutathione using poly(thionine) as a mediator in the presence of Fenton-type reaction”, Talanta, 170, pp. 399–405 (2017).