Covalent attachment of aminoferrocene to pseudo-graphite for selective sensing of H2O2
Original scientific paper
DOI:
https://doi.org/10.5599/jese.2705Keywords:
Peroxide detection, ferrocene, interference-free, diazonium, graphitic carbonAbstract
Hydrogen peroxide is an important analyte from both the standpoints of in vivo and in vitro analyses. Electrochemical methods offer good prospects, but present sensing methods suffer from inadequate limits of detection (LOD), low sensitivity, poor stability, and numerous interferents. To address these issues, we use the recently recognized peroxidase-like activity of ferrocene for sensing based on the reductive electrocatalysis of H2O2. This route obviates interferences from ascorbate, urea, urate, dopamine and glucose. Immobilization of ferrocene overcomes its low aqueous solubility. This is done by the chemical modification of a pseudo-graphite material rich in sp3 defects. The surface is modified for carboxylate features through a diazonium intermediate formed through 4-aminobenzoic acid. Aminoferrocene is attached to surface carboxylate groups through a N-ethyl-N’-(3-(dimethylamino)propyl) carbodiimide coupling agent. Cyclic voltametric studies indicate that the surface concentration of ferrocene is 1.06 nmol cm-2. Chronoamperometric response at -0.6 V vs. Ag/AgCl, gives a LOD of 0.070 µM with a high sensitivity of 553.2 μA mM-1 cm-2. This is the best sensitivity reported in the literature. Furthermore, the sensor is stable over 50 cyclic voltametric scans, and between the 3 calibration-curve studies.
Downloads
References
[1] N. Yang, W. Xiao, X. Song, W. Wang, X. Dong, Recent Advances in Tumor Microenvironment Hydrogen Peroxide-Responsive Materials for Cancer Photodynamic Therapy, Nano-Micro Letters 12 (2020) 15. https://doi.org/10.1007/s40820-019-0347-0 DOI: https://doi.org/10.1007/s40820-019-0347-0
[2] T. Ali, D. Li, T. N. F. Ponnamperumage, A. K. Peterson, J. Pandey, K. Fatima, J. Brzezinski, J. A. R. Jakusz, H. Gao, G. E. Koelsch, D. S. Murugan, X. Peng, Generation of Hydrogen Peroxide in Cancer Cells: Advancing Therapeutic Approaches for Cancer Treatment, Cancers 16 (2024) 2171. https://doi.org/10.3390/cancers16122171 DOI: https://doi.org/10.3390/cancers16122171
[3] K. Dhara, D. R. Mahapatra, Recent advances in electrochemical nonenzymatic hydrogen peroxide sensors based on nanomaterials: a review, Journal of Materials Science 54 (2019) 12319-12357. https://doi.org/10.1007/s10853-019-03750-y DOI: https://doi.org/10.1007/s10853-019-03750-y
[4] G. I. Berglund, G. H. Carlsson, A. T. Smith, H. Szöke, A. Henriksen, J. Hajdu, The catalytic pathway of horseradish peroxidase at high resolution, Nature 417 (2002) 463-468. https://doi.org/10.1038/417463a DOI: https://doi.org/10.1038/417463a
[5] C. Yang, M. E. Denno, P. Pyakurel, B. J. Venton, Recent trends in carbon nanomaterial-based electrochemical sensors for biomolecules: A review, Analytica Chimica Acta 887 (2015) 17-37. https://doi.org/10.1016/j.aca.2015.05.049 DOI: https://doi.org/10.1016/j.aca.2015.05.049
[6] X. Xu, S. Liu, H. Ju, A Novel Hydrogen Peroxide Sensor via the Direct Electrochemistry of Horseradish Peroxidase Immobilized on Colloidal Gold Modified Screen-printed Electrode, Sensors 3 (2003) 350-360. https://doi.org/10.3390/s30900350 DOI: https://doi.org/10.3390/s30900350
[7] S. M. Lewis, S. R. Dirksen, M. M. Heitkemper, L. Bucher, M. Harding, Medical-surgical nursing: assessment and management of clinical problems, 9th ed., Mosby, St. Louis, Missouri, 2013. https://www.zu.edu.jo/UploadFile/Library/E_Books/Files/LibraryFile_16951_48.pdf
[8] H. R. Bhoot, U. M. Zamwar, S. Chakole, A. Anjankar, Dietary Sources, Bioavailability, and Functions of Ascorbic Acid (Vitamin C) and Its Role in the Common Cold, Tissue Healing, and Iron Metabolism, Cureus 15 (11) (2023) e49308. https://doi.org/10.7759/cureus.49308 DOI: https://doi.org/10.7759/cureus.49308
.[9] L. Goldman, A. I Schafer, Goldman's Cecil Medicine 24Ed International edition, Saunders, Philadelphia, United States of America, 2012, p.1470-1475 https://doi.org/10.1016/B978-1-4377-1604-7.00235-9 DOI: https://doi.org/10.1016/B978-1-4377-1604-7.00235-9
[10] MedlinePlus, https://medlineplus.gov/ency/article/003487.htm (6/19/2025).
[11] R. Gaikwad, P.R. Thangaraj, A.K. Sen, Direct and rapid measurement of hydrogen peroxide in human blood using a microfluidic device, Scientific Reports 11 (2021) 2960. https://doi.org/10.1038/s41598-021-82623-4 DOI: https://doi.org/10.1038/s41598-021-82623-4
[12] R. N. Pittman, Regulation of Tissue Oxygenation, Colloquium Series on Integrated Systems Physiology: From Molecule to Function 3 (2011) 1-100. https://doi.org/10.4199/C00029ED1V01Y201103ISP017 DOI: https://doi.org/10.4199/C00029ED1V01Y201103ISP017
[13] A. J. Bard, L. R. Faulkner, H. S. White, Electrochemical Methods: Fundamentals and Appli-cations, 3rd ed., John Wiley & Sons, INC., Danvers, United States of America, 2022. ISBN 978-1-119-33405-7
[14] K. H. Rashid, A. A. Khadom, Sodium sulfite as an oxygen scavenger for the corrosion control of mild steel in petroleum refinery wastewater: optimization, mathematical modeling, surface morphology and reaction kinetics studies, Reaction Kinetics, Mechanisms and Catalysis 129 (2020) 1027-1046. https://doi.org/10.1007/s11144-020-01738-3 DOI: https://doi.org/10.1007/s11144-020-01738-3
[15] Sariga, A. Varghese, The Renaissance of Ferrocene-Based Electrocatalysts: Properties, Synthesis Strategies, and Applications, Topics in Current Chemistry 381 (2023) 32. https://doi.org/10.1007/s41061-023-00441-w DOI: https://doi.org/10.1007/s41061-023-00441-w
[16] Q. Hu, Y. Fang, X. Yu, J. Huang, L. Wang, A ferrocene-linked metal-covalent organic polymer as a peroxidase-enzyme mimic for dual channel detection of hydrogen peroxide, The Analyst 146 (2021) 487-494. https://doi.org/10.1039/D0AN01837F DOI: https://doi.org/10.1039/D0AN01837F
[17] A. Singh, D.R. Chowdhury, A. Paul, A kinetic study of ferrocenium cation decomposition utilizing an integrated electrochemical methodology composed of cyclic voltammetry and amperometry, The Analyst 139 (2014) 5747-5754. https://doi.org/10.1039/C4AN01325E DOI: https://doi.org/10.1039/C4AN01325E
[18] G. Zotti, G. Schiavon, S. Zecchin, D. Favretto, Dioxygen-decomposition of ferrocenium molecules in acetonitrile: The nat,ure of the electrode-fouling films during ferrocene electrochemistry, Journal of Electroanalytical Chemistry 456 (1998) 217-221. https://doi.org/10.1016/S0022-0728(98)00279-4 DOI: https://doi.org/10.1016/S0022-0728(98)00279-4
[19] R. Gracia, D. Mecerreyes, Polymers with redox properties: materials for batteries, biosensors and more, Polymer Chemistry 4 (2013) 2206. https://doi.org/10.1039/c3py21118e DOI: https://doi.org/10.1039/c3py21118e
[20] V. S. Tripathi, V. B. Kandimalla, H. Ju, Amperometric biosensor for hydrogen peroxide based on ferrocene-bovine serum albumin and multiwall carbon nanotube modified ormosil composite, Biosensors and Bioelectronics 21 (2006) 1529-1535. https://doi.org/10.1016/j.bios.2005.07.006 DOI: https://doi.org/10.1016/j.bios.2005.07.006
[21] N. A. Ertas, E. Kavak, F. Salman, H. C. Kazici, H. Kivrak, A. Kivrak, Synthesis of Ferrocene Based Naphthoquinones and its Application as Novel Non‐enzymatic Hydrogen Peroxide, Electroanalysis 32 (2020) 1178-1185. https://doi.org/10.1002/elan.201900715 DOI: https://doi.org/10.1002/elan.201900715
[22] G.-C. Zhao, M.-Q. Xu, Q. Zhang, A novel hydrogen peroxide sensor based on the redox of ferrocene on room temperature ionic liquid film, Electrochemistry Communications 10 (2008) 1924-1926. https://doi.org/10.1016/j.elecom.2008.10.005 DOI: https://doi.org/10.1016/j.elecom.2008.10.005
[23] H. Kabir, H. Zhu, J. May, K. Hamal, Y. Kan, T. Williams, E. Echeverria, D. N. McIlroy, D. Estrada, P. H. Davis, T. Pandhi, K. Yocham, K. Higginbotham, A. Clearfield, I.F. Cheng, The sp2-sp3 carbon hybridization content of nanocrystalline graphite from pyrolyzed vegetable oil, comparison of electrochemistry and physical properties with other carbon forms and allotropes, Carbon 144 (2019) 831-840. https://doi.org/10.1016/j.carbon.2018.12.058 DOI: https://doi.org/10.1016/j.carbon.2018.12.058
[24] H. Lim, J.S. Lee, H.-J. Shin, H. S. Shin, H. C. Choi, Spatially Resolved Spontaneous Reactivity of Diazonium Salt on Edge and Basal Plane of Graphene without Surfactant and Its Doping Effect, Langmuir 26 (2010) 12278-12284. https://doi.org/10.1021/la101254k DOI: https://doi.org/10.1021/la101254k
[25] J. Greenwood, T. H. Phan, Y. Fujita, Z. Li, O. Ivasenko, W. Vanderlinden, H. Van Gorp, W. Frederickx, G. Lu, K. Tahara, Y. Tobe, H. Uji-i, S.F.L. Mertens, S. De Feyter, Covalent Modification of Graphene and Graphite Using Diazonium Chemistry: Tunable Grafting and Nanomanipulation, ACS Nano 9 (2015) 5520-5535. https://doi.org/10.1021/acsnano.5b01580 DOI: https://doi.org/10.1021/acsnano.5b01580
[26] J. E. Johns, M. C. Hersam, Atomic Covalent Functionalization of Graphene, Accounts of Chemical Research 46 (2013) 77-86. https://doi.org/10.1021/ar300143e DOI: https://doi.org/10.1021/ar300143e
[27] B. D. L. Campéon, M. Akada, M. S. Ahmad, Y. Nishikawa, K. Gotoh, Y. Nishina, Non-destructive, uniform, and scalable electrochemical functionalization and exfoliation of graphite, Carbon 158 (2020) 356-363. https://doi.org/10.1016/j.carbon.2019.10.085 DOI: https://doi.org/10.1016/j.carbon.2019.10.085
[28] Y. Wang, C. Li, H. Meng, Y. Lu, H. Fan, Production of functionalized graphitic carbon materials via solvent-free mechanochemical method for supercapacitors and water treatment, Diamond and Related Materials 127 (2022) 109193. https://doi.org/10.1016/j.diamond.2022.109193 DOI: https://doi.org/10.1016/j.diamond.2022.109193
[29] D. Koirala, F. Dalbec, J. May, K. Hamal, P. B. Allen, F. I. Cheng, Biosensing with Polymerase Chain Reaction-Stable DNA-Functionalized Magnetically Susceptible Carbon-Iron Microparticles, Analytical Chemistry 95 (2023) 16631-16638. https://doi.org/10.1021/acs.analchem.3c02978.s001 DOI: https://doi.org/10.1021/acs.analchem.3c02978
[30] J. May, D. Koirala, F. Dalbec, J. Russell, H. Xiong, E. Echeverria, D. N. McIlroy, I. F. Cheng, Superhydrophilicity and Antifouling Behavior in Electrochemically Oxidized Nanocrystalline Pseudographite, Industrial & Engineering Chemistry Research 62 (2023) 6687-6696. https://doi.org/10.1021/acs.iecr.3c00140 DOI: https://doi.org/10.1021/acs.iecr.3c00140
[31] K. Hamal, J. May, D. Koirala, H. Zhu, H. Kabir, E. Echeverria, D.N. McIlroy, N. Nicholas, I.F. Cheng, Highly Stable, Low-Cost Metal-Free Oxygen Reduction Reaction Electrocatalyst Based on Nitrogen-Doped Pseudo-Graphite, Energy & Fuels 35 (2021) 10146-10155. https://doi.org/10.1021/acs.energyfuels.1c00658 DOI: https://doi.org/10.1021/acs.energyfuels.1c00658
[32] K. Hamal, D. Koirala, J. May, F. Dalbec, N. Nicholas, I. F. Cheng, An oxygen reduction reaction electrocatalyst tuned for hydrogen peroxide generation based on a pseudo-graphite doped with graphitic nitrogen, Journal of Electrochemical Science and Engineering 12 (2022) 1009-1023. https://doi.org/10.5599/jese.1407 DOI: https://doi.org/10.5599/jese.1407
[33] M. A. Isaacs, J. Davies-Jones, P.R. Davies, S. Guan, R. Lee, D. J. Morgan, R. Palgrave, Advanced XPS characterization: XPS-based multi-technique analyses for comprehensive understanding of functional materials, Materials Chemistry Frontiers 5 (2021) 7931-7963. https://doi.org/10.1039/D1QM00969A DOI: https://doi.org/10.1039/D1QM00969A
[34] S. Tougaard, Improved XPS analysis by visual inspection of the survey spectrum, Surface and Interface Analysis 50 (2018) 657-666. https://doi.org/10.1002/sia.6456 DOI: https://doi.org/10.1002/sia.6456
[35] C. D. Wagner, Sensitivity factors for XPS analysis of surface atoms, Journal of Electron Spectroscopy and Related Phenomena. 32 (1983) 99-102 https://doi.org/10.1016/0368-2048(83)85087-7 DOI: https://doi.org/10.1016/0368-2048(83)85087-7
[36] The International XPS Database of Monochromatic XPS Reference Spectra, Peak-fits, & Six B. E. Tables, https://xpsdatabase.net/carbon-spectra-graphene-fresh-peel-from-hopg (6/19/2025)
[37] X-ray Photoelectron Spectroscopy (XPS) Reference Pages, https://www.xpsfitting.com/2022/06/graphiticgraphenecarbon-nanotube-c-1s.html (6/19/2025).
[38] ThermoFischer Scientific, https://www.thermofisher.com/us/en/home/materials-science/learning-center/periodic-table/non-metal/carbon.html (6/19/2025).
[39] M. Toupin, D. Bélanger, Thermal Stability Study of Aryl Modified Carbon Black by in Situ Generated Diazonium Salt, The Journal of Physical Chemistry C 111 (2007) 5394-5401 https://doi.org/10.1021/jp066868e DOI: https://doi.org/10.1021/jp066868e
[40] M. Toupin, D. Bélanger, Spontaneous Functionalization of Carbon Black by Reaction with
4-Nitrophenyldiazonium Cations, Langmuir 24 (2008) 1910-1917. https://doi.org/10.1021/la702556n DOI: https://doi.org/10.1021/la702556n
[41] G. Pognon, T. Brousse, L. Demarconnay, D. Bélanger, Performance and stability of electrochemical capacitor based on anthraquinone modified activated carbon, Journal of Power Sources 196 (2011) 4117-4122. https://doi.org/10.1016/j.jpowsour.2010.09.097 DOI: https://doi.org/10.1016/j.jpowsour.2010.09.097
[42] G. Pognon, C. Cougnon, D. Mayilukila, D. Bélanger, Catechol-Modified Activated Carbon Prepared by the Diazonium Chemistry for Application as Active Electrode Material in Electrochemical Capacitor, ACS Applied Materials & Interfaces 4 (2012) 3788-3796. https://doi.org/10.1021/am301284n DOI: https://doi.org/10.1021/am301284n
[43] E. Lebègue, T. Brousse, J. Gaubicher, C. Cougnon, Spontaneous arylation of activated carbon from aminobenzene organic acids as source of diazonium ions in mild conditions, Electrochimica Acta 88 (2013) 680-687. https://doi.org/10.1016/j.electacta.2012.10.132 DOI: https://doi.org/10.1016/j.electacta.2012.10.132
[44] E. Lebègue, L. Madec, T. Brousse, J. Gaubicher, E. Levillain, C. Cougnon, Modification of activated carbons based on diazonium ions in situ produced from aminobenzene organic acid without addition of other acid, Journal of Materials Chemistry 21 (2011) 12221. https://doi.org/10.1039/c1jm11538c DOI: https://doi.org/10.1039/c1jm11538c
[45] J. A. Belmont, Process for preparing carbon materials with diazonium salts and resultant carbon products, US5554739A, 1994. https://patents.google.com/patent/US5554739A/en
[46] P. Salice, E. Fabris, C. Sartorio, D. Fenaroli, V. Figà, M. P. Casaletto, S. Cataldo, B. Pignataro, E. Menna, An insight into the functionalisation of carbon nanotubes by diazonium chemistry: Towards a controlled decoration, Carbon 74 (2014) 73-82. https://doi.org/10.1016/j.carbon.2014.02.084 DOI: https://doi.org/10.1016/j.carbon.2014.02.084
[47] J. L. Bahr, J. M. Tour, Highly Functionalized Carbon Nanotubes Using in Situ Generated Diazonium Compounds, Chemistry of Materials 13 (2001) 3823-3824. https://doi.org/10.1021/cm0109903 DOI: https://doi.org/10.1021/cm0109903
[48] X-ray Photoelectron Spectroscopy (XPS) Reference Pages, www.xpsfitting.com (6/19/2025).
[49] J. Gomez-Bolivar, I. P. Mikheenko, R. L. Orozco, S. Sharma, D. Banerjee, M. Walker, R. A. Hand, M. L. Merroun, L. E. Macaskie, Synthesis of Pd/Ru Bimetallic Nanoparticles by Escherichia coli and Potential as a Catalyst for Upgrading 5-Hydroxymethyl Furfural Into Liquid Fuel Precursors, Frontiers in Microbiology 10 (2019) 1276. https://doi.org/10.3389/fmicb.2019.01276 DOI: https://doi.org/10.3389/fmicb.2019.01276
[50] L. H. Grey, H.-Y. Nie, M. C. Biesinger, Defining the nature of adventitious carbon and improving its merit as a charge correction reference for XPS, Applied Surface Science 653 (2024) 159319. https://doi.org/10.1016/j.apsusc.2024.159319 DOI: https://doi.org/10.1016/j.apsusc.2024.159319
[51] J. Pinson, F. Podvorica, Attachment of organic layers to conductive or semiconductive surfaces by reduction of diazonium salts, Chemical Society Reviews 34 (2005) 429. https://doi.org/10.1039/b406228k DOI: https://doi.org/10.1039/b406228k
[52] A. Mattiuzzi, Q. Lenne, J. Carvalho Padilha, L. Troian-Gautier, Y. R. Leroux, I. Jabin, C. Lagrost, Strategies for the Formation of Monolayers from Diazonium Salts: Unconventional Grafting Media, Unconventional Building Blocks, Frontiers in Chemistry 8 (2020) 559. https://doi.org/10.3389/fchem.2020.00559 DOI: https://doi.org/10.3389/fchem.2020.00559
[53] C. Gautier, I. López, T. Breton, A post-functionalization toolbox for diazonium (electro)-grafted surfaces: review of the coupling methods, Materials Advances 2 (2021) 2773-2810. https://doi.org/10.1039/D1MA00077B DOI: https://doi.org/10.1039/D1MA00077B
[54] R.G. Compton, C.E. Banks, Understanding Voltammetry, 2nd ed., Imperial College Press, London, 2011. https://doi.org/10.1142/p726 DOI: https://doi.org/10.1142/p726
[55] M. Mattoussi, F. Matoussi, N. Raouafi, Non-enzymatic amperometric sensor for hydrogen peroxide detection based on a ferrocene-containing cross-linked redox-active polymer, Sensors and Actuators B 274 (2018) 412-418. https://doi.org/10.1016/j.snb.2018.07.145 DOI: https://doi.org/10.1016/j.snb.2018.07.145
[56] E. Theodorsson, Limit of detection, limit of quantification and limit of blank, European Federation of Clinical Chemistry and Laboratory Medicine. (n.d.).
[57] Limit of detection in analysis, in: The IUPAC Compendium of Chemical Terminology, , International Union of Pure and Applied Chemistry (IUPAC), Research Triangle Park, NC, 2014 https://doi.org/10.1351/goldbook.L03540 DOI: https://doi.org/10.1351/goldbook.L03540
[58] F. Allegrini, A. C. Olivieri, Comprehensive Chemometrics 2nded., Elsevier, Cambridge, United States of America, 2020, p. 441-463. https://doi.org/10.1016/B978-0-12-409547-2.14612-8 DOI: https://doi.org/10.1016/B978-0-12-409547-2.14612-8
[59] MilliporeSigma,https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/protein-biology/enzyme-activity-assays/peroxidase-enzymes?srsltid=AfmBOoqsKT40Z3ZbHo2kuYlfXI47VdQkzwSoUOA0fAt78sXWpkT60Vq7#horseradish (6/19/2025).
[60] J. Dou, G. Zhu, B. Hu, J. Yang, Y. Ge, X. Li, J. Liu, Wall thickness-tunable AgNPs-NCNTs for hydrogen peroxide sensing and oxygen reduction reaction, Electrochimica Acta 306 (2019) 466-476. https://doi.org/10.1016/j.electacta.2019.03.152 DOI: https://doi.org/10.1016/j.electacta.2019.03.152
[61] W. Hooch Antink, Y. Choi, K. Seong, Y. Piao, Simple synthesis of CuO/Ag nanocomposite electrode using precursor ink for non-enzymatic electrochemical hydrogen peroxide sensing, Sensors and Actuators B 255 (2018) 1995-2001. https://doi.org/10.1016/j.snb.2017.08.217 DOI: https://doi.org/10.1016/j.snb.2017.08.217
[62] M. Guler, V. Turkoglu, A. Bulut, M. Zahmakiran, Electrochemical sensing of hydrogen peroxide using Pd@Ag bimetallic nanoparticles decorated functionalized reduced graphene oxide, Electrochimica Acta 263 (2018) 118-126. https://doi.org/10.1016/j.electacta.2018.01.048 DOI: https://doi.org/10.1016/j.electacta.2018.01.048
[63] A. Mulchandani, S. Pan, Ferrocene-Conjugatedm-Phenylenediamine Conducting Polymer-Incorporated Peroxidase Biosensors, Analytical Biochemistry 267 (1999) 141-147. https://doi.org/10.1006/abio.1998.2983 DOI: https://doi.org/10.1006/abio.1998.2983
[64] H. Zhang, H. Hu, Y. Li, J. Wang, L. Ma, A ferrocene-based hydrogel as flexible electrochemical biosensor for oxidative stress detection and antioxidation treatment, Biosensors and Bioelectronics 248 (2024) 115997. https://doi.org/10.1016/j.bios.2023.115997 DOI: https://doi.org/10.1016/j.bios.2023.115997
[65] B. Wu, S. Yeasmin, Y. Liu, L.-J. Cheng, Ferrocene-grafted carbon nanotubes for sensitive non-enzymatic electrochemical detection of hydrogen peroxide, Journal of Electroanalytical Chemistry 908 (2022) 116101. https://doi.org/10.1016/j.jelechem.2022.116101 DOI: https://doi.org/10.1016/j.jelechem.2022.116101
[66] N. Sun, L. Guan, Z. Shi, N. Li, Z. Gu, Z. Zhu, M. Li, Y. Shao, Ferrocene Peapod Modified Electrodes: Preparation, Characterization, and Mediation of H2O2, Analytical Chemistry 78 (2006) 6050-6057. https://doi.org/10.1021/ac060396i DOI: https://doi.org/10.1021/ac060396i
Downloads
Published
Issue
Section
License
Copyright (c) 2025 Forrest Dalbec, Jeremy May, Dipak Koirala, Jhonnathan A. Plascencia , Micheal Okeke, Joshua Russell, Hui Xiong, I. Francis Cheng
This work is licensed under a Creative Commons Attribution 4.0 International License.
