{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,11]],"date-time":"2026-04-11T04:56:31Z","timestamp":1775883391896,"version":"3.50.1"},"reference-count":48,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2026,4,9]],"date-time":"2026-04-09T00:00:00Z","timestamp":1775692800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biosensors"],"abstract":"<jats:p>Neurofilament light chain (NfL) is a promising biomarker of axonal injury across acute and chronic neurodegeneration, which can improve drug discovery and disease monitoring models. Traditional in vivo animal models cannot fully mimic human pathophysiology of neurodegenerative diseases (NDDs), but in vitro models based on human cells solve this problem, reducing the time and cost of drug testing. We developed an electrochemical immunosensor for NfL detection in cell culture media to monitor acute neuronal injury in in vitro models. The biosensor was designed in two configurations: the label-free system, which directly detects NfL in the sample via the antibody\u2013antigen interaction, and the sandwich configuration, which incorporates two additional antibodies. Detection was examined using electrochemical techniques, including cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and chronoamperometry (CA). The sensor demonstrated a detection limit of 3\u20139 pg mL\u22121, and a dynamic working range spanning from 10 up to 107 pg mL\u22121. Importantly, NfL was successfully detected in physiological media collected from cultured neurons that were differentiated from the long-term human neuroepithelial-like stem cells. This discovery highlights the platform\u2019s applicability for in vitro neurodegenerative models. The immunosensor offers a sensitive, scalable, and cost-effective alternative for neurodegeneration detection in drug testing applications.<\/jats:p>","DOI":"10.3390\/bios16040212","type":"journal-article","created":{"date-parts":[[2026,4,10]],"date-time":"2026-04-10T07:55:12Z","timestamp":1775807712000},"page":"212","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Electrochemical Detection of Neuronal Injury in Cell Culture Samples: A Cost-Effective Biosensor for Neurofilament Light Sensing"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6616-9023","authenticated-orcid":false,"given":"Anna","family":"Panteleeva","sequence":"first","affiliation":[{"name":"Nanobioengineering Group, Institute for Bioengineering of Catalonia (IBEC), Barcelona Institute of Science and Technology (BIST), 08028 Barcelona, Spain"},{"name":"Department of Electronics and Biomedical Engineering, University of Barcelona, 08007 Barcelona, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sujey","family":"Palma-Florez","sequence":"additional","affiliation":[{"name":"Nanobioengineering Group, Institute for Bioengineering of Catalonia (IBEC), Barcelona Institute of Science and Technology (BIST), 08028 Barcelona, Spain"},{"name":"Department of Electronics and Biomedical Engineering, University of Barcelona, 08007 Barcelona, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-7482-3447","authenticated-orcid":false,"given":"Ashlyne M.","family":"Smith","sequence":"additional","affiliation":[{"name":"Nanobioengineering Group, Institute for Bioengineering of Catalonia (IBEC), Barcelona Institute of Science and Technology (BIST), 08028 Barcelona, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0648-8380","authenticated-orcid":false,"given":"Sara","family":"Palma-Tortosa","sequence":"additional","affiliation":[{"name":"Laboratory of Stem Cells and Restorative Neurology, Lund Stem Cell Center, Lund University, 221 84 Lund, Sweden"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2296-2449","authenticated-orcid":false,"given":"Zaal","family":"Kokaia","sequence":"additional","affiliation":[{"name":"Laboratory of Stem Cells and Restorative Neurology, Lund Stem Cell Center, Lund University, 221 84 Lund, Sweden"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1140-3679","authenticated-orcid":false,"given":"Josep","family":"Samitier","sequence":"additional","affiliation":[{"name":"Nanobioengineering Group, Institute for Bioengineering of Catalonia (IBEC), Barcelona Institute of Science and Technology (BIST), 08028 Barcelona, Spain"},{"name":"Department of Electronics and Biomedical Engineering, University of Barcelona, 08007 Barcelona, Spain"},{"name":"Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), 28029 Madrid, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1490-8373","authenticated-orcid":false,"given":"M\u00f2nica","family":"Mir","sequence":"additional","affiliation":[{"name":"Nanobioengineering Group, Institute for Bioengineering of Catalonia (IBEC), Barcelona Institute of Science and Technology (BIST), 08028 Barcelona, Spain"},{"name":"Department of Electronics and Biomedical Engineering, University of Barcelona, 08007 Barcelona, Spain"},{"name":"Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), 28029 Madrid, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,9]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Lamptey, R.N.L., Chaulagain, B., Trivedi, R., Gothwal, A., Layek, B., and Singh, J. (2022). A Review of the Common Neurodegenerative Disorders: Current Therapeutic Approaches and the Potential Role of Nanotherapeutics. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms23031851"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"255","DOI":"10.1016\/S1474-4422(19)30411-9","article-title":"The Global Burden of Neurological Disorders: Translating Evidence into Policy","volume":"19","author":"Feigin","year":"2020","journal-title":"Lancet Neurol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"a033118","DOI":"10.1101\/cshperspect.a033118","article-title":"Clinical Neurology and Epidemiology of the Major Neurodegenerative Diseases","volume":"10","author":"Erkkinen","year":"2018","journal-title":"Cold Spring Harb. Perspect. Biol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"83","DOI":"10.3233\/JAD-215699","article-title":"Alzheimer\u2019s Disease: Key Insights from Two Decades of Clinical Trial Failures","volume":"87","author":"Kim","year":"2022","journal-title":"J. Alzheimer\u2019s Dis."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"272","DOI":"10.1016\/j.trci.2019.05.008","article-title":"Alzheimer\u2019s Disease Drug Development Pipeline: 2019","volume":"5","author":"Cummings","year":"2019","journal-title":"Alzheimer\u2019s Dement. Transl. Res. Clin. Interv."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"40","DOI":"10.1016\/j.brainres.2016.03.051","article-title":"Drug Discovery Using Neurons Derived from Patients: Studying Human Disease Using Human Neurons","volume":"1656","author":"Ahfeldt","year":"2017","journal-title":"Brain Res."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Biffi, E., Regalia, G., Menegon, A., Ferrigno, G., and Pedrocchi, A. (2013). The Influence of Neuronal Density and Maturation on Network Activity of Hippocampal Cell Cultures: A Methodological Study. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0083899"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Kussauer, S., David, R., and Lemcke, H. (2019). HiPSCs Derived Cardiac Cells for Drug and Toxicity Screening and Disease Modeling: What Micro- Electrode-Array Analyses Can Tell Us. Cells, 8.","DOI":"10.3390\/cells8111331"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"486","DOI":"10.1007\/s00216-003-2149-x","article-title":"Biological Application of Microelectrode Arrays in Drug Discovery and Basic Research","volume":"377","author":"Stett","year":"2003","journal-title":"Anal. Bioanal. Chem."},{"key":"ref_10","unstructured":"Blum, L.J., and Coulet, P.R. (1991). What Is a Biosensor? (Biosensor Principles and Applications), CRC Press. [1st ed.]."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Sun, X., Sun, X., Wang, Q., Wang, X., Feng, L., Yang, Y., Jing, Y., Yang, C., and Zhang, S. (2022). Biosensors toward Behavior Detection in Diagnosis of Alzheimer\u2019s Disease. Front. Bioeng. Biotechnol., 10.","DOI":"10.3389\/fbioe.2022.1031833"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1237","DOI":"10.1021\/acssensors.2c00333","article-title":"Biosensors Integration in Blood\u2013Brain Barrier-on-a-Chip: Emerging Platform for Monitoring Neurodegenerative Diseases","volume":"7","author":"Mir","year":"2022","journal-title":"ACS Sens."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"20720","DOI":"10.1021\/acsami.2c02936","article-title":"Surface Resonance Immunosensor with Antibody-Functionalized Nanoparticles for Ultrasensitive Quantification of the CD5 Biomarker","volume":"14","author":"Popov","year":"2022","journal-title":"ACS Appl. Mater. Interfaces"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1475","DOI":"10.1039\/C8AY02594K","article-title":"A Sensitive and Regenerative Electrochemical Immunosensor for Quantitative Detection of: Escherichia Coli O157:H7 Based on Stable Polyaniline Coated Screen-Printed Carbon Electrode and RGO-NR-Au@Pt","volume":"11","author":"Mo","year":"2019","journal-title":"Anal. Methods"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1038\/nrneurol.2010.4","article-title":"Cerebrospinal Fluid and Plasma Biomarkers in Alzheimer Disease","volume":"6","author":"Blennow","year":"2010","journal-title":"Nat. Rev. Neurol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"954","DOI":"10.1038\/s41591-021-01382-x","article-title":"Biomarkers for Neurodegenerative Diseases","volume":"27","author":"Hansson","year":"2021","journal-title":"Nat. Med."},{"key":"ref_17","first-page":"1277","article-title":"Association of Cerebrospinal Fluid \u03b2-Amyloid 1-42, t-Tau, p-Tau 181, and \u03b1-Synuclein Levels with Clinical Features of Drug-Naive Patients with Early Parkinson Disease","volume":"70","author":"Kang","year":"2013","journal-title":"JAMA Neurol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1979","DOI":"10.1172\/JCI80743","article-title":"Quantification of Mutant Huntingtin Protein in Cerebrospinal Fluid from Huntington\u2019s Disease Patients","volume":"125","author":"Wild","year":"2015","journal-title":"J. Clin. Investig."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"143","DOI":"10.1016\/j.neurobiolaging.2020.07.018","article-title":"Blood and Cerebrospinal Fluid Neurofilament Light Differentially Detect Neurodegeneration in Early Alzheimer\u2019s Disease","volume":"95","author":"Andersson","year":"2020","journal-title":"Neurobiol. Aging"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"930","DOI":"10.1212\/WNL.0000000000003680","article-title":"Blood-Based NfL: A Biomarker for Differential Diagnosis of Parkinsonian Disorder","volume":"88","author":"Hansson","year":"2017","journal-title":"Neurology"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"2711","DOI":"10.1093\/brain\/awac394","article-title":"Neurofilament Light Chain in Drug Development for Amyotrophic Lateral Sclerosis: A Critical Appraisal","volume":"146","author":"Benatar","year":"2023","journal-title":"Brain"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"577","DOI":"10.1038\/s41582-018-0058-z","article-title":"Neurofilaments as Biomarkers in Neurological Disorders","volume":"14","author":"Khalil","year":"2018","journal-title":"Nat. Rev. Neurol."},{"key":"ref_23","first-page":"056","article-title":"Neurofilament Light (NF-L) Chain Protein from a Highly Polymerized Structural Component of the Neuronal Cytoskeleton to a Neurodegenerative Disease Biomarker in the Periphery","volume":"7","author":"Zhao","year":"2021","journal-title":"J. Alzheimer\u2019s Neurodegener. Dis."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2247","DOI":"10.1212\/WNL.0000000000001642","article-title":"Neurofilament Light Chain: A Prognostic Biomarker in Amyotrophic Lateral Sclerosis","volume":"84","author":"Lu","year":"2015","journal-title":"Neurology"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"673","DOI":"10.1016\/S1474-4422(16)00070-3","article-title":"CSF and Blood Biomarkers for the Diagnosis of Alzheimer\u2019s Disease: A Systematic Review and Meta-Analysis","volume":"15","author":"Olsson","year":"2016","journal-title":"Lancet Neurol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"114545","DOI":"10.1016\/j.mseb.2020.114545","article-title":"Immunoaffinity Biosensor for Neurofilament Light Chain Detection and Its Use in Parkinson\u2019s Diagnosis","volume":"256","author":"Uzun","year":"2020","journal-title":"Mater. Sci. Eng. B"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"137815","DOI":"10.1016\/j.electacta.2021.137815","article-title":"Electrochemical Immunoplatform to Unravel Neurodegeneration and Alzheimer\u2019s Disease through the Determination of Neurofilament Light Protein","volume":"371","author":"Valverde","year":"2021","journal-title":"Electrochim. Acta"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1365","DOI":"10.1002\/sctm.20-0134","article-title":"Grafted Human Pluripotent Stem Cell-Derived Cortical Neurons Integrate into Adult Human Cortical Neural Circuitry","volume":"9","author":"Laterza","year":"2020","journal-title":"Stem Cells Transl. Med."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1405","DOI":"10.1016\/j.bios.2005.07.012","article-title":"Enzyme Inhibition-Based Biosensors for Food Safety and Environmental Monitoring","volume":"21","author":"Amine","year":"2006","journal-title":"Biosens. Bioelectron."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"676","DOI":"10.1038\/nmeth.2019","article-title":"Fiji: An Open-Source Platform for Biological-Image Analysis","volume":"9","author":"Schindelin","year":"2012","journal-title":"Nat. Methods"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Borman, P., and Elder, D. (2017). Q2(R1) Validation of Analytical Procedures. ICH Quality Guidelines, Wiley.","DOI":"10.1002\/9781118971147.ch5"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Lichtenberg, J.Y., Ling, Y., and Kim, S. (2019). Non-Specific Adsorption Reduction Methods in Biosensing. Sensors, 19.","DOI":"10.3390\/s19112488"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"645","DOI":"10.3390\/s90100645","article-title":"Reduction of Non-Specific Protein Adsorption Using Poly(Ethylene) Glycol (PEG) Modified Polyacrylate Hydrogels in Immunoassays for Staphylococcal Enterotoxin B Detection","volume":"9","author":"Charles","year":"2009","journal-title":"Sensors"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"4072","DOI":"10.1021\/ie503897h","article-title":"Protein Sensing with Aptamer Immobilized on an Antifouling Binary Self-Assembled Monolayer","volume":"54","author":"Feyzizarnagh","year":"2015","journal-title":"Ind. Eng. Chem. Res."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"27925","DOI":"10.1039\/D1RA03785D","article-title":"Reducing the Resistance for the Use of Electrochemical Impedance Spectroscopy Analysis in Materials Chemistry","volume":"11","author":"Laschuk","year":"2021","journal-title":"RSC Adv."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"819","DOI":"10.3906\/kim-2012-69","article-title":"Development of a Functional Impedimetric Immunosensor for Accurate Detection of Thy- Roid-Stimulating Hormone","volume":"45","author":"Asav","year":"2021","journal-title":"Turk. J. Chem."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"126780","DOI":"10.1016\/j.snb.2019.126780","article-title":"An Insight into Impedimetric Immunosensor and Its Electrical Equivalent Circuit","volume":"297","author":"Saxena","year":"2019","journal-title":"Sens. Actuators B Chem."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"49260","DOI":"10.1021\/acsomega.5c02911","article-title":"Double Step Electrochemical Etching for Reuse of Gold Screen Printed Electrode","volume":"10","author":"Lee","year":"2025","journal-title":"ACS Omega"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"2834","DOI":"10.1039\/D3NR05456J","article-title":"Recent Progress in Biosensor Regeneration Techniques","volume":"16","author":"Jia","year":"2024","journal-title":"Nanoscale"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"6267","DOI":"10.1021\/la503533g","article-title":"Biosensor Regeneration: A Review of Common Techniques and Outcomes","volume":"31","author":"Goode","year":"2015","journal-title":"Langmuir"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1832","DOI":"10.1002\/acn3.51676","article-title":"Age- and Disease-specific Reference Values for Neurofilament Light Presented in an Online Interactive Support Interface","volume":"9","author":"Vermunt","year":"2022","journal-title":"Ann. Clin. Transl. Neurol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1007\/BF01324701","article-title":"Staurosporine Differentially Inhibits Glioma versus Non-Glioma Cell Lines","volume":"16","author":"Baltuch","year":"1993","journal-title":"J. Neurooncol."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Carles, A., Freyssin, A., Perin-Dureau, F., Rubinstenn, G., and Maurice, T. (2024). Targeting N-Methyl-d-Aspartate Receptors in Neurodegenerative Diseases. Int. J. Mol. Sci., 25.","DOI":"10.20944\/preprints202402.0679.v1"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1016\/j.ejphar.2013.04.054","article-title":"Modulation of NMDA Receptor at the Synapse: Promising Therapeutic Interventions in Disorders of the Nervous System","volume":"719","author":"Mellone","year":"2013","journal-title":"Eur. J. Pharmacol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"136850","DOI":"10.1016\/j.cej.2022.136850","article-title":"A Neurofilament-Light Chains Electrochemical Immunosensor Expected to Reveal the Early Stage of Neurodegenerative Diseases","volume":"446","author":"Li","year":"2022","journal-title":"Chem. Eng. J."},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Adil, O., and Shamsi, M.H. (2023). Electrochemical Impedance Immunoassay for ALS-Associated Neurofilament Protein: Matrix Effect on the Immunoplatform. Biosensors, 13.","DOI":"10.3390\/bios13020247"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"092510","DOI":"10.1149\/1945-7111\/acfb41","article-title":"Improvement of Low-Cost Commercial Carbon Screen-Printed Electrodes Conductivities with Controlled Gold Reduction Towards Thiol Modification","volume":"170","author":"Partanen","year":"2023","journal-title":"J. Electrochem. Soc."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"1548","DOI":"10.1177\/0963689718798564","article-title":"Differentiation of Sendai Virus-Reprogrammed IPSC into \u03b2 Cells, Compared with Human Pancreatic Islets and Immortalized \u03b2 Cell Line","volume":"27","author":"Pellegrini","year":"2018","journal-title":"Cell Transplant."}],"container-title":["Biosensors"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-6374\/16\/4\/212\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,4,11]],"date-time":"2026-04-11T04:27:13Z","timestamp":1775881633000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-6374\/16\/4\/212"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,4,9]]},"references-count":48,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2026,4]]}},"alternative-id":["bios16040212"],"URL":"https:\/\/doi.org\/10.3390\/bios16040212","relation":{},"ISSN":["2079-6374"],"issn-type":[{"value":"2079-6374","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,4,9]]}}}