{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,31]],"date-time":"2026-01-31T19:43:09Z","timestamp":1769888589259,"version":"3.49.0"},"reference-count":34,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2026,1,29]],"date-time":"2026-01-29T00:00:00Z","timestamp":1769644800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Funda\u00e7\u00e3o para a Ci\u00eancia e a Tecnologia","award":["2022.14536.BD"],"award-info":[{"award-number":["2022.14536.BD"]}]},{"name":"Luxembourg National Research Fund (FNR) PROFET-SEQ","award":["C22\/MS\/17380687"],"award-info":[{"award-number":["C22\/MS\/17380687"]}]},{"name":"la Caixa Banking Foundation NeuralGRAB","award":["LCF\/PR\/HR21-00410"],"award-info":[{"award-number":["LCF\/PR\/HR21-00410"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biosensors"],"abstract":"<jats:p>In this work, we present the introductory methodology for a graphene field-effect transistor (GFET)-based platform for probing the electrochemical fingerprints of amino acids, designed to enable stable and controlled surface chemistry and electrochemical measurements toward peptide and protein sequencing. We begin with a focused conceptual review that motivates electrochemical fingerprinting as a strategy for amino acid and peptide identification and contextualizes this approach within recent advances in protein manipulation relevant to sequencing. We then describe a graphene functionalization protocol that facilitates the directional attachment of amino acids onto the graphene surface. This surface chemistry is quantitatively characterized through surface plasmon resonance (SPR), yielding surface densities in the order of 1012 molecules\/cm2. The same functionalization protocol enables in situ peptide synthesis directly on graphene, as demonstrated by the successful synthesis of a model tripeptide. To support electrochemical interrogation, we developed three complementary platforms for sensor preconditioning, surface functionalization, and titration-based electrochemical measurements, compatible with both aqueous and organic solutions. Preliminary stability measurements indicate a Dirac point drift below 10 mV over 45 min. Altogether, this work establishes the experimental foundations for electrochemical amino acid and peptide fingerprinting using GFET sensors and provides a framework for the future development of electrochemically enabled protein sequencing technologies.<\/jats:p>","DOI":"10.3390\/bios16020083","type":"journal-article","created":{"date-parts":[[2026,1,29]],"date-time":"2026-01-29T16:08:17Z","timestamp":1769702897000},"page":"83","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A Graphene Field-Effect Transistor-Based Biosensor Platform for the Electrochemical Profiling of Amino Acids"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0009-0005-1751-0001","authenticated-orcid":false,"given":"Roanne Deanne","family":"Aves","sequence":"first","affiliation":[{"name":"Luxembourg Institute of Science and Technology (LIST), 28 Avenue des Hauts-Fourneaux, L-4362 Esch-sur-Alzette, Luxembourg"},{"name":"Faculty of Science, Technology, and Medicine, University of Luxembourg, 2 Place de l\u2019Universit\u00e9, L-4365 Esch-sur-Alzette, Luxembourg"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4903-9435","authenticated-orcid":false,"given":"Janwa","family":"El-Maiss","sequence":"additional","affiliation":[{"name":"Luxembourg Institute of Science and Technology (LIST), 28 Avenue des Hauts-Fourneaux, L-4362 Esch-sur-Alzette, Luxembourg"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5540-4633","authenticated-orcid":false,"given":"Divya","family":"Balakrishnan","sequence":"additional","affiliation":[{"name":"Luxembourg Institute of Science and Technology (LIST), 28 Avenue des Hauts-Fourneaux, L-4362 Esch-sur-Alzette, Luxembourg"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4765-1789","authenticated-orcid":false,"given":"Naveen","family":"Kumar","sequence":"additional","affiliation":[{"name":"DeepNano Group, James Watt School of Engineering, University of Glasgow, University Avenue, Glasgow G12 8QQ, UK"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7794-9746","authenticated-orcid":false,"given":"Mafalda","family":"Abrantes","sequence":"additional","affiliation":[{"name":"International Iberian Nanotechnology Laboratory (INL), Avenida Mestre Jos\u00e9 Veiga s\/n, 4715-330 Braga, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5622-7760","authenticated-orcid":false,"given":"J\u00e9r\u00f4me","family":"Borme","sequence":"additional","affiliation":[{"name":"International Iberian Nanotechnology Laboratory (INL), Avenida Mestre Jos\u00e9 Veiga s\/n, 4715-330 Braga, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6473-2508","authenticated-orcid":false,"given":"Vihar","family":"Georgiev","sequence":"additional","affiliation":[{"name":"DeepNano Group, James Watt School of Engineering, University of Glasgow, University Avenue, Glasgow G12 8QQ, UK"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9875-6188","authenticated-orcid":false,"given":"Pedro","family":"Alpuim","sequence":"additional","affiliation":[{"name":"International Iberian Nanotechnology Laboratory (INL), Avenida Mestre Jos\u00e9 Veiga s\/n, 4715-330 Braga, Portugal"},{"name":"Center of Physics of the Universities of Minho and Porto, University of Minho, 4710-057 Braga, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0526-2507","authenticated-orcid":false,"given":"C\u00e9sar Pascual","family":"Garc\u00eda","sequence":"additional","affiliation":[{"name":"Luxembourg Institute of Science and Technology (LIST), 28 Avenue des Hauts-Fourneaux, L-4362 Esch-sur-Alzette, Luxembourg"}]}],"member":"1968","published-online":{"date-parts":[[2026,1,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Zhang, G., Annan, R.S., Carr, S.A., and Neubert, T.A. (2010). Overview of Peptide and Protein Analysis by Mass Spectrometry. Curr. Protoc. Protein Sci., 62.","DOI":"10.1002\/0471140864.ps1601s62"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"4728","DOI":"10.1021\/acs.analchem.4c06750","article-title":"Recent Advances in Mass Spectrometry-Based Bottom-Up Proteomics","volume":"97","author":"Movassaghi","year":"2025","journal-title":"Anal. Chem."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"e202400824","DOI":"10.1002\/cbic.202400824","article-title":"Towards Next Generation Protein Sequencing","volume":"26","author":"Yi","year":"2025","journal-title":"ChemBioChem"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1038\/s41587-019-0401-y","article-title":"Reading Amino Acids in a Nanopore","volume":"38","author":"Howorka","year":"2020","journal-title":"Nat. Biotechnol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"662","DOI":"10.1038\/s41586-024-07935-7","article-title":"Multi-Pass, Single-Molecule Nanopore Reading of Long Protein Strands","volume":"633","author":"Motone","year":"2024","journal-title":"Nature"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"176","DOI":"10.1038\/s41587-019-0345-2","article-title":"Electrical Recognition of the Twenty Proteinogenic Amino Acids Using an Aerolysin Nanopore","volume":"38","author":"Ouldali","year":"2020","journal-title":"Nat. Biotechnol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1038\/s41592-023-02095-4","article-title":"Peptide Sequencing Based on Host\u2013Guest Interaction-Assisted Nanopore Sensing","volume":"21","author":"Zhang","year":"2024","journal-title":"Nat. Methods"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"103586","DOI":"10.1016\/j.isci.2021.103586","article-title":"ProtSeq: Toward High-Throughput, Single-Molecule Protein Sequencing via Amino Acid Conversion into DNA Barcodes","volume":"25","author":"Hong","year":"2022","journal-title":"iScience"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"186","DOI":"10.1126\/science.abo7651","article-title":"Real-Time Dynamic Single-Molecule Protein Sequencing on an Integrated Semiconductor Device","volume":"378","author":"Reed","year":"2022","journal-title":"Science"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"283","DOI":"10.3891\/acta.chem.scand.04-0283","article-title":"Method for Determination of the Amino Acid Sequence in Peptides","volume":"4","author":"Edman","year":"1950","journal-title":"Acta Chem. Scand."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2488","DOI":"10.1039\/b700200a","article-title":"Subfemtomole Level Protein Sequencing by Edman Degradation Carried out in a Microfluidic Chip","volume":"24","author":"Chen","year":"2007","journal-title":"Chem. Commun."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1076","DOI":"10.1038\/nbt.4278","article-title":"Highly Parallel Single-Molecule Identification of Proteins in Zeptomole-Scale Mixtures","volume":"36","author":"Swaminathan","year":"2018","journal-title":"Nat. Biotechnol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"13973","DOI":"10.1021\/jacs.5c03385","article-title":"After 75 Years, an Alternative to Edman Degradation: A Mechanistic and Efficiency Study of a Base-Induced Method for N-Terminal Peptide Sequencing","volume":"147","author":"Deol","year":"2025","journal-title":"J. Am. Chem. Soc."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"6","DOI":"10.1007\/s40828-018-0060-5","article-title":"Acid\u2013Base Equilibria of Amino Acids: Microscopic and Macroscopic Acidity Constants","volume":"4","author":"Scholz","year":"2018","journal-title":"ChemTexts"},{"key":"ref_15","unstructured":"Nelson, D.L., Cox, M.M., and Lehninger, A.L. (2017). Amino Acids, Peptides, and Proteins. Lehninger Principles of Biochemistry, W.H. Freeman and Company."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1001","DOI":"10.1021\/acs.jchemed.0c01144","article-title":"Analysis of Protonation Equilibria of Amino Acids in Aqueous Solutions Using Microsoft Excel","volume":"98","year":"2021","journal-title":"J. Chem. Educ."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"134085","DOI":"10.1109\/ACCESS.2024.3411168","article-title":"Discovery of Amphoteric Fingerprints of Amino Acids With Field-Effect Transistors","volume":"12","author":"Kumar","year":"2024","journal-title":"IEEE Access"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1016\/j.petrol.2012.09.003","article-title":"Physical Properties of Aqueous Glycerol Solutions","volume":"98\u201399","author":"Takamura","year":"2012","journal-title":"J. Pet. Sci. Eng."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Crist\u00f3bal-Lecina, E., El-Maiss, J., Figueras, E., Singh, A.C., Krishnamoorthy, S., \u00d8sterbye, T., Pascual Garc\u00eda, C., and Andreu, D. (2023). Acid-Modulated Peptide Synthesis for Application on Oxide Biosensor Interfaces. Nanomaterials, 13.","DOI":"10.3390\/nano13243092"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1759","DOI":"10.1002\/bip.1978.360170711","article-title":"Ellipsometry as a Tool to Study the Adsorption Behavior of Synthetic and Biopolymers at the Air\u2013Water Interface","volume":"17","author":"Benjamins","year":"1978","journal-title":"Biopolymers"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"18524","DOI":"10.1039\/C5CP02306H","article-title":"Adsorption of Bovine Serum Albumin on Silver Surfaces Enhances the Release of Silver at pH Neutral Conditions","volume":"17","author":"Wang","year":"2015","journal-title":"Phys. Chem. Chem. Phys."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2406","DOI":"10.1002\/pola.26016","article-title":"Synthesis and Characterization of Pyrene Bearing Amphiphilic Miktoarm Star Polymer and Its Noncovalent Interactions with Multiwalled Carbon Nanotubes","volume":"50","author":"Durmaz","year":"2012","journal-title":"J. Polym. Sci. Part Polym. Chem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"641","DOI":"10.1016\/S0969-2126(00)00065-4","article-title":"Volume Changes on Protein Folding","volume":"2","author":"Harpaz","year":"1994","journal-title":"Structure"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2309","DOI":"10.1016\/j.bpj.2011.03.004","article-title":"On the Distribution of Protein Refractive Index Increments","volume":"100","author":"Zhao","year":"2011","journal-title":"Biophys. J."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Cabral, P.D., Domingues, T., Machado, G., Chicharo, A., Cerqueira, F., Fernandes, E., Athayde, E., Alpuim, P., and Borme, J. (2020). Clean-Room Lithographical Processes for the Fabrication of Graphene Biosensors. Materials, 13.","DOI":"10.3390\/ma13245728"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"13059","DOI":"10.1021\/jacs.5c03552","article-title":"Graphene-Based Glucose Sensors with an Attomolar Limit of Detection","volume":"147","author":"Lopes","year":"2025","journal-title":"J. Am. Chem. Soc."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"403","DOI":"10.1039\/D0AN01661F","article-title":"Graphene Field-Effect Transistors as Bioanalytical Sensors: Design, Operation and Performance","volume":"146","author":"Sauvage","year":"2021","journal-title":"Analyst"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"495","DOI":"10.1186\/s12951-022-01695-0","article-title":"Ultrasensitive Dopamine Detection with Graphene Aptasensor Multitransistor Arrays","volume":"20","author":"Abrantes","year":"2022","journal-title":"J. Nanobiotechnol."},{"key":"ref_29","unstructured":"Zhang, X., Qi, Q., Jing, Q., Ao, S., Zhang, Z., Ding, M., Wu, M., Liu, K., Wang, W., and Ling, Y. (2020). Electrical Probing of COVID-19 Spike Protein Receptor Binding Domain via a Graphene Field-Effect Transistor. arXiv."},{"key":"ref_30","unstructured":"Kwong Hong Tsang, D. (2021, May 30). Chemically Functionalised Graphene Biosensor for the Label-Free Sensing of Exosomes, Imperial College London. Available online: http:\/\/spiral.imperial.ac.uk\/handle\/10044\/1\/71440."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"6487","DOI":"10.1021\/jp311504d","article-title":"Revisiting the Mechanism of Neutral Hydrolysis of Esters: Water Autoionization Mechanisms with Acid or Base Initiation Pathways","volume":"117","author":"Pliego","year":"2013","journal-title":"J. Phys. Chem. B"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"12868","DOI":"10.1021\/la503439g","article-title":"Succinimidyl Ester Surface Chemistry: Implications of the Competition between Aminolysis and Hydrolysis on Covalent Protein Immobilization","volume":"30","author":"Lim","year":"2014","journal-title":"Langmuir"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"263","DOI":"10.1021\/acs.langmuir.4c03420","article-title":"Tailoring DNA Surface Interactions on Single-Layer Graphene: Comparative Analysis of Pyrene, Acridine, and Fluorenyl Methyl Linkers","volume":"41","author":"Domingues","year":"2025","journal-title":"Langmuir"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"4911","DOI":"10.1038\/ncomms5911","article-title":"Chemical Sensing with Switchable Transport Channels in Graphene Grain Boundaries","volume":"5","author":"Yasaei","year":"2014","journal-title":"Nat. Commun."}],"container-title":["Biosensors"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-6374\/16\/2\/83\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,31]],"date-time":"2026-01-31T05:18:49Z","timestamp":1769836729000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-6374\/16\/2\/83"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,1,29]]},"references-count":34,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2026,2]]}},"alternative-id":["bios16020083"],"URL":"https:\/\/doi.org\/10.3390\/bios16020083","relation":{},"ISSN":["2079-6374"],"issn-type":[{"value":"2079-6374","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,1,29]]}}}