{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T04:34:19Z","timestamp":1787027659324,"version":"build-2736575974"},"reference-count":40,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2022,9,3]],"date-time":"2022-09-03T00:00:00Z","timestamp":1662163200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Natural Sciences and Engineering Research Council of Canada (NSERC)"},{"name":"Strathclyde\/Waterloo Joint Transatlantic Funding"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biosensors"],"abstract":"<jats:p>Tetracyclines are a widely used group of antibiotics, many of which are currently only used in veterinary medicine and animal husbandry due to their adverse side effects. For the detection of tetracyclines, we previously reported a DNA aptamer named OTC5 that binds to tetracycline, oxytetracycline, and doxycycline with similar KD\u2019s of ~100 nM. Tetracyclines have an intrinsic fluorescence that is enhanced upon binding to OTC5, which can be used as a label-free and dye-free sensor. In this work, the effect of pH and metal ions on the sensor was studied. Mg2+ ions are required for the binding of OTC5 to its target with an optimal concentration of 2 mM. Other metal ions including Ca2+ and Zn2+ can also support aptamer binding. Although Mn2+ barely supported binding, the binding can be rescued by Mg2+. ITC studies confirmed that OTC5 had a KD of 0.2 \u03bcM at a pH of 6.0 and 0.03 \u03bcM at a pH of 8.3. Lower pH (pH 6) showed better fluorescence enhancement than higher pH (pH 8.3), although a pH of 6.0 had slightly higher KD values. Under optimized sensing conditions, sensors with limit of detections (LODs) of 0.1\u20130.7 nM were achieved for tetracycline, oxytetracycline, and doxycycline, which are up to 50-fold lower than previously reported. Milk samples were also tested yielding an LOD of 16 nM oxytetracycline at a pH of 6.0.<\/jats:p>","DOI":"10.3390\/bios12090717","type":"journal-article","created":{"date-parts":[[2022,9,5]],"date-time":"2022-09-05T20:48:25Z","timestamp":1662410905000},"page":"717","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":30,"title":["Metal and pH-Dependent Aptamer Binding of Tetracyclines Enabling Highly Sensitive Fluorescence Sensing"],"prefix":"10.3390","volume":"12","author":[{"given":"Yichen","family":"Zhao","sequence":"first","affiliation":[{"name":"Department of Chemistry, Waterloo Institute for Nanotechnology, Water Institute, University of Waterloo, Waterloo, ON N2L 3G1, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Biwen","family":"Gao","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Waterloo Institute for Nanotechnology, Water Institute, University of Waterloo, Waterloo, ON N2L 3G1, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2005-8093","authenticated-orcid":false,"given":"Peihuan","family":"Sun","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Waterloo Institute for Nanotechnology, Water Institute, University of Waterloo, Waterloo, ON N2L 3G1, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiawen","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Waterloo Institute for Nanotechnology, Water Institute, University of Waterloo, Waterloo, ON N2L 3G1, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5918-9336","authenticated-orcid":false,"given":"Juewen","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Waterloo Institute for Nanotechnology, Water Institute, University of Waterloo, Waterloo, ON N2L 3G1, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,9,3]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"232","DOI":"10.1128\/MMBR.65.2.232-260.2001","article-title":"Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance","volume":"65","author":"Chopra","year":"2001","journal-title":"Microbiol. Mol. Biol. Rev."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"a025387","DOI":"10.1101\/cshperspect.a025387","article-title":"Tetracycline Antibiotics and Resistance","volume":"6","author":"Grossman","year":"2016","journal-title":"Cold Spring Harb. Perspect. Med."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"209","DOI":"10.1007\/s10311-013-0404-8","article-title":"Tetracycline Antibiotics in the Environment: A Review","volume":"11","author":"Daghrir","year":"2013","journal-title":"Environ. Chem. Lett."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"141975","DOI":"10.1016\/j.scitotenv.2020.141975","article-title":"Occurrence, Fate, and Risk Assessment of Typical Tetracycline Antibiotics in the Aquatic Environment: A Review","volume":"753","author":"Xu","year":"2021","journal-title":"Sci. Total Environ."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1329","DOI":"10.1016\/j.clinthera.2005.09.005","article-title":"Safety of Doxycycline and Minocycline: A Systematic Review","volume":"27","author":"Smith","year":"2005","journal-title":"Clin. Ther."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"145291","DOI":"10.1016\/j.scitotenv.2021.145291","article-title":"Tetracyclines in the Environment: An Overview on the Occurrence, Fate, Toxicity, Detection, Removal Methods, and Sludge Management","volume":"771","author":"Scaria","year":"2021","journal-title":"Sci. Total Environ."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1007\/s00604-005-0372-1","article-title":"Residue Analysis of Oxytetracycline in Water and Sediment Samples by High-Performance Liquid Chromatography and Immunochemical Techniques","volume":"151","author":"Himmelsbach","year":"2005","journal-title":"Microchim. Acta"},{"key":"ref_8","first-page":"575","article-title":"Nanomaterial-Based Fluorescent Biosensors for Veterinary Drug Detection in Foods","volume":"28","author":"Tao","year":"2020","journal-title":"J. Food Drug Anal."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"637","DOI":"10.1007\/s00604-019-3718-9","article-title":"A Highly Sensitive Tetracycline Sensor Based on a Combination of Magnetic Molecularly Imprinted Polymer Nanoparticles and Surface Plasmon Resonance Detection","volume":"186","author":"Gao","year":"2019","journal-title":"Microchim. Acta"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1353","DOI":"10.1016\/j.cclet.2021.08.095","article-title":"A New Eu-Mof for Ratiometrically Fluorescent Detection toward Quinolone Antibiotics and Selective Detection toward Tetracycline Antibiotics","volume":"33","author":"Wang","year":"2022","journal-title":"Chin. Chem. Lett."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Mehlhorn, A., Rahimi, P., and Joseph, Y. (2018). Aptamer-Based Biosensors for Antibiotic Detection: A Review. Biosensors, 8.","DOI":"10.20944\/preprints201804.0343.v1"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1016\/j.snb.2019.02.103","article-title":"A Reagentless Aptasensor Based on Intrinsic Aptamer Redox Activity for the Detection of Tetracycline in Water","volume":"288","author":"Alawad","year":"2019","journal-title":"Sens. Actuators B: Chem."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"16800","DOI":"10.1002\/anie.202008663","article-title":"Advances and Challenges in Small-Molecule DNA Aptamer Isolation, Characterization, and Sensor Development","volume":"60","author":"Yu","year":"2021","journal-title":"Angew. Chem. Int. Ed. Engl."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"115699","DOI":"10.1016\/j.trac.2019.115699","article-title":"Innovative Engineering and Sensing Strategies for Aptamer-Based Small-Molecule Detection","volume":"121","author":"Alkhamis","year":"2019","journal-title":"TrAC Trends Anal. Chem."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"14","DOI":"10.3389\/fchem.2016.00014","article-title":"Small-Molecule Binding Aptamers: Selection Strategies, Characterization, and Applications","volume":"4","author":"Ruscito","year":"2016","journal-title":"Front. Chem."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"130361","DOI":"10.1016\/j.foodchem.2021.130361","article-title":"Advances and Perspectives of Aptasensors for the Detection of Tetracyclines: A Class of Model Compounds of Food Analysis","volume":"364","author":"Wang","year":"2021","journal-title":"Food Chem."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"319","DOI":"10.1126\/science.aao6750","article-title":"Aptamer\u2013Field-Effect Transistors Overcome Debye Length Limitations for Small-Molecule Sensing","volume":"362","author":"Nakatsuka","year":"2018","journal-title":"Science"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"3103","DOI":"10.1021\/acschembio.7b00634","article-title":"High-Affinity Nucleic-Acid-Based Receptors for Steroids","volume":"12","author":"Yang","year":"2017","journal-title":"ACS Chem. Biol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"3172","DOI":"10.1021\/acs.analchem.0c04592","article-title":"Isolation of Natural DNA Aptamers for Challenging Small-Molecule Targets, Cannabinoids","volume":"93","author":"Yu","year":"2021","journal-title":"Anal. Chem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"3142","DOI":"10.1021\/acs.analchem.1c04349","article-title":"Selection of Aptamers for Sensing Caffeine and Discrimination of Its Three Single Demethylated Analog","volume":"94","author":"Huang","year":"2022","journal-title":"Anal. Chem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1986","DOI":"10.1039\/D0AN01967D","article-title":"The Isolation of a DNA Aptamer to Develop a Fluorescent Aptasensor for the Thiamethoxam Pesticide","volume":"146","author":"Luo","year":"2021","journal-title":"Analyst"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"168","DOI":"10.1016\/j.aca.2019.07.031","article-title":"Selection and Application of Ssdna Aptamers against Spermine Based on Capture-Selex","volume":"1081","author":"Tian","year":"2019","journal-title":"Anal. Chim. Acta"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"10175","DOI":"10.1021\/acs.analchem.2c01561","article-title":"Label-Free and Dye-Free Fluorescent Sensing of Tetracyclines Using a Capture-Selected DNA Aptamer","volume":"94","author":"Zhao","year":"2022","journal-title":"Anal. Chem."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"15580","DOI":"10.1039\/c0cp00044b","article-title":"Fast and Ultrafast Spectroscopic Investigation of Tetracycline Derivatives in Organic and Aqueous Media","volume":"12","author":"Carlotti","year":"2010","journal-title":"PCCP"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"2607","DOI":"10.1093\/nar\/gkl347","article-title":"Thermodynamic Characterization of an Engineered Tetracycline-Binding Riboswitch","volume":"34","author":"Weigand","year":"2006","journal-title":"Nucleic Acids Res."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1254","DOI":"10.1016\/j.bmc.2007.10.073","article-title":"Ssdna Aptamers That Selectively Bind Oxytetracycline","volume":"16","author":"Niazi","year":"2008","journal-title":"Bioorg. Med. Chem."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"7245","DOI":"10.1016\/j.bmc.2008.06.033","article-title":"Single-Stranded DNA Aptamers Specific for Antibiotics Tetracyclines","volume":"16","author":"Niazi","year":"2008","journal-title":"Bioorg. Med. Chem."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"2549","DOI":"10.1016\/S0968-0896(01)00063-3","article-title":"A Tetracycline-Binding RNA Aptamer","volume":"9","author":"Berens","year":"2001","journal-title":"Bioorg. Med. Chem."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"3473","DOI":"10.1021\/acs.biochem.0c00586","article-title":"Selection of High-Affinity RNA Aptamers That Distinguish between Doxycycline and Tetracycline","volume":"59","author":"Tickner","year":"2020","journal-title":"Biochemistry"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"428","DOI":"10.1021\/ic00056a015","article-title":"Metal Ion-Tetracycline Interactions in Biological Fluids. 10. Structural Investigations on Copper(Ii) Complexes of Tetracycline, Oxytetracycline, Chlortetracycline, 4-(Dedimethylamino)Tetracycline, and 6-Desoxy-6-Demethyltetracycline and Discussion of Their Binding Modes","volume":"32","author":"Lambs","year":"1993","journal-title":"Inorg. Chem."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"3001","DOI":"10.1021\/ic00290a022","article-title":"Metal Ion-Tetracycline Interactions in Biological Fluids. 9. Circular Dichroism Spectra of Calcium and Magnesium Complexes with Tetracycline, Oxytetracycline, Doxycycline, and Chlortetracycline and Discussion of Their Binding Modes","volume":"27","author":"Lambs","year":"1988","journal-title":"Inorg. Chem."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"743","DOI":"10.1007\/BF01978919","article-title":"Metal Ion-Tetracycline Interactions in Biological Fluids. Part 3. Formation of Mixed-Metal Ternary Complexes of Tetracycline, Oxytetracycline, Doxycycline and Minocycline with Calcium and Magnesium, and Their Involvement in the Bioavailability of These Antibiotics in Blood Plasma","volume":"14","author":"Lambs","year":"1984","journal-title":"Agents Actions"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1107","DOI":"10.1039\/b304523b","article-title":"Fluorescence Kinetics of Aqueous Solutions of Tetracycline and Its Complexes with Mg2+ and Ca2+","volume":"2","author":"Schneider","year":"2003","journal-title":"Photochem. Photobiol. Sci."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"973","DOI":"10.1021\/bi00170a016","article-title":"In Vitro Selection of RNA Aptamers Specific for Cyanocobalamin","volume":"33","author":"Lorsch","year":"1994","journal-title":"Biochemistry"},{"key":"ref_35","first-page":"e202200010","article-title":"Selection of DNA Aptamers for Sensing Uric Acid in Simulated Tears","volume":"2","author":"Liu","year":"2022","journal-title":"Anal. Sens."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"13822","DOI":"10.1007\/s11356-016-6513-8","article-title":"Dissipation Kinetics of Oxytetracycline, Tetracycline, and Chlortetracycline Residues in Soil","volume":"23","author":"Li","year":"2016","journal-title":"Environ. Sci. Pollut. Res."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1080\/03601230903404556","article-title":"Hydrolysis and Photolysis of Oxytetracycline in Aqueous Solution","volume":"45","author":"Xuan","year":"2009","journal-title":"J. Environ. Sci. Health B"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"188","DOI":"10.1016\/j.ccr.2016.04.009","article-title":"Impact of Metal Coordination on the Antibiotic and Non-Antibiotic Activities of Tetracycline-Based Drugs","volume":"327\u2013328","author":"Guerra","year":"2016","journal-title":"Coord. Chem. Rev."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"12985","DOI":"10.1002\/anie.202102330","article-title":"Freezing-Assisted Conjugation of Unmodified Diblock DNA to Hydrogel Nanoparticles and Monoliths for DNA and Hg2+ Sensing","volume":"60","author":"Li","year":"2021","journal-title":"Angew. Chem. Int. Ed."},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Ayodele, O.O., Adesina, A.O., Pourianejad, S., Averitt, J., and Ignatova, T. (2021). Recent Advances in Nanomaterial-Based Aptasensors in Medical Diagnosis and Therapy. Nanomaterials, 11.","DOI":"10.3390\/nano11040932"}],"container-title":["Biosensors"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-6374\/12\/9\/717\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T00:22:54Z","timestamp":1760142174000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-6374\/12\/9\/717"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,9,3]]},"references-count":40,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2022,9]]}},"alternative-id":["bios12090717"],"URL":"https:\/\/doi.org\/10.3390\/bios12090717","relation":{},"ISSN":["2079-6374"],"issn-type":[{"value":"2079-6374","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,9,3]]}}}