{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,1]],"date-time":"2026-05-01T22:12:13Z","timestamp":1777673533767,"version":"3.51.4"},"reference-count":44,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2020,6,2]],"date-time":"2020-06-02T00:00:00Z","timestamp":1591056000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e a Tecnologia","doi-asserted-by":"publisher","award":["PTDC\/MEDQUI\/28721\/2017"],"award-info":[{"award-number":["PTDC\/MEDQUI\/28721\/2017"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>The development of solid materials that deliver nitric oxide (NO) are of interest for several therapeutic applications. Nevertheless, due to NO\u2019s reactive nature, rapid diffusion and short half-life, reporting their NO delivery characteristics is rather complex. The full knowledge of this parameter is fundamental to discuss the therapeutic utility of these materials, and thus, the NO quantification strategy must be carefully considered according to the NO-releasing scaffold type, to the expected NO-releasing amounts and to the medium of quantification. In this work, we explore and discuss three different ways of quantifying the release of NO in different biological fluids: haemoglobin assay, Griess assay and NO electrochemical detection. For these measurements, different porous materials, namely zeolites and titanosilicates were used as models for NO-releasing platforms. The oxyhaemoglobin assay offers great sensitivity (nanomolar levels), but it is only possible to monitor the NO release while oxyhaemoglobin is not fully converted. On the other hand, Griess assay has low sensitivity in complex biological media, namely in blood, and interferences with media make NO measurements questionable. Nevertheless, this method can measure micromolar amounts of NO and may be useful for an initial screening for long-term release performance. The electrochemical sensor enabled real-time measurements in a variety of biological settings. However, measured NO is critically low in oxygenated and complex media, giving transient signals, which makes long-term quantification impossible. Despite the disadvantages of each method, the combination of all the results provided a more comprehensive NO release profile for these materials, which will help to determine which formulations are most promising for specific therapeutic applications. This study highlights the importance of using appropriate NO quantification tools to provide accurate reports.<\/jats:p>","DOI":"10.3390\/molecules25112580","type":"journal-article","created":{"date-parts":[[2020,6,3]],"date-time":"2020-06-03T04:12:09Z","timestamp":1591157529000},"page":"2580","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":24,"title":["A Comparison of Different Approaches to Quantify Nitric Oxide Release from NO-Releasing Materials in Relevant Biological Media"],"prefix":"10.3390","volume":"25","author":[{"given":"Rosana","family":"V. Pinto","sequence":"first","affiliation":[{"name":"CERENA. Departamento de Engenharia Qu\u00edmica, Instituto Superior T\u00e9cnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal"},{"name":"Centro de Qu\u00edmica Estrutural, Faculdade de Ci\u00eancias, Universidade de Lisboa, 1749-016 Lisboa, Portugal"}]},{"given":"Fernando","family":"Antunes","sequence":"additional","affiliation":[{"name":"Centro de Qu\u00edmica Estrutural, Faculdade de Ci\u00eancias, Universidade de Lisboa, 1749-016 Lisboa, Portugal"}]},{"given":"Jo\u00e3o","family":"Pires","sequence":"additional","affiliation":[{"name":"Centro de Qu\u00edmica Estrutural, Faculdade de Ci\u00eancias, Universidade de Lisboa, 1749-016 Lisboa, Portugal"}]},{"given":"Ana","family":"Silva-Herdade","sequence":"additional","affiliation":[{"name":"Instituto de Bioqu\u00edmica, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3061-9632","authenticated-orcid":false,"given":"Mois\u00e9s L.","family":"Pinto","sequence":"additional","affiliation":[{"name":"CERENA. Departamento de Engenharia Qu\u00edmica, Instituto Superior T\u00e9cnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2020,6,2]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"3742","DOI":"10.1039\/c2cs15273h","article-title":"Nitric oxide release: Part II. Therapeutic applications","volume":"41","author":"Carpenter","year":"2012","journal-title":"Chem. Soc. Rev."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"3731","DOI":"10.1039\/c2cs15272j","article-title":"Nitric oxide release: Part I. Macromolecular scaffolds","volume":"41","author":"Riccio","year":"2012","journal-title":"Chem. Soc. Rev."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"5135","DOI":"10.1002\/anie.201913135","article-title":"Tuning Cellular Biological Functions Through the Controlled Release of NO from a Porous Ti-MOF","volume":"59","author":"Pinto","year":"2020","journal-title":"Angew. Chem. Int. Ed."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"4966","DOI":"10.1002\/anie.200703934","article-title":"Gas storage in nanoporous materials","volume":"47","author":"Morris","year":"2008","journal-title":"Angew. Chem. Int. Ed."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1016\/j.niox.2019.05.010","article-title":"New generation of nitric oxide-releasing porous materials: Assessment of their potential to regulate biological functions","volume":"90","author":"Pinto","year":"2019","journal-title":"Nitric Oxide Biol. Chem."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1016\/j.mtcomm.2017.07.007","article-title":"Functionalised solids delivering bioactive nitric oxide gas for therapeutic applications","volume":"12","author":"Gregg","year":"2017","journal-title":"Mater. Today Commun."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"118","DOI":"10.1186\/1477-7819-11-118","article-title":"Nitric oxide and cancer: A review","volume":"11","author":"Choudhari","year":"2013","journal-title":"World J. Surg. Oncol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1007\/s12291-014-0446-0","article-title":"Free radicals: Properties, sources, targets, and their implication in various diseases","volume":"30","author":"Phaniendra","year":"2015","journal-title":"Indian J. Clin. Biochem."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"355","DOI":"10.1073\/pnas.98.1.355","article-title":"The biological lifetime of nitric oxide: Implications for the perivascular dynamics of NO and O2","volume":"98","author":"Thomas","year":"2001","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Pullano, S.A., Falcone, F., Critello, D.C., Bianco, M.G., Menniti, M., and Fiorillo, A.S. (2020). An Affordable Fabrication of a Zeolite-Based Capacitor for Gas Sensing. Sensors, 20.","DOI":"10.3390\/s20072143"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"3753","DOI":"10.1039\/c2cs15271a","article-title":"Nitric oxide release: Part III. Measurement and reporting","volume":"41","author":"Coneski","year":"2012","journal-title":"Chem. Soc. Rev."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"645","DOI":"10.1016\/j.freeradbiomed.2007.04.026","article-title":"Methods to detect nitric oxide and its metabolites in biological samples","volume":"43","author":"Bryan","year":"2007","journal-title":"Free Radic. Biol. Med."},{"key":"ref_13","first-page":"158","article-title":"Griess method for nitrite measurement of aqueous and protein-containing samples","volume":"Volume 359","author":"Kleinbongard","year":"2002","journal-title":"Nitric Oxide, Part D: Oxide Detection, Mitochondria and Cell Functions, and Peroxynitrite Reactions"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"276","DOI":"10.3390\/s30800276","article-title":"Measurement of Nitric Oxide Production in Biological Systems by Using Griess Reaction Assay","volume":"3","author":"Sun","year":"2003","journal-title":"Sensors"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"3475","DOI":"10.1007\/s00216-012-6671-6","article-title":"Overview of significant examples of electrochemical sensor arrays designed for detection of nitric oxide and relevant species in a biological environment","volume":"405","author":"Griveau","year":"2013","journal-title":"Anal. Bioanal. Chem."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"6396","DOI":"10.1021\/ja200663e","article-title":"Slow release of NO by microporous titanosilicate ETS-4","volume":"133","author":"Pinto","year":"2011","journal-title":"J. Am. Chem. Soc."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"224","DOI":"10.1039\/C3TB20929F","article-title":"Microporous titanosilicates Cu2+\u2013 and Co2+\u2013ETS-4 for storage and slow release of therapeutic nitric oxide","volume":"2","author":"Pinto","year":"2014","journal-title":"J. Mater. Chem. B"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"83","DOI":"10.1016\/j.micromeso.2016.04.021","article-title":"Storage and delivery of nitric oxide by microporous titanosilicate ETS-10 and Al and Ga substituted analogues","volume":"229","author":"Pinto","year":"2016","journal-title":"Microporous Mesoporous Mater."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"502","DOI":"10.1021\/ja0503579","article-title":"NO-releasing zeolites and their antithrombotic properties","volume":"128","author":"Wheatley","year":"2006","journal-title":"J. Am. Chem. Soc."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"195","DOI":"10.1016\/S1381-1169(03)00501-6","article-title":"Theoretical and experimental study of NO\/NO2 adsorption over Co-exchanged type-A zeolite","volume":"207","author":"Henao","year":"2004","journal-title":"J. Mol. Catal. A Chem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1203","DOI":"10.1021\/ja066098k","article-title":"High-capacity hydrogen and nitric oxide adsorption and storage in a metal-organic framework","volume":"129","author":"Xiao","year":"2007","journal-title":"J. Am. Chem. Soc."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"9027","DOI":"10.1073\/pnas.96.16.9027","article-title":"The oxyhemoglobin reaction of nitric oxide","volume":"96","author":"Gow","year":"1999","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_23","unstructured":"Feelisch, M., and Stamler, J.S. (1996). The Oxyhemoglobin Assay. Methods in Nitric Oxide Research, John Wiley & Sons, Ltd."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"240","DOI":"10.1016\/S0076-6879(94)33027-1","article-title":"Nitric oxide assay using hemoglobin method","volume":"Volume 233","author":"Murphy","year":"1994","journal-title":"Oxygen Radicals in Biological Systems Part C. Methods in Enzymology"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"426","DOI":"10.1002\/cber.187901201117","article-title":"Bemerkungen zu der Abhandlung der HH. Weselsky und Benedikt \u201eUeber einige Azoverbindungen\u201d","volume":"12","author":"Griess","year":"1879","journal-title":"Ber. Dtsch. Chem. Ges."},{"key":"ref_26","first-page":"237","article-title":"Quantitation of nitrate and nitrite in extracellular fluids","volume":"Volume 268","author":"Grisham","year":"1996","journal-title":"Nitric Oxide Part A: Sources and Detection of NO.; NO Synthase"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"FSO36","DOI":"10.4155\/fso.15.36","article-title":"Detecting and monitoring NO, SNO and nitrite in vivo","volume":"1","author":"Bellavia","year":"2015","journal-title":"Futur. Sci. OA"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1274","DOI":"10.1021\/ac5039779","article-title":"Practical Assay for Nitrite and Nitrosothiol as an Alternative to the Griess Assay or the 2,3-Diaminonaphthalene Assay","volume":"87","author":"Shen","year":"2015","journal-title":"Anal. Chem."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1620","DOI":"10.1111\/bph.12832","article-title":"Measurement of NO in biological samples","volume":"172","author":"Csonka","year":"2015","journal-title":"Br. J. Pharmacol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"89","DOI":"10.1016\/j.jacc.2013.03.070","article-title":"Effects of Nitric Oxide on Cell Proliferation: Novel Insights","volume":"62","author":"Napoli","year":"2013","journal-title":"J. Am. Coll. Cardiol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1515","DOI":"10.1016\/j.actbio.2009.10.038","article-title":"NO-loaded Zn2+-exchanged zeolite materials: A potential bifunctional anti-bacterial strategy","volume":"6","author":"Fox","year":"2010","journal-title":"Acta Biomater."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"5524","DOI":"10.1038\/s41598-017-05898-6","article-title":"A Drug Carrier for Sustained Zero-Order Release of Peptide Therapeutics","volume":"7","author":"Zhao","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1055","DOI":"10.1007\/s00216-004-2674-2","article-title":"Eliminating absorbing interference using the H-point standard addition method: Case of Griess assay in the presence of interferent heme enzymes such as NOS","volume":"379","author":"Indika","year":"2004","journal-title":"Anal. Bioanal. Chem."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1957","DOI":"10.1021\/ac303787p","article-title":"Inaccuracies of Nitric Oxide Measurement Methods in Biological Media","volume":"85","author":"Hunter","year":"2013","journal-title":"Anal. Chem."},{"key":"ref_35","first-page":"2621","article-title":"Quantification of nitric oxide by high-performance liquid chromatography-fluorometric method in subgenomic hepatitis C virus-replicon expressing Huh7 cells upon treatment with acetylsalicylic acid","volume":"16","year":"2018","journal-title":"Exp. Ther. Med."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"697","DOI":"10.1161\/01.ATV.0000204350.44226.9a","article-title":"Unraveling the Reactions of Nitric Oxide, Nitrite, and Hemoglobin in Physiology and Therapeutics","volume":"26","author":"Schechter","year":"2006","journal-title":"Arterioscler. Thromb. Vasc. Biol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"6850","DOI":"10.1021\/ac800185x","article-title":"Fluorinated xerogel-derived microelectrodes for amperometric nitric oxide sensing","volume":"80","author":"Shin","year":"2008","journal-title":"Anal. Chem."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"11494","DOI":"10.1021\/bi00036a023","article-title":"Reaction of Nitric Oxide with the Free Sulfhydryl Group of Human Serum Albumin Yields a Sulfenic Acid and Nitrous Oxide","volume":"34","author":"DeMaster","year":"1995","journal-title":"Biochemistry"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"444","DOI":"10.1073\/pnas.89.1.444","article-title":"S-nitrosylation of proteins with nitric oxide: Synthesis and characterization of biologically active compounds","volume":"89","author":"Stamler","year":"1992","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"4333","DOI":"10.1093\/jxb\/erz242","article-title":"Current approaches to measure nitric oxide in plants","volume":"70","author":"Vishwakarma","year":"2019","journal-title":"J. Exp. Bot."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"4704","DOI":"10.1021\/ja00534a024","article-title":"Accurate redetermination of crystal structure of dehydrated zeolite A. Absence of near zero coordination of sodium. Refinement of silicon, aluminum-ordered superstructure","volume":"102","author":"Pluth","year":"1980","journal-title":"J. Am. Chem. Soc."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"98","DOI":"10.1016\/j.micromeso.2007.10.055","article-title":"Mercury removal with titanosilicate ETS-4: Batch experiments and modelling","volume":"115","author":"Lopes","year":"2008","journal-title":"Microporous Mesoporous Mater."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"347","DOI":"10.1038\/367347a0","article-title":"Structure of the microporous titanosilicate ETS-10","volume":"367","author":"Anderson","year":"1994","journal-title":"Nature"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1016\/S0927-7757(00)00648-8","article-title":"Synthesis of microporous titano-alumino-silicate ETAS-10 with different framework aluminum contents","volume":"179","author":"Lin","year":"2001","journal-title":"Colloids Surf. A Physicochem. Eng. Asp."}],"container-title":["Molecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1420-3049\/25\/11\/2580\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T09:34:51Z","timestamp":1760175291000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1420-3049\/25\/11\/2580"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,6,2]]},"references-count":44,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2020,6]]}},"alternative-id":["molecules25112580"],"URL":"https:\/\/doi.org\/10.3390\/molecules25112580","relation":{},"ISSN":["1420-3049"],"issn-type":[{"value":"1420-3049","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,6,2]]}}}