{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,28]],"date-time":"2025-10-28T00:30:24Z","timestamp":1761611424586,"version":"build-2065373602"},"reference-count":35,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2017,7,15]],"date-time":"2017-07-15T00:00:00Z","timestamp":1500076800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Sensors"],"abstract":"<jats:p>A new fluorescent arrayed biosensor has been developed to discriminate species and concentrations of target proteins by using plural different phospholipid liposome species encapsulating fluorescent molecules, utilizing differences in permeation of the fluorescent molecules through the membrane to modulate liposome-target protein interactions. This approach proposes a basically new label-free fluorescent sensor, compared with the common technique of developed fluorescent array sensors with labeling. We have confirmed a high output intensity of fluorescence emission related to characteristics of the fluorescent molecules dependent on their concentrations when they leak from inside the liposomes through the perturbed lipid membrane. After taking an array image of the fluorescence emission from the sensor using a CMOS imager, the output intensities of the fluorescence were analyzed by a principal component analysis (PCA) statistical method. It is found from PCA plots that different protein species with several concentrations were successfully discriminated by using the different lipid membranes with high cumulative contribution ratio. We also confirmed that the accuracy of the discrimination by the array sensor with a single shot is higher than that of a single sensor with multiple shots.<\/jats:p>","DOI":"10.3390\/s17071630","type":"journal-article","created":{"date-parts":[[2017,7,18]],"date-time":"2017-07-18T03:45:16Z","timestamp":1500349516000},"page":"1630","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":11,"title":["A Label-Free Fluorescent Array Sensor Utilizing Liposome Encapsulating Calcein for Discriminating Target Proteins by Principal Component Analysis"],"prefix":"10.3390","volume":"17","author":[{"given":"Ryota","family":"Imamura","sequence":"first","affiliation":[{"name":"Graduate School of Science and Technology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan"}]},{"given":"Naoki","family":"Murata","sequence":"additional","affiliation":[{"name":"Graduate School of Science and Technology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan"}]},{"given":"Toshinori","family":"Shimanouchi","sequence":"additional","affiliation":[{"name":"Graduate School of Environmental and Life Science, Okayama University, 1-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan"}]},{"given":"Kaoru","family":"Yamashita","sequence":"additional","affiliation":[{"name":"Graduate School of Science and Technology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan"}]},{"given":"Masayuki","family":"Fukuzawa","sequence":"additional","affiliation":[{"name":"Graduate School of Science and Technology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan"}]},{"given":"Minoru","family":"Noda","sequence":"additional","affiliation":[{"name":"Graduate School of Science and Technology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan"}]}],"member":"1968","published-online":{"date-parts":[[2017,7,15]]},"reference":[{"key":"ref_1","first-page":"521","article-title":"Microchip-based immunoassay","volume":"56","author":"Tachi","year":"2007","journal-title":"Anal. Sci."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1093\/nar\/gkt755","article-title":"Heterogeneous staining: A tool for studies of how fluorescent dyes affect the physical properties of DNA","volume":"41","author":"Nyberg","year":"2013","journal-title":"Nucleic Acids Res."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"862","DOI":"10.1039\/c2mb25422k","article-title":"Chemical biology-based approaches on fluorescent labeling of proteins in live cells","volume":"9","author":"Jung","year":"2013","journal-title":"Mol. BioSyst."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"618","DOI":"10.1016\/j.bios.2008.06.018","article-title":"A fluorescence detection platform using spatial electroluminescent excitation for measuring botulinum neurotoxin A activity","volume":"24","author":"Sapsford","year":"2008","journal-title":"Biosens. Bioelectron."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"232","DOI":"10.1016\/0014-5793(71)80319-8","article-title":"Immunoassay using antigen-enzyme conjugates","volume":"15","author":"Schuurs","year":"1971","journal-title":"FEBS Lett."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"279","DOI":"10.1081\/IAS-200028084","article-title":"Development of liposome immune lysis assay for the herbicide atrazine","volume":"25","author":"Hendrickson","year":"2004","journal-title":"J. Immunoass. Immunochem."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"346","DOI":"10.1252\/kakoronbunshu.31.346","article-title":"Characterization of competitive measurement of antigens by use of antigen-coupled liposomes","volume":"31","author":"Tomioka","year":"2005","journal-title":"Kagaku Kogaku Ronbunnshu"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"943","DOI":"10.1252\/jcej.34.943","article-title":"Characteristics of liposome immunosorbent assay (LISA) using liposomes encapsulating cpenzyme \u03b2\u2013NAD+","volume":"34","author":"Kumada","year":"2001","journal-title":"J. Chem. Eng. Jpn."},{"key":"ref_9","unstructured":"Takada, K., Fujimoto, T., Shimanouchi, T., Fukuzawa, M., Yamashita, K., and Noda, M. (2013, January 27\u201331). A new discrimination method of target biomolecules with miniaturized sensor array utilizing liposome encapsulating fluorescent molecules with time course analysis. Proceedings of the 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS, Freiburg, Germany."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1","DOI":"10.5360\/membrane.34.342","article-title":"Membranomics research on interactions between liposome membranes with membrane chip analysis","volume":"34","author":"Shimanouchi","year":"2009","journal-title":"Membrane"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"45","DOI":"10.5004\/dwt.2010.1697","article-title":"Monitoring of membrane damages by dialysis treatment: Study with membrane chip analysis","volume":"17","author":"Shimanouchi","year":"2010","journal-title":"Desalination Water Treat."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"156","DOI":"10.1016\/j.colsurfb.2009.05.014","article-title":"Calcein Permeation across phosphatidylcholine bilayer membrane: Effects of membrane fluidity, liposome size, and immobilization","volume":"73","author":"Shimanouchi","year":"2009","journal-title":"Colloids Surf. B"},{"key":"ref_13","first-page":"241","article-title":"Detection of protein conformation under stress conditions using liposomes as sensor materials","volume":"16","author":"Kuboi","year":"2004","journal-title":"Sens. Mater."},{"key":"ref_14","first-page":"556","article-title":"Interactions of histatin 5 and histatin 5-derived peptides with liposome membranes: Surface effects, translocation and permeabilization","volume":"379","author":"Hertog","year":"2004","journal-title":"Biochem. J."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1016\/S0005-2736(02)00659-4","article-title":"Secreted phospholipase A2 as a new enzymatic trigger mechanism for localised liposomal drug release and absorption in diseased tissue","volume":"1609","author":"Davidsen","year":"2003","journal-title":"Biochim. Biophys. Acta Biomembr."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"517","DOI":"10.2116\/analsci.23.517","article-title":"Simultaneous measurements of K+ and calcein release from liposomes and the determination of pore size formed in a membrane","volume":"23","author":"Katsu","year":"2007","journal-title":"Anal. Sci."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"663","DOI":"10.1111\/jam.12262","article-title":"Investigation of the role of tryptophan residues in cationic antimicrobial peptides to determine the mechanism of antimicrobial action","volume":"115","author":"Bi","year":"2013","journal-title":"J. Appl. Microbiol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"2190","DOI":"10.3390\/ijms14012190","article-title":"Effect of repetitive lysine-tryptophan motifs on the eukaryotic membrane","volume":"14","author":"Gopal","year":"2013","journal-title":"Int. J. Mol. Sci."},{"key":"ref_19","first-page":"1","article-title":"Physics of Heterogeneous structures in biomembranes","volume":"68","author":"Komura","year":"2013","journal-title":"J. Phys. Soc. Jpn."},{"key":"ref_20","first-page":"128","article-title":"Phase transition and phase separation in biomembranes","volume":"60","author":"Komura","year":"2005","journal-title":"J. Phys. Soc. Jpn."},{"key":"ref_21","first-page":"301","article-title":"Incorporating Data Mining Techniques on Software Cost Estimation: Validation and Improvement","volume":"2","author":"Sharma","year":"2012","journal-title":"IJETAE"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"40","DOI":"10.1016\/j.snb.2008.07.022","article-title":"A bio-thermochemical microbolometer with immobilized intact liposome on sensor solid surface","volume":"135","author":"Noda","year":"2008","journal-title":"Sens. Actuators B Chem."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Murata, N., Imamura, R., Du, W., Fukuzawa, M., and Noda, M. (2015, January 12\u201316). An image processing platform for expendable bio-sensing system with arrayed biosensor and embedded imager. Proceedings of the 3rd International Conference on Applied Computing and Information Technology\/2nd International Conference on Computational Science and Intelligence, Okayama, Japan.","DOI":"10.1109\/ACIT-CSI.2015.48"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"139","DOI":"10.1023\/A:1016832027325","article-title":"Measurement of cell volume changes by fluorescence self-quenching","volume":"12","author":"Hamann","year":"2002","journal-title":"J. Fluoresc."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"513","DOI":"10.1023\/B:JOFL.0000008062.85865.c6","article-title":"Selective dequenching by photobleaching increases fluorescence probe visibility","volume":"13","author":"Roberts","year":"2003","journal-title":"J. Fluoresc."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"4122","DOI":"10.1016\/j.bpj.2009.02.050","article-title":"Lipid mixing and content release in single-vesicle SNARE-driven fusion assay with 1\u20135 ms resolution","volume":"96","author":"Wang","year":"2009","journal-title":"Biophys. J."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1365","DOI":"10.1002\/btpr.93","article-title":"Different effects of l-arginine on protein refolding: Suppressing aggregates of hydrophobic interaction, not covalent binding","volume":"24","author":"Chen","year":"2008","journal-title":"Biotechnol. Prog."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"5154","DOI":"10.1021\/jf021005d","article-title":"Preparation of antimicrobial reduced lysozyme compatible in food applications","volume":"51","author":"Touch","year":"2003","journal-title":"J. Agric. Food Chem."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"620","DOI":"10.1002\/elan.201500412","article-title":"A micromechanical cantilever-based liposome biosensor for characterization of protein-membrane interaction","volume":"28","author":"Zhang","year":"2016","journal-title":"Electroanalysis"},{"key":"ref_30","first-page":"7","article-title":"Novel development of liposome application for development of artificial cell","volume":"2005","author":"Oku","year":"2005","journal-title":"Anal. NTS Press."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"174","DOI":"10.1016\/j.bej.2005.08.030","article-title":"Evaluation of temperature and guanidine hydrochloride-induced protein\u2013liposome interactions by using immobilized liposome chromatography","volume":"29","author":"Yoshimoto","year":"2006","journal-title":"Biochem. Eng. J."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"343","DOI":"10.1016\/j.colsurfb.2013.07.028","article-title":"Relationship between the mobility of phosphocholine headgroup and the protein-liposome interacation: A dielectric sperctoscopic study","volume":"116","author":"Shimanouchi","year":"2014","journal-title":"Colloids Surf. B"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/1471-2105-15-51","article-title":"Statistical Hypothesis Testing of Factor Loading in Principal Component Analysis and Its Application to Metabolite Set Enrichment Analysis","volume":"15","author":"Yamamoto","year":"2014","journal-title":"BMC Bioinform."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"714","DOI":"10.1093\/bib\/bbq090","article-title":"Principal component analysis based methods in bioinformatics studies","volume":"12","author":"Ma","year":"2011","journal-title":"Brief. Bioinform."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"742","DOI":"10.1590\/S0103-50532002000600004","article-title":"Multivariate QSAR","volume":"13","author":"Ferreira","year":"2002","journal-title":"J. Braz. Chem. Soc."}],"container-title":["Sensors"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1424-8220\/17\/7\/1630\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T18:42:48Z","timestamp":1760208168000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1424-8220\/17\/7\/1630"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2017,7,15]]},"references-count":35,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2017,7]]}},"alternative-id":["s17071630"],"URL":"https:\/\/doi.org\/10.3390\/s17071630","relation":{},"ISSN":["1424-8220"],"issn-type":[{"type":"electronic","value":"1424-8220"}],"subject":[],"published":{"date-parts":[[2017,7,15]]}}}