title: Amplified and selective assay of collagens by enzymatic and fluorescent reactions.
abstract: Sensitive and selective assay of collagen is of substantial importance to the diagnostic study of health- and aging-related failures. In this paper, we describe a highly specific and sensitive method for the assay of whole collagens in biological samples using a novel fluorogenic reagent, 3,4-dihydroxyphenylacetic acid (3,4-DHPAA). The 3,4-DHPAA reagent can selectively detect N-terminal Gly-containing peptides (NGPs) in the presence of sodium borate and NaIO4. Under conditions optimized, this assay format for collagen, termed 3,4-DHPAA assay method showed a good linear relationship between the amplified FL signals and the collagen concentrations from 0.18 to 12 μg/ml. Therefore the sensitive determination of intracellular collagens in cheek tissue and HeLa cells was individually possible without any separation protocol. The dual recognitions of the collagens in the samples could be performed by the enzymatic digestion and the FL reaction. The proposed assay method enables the determination facile, specific, sensitive and quantitative for biogenic collagens.
authors.affiliation: 1] Faculty of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-Machi, Nagasaki 852-8521, Japan [2] Department of Pharmacy, State University of Bangladesh, 77 Shatmosjid road, Dhanmondi, Dhaka-1205, Bangladesh.
authors.forename: Hasina
authors.initials: H
authors.lastname: Yasmin
authors.affiliation: Faculty of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-Machi, Nagasaki 852-8521, Japan.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Kabashima
authors.affiliation: 1] Faculty of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-Machi, Nagasaki 852-8521, Japan [2] Department of Pharmacy, State University of Bangladesh, 77 Shatmosjid road, Dhanmondi, Dhaka-1205, Bangladesh.
authors.forename: Mohammed Shafikur
authors.initials: MS
authors.lastname: Rahman
authors.affiliation: Faculty of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-Machi, Nagasaki 852-8521, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Shibata
authors.affiliation: Faculty of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-Machi, Nagasaki 852-8521, Japan.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Kai
mesh_terms: D000818:Animals; D002417:Cattle; D002460:Cell Line; D003094:Collagen / Q000378:metabolism*; D004789:Enzyme Activation; D057075:Enzyme Assays / Q000379:methods*; D006801:Humans; D011933:Reagent Kits, Diagnostic; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D011933:Reagent Kits, Diagnostic; D003094:Collagen