title: Development of an enzymatic chromatography strip with nicotinamide adenine dinucleotide-tetrazolium coupling reactions for quantitative l-lactate analysis.
abstract: In this study, a dry assay of l-lactate via the enzymatic chromatographic test (ECT) was developed. An l-lactate dehydrogenase plus a nicotinamide adenine dinucleotide (NADH) regeneration reaction were applied simultaneously. Various tetrazolium salts were screened to reveal visible color intensities capable of determining the lactate concentrations in the sample. The optimal analysis conditions were as follows. The diaphorase (0.5 μl, 2(-6)U/μl) was immobilized in the test line of the ECT strip. Nitrotetrazolium blue chloride (5 μl, 12 mM), l-lactate dehydrogenase (1 μl, 0.25U/μl), and NAD(+) (2μl, 1.5×10(-5)M) were added into the mobile phase (100 μl) composed of 0.1% (w/w) Tween 20 in 10mM phosphate buffer (pH 9.0), and the process was left to run for 10 min. This detection had a linear range of 0.039 to 5mM with a detection limit of 0.047 mM. This quantitative analysis process for l-lactate was easy to operate with good stability and was proper for the point-of-care testing applications.
authors.affiliation: Department of Chemical Engineering, National Chung Hsing University, Taichung 402, Taiwan.
authors.forename: Shu-Chen
authors.initials: SC
authors.lastname: Kan
authors.affiliation: Department of Chemical Engineering, National Chung Hsing University, Taichung 402, Taiwan.
authors.forename: Wei-Feng
authors.initials: WF
authors.lastname: Chang
authors.affiliation: Department of Chemical Engineering, National Chung Hsing University, Taichung 402, Taiwan.
authors.forename: Min-Chi
authors.initials: MC
authors.lastname: Lan
authors.affiliation: Department of Chemical Engineering, National Chung Hsing University, Taichung 402, Taiwan.
authors.forename: Chia-Chi
authors.initials: CC
authors.lastname: Lin
authors.affiliation: Department of Chemical Engineering, National Chung Hsing University, Taichung 402, Taiwan.
authors.forename: Wei-Shiang
authors.initials: WS
authors.lastname: Lai
authors.affiliation: Biotechnology Center, National Chung Hsing University, Taichung 402, Taiwan.
authors.forename: Chwen-Jen
authors.initials: CJ
authors.lastname: Shieh
authors.affiliation: Research and Development Department, Taiwan Unison Biotech, Hsinchu 300, Taiwan.
authors.forename: Kuang-Pin
authors.initials: KP
authors.lastname: Hsiung
authors.affiliation: Department of Chemical Engineering, National Chung Hsing University, Taichung 402, Taiwan. Electronic address: ycliu@dragon.nchu.edu.tw.
authors.forename: Yung-Chuan
authors.initials: YC
authors.lastname: Liu
mesh_terms: D000818:Animals; D002845:Chromatography / Q000379:methods*; D047010:Clostridium kluyveri / Q000201:enzymology; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000378:metabolism; D019344:Lactic Acid / Q000032:analysis* / Q000097:blood / Q000737:chemistry*; D057230:Limit of Detection; D009243:NAD / Q000737:chemistry*; D011817:Rabbits; D011934:Reagent Strips / Q000737:chemistry*; D013778:Tetrazolium Salts / Q000737:chemistry*
chemical_list: D004800:Enzymes, Immobilized; D011934:Reagent Strips; D013778:Tetrazolium Salts; D009243:NAD; D019344:Lactic Acid; D007770:L-Lactate Dehydrogenase
keywords: Chromatographic strip; Point-of-care testing; Tetrazolium salts; l-Lactate; l-Lactate dehydrogenase