Spectrophotometric-Dual-Enzyme-Simultaneous Assay in One Reaction Solution: Chemometrics and Experimental Models

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• 作者：Hongbo Liu ; Xiaolan Yang ; Lin Liu ; Jizheng Dang ; Yanling Xie ; Yi Zhang ; Jun Pu ; Gaobo Long ; Yuanli Li ; Yonghua Yuan ; Juan Liao ; Fei Liao
• 刊名：Analytical Chemistry
• 出版年：2013
• 出版时间：February 19, 2013
• 年：2013
• 卷：85
• 期：4
• 页码：2143-2154
• 全文大小：539K
• 年卷期：v.85,no.4(February 19, 2013)
• ISSN：1520-6882

文摘

Spectrophotometric-dual-enzyme-simultaneous assay in one reaction solution (SDESA) is proposed. SDESA requires the following: (a) Enzyme A acts on Substrate A to release Product A bearing the longest difference absorbance peak (位_A) much larger than that of Product B (位_B) formed by Enzyme B action on Substrate B; 位_B is close to the longest isoabsorbance wavelength of Product A and Substrate A (位₀); (b) absorbance at 位_A and 位₀ is quantified via swift alternation of detection wavelengths and corrected on the basis of absorbance additivity; (c) inhibition/activation on either enzyme by any substance is eliminated; (d) Enzyme A is quantified via an integration strategy if levels of Substrate A are lower than the Michaelis constant. Chemometrics of SDESA was tested with 纬-glutamyltransferase and lactate-dehydrogenase of complicated kinetics. 纬-Glutamyltransferase releases p-nitroaniline from 纬-glutamyl-p-nitroaniline with 位₀ at 344 nm and 位_A close to 405 nm, lactate-dehydrogenase consumes reduced nicotinamide dinucleotide bearing 位_B at 340 nm. Kinetic analysis of reaction curve yielded lactate-dehydrogenase activity free from inhibition by p-nitroaniline; the linear range of initial rates of 纬-glutamyltransferase via the integration strategy, and that of lactate-dehydrogenase after interference elimination, was comparable to those by separate assays, respectively; the quantification limit of either enzyme by SDESA at 25-fold higher activity of the other enzyme remained comparable to that by a separate assay. To test potential application, SDESA of alkaline phosphatase (ALP) and 尾-d-galactosidase as enzyme-linked-immunoabsorbent assay (ELISA) labels were examined. ALP releases 4-nitro-1-naphthol from 4-nitronaphthyl-1-phosphate with 位₀ at 405 nm and 位_A at 458 nm, 尾-d-galactosidase releases 4-nitrophenol from 尾-d-(4-nitrophenyl)-galactoside with 位_B at 405 nm. No interference from substrates/products made SDESA of 尾-galactosidase and ALP simple for ELISA of penicillin G and clenbuterol in one well, and the quantification limit of either hapten was comparable to that via a separate assay. Hence, SDESA is promising.