title: Paper microfluidic-based enzyme catalyzed double microreactor.
abstract: We describe a paper microfluidic-based enzyme catalyzed double microreactor assay using fluorescent detection. Here, solutions of lactate dehydrogenase (LDH) and diaphorase (DI) were directly spotted onto the microfluidic paper-based analytical device (μPAD). Samples containing lactic acid, resazurin, and nicotinamide adenine dinucleotide oxidized form (NAD(+) ), potassium chloride (KCl), and BSA, in MES buffer were separately spotted onto the μPAD and MES buffer flowed through the device. A cascade reaction occurs upon the sample spot overlapping with LDH to form pyruvate and nicotinamide adenine dinucleotide reduced form (NADH). Subsequently, NADH is used in the conversion of resazurin to fluorescent resorufin by DI. The μPAD avoids the need of surface functionalization or enzyme immobilization steps. These microreactor devices are low cost and easy to fabricate and effect reaction based solely on buffer capillary action.
authors.affiliation: Department of Chemistry and Biochemistry, California State University, Los Angeles, CA, USA.
authors.forename: Ivonne M
authors.initials: IM
authors.lastname: Ferrer
authors.forename: Hector
authors.initials: H
authors.lastname: Valadez
authors.forename: Lissette
authors.initials: L
authors.lastname: Estala
authors.forename: Frank A
authors.initials: FA
authors.lastname: Gomez
mesh_terms: D000818:Animals; D019149:Bioreactors / Q000191:economics; D002417:Cattle; D008058:Dihydrolipoamide Dehydrogenase / Q000378:metabolism*; D004800:Enzymes, Immobilized / Q000378:metabolism*; D004867:Equipment Design; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000032:analysis* / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D019344:Lactic Acid / Q000378:metabolism; D046210:Microfluidic Analytical Techniques / Q000191:economics / Q000295:instrumentation*; D009243:NAD / Q000378:metabolism; D010078:Oxazines / Q000032:analysis* / Q000378:metabolism; D010209:Paper; D011189:Potassium Chloride / Q000378:metabolism; D019289:Pyruvic Acid / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000378:metabolism; D014966:Xanthenes / Q000032:analysis* / Q000378:metabolism
chemical_list: D004800:Enzymes, Immobilized; D005456:Fluorescent Dyes; D010078:Oxazines; D014966:Xanthenes; D009243:NAD; C005843:resazurin; D012710:Serum Albumin, Bovine; D019344:Lactic Acid; C014180:resorufin; D011189:Potassium Chloride; D019289:Pyruvic Acid; D007770:L-Lactate Dehydrogenase; D008058:Dihydrolipoamide Dehydrogenase
keywords: Diaphorase; Double microreactor; Fluorescence; Microfluidics; Paper-based chip; Resorufin