title: Detection of Enzyme Inhibitors in Crude Natural Extracts Using Droplet-Based Microfluidics Coupled to HPLC.
abstract: Natural product screening for new bioactive compounds can greatly benefit from low reagents consumption and high throughput capacity of droplet-based microfluidic systems. However, the creation of large droplet libraries in which each droplet carries a different compound is a challenging task. A possible solution is to use an HPLC coupled to a droplet generating microfluidic device to sequentially encapsulate the eluting compounds. In this work we demonstrate the feasibility of carrying out enzyme inhibiting assays inside nanoliter droplets with the different components of a natural crude extract after being separated by a coupled HPLC column. In the droplet formation zone, the eluted components are mixed with an enzyme and a fluorogenic substrate that permits to follow the enzymatic reaction in the presence of each chromatographic peak and identify those inhibiting the enzyme activity. Using a fractal shape channel design and automated image analysis, we were able to identify inhibitors of Clostridium perfringens neuraminidase present in a root extract of the Pelargonium sidoides plant. This work demonstrates the feasibility of bioprofiling a natural crude extract after being separated in HPLC using microfluidic droplets online and represents an advance in the miniaturization of natural products screening.
authors.affiliation: Laboratorio de Biofisicoquímica, Facultad de Química, Universidad Nacional Autónoma de México , Ciudad de México 04510, México.
authors.forename: Abraham
authors.initials: A
authors.lastname: Ochoa
authors.affiliation: Laboratorio de Biofisicoquímica, Facultad de Química, Universidad Nacional Autónoma de México , Ciudad de México 04510, México.
authors.forename: Enrique
authors.initials: E
authors.lastname: Álvarez-Bohórquez
authors.affiliation: Laboratorio de Biofisicoquímica, Facultad de Química, Universidad Nacional Autónoma de México , Ciudad de México 04510, México.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Castillero
authors.affiliation: Laboratorio de Biofisicoquímica, Facultad de Química, Universidad Nacional Autónoma de México , Ciudad de México 04510, México.
authors.forename: Luis F
authors.identifier: 0000-0002-7199-1740
authors.initials: LF
authors.lastname: Olguin
mesh_terms: D001688:Biological Products / Q000032:analysis* / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003016:Clostridium perfringens / Q000201:enzymology; D057075:Enzyme Assays; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000737:chemistry; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D009439:Neuraminidase / Q000037:antagonists & inhibitors*; D031316:Pelargonium / Q000737:chemistry; D010936:Plant Extracts / Q000032:analysis* / Q000737:chemistry; D018517:Plant Roots / Q000737:chemistry; D053243:Zanamivir / Q000032:analysis / Q000737:chemistry
chemical_list: D001688:Biological Products; D004791:Enzyme Inhibitors; D010936:Plant Extracts; D009439:Neuraminidase; D053243:Zanamivir