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Detection of enzyme inhibitors in crude natural extracts using droplet-based microfluidics coupled to HPLC Abraham Ochoa, Enrique Álvarez-Bohórquez, Eduardo Castillero, and Luis F. Olguin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04988 • Publication Date (Web): 04 Apr 2017 Downloaded from http://pubs.acs.org on April 5, 2017
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Analytical Chemistry
Detection of enzyme inhibitors in crude natural extracts using droplet-based microfluidics coupled to HPLC
Abraham Ochoa, Enrique Álvarez-Bohórquez, Eduardo Castillero and Luis F. Olguin*
Laboratorio de Biofisicoquímica, Facultad de Química, Universidad Nacional Autónoma de México, Ciudad de México 04510, México
*Corresponding author E-mail address:
[email protected]; Tel: +52 55 56223899 Ext 44434
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Analytical Chemistry
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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 nano-liter 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 on-line and represents an advance in the miniaturization of natural products screening.
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Analytical Chemistry
Introduction New drug lead discovery is normally a time consuming and expensive task, requiring the screening of a vast number of different compounds. In particular, natural product screening is challenging, as it involves careful purification and identification of the bioactive molecules present in the sea of compounds that constitute a natural crude extract. Despite this cumbersome process, the reward is the finding of diverse, and sometimes new, molecular scaffolds not present in synthetic libraries. These natural chemical structures, or direct derivatives of them, have constituted ~40% of all molecules approved by the FDA from 1981 to 2014.1 Hence, the development of new techniques that accelerate the identification of natural compounds with interesting biological activities at lower costs and greater efficiency is a compelling task.2,3 Microfluidic droplets technology can carry out massive biochemical assays employing nano- to femtoliter-size monodisperse emulsions in which each droplet serves as one microreactor.4,5 Using these low volumes of reagents and samples, together with existing automated interrogation and selection of droplets at frequencies of kHz, make this platform extremely useful for carrying out high-throughput assays. Examples of this power are experiments of directed evolution in which 107-108 different cells or genes have been screened in 3-10 h employing
