Microfluidic triple-gradient generator for efficient screening of chemical space

Abstract

Generation of a combinatorial gradient for multiple chemicals is essential for studies of biochemical stimuli, chemoattraction, protein crystallization and others. While currently available platforms require complex design/settings to obtain a double-gradient chemical matrix, we herein report for the first time a simple triple-gradient matrix (TGM) device for efficient screening of chemical space. The TGM device is composed of two glass slides and works following the concept of SlipChip. The device utilizes XYZ space to distribute three chemicals and establishes a chemical gradient matrix within 5 min. The established matrix contains 24 or 104 screening conditions depending on the device used, which covers a concentration range of [0.117–1, 0.117–1 and 0.686–1] and [0.0830–1, 0.0830–1, 0.686–1] respectively for the three chemicals. With the triple gradients built simultaneously, this TGM device provides order-of-magnitude improvement in screening efficiency over existing single- or double-gradient generators. As a proof of concept, we applied the device to screen the crystallization conditions for two model proteins of lysozyme and trypsin and confirmed the crystal structures using X-ray diffraction. Furthermore, we successfully obtained the crystallization condition of adhesin competence repressor, a protein that senses the alterations in intracellular zinc concentrations. We expect the TGM system to be widely used as an analytical platform for material synthesis and chemical screening beyond for protein crystallization.