title: 3D-Printed High-Density Droplet Array Chip for Miniaturized Protein Crystallization Screening under Vapor Diffusion Mode.
abstract: Here we describe the combination of three-dimensional (3D) printed chip and automated microfluidic droplet-based screening techniques for achieving massively parallel, nanoliter-scale protein crystallization screening under vapor diffusion mode. We fabricated high-density microwell array chips for sitting-drop vapor diffusion crystallization utilizing the advantage of the 3D-printing technique in producing high-aspect-ratio chips. To overcome the obstacle of 3D-printed microchips in performing long-term reactions caused by their porousness and gas permeability properties in chip body, we developed a two-step postprocessing method, including paraffin filling and parylene coating, to achieve high sealability and stability. We also developed a simple method especially suitable for controlling the vapor diffusion speed of nanoliter-scale droplets by changing the layer thickness of covering oil. With the above methods, 84 tests of nanoliter-scale protein crystallization under vapor diffusion mode were successfully achieved in the 7 × 12 droplet array chip with a protein consumption of 10 nL for each test, which is 20-100 times lower than that in the conventional large-volume screening system. Such a nanoliter-scale vapor diffusion system was applied to two model proteins with commercial precipitants and displayed advantages over that under microbatch mode. It identified more crystallization conditions, especially for the protein samples with lower concentrations.
authors.affiliation: Institute of Microanalytical Systems, Department of Chemistry and Innovation Center for Cell Signaling Network, and ‡Life Sciences Institute and Innovation Center for Cell Signaling Network, Zhejiang University , Hangzhou, 310058, China.
authors.forename: Yi-Ran
authors.initials: YR
authors.lastname: Liang
authors.affiliation: Institute of Microanalytical Systems, Department of Chemistry and Innovation Center for Cell Signaling Network, and ‡Life Sciences Institute and Innovation Center for Cell Signaling Network, Zhejiang University , Hangzhou, 310058, China.
authors.forename: Li-Na
authors.initials: LN
authors.lastname: Zhu
authors.affiliation: Institute of Microanalytical Systems, Department of Chemistry and Innovation Center for Cell Signaling Network, and ‡Life Sciences Institute and Innovation Center for Cell Signaling Network, Zhejiang University , Hangzhou, 310058, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Gao
authors.affiliation: Institute of Microanalytical Systems, Department of Chemistry and Innovation Center for Cell Signaling Network, and ‡Life Sciences Institute and Innovation Center for Cell Signaling Network, Zhejiang University , Hangzhou, 310058, China.
authors.forename: Hong-Xia
authors.initials: HX
authors.lastname: Zhao
authors.affiliation: Institute of Microanalytical Systems, Department of Chemistry and Innovation Center for Cell Signaling Network, and ‡Life Sciences Institute and Innovation Center for Cell Signaling Network, Zhejiang University , Hangzhou, 310058, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: Institute of Microanalytical Systems, Department of Chemistry and Innovation Center for Cell Signaling Network, and ‡Life Sciences Institute and Innovation Center for Cell Signaling Network, Zhejiang University , Hangzhou, 310058, China.
authors.forename: Sheng
authors.initials: S
authors.lastname: Ye
authors.affiliation: Institute of Microanalytical Systems, Department of Chemistry and Innovation Center for Cell Signaling Network, and ‡Life Sciences Institute and Innovation Center for Cell Signaling Network, Zhejiang University , Hangzhou, 310058, China.
authors.forename: Qun
authors.identifier: http://orcid.org/0000-0002-6250-252X
authors.initials: Q
authors.lastname: Fang
mesh_terms: D003460:Crystallization; D004058:Diffusion; D005740:Gases; D044085:Microfluidics; D011506:Proteins / Q000737:chemistry*
chemical_list: D005740:Gases; D011506:Proteins
keywords: 3D-printed chip; droplet array; high density; protein crystallization; surface modification