A robust and scalable microfluidic metering method that allows protein crystal growth by free interface diffusion

Abstract

Producing robust and scalable fluid metering in a microfluidic device is a challenging problem. We developed a scheme for metering fluids on the picoliter scale that is scalable to highly integrated parallel architectures and is independent of the properties of the working fluid. We demonstrated the power of this method by fabricating and testing a microfluidic chip for rapid screening of protein crystallization conditions, a major hurdle in structural biology efforts. The chip has 480 active valves and performs 144 parallel reactions, each of which uses only 10 nl of protein sample. The properties of microfluidic mixing allow an efficient kinetic trajectory for crystallization, and the microfluidic device outperforms conventional techniques by detecting more crystallization conditions while using 2 orders of magnitude less protein sample. We demonstrate that diffraction-quality crystals may be grown and harvested from such nanoliter-volume reactions.

Digital microfluidics using soft lithography

“…At the device level, microfluidics is becoming a mature technology. [1][2][3][4][5] Although complex application-driven microfluidic devices have been developed, parallelizing processes such as protein crystallization 6,7 and proteomic analysis, 8,9 there has been little progress in the development of scalable and programmable microfluidic systems. Even recent advances in modular microfluidic breadboards 10 adopt a model where distinct interconnection networks are manufactured for each application.…”

Screening of Protein Crystallization Conditions on a Microfluidic Chip Using Nanoliter-Size Droplets

“…Such screening would also provide multidimensional phase diagrams for protein crystallization, important for fundamental understanding of protein crystallization. This system should be applicable to crystallizing other classes of molecules.In principle, crystals that we obtained during screening are large enough for structural determination by synchrotron radiation, and we are currently optimizing crystal harvesting and manipulation, already demonstrated in PDMS-based microfluidics by Hansen et al 10 We believe that this simple system will be used in individual research labs to answer fundamental questions in crystallization and will be used for crystallization of new targets. This system also has the potential to serve as the basis of high-throughput, automated crystallization systems.…”

“…9 Microfluidic platforms, therefore, are an attractive choice for macromolecular crystallization, 6 as was clearly demonstrated by Hansen et al 10 These authors have crystallized proteins on a microfluidic device by free interface diffusion, a method that was previously possible only in microgravity. 10 Only ∼10 nL of the protein solution was used for each of 144 trials, which were conducted inside microfabricated chambers controlled by pressure-operated valves.The system described here (Figure 1) used three steps to crystallize proteins inside droplets, implemented using PDMS microfluidic devices with channels of 150 × 100 µm 2 crosssectional dimensions: 1,2 (1) Aqueous stock solutions were loaded into syringes, and syringes were connected to the convening channels of a microfluidic device. Only one stock solution and one syringe were required for each reagent or protein.…”

A Microfluidic Device for Kinetic Optimization of Protein Crystallization and In Situ Structure Determination

“…The device was further evaluated in its ability to scale-up crystallization conditions found in 10 nL FID reactors screening chips. 1 We attempted to scale-up crystallization of a total of 14 targets, including eight well-characterized crystallization standards, three previously crystallized integral membrane proteins, one nucleic acid/protein complex, and two targets of unknown structure that only had been crystallized in 10 nL FID reactors. All 14 targets tested were successfully crystallized using chemical conditions identified in 10 nL volume reactors.…”