Passive Picoinjection Enables Controlled Crystallization in a Droplet Microfluidic Device

Abstract

in parallel, enabling large data-sets to be generated for statistical analysis. One of the most convenient ways of generating large numbers of identical droplets is using microfluidic devices. [2] These can be used to create static arrays and continuously flowing droplets, and a wide range of techniques are available for onchip analysis. [3,4] Microfluidic systems are therefore attracting increasing attention for applications such as studying nucleation kinetics, [5,6] for screening protein crystallization conditions and generating large protein crystals for structural analysis, [7][8][9] and for exploring polymorphism in organic [10,11] and inorganic crystal systems. [12][13][14] A range of strategies have been used to achieve on-chip crystallization. Protein crystals are often highly soluble such that precipitation can be effectively achieved by combining protein and precipitant solutions at the point of droplet formation, [6,8] or controlling water removal from the plugs of protein solution. [15] More complex strategies have also been explored to promote the formation of high quality crystals including valve-based systems, [9,16] merging of alternate droplets containing protein and precipitant, and separation of nucleation and growth events, [17] where additional protein/ precipitant is added to existing plugs downstream of the point of initial droplet formation. Crystallization of soluble organics and inorganics has also been achieved via droplet-shrinkage [18] and on-chip temperature control. [11,13] Many crystal systems that are important to industry, the environment, and biology-such as calcium carbonate, sulfate, and phosphate-are highly insoluble, however, which results in short induction times and makes on-chip study of their precipitation more challenging due to problems with device fouling. [12] While precipitation within droplets rather than single-phase systems greatly reduces channel fouling, [19] the formation of supersaturated droplets by combining cation and anion solutions using a conventional "Y-shaped" channel or equivalent inevitably leads to precipitation at this junction. This can give rise to a range of problems including the introduction of crystal seeds into droplets. These issues can be minimized, or even eliminated, using droplet fusion-where pairs of droplets are fused using special channel architectures or applied fields [20] -or direct injection strategies, where use of a "picoinjector" to add solution to flowing droplets [21,22] offers Segmented flow microfluidic devices offer an attractive means of studying crystallization processes. However, while they are widely employed for protein crystallization, there are few examples of their use for sparingly soluble compounds due to problems with rapid device fouling and irreproducibility over longer run-times. This article presents a microfluidic device which overcomes these issues, as this is constructed around a novel design of "picoinjector" that facilitates direct injection into flowing droplets. Exploiting a Venturi junction to ...

Crystallization in Confinement

“…Poorly soluble compounds have been generated by a range of methods including combining reactant solutions at the point of droplet formation, [137] creating streams of droplets that alternate in content and subsequently merging pairs of droplets, [138] or directly injecting one solution into flowing droplets. [139] Initial studies of crystallization in droplet microfluidic devices focused on the development of strategies to generate Reproduced with permission. [133] Copyright 2013, Elsevier Masson SAS.…”

“…This also contrasts with other studies of calcium carbonate precipitation in microfluidic droplets where ACC was clearly demonstrated as the initial precipitated phase. [137,139] Notably, these employed lower reagent concentrations, and also more sophisticated strategies for reagent mixing that precluded precipitation at the point of droplet mixing.…”

“…The role of mixing in determining the outcome of crystallization reactions has also been seen in the precipitation of calcium carbonate. [139] A picoinjector was used to prepare droplets of 2.5 × 10 −3 m calcium carbonate solution in oil by injecting Na 2 CO 3 or CaCl 2 solution into flowing droplets of the counterion. The droplets were then passed along a straight or serpentine channel prior to collection of the product crystals by Figure 11.…”

Droplet microfluidics: from proof-of-concept to real-world utility?

“…Since the entire process takes only a few milliseconds, pico-injectors have been used to excellent effect in the multiplexed analysis of enzyme kinetics, 24 digital detection of RNA, 25 and protein crystallisation screening. 26 Finally, the ability to sort droplets based on their content is a necessary component of many biochemical assays, since sorting allows the selective capture and isolation of relevant droplets from extremely large populations. Critically, recent developments in droplet-sorting technologies have leveraged both passive and active strategies, including the use of viscoelastic fluid properties, 27 electric fields, 20 electric capacitance 28 and magnetic rails, 29 to allow for the rapid and robust selection of droplets with user-defined properties.…”

Evaluation of microflow configurations for scale inhibition and serial X-ray diffraction analysis of crystallization processes

“…[13][14][15] Thanks to the ever-improving accessibility of microfabrication techniques, microfluidic devices are now drawing increasing attention as a means of performing crystallization, where these offer clean and controllable reaction environments. [16][17][18][19][20] An additional benefit of utilizing microfluidics is the ability of lab-on-a-chip devices to be coupled to characterization techniques, such as X-ray scattering and diffraction, which can facilitate in situ analysis of crystallization and other assembly processes. Importantly, performing these types of analyses require highly controlled and low X-ray absorbing sample environments, where microfluidic devices seem like a natural choice, since their small channel size provides efficient and welldefined heat and mass transport and minimises the required beam path through a sample.…”