Microfluidic platform enables tailored translocation and reaction cascades in nanoliter droplet networks.

In the field of bottom-up synthetic biology, lipid membranes are the scaffold to create minimal cells and mimic reactions and processes at or across the membrane. In this context, we employ here a versatile microfluidic platform that enables precise positioning of nanoliter droplets with user-specified lipid compositions and in a defined pattern. Adjacent droplets make contact and form a droplet interface bilayer to simulate cellular membranes. Translocation of molecules across membranes are tailored by the addition of alpha-hemolysin to selected droplets. Moreover, we developed a protocol to analyze the translocation of non-fluorescent molecules between droplets with mass spectrometry. Our method is capable of automated formation of one- and two-dimensional droplet networks, which we demonstrated by connecting droplets containing different compound and enzyme solutions to perform translocation experiments and a multistep enzymatic cascade reaction across the droplet network. Our platform opens doors for creating complex artificial systems for bottom-up synthetic biology.

Manipulation of single cells inside nanoliter water droplets using acoustic forces.

Droplet microfluidics enables high-throughput screening of single cells and is particularly valuable for applications, where the secreted compounds are analyzed. Typically, optical methods are employed for analysis, which are limited in their applicability as labeling protocols are required. Alternative label-free methods such as mass spectrometry would broaden the range of assays but are harmful to the cells, which is detrimental for some applications such as directed evolution. In this context, separation of cells from supernatant is beneficial prior to the analysis to retain viable cells. In this work, we propose an in-droplet separation method based on contactless and label-free acoustic particle manipulation. In a microfluidic chip, nanoliter droplets containing particles are produced at a T-junction. The particles are trapped in the tip of the droplet by the interplay of acoustic forces in two dimensions and internal flow fields. The droplets are subsequently split at a second T-junction into two daughter droplets-one containing the supernatant and the other containing the corresponding particles. The separation efficiency is measured in detail for polystyrene (PS) beads as a function of droplet speed, size, split ratio, and particle concentration. Further, single-bead (PS) and single-cell (yeast) experiments were carried out. At a throughput of 114 droplets/min, a separation efficiency of 100% ± 0% was achieved for more than 150 droplets. Finally, mammalian cells and bacteria were introduced into the system to test its versatility. This work demonstrates a robust, non-invasive strategy to perform single yeast cell-supernatant sampling in nanoliter volumes.

Parallel Sampling of Nanoliter Droplet Arrays for Noninvasive Protein Analysis in Discrete Yeast Cultivations by MALDI-MS.

Miniaturization of cell-based assays enables the analysis of secreted compounds from low cell numbers down to a single cell. Droplet microfluidics is a well-established tool for high-throughput single-cell analysis. Nevertheless, the integration of label-free bioanalytical techniques like mass spectrometry is still ongoing. For example, without additional separation steps, droplet-enclosed cells do not survive the analysis. Cell separation techniques for droplets have been reported, but could not yet be coupled to mass spectrometric analysis. Here, we present a simple approach for high-throughput cell separation in parallel in nanoliter droplets and demonstrate that it can be used for qualitative analysis of protein secretion by the yeast Komagataella phaffii. Using a custom-made droplet spotter, we generated an array of 200 droplets of nanoliter volumes on a glass plate, each containing approximately 500 cells. After cultivation for 24 h, a second plate was placed above the droplet array and brought in contact with the droplets. All droplets were sampled in parallel by plate-based droplet splitting. The nanoliter samples of the supernatant could be interfaced with mass spectrometry and we were able to detect the protein brazzein (his-tagged, 7445 Da) in all but two droplets. Additionally, we show that the cells were viable after the cell separation and a sample from one spot could be transferred to a cultivation tube. An advantage of our protocol is that each cell suspension is directly linked to the analysis result by its position. Furthermore, we demonstrate that our method is capable of splitting around 6000 droplets in a few seconds. In the future, additional processing steps on a small scale, such as desalting and protein digestion, could be developed and will enable structural proteomics in nanoliter volumes.

Microfluidic Platform for Multimodal Analysis of Enzyme Secretion in Nanoliter Droplet Arrays.

High-throughput screening of cell-secreted proteins is essential for various biotechnological applications. In this article, we show a microfluidic approach to perform the analysis of cell-secreted proteins in nanoliter droplet arrays by two complementary methods, fluorescence microscopy and mass spectrometry. We analyzed the secretion of the enzyme phytase, a phosphatase used as an animal feed additive, from a low number of yeast cells. Yeast cells were encapsulated in nanoliter volumes by droplet microfluidics and deposited on spatially defined spots on the surface of a glass slide mounted on the motorized stage of an inverted fluorescence microscope. During the following incubation for several hours to produce phytase, the droplets can be monitored by optical microscopy. After addition of a fluorogenic substrate at a defined time, the relative concentration of phytase was determined in every droplet. Moreover, we demonstrate the use of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to monitor the multistep conversion of the native substrate phytic acid by phytase secreted in 7 nL droplets containing 50-100 cells. Our method can be adapted to various other protocols. As the droplets are easily accessible, compounds such as assay reagents or matrix molecules can be added to all or to selected droplets only, or part of the droplet volume could be removed. Hence, this platform is a versatile tool for questions related to cell secretome analysis.