Coupling of droplet-on-demand microfluidcs with ESI/MS to study single-cell catalysis

Abstract

Droplet microfluidics provides an efficient method for analysing reactions within the range of nanoliters to picoliters. However, the sensitive, label-free and versatile detection with ESI/MS poses some difficulties. One challenge is the difficult association of droplets with the MS signal in high-throughput droplet analysis. Hence, a droplet-on-demand system for the generation of a few droplets can address this and other problems such as surfactant concentration or cross-contamination. Accordingly, the system has been further developed for online coupling with ESI/MS. To achieve this, we developed a setup enabling on-demand droplet generation by hydrodynamic gating, with downstream microscopic droplet detection and MS analysis. This facilitated the incorporation of 1-9 yeast cells into individual 1-5 nL droplets and the monitoring of yeast-catalysed transformation from ketoester to ethyl-3-hydroxybutyrate by MS. With our method a mean production rate of 0.035 ± 0.017 fmol per cell per h was observed with a detection limit of 0.30 μM. In conclusion, our droplet-on-demand method is a versatile and advantageous tool for cell encapsulation in droplets, droplet imaging and reaction detection using ESI/MS.

Fig. 1. (A–C) A schematic illustration of the script programmed into the MAT software. (D) The microscopic images illustrate the various states through the use of a coloured water phase containing Fe(SCN)₃. Initially, state 1 is established, followed by the configuration of automated pressure pulsing. During this process, the pressure of pump 1 (cont. phase) is decreased (a). Subsequently, after a defined waiting period (b), the pressure of pump 1 is returned to its initial value (c). Then, after another waiting period (d), multiple cycles can be executed to generate additional droplets, as seen in states 2 and 3. Finally, the pump pressures can be adjusted differently, such as setting them to ambient pressure to cease droplet generation.

Fig. 2. Schematic of the steps for direct coupling of active droplet generation to ESI/MS of caffeine droplets. (A) droplet generation; (B) droplet imaging and storage; (C) transition of droplets and (D) mass spectrometric detection. Each step was observed under the microscope and reflected the respective stage's image. The channels at the cross in A had a diameter of 60 μm and the introduced capillaries was fused silica, PEEK and HPFA.

Fig. 3. Mass trace of DoD generated water droplets with 10 μM caffeine with direct mass spectrometric coupling through the connector in MRM mode. Cont. phase: FC-40; sheath liquid: MeOH/H₂O + 0.1% FA. Droplet size from first MS signal 7.2 nL, 10.1 nL; 5.2 nL 3.0 nL to last signal 4.3 nL.

Fig. 4. Schematic illustration of the yeast-catalysed reaction in an aqueous droplet (in blue) surrounded by the cont. phase (in green) in a HPFA capillary. Within the droplet, EAA is converted to EHB, for which at least one baker's yeast must be present in the droplet.

Fig. 5. Evaluation of the DoD-generated droplets by ESI/MS. (A) mass trace of 5 droplets in MRM mode, each generated by one pressure pulse. The distribution of cells in the droplets is statistically distributed. The droplets of the corresponding signals are shown in (C). The number of cells (highlighted in orange) in the droplets can be counted. The calibration series in (B) was used for the evaluation. Each point in the calibration was obtained from a minimum of 31 droplets in MRM mode, and a Grubbs outlier test was performed to detect outliers. In (D), the product formation rate for each cell number is shown. In addition, the droplets in Fig. S15 and S16 from other storage capillaries were included.