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Automated droplet manipulation in microfluidic systems

Christoph Merten, Jatin Panwar

Disclosed herein is a method of operating a microfluidic system, a microfluidic system for manipulating sample objects, and a control system for operating a microfluidic system. The method according to the invention is for operating a microfluidic system that comprises a manipulation zone for manipulating sample objects flowing through the manipulation zone, a first detection zone arranged upstream of or in the manipulation zone and a second detection zone arranged upstream of, in or downstream of the manipulation zone. A first count is determined that characterizes a number of sample objects flowing through the first detection zone. Sample objects are manipulated in the manipulation zone. A second count is determined that characterizes a number of sample objects flowing through the second detection zone. One or more manipulation parameters for manipulating the sample objects in the manipulation zone are adjusted based on the first count and the second count.

2023

Design and construction of a microfluidics workstation for high-throughput multi-wavelength fluorescence and transmittance activated droplet analysis and sorting

Alexis Louis Autour, Christoph Merten, Jatin Panwar

Droplet microfluidics has revolutionized quantitative high-throughput bioassays and screening, especially in the field of single-cell analysis where applications include cell characterization, antibody discovery and directed evolution. However, droplet microfluidic platforms capable of phenotypic, fluorescence-based readouts and sorting are still mostly found in specialized labs, because their setup is complex. Complementary to conventional FACS, microfluidic droplet sorters allow the screening of cell libraries for secreted factors, or even for the effects of secreted or surface-displayed factors on a second cell type. Furthermore, they also enable PCR-activated droplet sorting for the isolation of genetic material harboring specific markers. In this protocol, we provide a detailed step-by-step guide for the construction of a high-throughput droplet analyzer and sorter, which can be accomplished in similar to 45 working hours by nonspecialists. The resulting instrument is equipped with three lasers to excite the fluorophores in droplets and photosensors that acquire fluorescence signals in the blue (425-465 nm), green (505-545 nm) and red (580-630 nm) spectrum. This instrument also allows transmittance-activated droplet sorting by analyzing the brightfield light intensity transmitting through the droplets. The setup is validated by sorting droplets containing fluorescent beads at 200 Hz with 99.4% accuracy. We show results from an experiment where droplets hosting single cells were sorted on the basis of increased matrix metalloprotease activity as an application of our workstation in single-cell molecular biology, e.g., to analyze molecular determinants of cancer metastasis.

NATURE PORTFOLIO2023

Fluorescence crosstalk reduction by modulated excitation-synchronous acquisition for multispectral analysis in high-throughput droplet microfluidics

Christoph Merten, Jatin Panwar

Crosstalk between fluorescent biomarkers significantly limits the resolution of multispectral fluorescence analysis in real-time droplet-microfluidics applications. The crosstalk is a result of overlapping emission and excitation spectra of different fluorophores in multiplexed analyses. To mitigate this crosstalk, we present a method that modulates multiple laser beams to selectively and sequentially excite the fluorophores by a single beam of a particular wavelength using acousto-optic modulators at a frequency of 0.1 MHz. An FPGA based data acquisition algorithm synchronized with the modulation signal then acquires the emission signals only from the fluorescence channel that corresponds to the excitation wavelength provided in that particular time window. We applied our method for fluorescence-based droplet analysis in microfluidics and demonstrate that the method is able to reduce crosstalk contribution between channels by >97% and can resolve fluorescence populations that are indistinguishable with conventional droplet analysis methods.

ROYAL SOC CHEMISTRY2023