Research background:

Droplet microfluidics, as a high-throughput detection and screening technology developed in recent years, is widely used in fields such as enzyme directed evolution, antibody development, and high-yield strain screening due to its advantages of low cost and ultra-high throughput. Fluorescence activated droplet sorting is currently the mainstream microfluidic screening method, while multi-color fluorescence detection provides important support for more flexible selection of fluorescent labels and construction of more complex multi parameter fluorescence systems.

This experiment involves mixing green and red fluorescent microspheres for droplet encapsulation, followed by dual laser excitation and detection of the droplets to obtain data on the encapsulation state of the microspheres within the droplets. This validates the functional applicability of the DREM cell platform for multi-color fluorescence detection and screening, laying the foundation for related applications.

Experimental process:

1. Sample processing: Adjust the concentration of green fluorescent microspheres (λ ex=488nm; λ em=520nm; particle size 5um) and red fluorescent microspheres (λ ex=620nm; λ em=680nm; particle size 5um) to 5x10 ^ 6/ml; Mix the two in a 1:1 ratio;

2. Use a droplet microfluidic cell sorter (DREM cell) to encapsulate the above samples into microdroplets, collect fluorescence signals from the 520nm channel (PMT3) and 670nm channel (PMT1), respectively, and sort the target droplet population after gating; The usage process of the DREAM cell instrument is as follows:

Experimental results:

1. Collect signals from the 520nm channel (PMT3) and 670nm channel (PMT1) separately, and in the real-time signal maps of the two channels, identify and distinguish droplets containing green fluorescent microspheres, those containing red microspheres, and those co encapsulated with red and green fluorescent microspheres;

2. In the two-dimensional scatter plot, P1, P2, and P3 droplet populations were subjected to gating and sorting, and the collected droplets were observed under a microscope, as shown in the following figure: the P1 population sorted out red fluorescent microspheres, the P2 population sorted out green fluorescent microspheres, and the P3 population sorted out droplets co encapsulating red and green fluorescent microspheres, which met expectations;

Experimental conclusion:

In this experiment, the droplet microfluidic cell sorter (DREM cell) developed by Tianmu Biotechnology was used to identify and distinguish droplets containing red fluorescent microspheres, droplets containing green fluorescent microspheres, and droplets co encapsulating two types of microspheres. Based on the signals from the dual fluorescence detection channels, a ring gate was used to successfully screen the three types of droplet populations in the mixed droplets, demonstrating the accuracy of the system sorting.

Application prospects:

The simultaneous detection and analysis of multiple different fluorescent signals of target samples play an important role in high-throughput screening research, such as multifunctional enzyme evolution research, protein interaction research, immune cell screening research, multiple fluorescence PCR research, antibody screening, etc. A typical case of enzyme evolution research is shown in the following figure:

This study achieved the modification of the complex trait of stereoselectivity towards ibuprofen esterase by using two different fluorescently labeled substrates. After five rounds of mutation and screening, the high-quality mutant obtained showed a 700 fold increase in selectivity towards S-ibuprofen substrate (DOI: 10.1038/s41467-018-03492-6)