With droplet-based microfluidic systems, researchers can rapidly screen compound and reaction libraries. These systems often use fluorescent reporters to help sort out the compounds and reaction conditions researchers want from the ones they don’t. The problem with relying on fluorescence is that most industrially relevant reactions don’t involve fluorescent reactants or products.
Researchers led by Shuwen Sun and Jeffrey C. Moore of Merck & Co. and Robert T. Kennedy of the University of Michigan have now combined droplet-based screening systems with mass spectrometry–activated sorting to overcome that constraint (Angew. Chem. Int. Ed. 2019, DOI: 10.1002/anie.201913203).
“Merck has had a profound interest in high-throughput screening in various forms for a long time,” Moore says. The company is especially interested in using such systems to screen for new enzyme catalysts as part of a directed evolution program. But few of the compounds in such a program are suitable for fluorescence-based sorting.
The new screening system consists of a microfluidic chip connected to a mass spectrometer and a sorting region monitored by a droplet-counting camera. Injected droplets are split into two unequal daughter droplets, with the larger droplet going to the mass spectrometer and the smaller droplet going to the sorting region.
The system has a delay line to make sure that the smaller droplet doesn’t reach the sorting region before its sibling has been analyzed by the mass spectrometer. The system uses the mass spectral signal to make the sorting decision and then uses an electric field to push the smaller droplet down one of two paths. Droplets containing the desired product are saved; other droplets are discarded.
But keeping the pairs of droplets in register is complicated, Kennedy says. Droplets can split or merge, or a bubble in the device can fool the camera. So the researchers can’t rely just on counting the droplets. “If you miss one, everything’s going to be out of register, and you’re going to be sorting the wrong droplet,” Kennedy says.
To overcome such potential problems, the researchers included marker droplets in the system. These droplets contain a dye that can be detected by the camera and a compound with a distinct mass signal. The system only sorts sample droplets when the marker droplets in both parts of the system are in sync.
Using the system, the researchers were able to sort 0.7 droplets per second. They screened a library of about 15,000 samples in 6 hours. Kennedy thinks the rate could be increased 10-fold. But the Merck scientists say the current speed is good enough for their needs.
“Mass-activated droplet sorting completely opens the field to screening based on any type of assay signal where the products of the desired reaction exhibit a unique mass signature—pretty much all reactions—and are present at a level that is detectable by mass spectrometry,” says Brian M. Paegel, an expert on high-throughput screening and microfluidics at the University of California, Irvine. “The team overcame numerous technical difficulties to deliver this technology, not the least of which being synchronization of sorting with detection.”