Mass Activated Droplet Sorting (MADS) Enables Highâ Throughput Screening of Enzymatic Reactions at Nanoliter Scale

Microfluidic droplet sorting enables the highâ throughput screening and selection of waterâ inâ oil microreactors at speeds and volumes unparalleled by traditional wellâ plate approaches. Most such systems sort using fluorescent reporters on modified substrates or reactions that are rarely industrially relevant. We describe a microfluidic system for highâ throughput sorting of nanoliter droplets based on direct detection using electrospray ionization mass spectrometry (ESIâ MS). Droplets are split, one portion is analyzed by ESIâ MS, and the second portion is sorted based on the MS result. Throughput of 0.7â samplesâ sâ 1 is achieved with 98â % accuracy using a selfâ correcting and adaptive sorting algorithm. We use the system to screen â 15â 000â samples in 6â h and demonstrate its utility by sorting 25â nL droplets containing transaminase expressed in vitro. Labelâ free ESIâ MS droplet screening expands the toolbox for droplet detection and recovery, improving the applicability of droplet sorting to protein engineering, drug discovery, and diagnostic workflows.A microfluidic system for sorting nanoliter droplets based on mass spectrometry is presented. Fully automated, labelâ free sorting at 0.7â samplesâ sâ 1 is achieved with 98â % accuracy. In vitro transcription and translation (ivTT) of a transaminase enzyme in ca.â 25â nL samples is demonstrated and samples are sorted on the basis of enzyme activity.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154315/1/anie201913203.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154315/2/anie201913203-sup-0001-misc_information.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154315/3/anie201913203_am.pd

Mass Activated Droplet Sorting (MADS) Enables Highâ Throughput Screening of Enzymatic Reactions at Nanoliter Scale

Microfluidic droplet sorting enables the highâ throughput screening and selection of waterâ inâ oil microreactors at speeds and volumes unparalleled by traditional wellâ plate approaches. Most such systems sort using fluorescent reporters on modified substrates or reactions that are rarely industrially relevant. We describe a microfluidic system for highâ throughput sorting of nanoliter droplets based on direct detection using electrospray ionization mass spectrometry (ESIâ MS). Droplets are split, one portion is analyzed by ESIâ MS, and the second portion is sorted based on the MS result. Throughput of 0.7â samplesâ sâ 1 is achieved with 98â % accuracy using a selfâ correcting and adaptive sorting algorithm. We use the system to screen â 15â 000â samples in 6â h and demonstrate its utility by sorting 25â nL droplets containing transaminase expressed in vitro. Labelâ free ESIâ MS droplet screening expands the toolbox for droplet detection and recovery, improving the applicability of droplet sorting to protein engineering, drug discovery, and diagnostic workflows.Ein Mikrofluidiksystem zur Sortierung von Nanolitertröpfchen basierend auf Massenspektrometrie erreicht eine vollautomatische markierungsfreie Sortierung bei 0.7 Probenâ sâ 1 mit 98â % Genauigkeit. Die Inâ vitroâ Transkription und â Translation (ivTT) eines Transaminaseâ Enzyms in Proben von etwa 25â nL wird demonstriert, und die Proben werden nach ihrer Enzymaktivität sortiert.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154446/1/ange201913203-sup-0001-misc_information.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154446/2/ange201913203.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154446/3/ange201913203_am.pd

Droplet Microfluidics Coupled to Microchip Electrophoresis for High-Throughput Enzyme Modulator Screening.

High-throughput screening (HTS) represents a powerful tool for drug discovery by allowing tens of thousands of assays to be completed within a single day. Typically, assays are performed in multiwell plates (MWPs) utilizing fluorogenic substrates for detection. These substrates increase development time and can introduce artifactual results. Therefore, alternate screening strategies based around natural substrates are necessary. We developed a screening platform that couples nanoliter volume samples to microchip electrophoresis for analysis using a novel polydimethylsiloxane (PDMS)-and-glass microfluidic device. The system was demonstrated by screening a small library against protein kinase A (PKA), which regulates metabolism within the cell. It was chosen for its well-characterized kinetic parameters and commercially available peptide substrates. Sample throughput of 0.16 Hz was achieved allowing at least 6 replicate MCE injections from each sample in a high quality assay (Z’-factor = 0.8). To demonstrate the ability to screen larger libraries, we developed a novel assay for sirtuin 5 (SIRT5) using a naturally derived peptide substrate. SIRT5 impacts metabolism and has reported oncogenic functions making inhibitor identification of clinical importance. Compared to the PKA assay previously developed, assay throughput was increased 3-fold and 1406 samples were analyzed within 46 minutes (0.5 Hz). Using a 250 ms separation, each assay sample could be analyzed 8 times by MCE generating over 11,000 electropherograms. Several previously unreported inhibitors of SIRT5 were identified and verified by dose-response analysis. Finally, work toward miniaturization of high-throughput assays was demonstrated by performing sample preparation and analysis completely within nanoliter volume droplets. A simple to use and easy to fabricate PDMS microfluidic device was developed to allow addition of assay reagents to nanoliter volume samples. Reagent use, relative to assays performed in 384 well plates, could be reduced 1,000-fold and sample-to-sample carryover was less than 5 percent under typical experimental conditions. Analysis of samples prepared in droplet format was demonstrated with droplets containing a fluorescent dye and addition of a fluorescent peptide. These samples could be analyzed by MCE at 0.33 samples per second but increased throughput should be possible by using higher flow rates.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/120766/1/edguetsc_1.pd

Enabling Biocatalysis by High-Throughput Protein Engineering Using Droplet Microfluidics Coupled to Mass Spectrometry

Directed Evolution is a key technology driving the utility of biocatalysis in pharmaceutical synthesis. Conventional approaches to Directed Evolution are conducted using bacterial cells expressing enzymes in microplates, with catalyzed reactions measured by HPLC, high-performance liquid chromatography-mass spectrometry (HPLC-MS), or optical detectors, which require either long cycle times or tailor-made substrates. To better fit modern, fast-paced process chemistry development where solutions are rapidly needed for new substrates, droplet microfluidics interfaced with electrospray ionization (ESI)-MS provides a label-free high-throughput screening platform. To apply this method to industrial enzyme screening and to explore potential approaches that may further improve the overall throughput, we optimized the existing droplet–MS methods. Carryover between droplets, traditionally a significant issue, was reduced to undetectable level by replacing the stainless steel ESI needle with a Teflon needle within a capillary electrophoresis (CE)–MS source. Throughput was improved to 3 Hz with a wide range of droplet sizes (10–50 nL) by tuning the sheath flow within the CE–MS source. The optimized method was demonstrated by screening reactions using two different transaminase libraries. Good correlations (<i>r</i>² ∼ 0.95) were found between the droplet–MS and LC–MS methods, with 100% match on hit variants. We further explored the capability of the system by performing in vitro transcription–translation inside the droplets and directly analyzing the intact reaction mixture droplets by MS. The synthesized protein attained comparable activity to the protein standard, and the complex samples appeared well tolerated by the MS. The success of the above applications indicates that the MS analysis of the microfluidic droplets is an available option for considerably accelerating the screening of enzyme evolution libraries

Identification of sirtuin 5 inhibitors by ultrafast microchip electrophoresis using nanoliter volume samples

Sirtuin 5 (SIRT5) is a member of the sirtuin family of protein deacylases that catalyzes removal of post-translational modifications, such as succinyl and malonyl moieties, on lysine residues. In light of SIRT5’s roles in regulating metabolism, and its reported oncogenic functions, SIRT5 modulators would be valuable tools for basic biological research and perhaps clinically. Several fluorescence assays for sirtuin modulators have been developed; however, the use of fluorogenic substrates has the potential to cause false positive results due to interactions of engineered substrates with enzyme or test compounds. Therefore, development of high-throughput screening (HTS) assays based on other methods is valuable. In this study, we report the development of a SIRT5 assay using microchip electrophoresis (MCE) for identification of SIRT5 modulators. A novel SIRT5 substrate based on succinate dehydrogenase (SDH) was developed to allow rapid and efficient separation of substrate and product peptide. To achieve high throughput, samples were injected onto the microchip using a droplet-based scheme. By coupling this approach to existing HTS sample preparation workflows, 1408 samples were analyzed at 0.5 Hz in 46 min. Using a 250 ms separation time, 8 MCE injections could be made from each sample generating >11,000 electropherograms during analysis. Of the 1280 chemicals tested, eight were identified as inhibiting SIRT5 activity by at least 70 percent and verified by dose-response analysis