Droplet Channel
Reagent Channel
A)
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To reduce the carryover, chip geometry, properties of channels‟ inner surface, and connections between channels. Potential solutions are: 1) Teflon is very resistant to carryover. So it could be beneficial to build a whole Teflon droplet channel with a wider outlet; 2) connections between channels should be minimized. Our first design was built by inserting 2 pieces of capillaries and 1 piece of Teflon tubing into a PDMS mold. The small gaps at the junction contributed to the carryover. For that, one-piece mold will be a better design; 3) the competition between convection and diffusion has been used to explain the carryover in reagent addition.32 Convection injects the reagent into the droplet, while carryover is caused by the reagent diffusing out of the droplet. Increasing the linear flow rate of the reagent can improve convection and reduce the effective diffusion, and thus reduce carryover. This can be achieved by narrowing the reagent channel.
Large-scale All-droplet Enzyme Modulator Screening
The miniaturizing feature of the all-droplet system will be especially beneficial for screenings which involve a large number of assays. Because the cost of reagent always limits the scale, conducting all reactions inside droplets can be considerable economical. SIRT6 is an expensive and low activity enzyme. It works at micromolar level, which is nearly 100-fold higher than the normal concentration of an enzyme in a screening. The traditional MWP-based screening usually requires at least 5 L for each reaction. Such low volume not only heavily demands the precision and the speed of the liquid handling equipment, but also exacerbates the evaporation. Considering the
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milliliter level dead volume of most automatic liquid dispenser, the expense for SIRT6 is forbidden. Besides, transferring reagents and samples by MWP-based HTS equipment costs a large amount of pipette tips. In contrast, a built-in-house all-droplet system requires only nanoliters of reaction volume, does not have evaporation issue, has no dead volume and needs no transfer. Table 6-1 compares the reagent consumption and cost for an MWP-based SIRT6 modulator screening and an all-droplet-system-based screen.
Table 6-1. Comparison between MWP-based SIRT6 modulator screening and all- droplet-system-based screening. (SIRT6 is 3 M/reaction, ~$270/nanomole; H3K9(Ac) is 50 M/reaction, ~$0.5/nanomole; H3K9* is 0.5 M/diluted reaction, ~$1.5/nanomole).
MWP-based screen All-droplet screen
Number of reactions 10000 10000
Reaction Volume 4 L 10 nL
Dead volume of dispenser 3 mL 0
SIRT6 (4 M stock) consumption 33 mL 75 L H3K9(Ac) (200 M stock) consumption 13 mL 25 L H3K9* (200 M stock) consumption 2 mL 5 L
Total cost of SIRT6 $36000 $81
Total cost of H3K9(Ac) $1300 $2.5
Total cost of H3K9* $600 $1.5
Cost of pipette tips $2000 0
Total cost $40000 $85
Currently, we are exploring miniaturizing SIRT6 modulator screening by implementing a microfabricated multi-step reaction addition device. A temporary design is a multi-channel reagent addition PDMS chip. The droplet channel will be fluorinated
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polymer-coated PDMS. The screening will be explored either by a fluorescent assay (with a fluorescent label far away from the acetylated lysine) or a MS assay.
To save the compounds from the libraries, a droplet splitter31 can be hyphenated to the droplet flow. Samples can be aspirated from MWPs into large plugs and then split into two daughter plugs with designated splitting ratio. One of the daughter plugs can be introduced into the all-droplet system for enzymatic assay. The other plug can go through a different assay which yields complementary results, or be stored as droplet library for future use.
Automation of Mass Spectrometry Plate Reader
To realize large-scale high throughput screening by the MS plate reader, automation of the system will be explored. In sample-droplet reformatting step, tube alignment should be precisely controlled by a computer. Because the perfluorinated oil on top of the plate is very thin, it is necessary to ensure all sippers start from the right height. For infusing sample droplets into ESI-MS, sample tubes (FEP) are now switched by hand, and the interface between the sample tube and the ESI housing requires screwing in a union head. A smoother interface can reduce the force applied on the sample tube, which lowers the possibility to crash the droplets inside and also facilitates automatic manipulator for tube switching. With all steps fully automated, the MS plate reader has a potential to analyzed nearly 150,000 samples in 24 hours.
115 Sample Cleanup
Droplet-ESI-MS systems can be improved by adding a step of sample preparation prior to either droplet generation or MS analysis. Removing the salts and high concentration buffer components can improve the quality of mass spectra, increase the signal to noise ratio of the target analytes, lower the demand on high reagent concentration, and prolong the lifetime of the instrument.
One way to clean up the sample is utilizing high throughput solid phase extraction (SPE) plates before droplet generation. There are 96 and 384-well SPE plates commercially available. The extraction phase is usually reverse phase material, either hydrophobic filters or common separation media (Figure 6-2A, B). Coated-magnetic beads have also been proved efficacious (Figure 6-2C).168
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Figure 6-2. Solid phase extraction plates. A) Filter-based SPE plate (3M EmporeTM: each well of the plate contains a standard density EmporeTM polypropylene membrane for efficient sample extraction). B) Particle-based SPE plate (Glysci Slit PlateTM: separation media is filter-less chromatographic particles. A 1-2 m slit at the bottom of each well in Slit Plate permits liquid to pass through). C) Magnetic beads-based SPE (Xiril AG Magnetic Plate-X: separation particles are coated on magnetic beads residing in each well. Separation is realized by attracting beads to the corner of each well of the assay plate by the magnets array inserted under the assay plate).
A) B) C) 3M Patent Polypropylene Filter Slit Slit Sample Chromatographic Media
Coated Magnetic Beads Magnets
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We have tried parallel sample preparation using multiple C18 spin columns. The whole process was finished in 3 min (1 min spin for binding, 1 min for washing and 1 min for eluting). High quality mass spectra were obtained. Product peaks from a low- yield reactions were observed, which would be very difficult for direct infusion of the untreated reaction mixture (Figure 6-3)
Figure 6-3. Comparison of mass spectra of the desalted reaction (left, desalted by C-18 spin column, undiluted) and the intact reaction mixture (right, diluted by 20 folds in order to observe peptides). The noise level of treated reaction is significantly lower. Despite the low yield, product peaks are observed in the desalted reaction mass spectrum.
Another sample cleaning method is to engineering an extraction bed inside the needle. Our group has explored the possibility of packing a short C18 chromatographic bed into the rear end of a nanospray tip. The target analyte partitions into the stationary phase when a sample plug passes through the bed. The water droplet coming later washes off the soluble impurities. Then an eluting droplet containing organic solvent elutes the
360 400 440 480 520 560 0 50 100 m/z H3K9 H3K9(Ac) [M+6H]6+ [M+5H]5+ [M+4H]4+ 360 400 440 480 520 560 0 50 100 m/z [M+6H]6+ H3K9 H3K9(Ac) [M+5H]5+