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Microfluidic platform for multimodal analysis of enzyme secretion in nanoliter droplet arrays Dominik Huemmer, Simon Bachler, Martin Köhler, Lars M. Blank, Renato Zenobi, and Petra S. Dittrich Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04506 • Publication Date (Web): 20 Dec 2018 Downloaded from http://pubs.acs.org on December 21, 2018

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Analytical Chemistry

Microfluidic platform for multimodal analysis of enzyme secretion in nanoliter droplet arrays Dominik Hümmer1, Simon Bachler1, Martin Köhler2, Lars M. Blank3, Renato Zenobi2, Petra S. Dittrich1* Department of Biosystems Science and Engineering, ETH Zürich, Mattenstr. 26, 4058 Basel, Switzerland Department of Chemistry and Applied Biosciences, ETH Zürich, Vladimir-Prelog-Weg 3, 8093 Zürich, Switzerland 3 Institute of Applied Microbiology, Aachen Biology and Biotechnology, RWTH Aachen University, Worringer Weg 1, 52074 Aachen, Germany 1 2

ABSTRACT: High-throughput screening of cell-secreted proteins is essential for various biotechnological applications. In this article, we show a microfluidic approach to perform the analysis of cell-secreted proteins in nanoliter droplet arrays by two complementary methods, fluorescence microscopy and mass spectrometry. We analyzed the secretion of the enzyme phytase, a phosphatase used as an animal feed additive, from a low number of yeast cells. Yeast cells were encapsulated in nanoliter volumes by droplet microfluidics and deposited on spatially-defined spots on the surface of a glass slide mounted on the motorized stage of an inverted fluorescence microscope. During the following incubation for several hours to produce phytase, the droplets can be monitored by optical microscopy. After addition of a fluorogenic substrate at a defined time, the relative concentration of phytase was determined in every droplet. Moreover, we demonstrate the use of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to monitor the multi-step conversion of the native substrate phytic acid by phytase secreted in 7 nL droplets containing 50-100 cells. Our method can be adapted to various other protocols. As the droplets are easily accessible, compounds such as assay reagents or matrix molecules can be added to all or to selected droplets only, or part of the droplet volume could be removed. Hence, this platform is a versatile tool for questions related to cell secretome analysis.

High-throughput screening of cells is required for many bioanalytical, biotechnological and diagnostic applications. In areas such as discovery of drugs and other active compounds,1 protein engineering2 or strain optimization,3 often target molecules such as antibodies,4–6 hormones7 or enzymes1–3 are secreted by the cell. In this context, it is of particular benefit to perform experimental procedures in miniaturized systems. Miniaturization not only reduces the consumption of resources, but also helps to accumulate the secreted compounds at sufficiently high concentrations for analytical measurements. Recently, various microfluidic methods have been developed to isolate and monitor single or few cells in confined volumes of nanoliters or even picoliters.8,9 Common approaches include the immobilization of cells in micro-fabricated wells10–12 or microchambers.13,14 For high throughput applications, droplet microfluidics proves to be particularly useful. In this technique, cells are encapsulated in aqueous droplets separated by a water-immiscible fluid (e.g., perfluorinated oil). Similar to fluorescence activated cell sorting (FACS),

these droplets can be analyzed and sorted by defined parameters.15,16 Although typically slower than commercial cytometers, droplet microfluidics-based approaches enable the integration of additional procedures, such as the injection of further components,17,18 the splitting of droplets,19,20 or droplet storage and incubation for cell culture on time scales up to several days.21,22 As alternative to the closed channel systems employed for droplet generation and manipulation, microdroplet arrays have been introduced, where droplets are positioned on a patterned surface at high densities.23–25 The major advantage of an open platform is the convenient accessibility of droplets. Droplet printing/spotting,26,27 dispensing28 or FACS29 can be applied to add components at a defined time point, or to selected droplets only. Likewise, designated droplets can be sampled for and after analysis, e.g. to retrieve cells with desired properties.25,28,30 Besides facile fluid handling and spatial labeling, microdroplet arrays offer great flexibility with respect to the coupled detection methods.

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photoresist (ma-P 1240, micro resist technology GmbH, Berlin, Germany) was spin-coated on top of the polysilazane, baked, exposed to UV light through a foil mask, and developed (ma-D 531, micro resist technology GmbH). Reactive ion etching was employed to remove residual photo resist in the spots and thereafter selectively etch the polysilazane in unprotected areas. Finally, the remaining resist was removed with acetone/propan-2-ol (Technic Inc., Rhode Island, USA) and the wafer was diced to obtain individual plates with dimensions of 75 mm x 25 mm. Twelve arrays of hydrophilic spots are located on each plate. One array consists of 200 hydrophilic spots with a diameter of 250 µm, equally spaced by 500 µm in all directions. (For the design, see Fig. S1, SI).

Fluorescence assays are widely applied in droplet analysis as they allow for fast and sensitive detection of a certain compound or enable the monitoring of reactions.1,31 However, fluorescence microscopy requires either labeling of target compounds or enzymatic conversions that involve fluorophores, which strongly limits its applicability. Mass spectrometry, on the other hand, provides label-free multiplexed analysis with additional structural information, but it usually lacks temporal resolution due to its destructive nature. Substantial improvements in instrumentation has made single-cell analysis by mass spectrometry feasible nowadays.32–36 37,38 Biomolecules such as peptides, metabolites,34,39 40 36 phospholipids or metal ions have been analyzed at the single-cell level. Techniques for integration of droplet microfluidics with mass spectrometry were developed in the past,27,41–43 and a few studies demonstrated the detection of cell-released compounds that accumulated in droplets.44,45

Droplet Spotting and Analysis Platform. The droplet spotting and analysis platform is illustrated in Figure 1, and photographs are shown in Fig. S2 (SI). It consists of an inverted fluorescence microscope (Olympus IX73) equipped with a motorized stage (HLD117, Prior) with a removable holder for the microarray plate. The plate features a built-in temperature control, walls to prevent oil leakage and a transparent bottom. In addition, a custom-made capillary holder with an integrated droplet sensor27 and a custom-made transparent micro tee for droplet generation were mounted on the microscope. All components of the microscope and the capillary holder stage were controlled via the software “Youscope” for automated microscopy.49 Before the spotting procedure starts, 1 mL syringes (309628, BD) were filled separately with the reagents and fluorinated oil (HFE 7500, 3M), and mounted on syringe pumps (Nem-B101-03A, Cetoni, Korbußen, Germany). Droplets were generated in the micro tee by injecting the aqueous phase (flow rate: 0.5 µL/min) into the nonmiscible fluorinated oil (flow rate: 2 µL/min). This process was monitored by a CCD camera (AD-3713TL, Dino-Lite). Droplet volume was determined from these images. The droplets are transported via HPFA capillary (1933, IDEX) through the droplet sensor to the microarray plate. Before and after spotting, the capillary was moved orthogonal to the plate by a motorized stage (M403.2PD, Physik Instrumente, Karlsruhe, Germany). Once the droplets were produced stably, the optical droplet sensor detects passing droplets by a change in the intensity of transmitted light, created by the change in refraction between oil and aqueous droplet. Signals were processed by a realtime data acquisition system (AdWin Gold II, Jäger Computergesteuerte Messtechnik GmbH, Lorsch, Germany). A MATLAB script (Mathworks), embedded in the Youscope software, detected a change in the trigger signal and the motorized microscope stage moves the plate. Typical spotting frequency was 1-2 Hz (video, SI).

In this study, we combine the benefits of microfluidic fluid handling as well as both complementary detection methods to quantify enzyme secretion from yeast encapsulated in nanoliter droplets and its enzymatic activity. Recently introduced methods to achieve dual fluorescence and MALDI MS read-out46 as well as our own previously developed method for MALDI-MS analysis of nL-droplets27,47 are not suitable for monitoring cell-based assays. Therefore, we reengineered the method to encapsulate yeast cells, incubate them over several hours on a temperaturecontrolled plate and perform multistep cell-based assays. We use transparent, ITO-coated plates for the deposition of the droplets and perform the spotting process directly on a fluorescence microscope for subsequent automated image acquisition. Aiming at strain characterization and optimization, we established protocols to measure the secretion of the enzyme phytase by the yeast Komagataella phaffii (also known as Pichia pastoris). Phytase is a phosphatase that serves as an important animal feed additive. It catalyzes the hydrolysis of indigestible organic phosphorous in plants and hence, increases the phosphorous availability for the animals. We measured the concentration of the enzyme by fluorescence spectroscopy using a synthetic substrate, while the multistep reaction with the native substrate phytic acid was characterized in situ by matrix-assisted laser desorption/ionization mass spectrometry (MALDIMS).

EXPERIMENTAL SECTION Fabrication of the microarray plates. The fabrication process of the microarray plate was adapted from a formerly presented protocol for microarrays for mass spectrometry (MAMS)48. In brief, a 4-inch borofloat glass wafer coated with indium tin oxide (ITO) with a resistance below 10 ohms/square (Siegert Wafer, Aachen, Germany) was cleaned in oxygen plasma. Subsequently, the hydrophobic substrate polysilazane (CAG37, Merck KGaA, Darmstadt, Germany) was spin-coated and cured on a hotplate at 300 °C for 2 h. As a protective layer, the positive

After droplet spotting, fluorescence and brightfield pictures were recorded using a light source (Lumen 300, Prior) and a CMOS camera (Zyla 4.2, Andor). The cell number per droplet was derived from the brightfield images (see Fig. S3). Automated image analysis and quality control was done by a custommade MATLAB script. Spots with a wrong number of


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30 °C was stopped 20 min after spotting the last droplet by quickly removing the surrounding oil and drying the droplets.

droplets are determined by fluorescence or size threshold. Yeast Strains and Cultivation. K. phaffii X33 was transformed with a linearized plasmid (pGAPzαB, Thermo Fisher Scientific) containing a zeocin resistence, a constitutive GAP-promoter and the Saccharomyces cerevisiae α-mating factor to trigger secretion. Downstream of the factor a codon-optimized appA gene from Escherichia coli MG1655 (encoding a phytase) was inserted. Yeast cells were precultured from cryo-stocks for 24 h at 30 °C at 220 rpm in 2 mL Yeast Extract-Peptone-Dextrose (YPD) broth (Sigma) in 12 mL culture tubes. After dilution (1:20) in YPD media, the cells were cultured again overnight under the same conditions. Prior to the experiments, cells were washed twice with the medium used in the assay. The optical density at 600 nm of the cell suspension was measured and adjusted to 2 AU with medium.

MALDI-MS Analysis. Prior to the MS analysis, the oil covering the droplets was removed by decantation and the cells and supernatant dried rapidly. A binary matrix was prepared and 7 nL were spotted onto each sample spot. The matrix solution consisted of 5 g/L alpha-cyano-4-hydroxycinnamic acid (CHCA) and 5 g/L 9-aminoacridine (9-AA) dissolved in 50% (v/v) acetonitrile/water (all chemicals from Sigma Aldrich). The addition of CHCA enabled the measurement despite high salt concentrations present in the sample.50 Spectra were acquired using an ABI SCIEX 4800 MALDI TOF/TOF analyzer. A ring-shaped ablation pattern with 15 equally spaced points was set to achieve a homogenous distribution over the whole spot surface. Measurements were conducted in negative reflector mode with a laser power of 4000 AU and 15 shots per sub-spectra. Peak intensities were normalized by the sum of the intensities of IP6-IP2. The number after IP indicates the amount of phosphates. To calculate absolute concentrations, it was assumed that the response factors of all inositol phosphates are similar. Experimental determination of these factors was not feasible since pure standards were not available. If the reaction did not progress further than IP2, the concentration of analyte i on a spot k can be derived from the initial amount of inositol phosphates.

Assays for Phytase Analysis. Supernatant of an overnight culture of K. phaffii in YPD medium was harvested after centrifugation (5 min, 1677  g) and diluted with YPD media with the same pH (4.8) as the supernatant. Five sets of 40 droplets (7 nL) with different phytase concentrations (0, 25, 50, 75 and 100%), relative to the initial phytase concentration, were spotted onto the microarray plate. A second droplet with the same volume, containing 100 µM fluorescein monophosphate (FMP, Santa Cruz Biotechnology) in 0.1 M ammonium acetate (pH 5, Sigma Aldrich), was added to every. The conversion of FMP to fluorescein was recorded at 30 °C with automated fluorescence microscopy at 10x magnification every 30 s for 450 s with blue excitation. Phytase secretion was calculated on the basis of the fluorescence increase per time received by linear regression of the fluorescence data. For reaction monitoring by mass spectrometry, supernatant of an overnight culture of K. phaffii in Difco Yeast Nitrogen Base with amino acids (YNB, 20 g/L glucose, BD) was harvested after centrifugation (5 min, 1677  g), mixed (1:1) with 1 mM IP6 in ammonium acetate (0.1 M, pH 5, both Sigma Aldrich) and filled into a 1 mL syringe. Every 10 min, for up to a total of 60 min 20 droplets of the reaction mixture were spotted at room temperature. As a reference point (0 min), denatured supernatant (95 °C, 5 min) was mixed (1:1) with 1 mM IP6 and 200 droplets were spotted on a different array. Due to the small amount of surrounding oil, the droplets dried immediately on the plate and the reaction stopped.

𝑐𝑖, 𝑘 = 𝑥𝑖, 𝑘 ∗ 𝑐𝐼𝑃6, 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 = 𝐼𝑖, 𝑘, 𝑟𝑒𝑙 ∗ 𝑐𝐼𝑃6, 𝑖𝑛𝑖𝑡𝑖𝑎𝑙

(1)

The concentration of all compounds was referenced to a negative control without cells (𝑐𝑖,𝑌𝑁𝐵) to subtract the effect of auto-catalysis. ∆𝑐𝑖,𝑘 = 𝑐𝑖, 𝑘 ― 𝑐𝑖,𝑌𝑁𝐵

(2)

Phosphate production per time (activity) was calculated by eq 3 derived from reaction stoichiometry. 𝑎𝑃𝑂42 ― , 𝑘 =

(4 ∗ ∆𝑐𝐼𝑃6, 𝑘 + 3 ∗ ∆𝑐𝐼𝑃5, 𝑘 + 2 ∗ ∆𝑐𝐼𝑃4, 𝑘 + ∆𝑐𝐼𝑃3, 𝑘) ―∆t𝑘

(3)

RESULTS AND DISCUSSION Platform Performance. First, the reliable operation of the spotting unit was confirmed. After droplet formation in the micro tee, the droplets were transferred through a capillary and deposited on a transparent glass slide that was mounted on an inverted microscope. The end of the capillary was positioned approximately 80 µm above the plate. Due to the custom-made hydrophilic/ hydrophobic pattern on the glass slide, the droplets were always guided exactly to a predefined hydrophilic position, and droplet spreading on the plate was prevented (Figure 2b). The reliable deposition of droplets is of particular importance for multiple spotting runs, i.e. it facilitates multistep assays and the spotting of matrix compounds for MALDI-MS. To avoid cross contamination between following spotting rounds, the system is flushed with water and oil in between. Furthermore, the end of the capillary is

Cell-based Phytase Assays. Yeast cells in YNB or YPD were encapsulated in 7 nL droplets and spotted on the microarray submersed in 3 mL fluorinated oil. As a reference, droplets containing only media were spotted in the last two rows. Droplets were imaged subsequently with 20 magnification in brightfield illumination. After seven hours of cultivation and phytase production at 24 °C, the enzymatic assay was conducted with FMP as described before. For the phytase activity assay in droplets, a 7 nL droplet of 1 mM IP6 was added to the enzyme-containing droplets (only YNB culture) instead of FMP. The reaction at



within several seconds and the reaction was stopped. After deposition of the matrix, MALDI-MS measurements were performed. Figure 4a shows a spectrum at the start of the reaction with the inositol phosphates (IP6-2). IP1 and inositol were not measured since the background in the medium was too high. As can be derived from the spectrum, partially dephosphorylated IP6 was already present at the start of the reaction presumably due to hydrolyzation during storage. To account for signal variations due to crystallization behavior, the intensity of each species was normalized by the sum of the intensities of all inositol phosphates measured in the respective nanoliter droplet. Figure 4b shows the distribution of relative intensity for the compound IP6, here measured in 160 different droplets and divided by its mean value. Although the measurements were performed in YNB medium, a low variation of 7.3% was achieved. Finally, the data analysis on all time points of the reaction revealed the course of the reaction. As shown in Figure 4c, the relative intensity of inositol phosphates with a higher phosphate content decreased over time. At the same time, the curves of intermediate products (IP3 and IP4) show a peak in the middle of the reaction time and IP2 increases over time. The results demonstrate the capability of our method to follow multi-step enzymatic reactions in nanoliter droplets label-free by MALDIMS.

positioned 130 µm above the plate for the second run so that it is not in contact with the already deposited first droplet. Continuous flow of droplets and oil spacer further prevents any carryover of compounds from one to the next droplet. Another critical performance parameter is the reproducibility of the volume of the aqueous droplets. Droplet generation was constantly monitored via an USB camera (volume, off-line) and a droplet detector (frequency, online). Additionally, the droplet diameter on the plate was automatically extracted during image processing to find spotting errors. Figure 2a shows a typical volume distribution in our experiments. With a coefficient of variation (CV) of 2.8% in droplet volume our platform performs similar to other droplet-based systems.36 To test the performance of the fluorescence measurement, droplets containing 100 µM fluorescein were spotted (Figure 2b). As shown in Figure 2c, the CV of the measured fluorescence is only 4.8%. The difference in variance between droplet volume and fluorescence measurements could be caused by small deviations in droplet geometry affected by droplet volume and wetting behavior. Fluorescence Assay to Determine Relative Phytase Concentration. Droplets with nanoliter volumes can be easily merged by spotting them on top of each other. This basic operation in fluidic handling in combination with a microscope enables various assays. Here, we demonstrate the use of the platform for enzymatic assays by analyzing the supernatant of phytase-secreting yeast. First, we deposited droplets containing dilutions of the fermentation supernatant under oil. In a second step, another droplet containing the substrate fluorescein monophosphate was added. The addition of the second droplet is the starting point for the enzymatic conversion of the substrate to the fluorescent product fluorescein, which was monitored on the fluorescence microscope (Figure 3). The slope in the linear region correlates with the activity of the enzyme and was used to determine relative enzyme concentration in the following experiments. At higher enzyme concentration, a plateau is reached due to the lack of substrate. Therefore, the time range of 250 s was used for data fitting in the following experiments.

Cell-based Assay with Fluorescence Readout. Our goal was to perform analysis of protein secretion directly in nanoliter droplets. Therefore, we encapsulated the cells during droplet formation and cultivated them in the spotted nanoliter droplets at 24 °C for seven hours. (Figure S3, SI) The estimated number of cells per droplet was 50-100 cells/droplet. After spotting the droplets on the plate, the yeast cells were cultured for 7 h at 24 °C. During the time of incubation, the cells proliferated (typically divided 1-2 times) and secreted the enzyme phytase, which accumulated in the droplet. Droplet volumes were stable during the incubation and we assumed that the enzyme does not diffuse into the oil. Afterwards, the substrate FMP was added to 80 droplets containing yeast cells. As a control, 20 droplets of YNB medium without cells were subjected to same procedure as the cell-containing droplets. Figure 5 depicts the relative phytase concentrations across the spotting area of 10 × 10 spots. The enzyme concentration in the cellcontaining droplets reached a level, at which its activity could clearly be distiguished from the background in YNB medium. The same experiment in YPD medium resulted in a three times higher phytase concentration due to higher expression rates in the complex medium YPD than in the mimimal medium YNB. Hence, we can clearly distinguish altered expression levels under different conditions. In both cases, the concentration was significantly below the concentration of enzyme in the supernatant of the overnight culture, most pobably because the cells were still in the lag phase. The heatmap in Figure 5b shows the relative phytase concentration per spot. Empty spots indicate excluded experiments, where e.g. the deposition of the droplets in the first or second spotting round did not occur correctly. The additional location information can be

Phytase Reaction Monitoring by MALDI-MS. The native substrate of phytase is D-myo-inositol hexakisphosphate (IP6), also referred to as phytic acid. The enzyme catalyzes the sequential release of phosphate from the highly phosphorylated IP6 to lower inositol phosphates (e.g. IP4) and finally inositol. The last step towards inositol is not favored in the case of E. coli phytase.51 By analyzing the nanoliter droplets with MALDI-MS, we were able to monitor this reaction and distinguish the various inositol phosphates even in YNB medium. Therefore, fermentation supernatant of phytase-producing yeast and 1 mM IP6 were mixed (1:1) and every 10 min 20 droplets were deposited on the plate. For the measurement representing the start of the reaction, the supernatant was incubated at 95°C for 5 min before mixing with IP6 to denature the enzymes and 200 droplets were spotted. Due to the low volume and the missing oil cover, the droplets evaporated


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analyze the phytase secretion of 50-100 cells. To reach our ultimate goal of single-cell analysis, further downscaling of droplet volume is required, where a better accumulation of enzymes can be achieved. Furthermore, using MALDI-MS a direct identification and structural analysis of secreted compounds will be accomplished. Since the droplets are located at defined positions and can be addressed individually, many different orthogonal analytical strategies could be applied to combine multi-omic analysis on the same cell (e.g., secretome and transcriptome). A cell with desired properties could be transferred to larger cultivation chambers. We believe that our platform is a versatile tool for various single-cell assays with a broad variety of applications.

used in future to combine results from different assays and link them directly to the cells. Furthermore target cells with high production could be identified and importantly recovered, which is essential for cell screening. To demonstrate that our method is suitable to perform more experiments in parallel, we performed the same assay in 3000 droplets (Figure S4, SI). However, due to the relatively slow spotting frequency a significant influence of the encapsulation order on the cultivation time and the secretion can be seen. Future efforts are directed towards increasing the spotting frequency. As an alternative, constructs with inducible promotors could be used in order to ensure a similar secretion time. Cell-based Assay with MALDI-MS Readout. Like in the fluorescence assay, we cultivated yeast in the micro-droplets for seven hours. For a better compatibility with the MALDI-MS measurements, only the minimal medium YNB was used. To measure phytase activity, IP6 was added to the droplets and incubated for 20 min. We analyzed again 80 droplets containing cells and 20 droplets with YNB medium only (after drying) and determined the change in the concentration of phytase for each species (Figure 6a). Almost all IP6 and IP5 present at the beginning was converted to IP4 as well as IP3, while IP2 remained almost unchanged. The changes fit well with the expected course of the reaction as derived from Figure 4c. The enzyme activity, meaning phosphate production or IP6 conversion per time, was calculated from the inositol phosphate concentrations (Figure 6b). Visualization of the reaction chambers indicates a small gradient of enzyme activity from the top to the bottom of the plate (Figure 6c), which we attribute to slightly lower cell numbers in the droplets of the bottom rows potentially caused by cell clustering and settling in the tubing. Phytase activity in animal feed is defined as the increase in phosphate over time during the conversion of phytic acid (IP6) to smaller inositol phosphates.52 As stated by Qvirist et al.,53 this definition does not represent the actual phytase activity (IP6 conversion) and furthermore is biased by phosphate production from different substrates and by different enzymes. With our method it is possible to measure the change in inositol phosphate concentrations and thus determine the sum of all phosphate molecules cleaved enzymatically from inositol phosphates. Methods like liquid chromatography, which have been used before53 cannot be applied for nanoliter volumes, and thus are not suitable for few-cell applications. In our case, we are not only able to derive the changes of each species, but also the total production of phosphate using eq 3.

ASSOCIATED CONTENT Supporting information The Supporting Information shows a photograph of the experimental setup, the CAD drawing of the microarray plate design, a micrograph of yeast cell cultivation in nanoliter droplets, and the result of upscaling the fluorescence assay. Supporting video: Droplet spotting This material is available free of charge via the Internet at http:// pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Author Contributions L.M.B. and P.S.D. designed the work, D.H. performed and analyzed the fluorescence experiments, D.H., M.K., R.Z. performed and analyzed the MS experiments, S.B. fabricated the microscopy slides, D.H. and P.S.D. wrote the manuscript, which all authors approved. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENT We gratefully acknowledge funding from the Swiss National Science Foundation (NCCR Molecular Systems Engineering and Systems X program, Grant No. 324). Dr. Anna Joelle Ruff and Dr. Stefanie Hamer of the Chair of Biotechnology at RWTH Aachen University are acknowledged for strain construction. Furthermore, the authors thank Christoph Bärtschi (ETH Zürich) for the fabrication of the device parts as well as Alexander Stettler (ETH Zürich) for help with cleanroom processes. Dr. Sandra Schulte and Jan Förster (both RWTH Aachen University) are acknowledged for helpful discussions and support in the project regarding yeast culture. Bill Jia (University of Cambridge) participated in the assay development and automated data analysis. We thank Darius Rackus (ETH Zürich) for proofreading.

CONCLUSION We established a new method for cell-based measurements in nanoliter volumes. Optical microscopy, fluorescence microscopy as well as MALDI-MS were employed on a single platform to quantify phytase secretion and characterize the reaction of the secreted enzyme. We were able to distinguish individual phytase reaction steps and analyze the enzyme activity more precisely than with commercially available phosphate assays.52 So far, we are able to

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Figure captions Figure 1. Overview of the droplet spotting and analysis platform. (a) Schematic drawing of the platform. Nanoliter droplets are generated in a custom-made transparent micro tee. Volume assessment and quality control are conducted by an integrated USB microscope. Droplets are transferred via capillary to an ITO-coated glass plate with hydrophilic spots (diameter of 250 µm) submersed in fluorinated oil. The plate is mounted on the motorized stage of an inverted fluorescence microscope and stage movement is triggered by an integrated optical sensor. (b) Schematics of the assays. (I) After spotting, the yeast cells are cultivated in the droplets and secrete the enzyme phytase. (II) A droplet with fluorescein monophosphate (FMP) is added to each droplet and the conversion to fluorescein by phytase is monitored by fluorescence time-lapse microscopy. (III) D-myo-inositol hexaphosphate (IP6) is added to another subset of droplets and the multistep reaction of IP6 to partially hydrolyzed inositol phosphates is analyzed by MALDI-MS.

Figure 2. Platform performance. (a) Histogram of droplet volume determined from the images taken with CCD camera directly after generation of YPD media droplets. (b) Fluorescence microscopy picture of an array of fluorescein droplets (100 µM) on a glass plate. (scale bar: 1 mm) c) Histogram of median fluorescence intensity per droplet normalized to the average fluorescence intensity.

Figure 3. Fluorescence assay to determine relative phytase concentration. Different concentrations of phytase were spotted on a microarray and a droplet of fluorescein monophosphate was added to each spot to start the assay. (a) Fluorescence increase over time caused by different concentrations of phytase fitted by linear regression. (b) Dependency of acid phosphatase activity on relative concentration of phytase. 0% phytase corresponds to YPD only (n=27-36).

Figure 4. Phytase reaction monitoring by MALDI-MS. (a) Mass spectrum at the reaction start with labeled peaks of inositol phosphates. (b) Histogram of relative intensity of IP6 at the beginning of the reaction normalized to its mean. (c) Relative intensities of IP6, IP5, IP4, IP3 and IP2 over the course of the reaction. The error bars correspond to the deviation of the aliquots at the same time point. (n = 7-15) Figure 5. Cell-based assay with fluorescence readout. Relative phytase concentration produced by K. phaffii after 7h of incubation in YNB or YPD in nanoliter droplets was measured. (a) Relative phytase concentration - comparison between droplets with and without yeast cells cultured in YNB and YPD. (b) Relative phytase concentration per spot of the cultivation in YNB. The two bottom rows contain only YNB medium. Positions without dots indicate that the results were excluded due to spotting errors.

Figure 6. Cell-based assay with MALDI-MS readout. (a) Difference in concentration between medium only and cellcontaining droplets after 20 min of enzymatic reaction. (b) Comparison of different phytase activities, based on phosphate production or IP6 reduction, calculated from concentration differences of inositol phosphates between medium and cellcontaining droplets. (c) Phytase activity based on phosphate production per spot. The two bottom rows contain only YNB medium. Positions without dots indicate that the results were excluded due to spotting errors.


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Overview of the droplet spotting and analysis platform. (a) Schematic drawing of the platform. Nanoliter droplets are generated in a custom-made transparent micro tee. Volume assessment and quality control are conducted by an integrated USB microscope. Droplets are transferred via capillary to an ITO-coated glass plate with hydrophilic spots (diameter of 250 µm) submersed in fluorinated oil. The plate is mounted on the motorized stage of an inverted fluorescence microscope and stage movement is triggered by an integrated optical sensor. (b) Schematics of the assays. (I) After spotting, the yeast cells are cultivated in the droplets and secrete the enzyme phytase. (II) A droplet with fluorescein monophosphate (FMP) is added to each droplet and the conversion to fluorescein by phytase is monitored by fluorescence time-lapse microscopy. (III) D-myo-inositol hexaphosphate (IP6) is added to another subset of droplets and the multistep reaction of IP6 to partially hydrolyzed inositol phosphates is analyzed by MALDI-MS.

ACS Paragon Plus Environment


Platform performance. (a) Histogram of droplet volume determined from the images taken with CCD camera directly after generation of YPD media droplets. (b) Fluorescence microscopy picture of an array of fluorescein droplets (100 µM) on a glass plate. (scale bar: 1 mm) c) Histogram of median fluorescence intensity per droplet normalized to the average fluorescence intensity


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Fluorescence assay to determine relative phytase concentration. Different concentrations of phytase were spotted on a microarray and a droplet of fluorescein monophosphate was added to each spot to start the assay. (a) Fluorescence increase over time caused by different concentrations of phytase fitted by linear regression. (b) Dependency of acid phosphatase activity on relative concentration of phytase. 0% phytase corresponds to YPD only (n=27-36).



Phytase reaction monitoring by MALDI-MS. (a) Mass spectrum at the reaction start with labeled peaks of inositol phosphates. (b) Histogram of relative intensity of IP6 at the beginning of the reaction normalized to its mean. (c) Relative intensities of IP6, IP5, IP4, IP3 and IP2 over the course of the reaction. The error bars correspond to the deviation of the aliquots at the same time point. (n = 7-15)


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Cell-based assay with fluorescence readout. Relative phytase concentration produced by K. phaffii after 7h of incubation in YNB or YPD in nanoliter droplets was measured. (a) Relative phytase concentration comparison between droplets with and without yeast cells cultured in YNB and YPD. (b) Relative phytase concentration per spot of the cultivation in YNB. The two bottom rows contain only YNB medium. Positions without dots indicate that the results were excluded due to spotting errors.



Cell-based assay with MALDI-MS readout. (a) Difference in concentration between medium only and cellcontaining droplets after 20 min of enzymatic reaction. (b) Comparison of different phytase activities, based on phosphate production or IP6 reduction, calculated from concentration differences of inositol phosphates between medium and cell-containing droplets. (c) Phytase activity based on phosphate production per spot. The two bottom rows contain only YNB medium. Positions without dots indicate that the results were excluded due to spotting errors.


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