Thomas Beneyton

Stéphane Thomas

Andrew D Griffiths

Jean-Marc Nicaud

Antoine Drevelle

Tristan Rossignol
email: tristan.rossignol@inra.fr

Droplet-based microfluidic high-throughput screening of heterologous enzymes secreted by the yeast Yarrowia lipolytica

Keywords: High-throughput enzymatic screening, Droplet-based microfluidics, Heterologous expression, Yarrowia lipolytica, Recombinant fungal protein Microbial Cell Factories

Background: Droplet-based microfluidics is becoming an increasingly attractive alternative to microtiter plate techniques for enzymatic high-throughput screening (HTS), especially for exploring large diversities with lower time and cost footprint. In this case, the assayed enzyme has to be accessible to the substrate within the water-in-oil droplet by being ideally extracellular or displayed at the cell surface. However, most of the enzymes screened to date are expressed within the cytoplasm of Escherichia coli cells, which means that a lysis step must take place inside the droplets for enzyme activity to be assayed. Here, we take advantage of the excellent secretion abilities of the yeast Yarrowia lipolytica to describe a highly efficient expression system particularly suitable for the droplet-based microfluidic HTS.

Results: Five hydrolytic genes from Aspergillus niger genome were chosen and the corresponding five Yarrowia lipolytica producing strains were constructed. Each enzyme (endo-β-1,4-xylanase B and C; 1,4-β-cellobiohydrolase A; endoglucanase A; aspartic protease) was successfully overexpressed and secreted in an active form in the crude supernatant. A droplet-based microfluidic HTS system was developed to (a) encapsulate single yeast cells; (b) grow yeast in droplets; (c) inject the relevant enzymatic substrate; (d) incubate droplets on chip; (e) detect enzymatic activity; and (f ) sort droplets based on enzymatic activity. Combining this integrated microfluidic platform with gene expression in Y. lipolytica results in remarkably low variability in the enzymatic activity at the single cell level within a given monoclonal population (<5%). Xylanase, cellobiohydrolase and protease activities were successfully assayed using this system. We then used the system to screen for thermostable variants of endo-β-1,4-xylanase C in error-prone PCR libraries. Variants displaying higher thermostable xylanase activities compared to the wild-type were isolated (up to 4.7-fold improvement).

Conclusions: Yarrowia lipolytica was used to express fungal genes encoding hydrolytic enzymes of interest. We developed a successful droplet-based microfluidic platform for the high-throughput screening (10 5 strains/h) of Y. lipolytica based on enzyme secretion and activity. This approach provides highly efficient tools for the HTS of recombinant enzymatic activities. This should be extremely useful for discovering new biocatalysts via directed evolution or protein engineering approaches and should lead to major advances in microbial cell factory development.

Background

The lack of rapid and generalized high-throughput screening (HTS) systems has proven to be a major bottleneck when it comes to creating biocatalysts via directed evolution and protein engineering approaches. Currently, the most flexible and widely used HTS methods involve isolating monoclonal populations in the wells of microtiter plates. These techniques require a significant investment in terms of money, time, and space, which severely limits the number of enzymes that can be screened and, consequently, the discovery of better biocatalysts. Droplet-based microfluidics has recently emerged as an attractive alternative to conventional HTS techniques because it has significantly higher throughput and significantly lower assay volumes [START_REF] Price | Discovery in droplets[END_REF]. In this approach, cells are compartmentalized in water-in-oil (w/o) picoliter droplets that can be individually manipulated at kHz frequencies (e.g., created, fused, injected, incubated, or sorted based on fluorescence) [START_REF] Seemann | Droplet based microfluidics[END_REF][START_REF] Theberge | Microdroplets in microfluidics: an evolving platform for discoveries in chemistry and biology[END_REF][START_REF] Sciambi | Accurate microfluidic sorting of droplets at 30 kHz[END_REF]. Typically, single bacteria or yeast are compartmentalized in 10 pl droplets allowing the screening of libraries with a 1.000 fold-increase in speed and 1-million-fold reduction in volume compared to robotic microtiter plate-based systems [START_REF] Agresti | Ultrahigh-throughput screening in drop-based microfluidics for directed evolution[END_REF].

Most of the time, in droplet-based microfluidic systems, enzymatic activities are detected using fluorogenic substrates especially adapted to the droplet format. In particular, the fluorescent probe product should not be transported from one compartment to another one over the time scale of the assay [START_REF] Gruner | Controlling molecular transport in minimal emulsions[END_REF][START_REF] Woronoff | New generation of amino coumarin methyl sulfonate-based fluorogenic substrates for amidase assays in dropletbased microfluidic applications[END_REF], and the substrate should be accessible to the expressed enzymes within the w/o droplet. Consequently, the expression system must be carefully chosen and, ideally, should involve enzymes that are expressed extracellularly or at the cell surface. To date, most of the protein libraries screened have relied on cytoplasmic or periplasmic expression in Escherichia coli, which implies that both the substrate and the product travel through the cell membrane [START_REF] Beneyton | CotA laccase: high-throughput manipulation and analysis of recombinant enzyme libraries expressed in E. coli using droplet-based microfluidics[END_REF][START_REF] Hosokawa | Droplet-based microfluidics for high-throughput screening of a metagenomic library for isolation of microbial enzymes[END_REF] or that an additional lysis step is needed to perform the enzymatic assay [START_REF] Romero | Dissecting enzyme function with microfluidic-based deep mutational scanning[END_REF][START_REF] Kintses | Picoliter cell lysate assays in microfluidic droplet compartments for directed enzyme evolution[END_REF][START_REF] Colin | Ultrahigh-throughput discovery of promiscuous enzymes by picodroplet functional metagenomics[END_REF]. While horseradish peroxidase libraries were screened displayed at the surface of Saccharomyces cerevisiae cells [START_REF] Agresti | Ultrahigh-throughput screening in drop-based microfluidics for directed evolution[END_REF], none extracellular recombinant expression systems were described up to now in combination with droplet-based microfluidics.

Yeasts have the advantage of being able to perform eukaryotic post-translational processing (e.g., glycosylation, folding, sequence cleavage, etc.) and can therefore produce and secrete heterologous eukaryotic proteins in their active form. Expression and secretion of heterologous proteins have been widely studied in yeast, for which several expression systems have been commercialized. In particular, the nonconventional yeast Yarrowia lipolytica, has been used as a model organism for protein secretion [START_REF] Nicaud | Protein expression and secretion in the yeast Yarrowia lipolytica[END_REF][START_REF] Nicaud | Yarrowia lipolytica[END_REF][START_REF] Madzak | Yarrowia lipolytica: recent achievements in heterologous protein expression and pathway engineering[END_REF], as it has a natural predisposition for protein secretion [START_REF] Barth | Physiology and genetics of the dimorphyc fungus Yarrowia lipolytica[END_REF]. It is potentially better at producing heterologous proteins than are many other commonly used yeast [START_REF] Müller | Comparison of expression systems in the yeasts Saccharomyces cerevisiae, Hansenula polymorpha, Klyveromyces lactis, Schizosaccharomyces pombe and Yarrowia lipolytica. Cloning of two novel promoters from Yarrowia lipolytica[END_REF], including Pichia pastoris, or even E. coli the most commonly exploited heterologous protein biofactory [START_REF] Boonvitthya | Comparison of the heterologous expression of Trichoderma reesei endoglucanase II and cellobiohydrolase II in the yeasts Pichia pastoris and Yarrowia lipolytica[END_REF][START_REF] Nars | Production of stable isotope labelled lipase Lip2 from Yarrowia lipolytica for NMR: investigation of several expression systems[END_REF]. Y. lipolytica presents several major advantages as a model: its genome has been fully sequenced and annotated [START_REF] Dujon | Genome evolution in yeasts[END_REF]; it is a species for which numerous genetic tools [START_REF] Barth | Physiology and genetics of the dimorphyc fungus Yarrowia lipolytica[END_REF][START_REF] Fickers | Selection of new over-producing derivatives for the improvement of extracellular lipase production by the non-conventional yeast Yarrowia lipolytica[END_REF] and high-throughput cloning methods [START_REF] Leplat | High-throughput transformation method for Yarrowia lipolytica mutant library screening[END_REF] are available; and an ever increasing number of its promoters and secretion signal sequences have been studied, described, and evaluated [START_REF] Madzak | Yarrowia lipolytica: recent achievements in heterologous protein expression and pathway engineering[END_REF][START_REF] Madzak | Strong hybrid promoters and integrative expression/secretion vectors for quasi-constitutive expression of heterologous proteins in the yeast Yarrowia lipolytica[END_REF][START_REF] Blazeck | Generalizing a hybrid synthetic promoter approach in Yarrowia lipolytica[END_REF].

Moreover, there are many examples of successful heterologous protein expression using Y. lipolytica [START_REF] Madzak | Heterologous protein expression and secretion in Yarrowia lipolytica[END_REF]. Because its genetic manipulation is easy to perform and its high protein yields can easily be scaled up, Y. lipolytica has become a cell factory of choice for producing heterologous proteins. It would thus be highly beneficial to develop a droplet-based microfluidic HTS system that uses Y. lipolytica because it would allow for the more efficient screening of enzyme libraries and undoubtedly advance the development of microbial cell factories in major ways.

One important aspect of enzyme production requirement today is the production of cellulolytic enzymes for the conversion of the lignocellulosic biomasses into fuels and chemicals. Cellulases are multienzyme complexes (endoglucanases, cellobiohydrolases and glucosidase) that catalyze the hydrolysis of β-1-4 linkages in cellulose, while xylanases cleave internal β-1,4 linkages of the xylan backbone. Nowadays, commercially available cellulases are produced by filamentous fungi of the genera Trichoderma and Aspergillus [START_REF] Bisaria | Bioprocessing of renewable resources to commodity bioproducts[END_REF][START_REF] Farinas | Developments in solid-state fermentation for the production of biomass-degrading enzymes for the bioenergy sector[END_REF]. However, improvement of catalytic efficiency, thermal stability or end-product inhibition resistance will have a great potential for sustainable biorefinery process for biomass conversion [START_REF] Bisaria | Bioprocessing of renewable resources to commodity bioproducts[END_REF]. Proteases, which are used to make detergent, leather, pharmaceuticals, and food, account for more than a half of the worldwide enzyme sales and therefore represent another major industrial and commercial interest [START_REF] Rao | Molecular and biotechnological aspects of microbial proteases[END_REF]. Fungal proteases are less pH sensitive and have broader substrate specificity than do bacterial equivalents. However, they exhibit lower reaction rates and heat tolerance [START_REF] Rao | Molecular and biotechnological aspects of microbial proteases[END_REF]. Addressing these weaknesses should help enhance their commercial potential.

In this study, we took advantage of Y. lipolytica's excellent secretion abilities to build extracellular expression systems for droplet-based microfluidic enzymatic screening applications. Five strains were constructed to produce five industrially relevant Aspergillus niger hydrolytic enzymes (two xylanases, one cellobiohydrolase, one endoglucanase and one aspartic protease); the enzymes were overexpressed and secreted in an active form. We developed a droplet-based microfluidic HTS system to encapsulate, cultivate, assay and sort yeast strains based on encoded enzymatic activities (10 5 strains/h). The activity levels of the xylanases, cellobiohydrolase, and protease were successfully detected in droplets with a remarkably low variability within monoclonal populations. Combining droplet-based microfluidic technology with the Y. lipolytica expression system provides a highly efficient and reliable platform for the high-throughput screening of recombinant enzyme activities.

Results and discussion

Expression of fungal genes in Yarrowia lipolytica

Five genes from Aspergillus niger (CBS513.88 strain [START_REF] Pel | Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513[END_REF]) encoding industrially relevant hydrolytic enzymes were used: xlnb (endo-β-1,4-xylanase B), xlnc (endo-β-1,4xylanase C), cbha (1,4-β-cellobiohydrolase A), egla (endoglucanase A), and pep1 (aspartic protease). The five genes were codon optimized and overexpressed in Y. lipolytica strain JMY2566 (see "Methods" section). This strain possesses a zeta docking platform that favors integration at that specific locus [START_REF] Leplat | High-throughput transformation method for Yarrowia lipolytica mutant library screening[END_REF][START_REF] Bordes | Isolation of a thermostable variant of Lip2 lipase from Yarrowia lipolytica by directed evolution and deeper insight into the denaturation mechanisms involved[END_REF], which allows for the efficient comparison of enzymatic activity among strains. The five resulting producing strains-JMY4346 (xlnb), JMY4510 (xlnc), JMY4469 (ccbha), JMY4343 (egla) and JMY4407 (pep1)-were cultivated in 20 ml of YNB glucose medium for 24 h and the corresponding supernatants were profiled using SDS-PAGE (Fig. 1). Each of the target enzymes was successfully overproduced and secreted as a major protein in the crude supernatant. All the enzymes demonstrated high expression levels without requiring any extensive optimization of growth conditions.

Enzymatic activity of the secreted proteins

To determine if the encoded proteins were produced in an active form, the xylanase, cellulase or protease activity was assayed in crude supernatants using fluorogenic substrates (Fig. 2). The xylanase and cellulase substrates were synthesized as previously described in [START_REF] Najah | New glycosidase substrates for droplet-based microfluidic screening[END_REF], using a sulfonated 7-hydroxycoumarin scaffold linked to a saccharide recognition unit. Xylanase activity was detected by monitoring the hydrolysis of a xylobiose-based coumarin (β-d-xylobioside-6,8-difluoro-7-hydroxycoumarin-4-methansulfonate, inset Fig. 2a) while cellulase (i.e., cellobiohydrolase) activity was detected by monitoring the hydrolysis of a cellobiose-based coumarin (β-d-cellobioside-6,8-difluoro-7hydroxycoumarin-4-methansulfonate, inset Fig. 2b). The protease substrate is commercially available and consists of a casein scaffold labeled with quenched green-fluorescent BODIPY ® FL fluorophores. Protease-catalyzed hydrolysis releases the fluorophores, revealing enzyme activity. All encoded proteins were found to be active after secretion.

The JMY4346 (xlnb) and JMY4510 (xlnc) strains exhibited similar levels of endo-β-1,4-xylanase activity, corresponding to endo-β-1,4-xylanase B and endoβ-1,4-xylanase C, respectively (Fig. 2a). Strain JMY4469 (cbha) displayed cellobiohydrolase activity corresponding to 1,4-β-cellobiohydrolase A (Fig. 2b). While the cellulase substrate was designed to reveal cellobiohydrolase activity, JMY4343 (egla) displayed a low activity with this substrate, seemingly due to the endocellulase activity of endoglucanase A (Fig. 2b). Lastly, strain JMY4407 (pep1) showed protease activity corresponding to aspartic protease (Fig. 2c). These results indicate that each of the five fungal hydrolytic genes was successfully overexpressed and that the corresponding recombinant enzymes were secreted in an active form.

Droplet-based microfluidic HTS platform

A droplet-based microfluidic system was developed for the enzymatic screening of recombinant proteins secreted by Y. lipolytica (Fig. 3). The platform was composed of two distinct microfluidic devices: a dropmaker and an integrated screening device. The dropmaker encapsulated single yeast cells in 20-pl droplets (Additional files 1, 2 and 3). The cells were grown in the droplets off-chip for 16 h at 28 °C. The droplets were then loaded in an integrated screening device (Additional files 4 and 5) to: (1) picoinject [START_REF] Abate | High-throughput injection with microfluidics using picoinjectors[END_REF] the fluorogenic substrate (Additional file 6), ( 2) incubate the droplets on-chip as the enzymatic reaction took place (Additional file 7) and (3) analyze the fluorescence (i.e., enzymatic activity) of each droplets, which could then be sorted based on fluorescence [START_REF] Baret | Fluorescence-activated droplet sorting (FADS): efficient microfluidic cell sorting based on enzymatic activity[END_REF].

Encapsulation and growth of Y. lipolytica in 20 pl droplets

Droplet-based microfluidic approaches have previously been used with S. cerevisiae [START_REF] Agresti | Ultrahigh-throughput screening in drop-based microfluidics for directed evolution[END_REF][START_REF] Boitard | Monitoring single-cell bioenergetics via the coarsening of emulsion droplets[END_REF][START_REF] Sjostrom | High-throughput screening for industrial enzyme production hosts by droplet microfluidics[END_REF][START_REF] Wang | Microfluidic high-throughput culturing of single cells for selection based on extracellular metabolite production or consumption[END_REF][START_REF] Huang | Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast[END_REF] but never with Y. lipolytica. The encapsulation procedure had to be optimized to achieve reliable single cell encapsulation. First of all, microfluidic channels are usually treated to be both hydrophobic and fluorophilic to have proper surface channel wettability for droplets formation. However, Y. lipolytica displays an affinity for hydrophobic substrates [START_REF] Fickers | Hydrophobic substrate utilisation by the yeast Yarrowia lipolytica, and its potential applications[END_REF] and tended to adhere to and aggregate on channel walls when flowed in the dropmaker device. To prevent adhesion, cell suspensions were supplemented with 0.1% TWEEN ® 20 non-ionic detergent. In addition, tubing with a small inner diameter (100 μm)was used to increase shear stress and prevent the formation of cell aggregates prior to encapsulation. The cell suspensions were also continuously stirred during the encapsulation process to prevent cell sedimentation and variation in local cell density. Following these modifications, single Y. lipolytica cells were successfully encapsulated in 20 pl droplets. Droplets were produced at a rate of 2500 to 3000 per second via hydrodynamic flow focusing [START_REF] Anna | Formation of dispersions using flow focusing in microchannels[END_REF] of a cell suspension with a fluorinated oil phase containing 2.5% w/w KryJeffD 900 fluorosurfactant [START_REF] Beneyton | High-throughput screening of filamentous fungi using nanoliter-range droplet-based microfluidics[END_REF]. The number of yeast cells per droplet followed a Poisson distribution [START_REF] Clausell-Tormos | Droplet-based microfluidic platforms for the encapsulation and ccreening of mammalian cells and multicellular organisms[END_REF] and was controlled by adjusting the initial density of the cell suspensions to give an average number of cells per droplets, λ, of 0.03-0.1 (Additional file 8). Droplets were collected off chip in a glass vial and incubated for 16 h at 28 °C to allow cell growth and enzyme secretion. Based on image analysis, the droplets contained dozens of cells after incubation, showing that Y. lipolytica was able to replicate in the droplets.

Detection of enzymatic activities in droplets

In the first set of experiments, the two xylanase strains JMY4346 (xlnb) and JMY4510 (xlnc) were assayed. Two strain-specific emulsions were produced sequentially with a λ equal to 0.03. The strains were encapsulated in growth medium containing strain-specific concentrations of sulforhodamine B red fluorophore to optically encode droplets (10 and 50 μM for JMY4510 [xlnc] and JMY4346 [xlnb], respectively). The binary emulsion was collected in a glass vial, strains were grown for 16 h at 28 °C, and the droplets were then loaded into the integrated screening device. The xylobiose-based coumarin substrate was picoinjected into every droplet, and fluorescence was measured immediately after injection (t 0 , Fig. 4 a) and after 10 min of on-chip incubation (t outlet , Fig. 4 b). As expected, both strains displayed xylanase activity. For JMY4346 (xlnb) strain, a positive droplet population showing xylanase activity (2.7% of the droplets [theoretically 3% if λ = 0.03]; RFU: 18.4 ± 3.1%) clearly demarcated from the empty droplet main population (RFU: 1 ± 5.9%). In the same way, for JMY4510 (xlnc) strain, a positive population (2.4% of the droplets; RFU: 16.4 ± 3%) was well separated from the empty droplet population (RFU: 1 ± 7.1%). Both strains showed remarkably low variability in their signal distributions (coefficient of variation [CV] of 3%). This narrow phenotypic distribution at the single cell level within a monoclonal population is highly desirable for enzymatic screening applications and is due to a combination of two factors. First, the expression system developed in Y. lipolytica under a strong constitutive promoter is characterized by low variability in expression levels [START_REF] Cambon | A new Yarrowia lipolytica expression system: an efficient tool for rapid and reliable kinetic analysis of improved enzymes[END_REF]. Second, the integrated screening device allows for a high degree of control. The injection of the substrate to initiate the enzymatic reaction and the on-chip incubation along the delay line insure a precise control of the incubation time in each droplet. More specifically, the delay line structure design, made of constrictions and expansions, ensured that average droplet residence time, and hence incubation time, displayed little variance [START_REF] Frenz | Reliable microfluidic on-chip incubation of droplets in delay-lines[END_REF].

In a second set of experiments, strains JMY4346 (xlnb), JMY4469 (cbha), and JMY4407 (pep1) were assayed for xylanase, cellobiohydrolase, and protease activity, respectively. Three strain-specific emulsions were produced sequentially with a λ equal to 0.04, 0.08 and 0.03 for JMY4346 (xlnb), JMY4469 (cbha), and JMY4407 (pep1), respectively. The strains were encapsulated in growth medium containing strain-specific concentrations of sulforhodamine B red fluorophore to optically Following off-chip incubation, the droplets were loaded into the integrated screening device and were picoinjected with either the xylobiose-based coumarin substrate (Fig. 5a), the cellobiose-based coumarin substrate (Fig. 5b), or the protease substrate (Fig. 5c). Each strain showed hydrolytic activity against the respective substrate with comparable narrow signal distribution, while no activity was detected for the other strains.

These results demonstrate that our droplet-based microfluidic HTS system is an efficient and reliable method for detecting endoxylanase, cellobiohydrolase, and protease activity. The low variability obtained within monoclonal populations makes this approach very useful for screening enzyme libraries in the context of protein engineering or directed evolution.

Screening for thermostable mutants of the endo-β-1,4-xylanase C

Improving the thermostability of the enzymatic cocktail composed of endo-β-1,4-xylanase B and C would be of particular interest from a biotechnological perspective in the field of animal feed. The thermostability of both xylanases against a heat shock of 90 °C for 30 s was investigated (Fig. 6a). Crude supernatant from strains JMY4346 (xlnb) and JMY4510 (xlnc) were assayed in bulk with the xylanase substrate before and after the heat shock. Endo-β-1,4-xylanase C was found to be less thermostable than endo-β-1,4-xylanase B with residual activities of 3.7 and 71.7%, respectively.

We therefore focused on improving endo-β-1,4xylanase C thermostability and set up a screen for thermostable variants as proof of concept of our droplet-based microfluidic HTS system. Libraries were generated from xlnc gene using error-prone PCR. To evaluate how mutation frequency affects a library phenotypic distribution, we constructed two small libraries with different mutation frequencies: library A had a low mutation frequency (2 per kb) and library B had a medium mutation frequency (>4 per kb). Gene libraries were transformed in Y. lipolytica, yielding 800 clones for library A and 600 clones for library B. Using the dropletbased microfluidic HTS system described above, both libraries were analyzed before and after a heat shock of 90 °C for 30 s (Fig. 6b,c; Additional file 9). Before exposure to heat shock, 27% of the clones (216 clones) in library A and 9% of the clones (54 clones) in library B displayed xylanase activity. None of these clones were found to have greater activity than wild-type xylanase (>[mean wild-type activity + 3σ]). Those library phenotypic distributions were associated with mutation frequency: the higher mutation rate led to a lower number of active clones. After exposure to heat shock, 1% of the clones (8 clones) in library A were found to have higher activities than the wild-type xylanase (>[mean wild-type activity + 3σ]), while this percentage was 0.17% (1 clone) for library B. Therefore, our droplet-based microfluidic HTS approach revealed that 8 and 1 potential thermostable variants could be found in library A and B respectively.

To evaluate the sensitivity of our droplet-based microfluidic HTS system as compared to more classical, lowerthroughput microtiter plate methods, 480 clones were isolated from library A and placed in 96-well plates. Each was individually screened for xylanase thermostable activity. As above, prior to the heat shock, none of the clones were found to have higher levels of activity than wild-type xylanase (>[mean wild-type activity + 3σ]; data not shown). Following the heat shock, six clones demonstrated activity greater than that in the wild-type (>[mean wild-type activity + 3σ]), which corresponded to 1.25% of the library. These results are very similar to those obtained using the droplet-based microfluidic HTS approach. The six mutants with thermostable xylanase were individually grown in test tubes and enzyme activity levels in the supernatants were evaluated in duplicate before and after a heat shock (Additional file 10). All the mutants had an absolute xylanase activity level higher than that of the wild-type after a heat shock. Four of the six clones had more than 30% residual activity; the wildtype had only 5%. The sequencing of the mutated gene in the six clones reveals that they contain from 1 to 6 point mutations (average of 2.5) at the amino acid level (Additional file 11). Mutations are located all along the protein, with a higher density at the N-terminus (Additional file 11). Thus, the thermoresistance observed for these clones is likely to be a consequence of these mutations.

Conclusion

These results show that using a droplet-based microfluidic HTS approach with the Y. lipolytica expression system allowed for a rapid, efficient, and reliable kinetic analysis of recombinant enzymes at the single cell level. This study focused on the expression of fungal genes encoding hydrolytic enzymes of biotechnological interest, but the technique could be extended to any gene encoding for an enzymatic activity that could be detected using a fluorescent assay compatible with droplet-based microfluidics. Another application will be the screening of strains having altered metabolisms using fluorescent reporter systems. We have developed a precise, highthroughput and low-cost screening technique that can be used to compare and isolate improved enzymes within libraries. This will undoubtedly help to speed up the discovery of new enzymes via directed evolution or protein engineering strategies.

Methods

Strains and plasmids

The five fungal heterologous genes from A. niger strain CBS 513.88

-xlnb (endo-β-1,4-xylanase B), xlnc (endo- β-1,4-xylanase C), cbha (1,4-β-cellobiohydrolase A)
, egla (endoglucanase A), and pep1 (aspartic protease)-were synthesized with codon optimization by GenScript (Piscataway, NJ, USA) and in accordance with the codon usage in Y. lipolytica. Codon optimized genes were cloned into a JMP62-type vector [START_REF] Nicaud | Protein expression and secretion in the yeast Yarrowia lipolytica[END_REF][START_REF] Pignède | Autocloning and amplification of LIP2 in Yarrowia lipolytica[END_REF] under the control of the strong and constitutive pTEF promoter [START_REF] Müller | Comparison of expression systems in the yeasts Saccharomyces cerevisiae, Hansenula polymorpha, Klyveromyces lactis, Schizosaccharomyces pombe and Yarrowia lipolytica. Cloning of two novel promoters from Yarrowia lipolytica[END_REF]. The vectors used in this study are listed in Additional file 12. Expression vectors were digested with the restriction enzyme NotI (New England Biolabs, Evry, France) to release the expression cassettes. The gel-purified expression cassettes were then used to transform Y. lipolytica strain JMY2566 [START_REF] Leplat | High-throughput transformation method for Yarrowia lipolytica mutant library screening[END_REF] using the lithium acetate method [START_REF] Dall | Multiple-copy integration in the yeast Yarrowia lipolytica[END_REF],

giving rise to strains JMY4346 (xlnb), JMY4510 (xlnc), JMY4469 (cbha), JMY4343 (egla), and JMY4407 (pep1).

Transformants were selected on YNB plates. Genomic DNA from the positive transformants was prepared as described elsewhere [START_REF] Querol | Molecular monitoring of wine fermentations conducted by active dry yeast strains[END_REF], and integration of the overexpression cassette at the specific zeta locus was confirmed by PCR using gene-specific primers (Additional file 13) and a primer located in the zeta locus (5'-TCTTCTGC CTCCAGGAAGTC-3'). DNA fragments were recovered from agarose gels using a QIAquick Gel Extraction Kit (Qiagen, Hikden, Germany). The QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany) was used for plasmid purification. All the reactions were performed in accordance with the manufacturer's instructions.

Growth conditions

Escherichia coli strains were grown according to standard protocols [START_REF] Sambrook | Molecular cloning: a laboratory manual[END_REF] in Luria-Bertani broth medium containing 50 μg/ml kanamycin. For the yeast inoculum, rich YPD medium containing 1% (w/v) yeast extract, 1% (w/v) peptone, and 2% (w/v) glucose was used. Transformants were selected on minimal YNB medium, composed of 0.17% (w/v) yeast nitrogen base (without amino acids and ammonium sulfate; BD Difco), 0.5% (w/v) NH4Cl, 50 mM phosphate buffer (pH 6.8), and 2% (w/v) glucose. When necessary, leucine and uracil were added to a final concentration of 0.1 g l. Solid media were complemented with 1.6% agar. Single colonies were grown for 24 h at 28 °C in 2 ml of YNB glucose medium (YNB without amino acids [1.7 g/l], yeast extract [1.5 g/l], glucose [10 g/l], NH4Cl [100 mM], and KH 2 P0 4 /Na 2 HPO 4 buffer [50 mM, pH 6.8]). 20 ml of fresh YNB glucose medium were inoculated at an OD 600 value of 0.05. The culture was grown at 28 °C until it reached an OD 600 value of 0.4-0.6, in the microfluidic experiments, or for 24 h for bulk measurements of hydrolytic activities and SDS analysis.

In the mutant library experiment, fresh YNB glucose medium was inoculated with -80 °C glycerol cell suspensions with an OD 600 value of 0.05.

For growth in 96-well plates, yeasts were precultivated for 48 h in YPD medium in microtiter plates. 5 μl of the precultures were then transferred to 120 μl of fresh YNB medium and grown for 30 h at 28 °C on an orbital shaker at 170 rpm.

SDS-PAGE analysis

20 μl of crude supernatant were analyzed electrophoresis on a 10% NuPAGE Bis-Tris-acetate sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) (Thermo Fisher Scientific, Villebon-sur-Yvette, France). After migration, the gel was subject to colloidal blue staining.

Primary enzymatic activity detection on yeast transformants

Cells were grown for 24 h and then removed via centrifugation (6000 rpm for 20 min, 28 °C). Xylanase, cellulase, or protease activity levels in the crude supernatants were monitored using adapted fluorescence assays in a Spectramax M5 spectrofluorometer (Molecular Devices, Wokingham, UK).

Xylanase assay

25 μl of crude supernatant was added to 25 μl of a 300 μM solution of β-d-xylobioside-6,8-difluoro-7hydroxycoumarin-4-methansulfonate in acetate buffer (100 mM, pH 4.5). The reaction was monitored at 25 °C for 30 min (λ ex = 360 nm, λ em = 460 nm).

Cellobiohydrolase assay

25 μl of crude supernatant was added to 25 μl of a 300 μM solution of β-d-cellobioside-6,8-difluoro-7hydroxycoumarin-4-methansulfonate in acetate bufffer (100 mM, pH 4.5). The reaction was monitored at 25 °C for 30 min (λ ex = 360 nm, λ em = 460 nm).

Protease assay

25 μl of crude supernatant was added to 25 μl of a 50 μg/ ml solution of Enzchek Protease Assay substrate (Thermo Fisher Scientific, Villebon-sur-Yvette, France) in acetate buffer (100 mM, pH 4.0). The reaction was monitored at 25 °C for 30 min (λ ex = 480 nm, λ em = 518 nm).

Droplet-based microfluidics Fabrication of microfluidic devices

Poly-(dimethylsiloxane) (PDMS) microfluidic devices were fabricated as previously described [START_REF] Beneyton | CotA laccase: high-throughput manipulation and analysis of recombinant enzyme libraries expressed in E. coli using droplet-based microfluidics[END_REF] from 20 or 70 μm-deep molds of SU-8 2025 and SU-8 2050 negative photoresists (MicroChem Corp), respectively.

Optical setup, data acquisition and control system for droplet analysis

The optical setup used to monitor microfluidic experiments has previously been described, as well as the data acquisition and control system [START_REF] Najah | New glycosidase substrates for droplet-based microfluidic screening[END_REF]. In addition, to allow the sorting of a particular droplet, the data acquisition card provided a signal to a model 623B high-voltage amplifier (Trek Inc.) connected to the electrodes of the microfluidic device.

Surfactant synthesis

We used aqueous droplets in Novec7500 fluorinated oil (3M) stabilized against coalescence by a triblock copolymer fluorosurfactant comprising two perfluoropolyether (PFPE) chains linked by one Jeffamine ® polyetheramine chain (PEA), KryJeffD 900 . KryJeffD 900 surfactant was prepared in house as previously described [START_REF] Beneyton | High-throughput screening of filamentous fungi using nanoliter-range droplet-based microfluidics[END_REF].

Microfluidic chip operation

Droplets were produced using a dropmaker device (Additional file 1) by flow-focusing the cell suspension stream with two streams of Novec7500 oil containing 2.5% (w/w) of KryJeffD 900 surfactant. The liquids were pumped into the dropmaker using standard infusion/ withdraw PHD 22/2000 syringe pumps (Harvard Apparatus Inc.). For the oil steams, a syringe (Injekt ® ; BBRAUN) was connected to the dropmaker using a 0.6 × 25 mm needle (Terumo) and PTFE tubing (Fisher Scientific) with an inner diameter (ID) of 0.56 mm and an outer diameter (OD) of 1.07 mm. For the cell suspension stream, a glass syringe (Hamilton Gastight 1002) was connected to the dropmaker using 1/32" fluidic connections with FEP tubing (ID 102 μm, OD 0.79 mm). The dropmaker was used to produce 20 pl droplets (Q aqueous 150 μl/h, Q oil 400 μl/h, 2000-3000 droplets/s). The droplets flowed off chip through PTFE tubing (IDEX) to a glass vial attached to a Peltier device, where they were incubated at 28 °C [START_REF] Mazutis | Droplet-based microfluidic systems for high-throughput single DNA molecule isothermal amplification and analysis[END_REF]. The droplets were then loaded into the integrated screening device (Additional file 4). The liquids were pressure driven into the device via PTFE tubing (ID 0.56 mm, OD 1.07 mm) using a microfluidic flow control system (MFCS [2 × 2 bar inlets, 2 × 1 bar inlets], Fluigent). Droplets were reloaded (P droplets 500-700 mbar) and spaced with Novec7500 oil (P oil 500-700 mbar). The substrate was picoinjected (P substrate 500-700 mbar, 200-300 droplets/s) by applying an AC field (20kHz, 200 V pp ), and the droplets were incubated on chip after oil extraction (Q extrac -25 to -100 μl/h). The droplets were spaced after incubation (Q spacing 200-1000 μl/h) and analyzed by the optical setup. Fluorescent droplets could be sorted at a rate of 300 droplets/s by applying a AC field pulses (30 kHz; 1200 V pp ; 0.3-0.6 ms).

Strain activity in droplets

Yeast suspension 20 ml of fresh YNB glucose medium was inoculated at an OD 600 value of 0.01. The strains were cultured at 28 °C until an OD 600 value of 0.4-0.6 was reached. 15 ml of culture were then centrifuged (6000 rpm, 10 min, 4 °C), and the resulting pellet was washed with fresh YNB glucose medium. This washing step was repeated three times, and the pellet was resuspended in 1 ml of fresh YNB glucose containing 0.1% TWEEN ® 20 and 10 μM sulforhodamine B (barcoding dye). The OD 600 was measured, and the yeast suspension was then diluted to achieve the desired number of cells per droplet volume (an OD 600 of 1 corresponds to 3.4 × 10 7 cells per ml).

Yeast encapsulation

The cell suspension was encapsulated in 20-pl droplets using the dropmaker. A cylindrical PTFE magnetic stirring bar was used to avoid cell sedimentation in the syringe during the encapsulation process. The droplets were collected in a glass vial. For multiple strains emulsions, the strains were encapsulated sequentially using different level of coding dye and collected together in a single glass vial. The yeasts were cultured in the droplets at 28 °C for 24 h.

Assays in droplets

The droplets were loaded into the integrated screening device, and the substrate was picoinjected into every droplets (typically 5 pl in 20 pl droplets). To assay xylanase activity, the xylobiose-based substrate (150 μM in acetate buffer [50 mM, pH 4.5]) was used. To assay cellobiohydrolase activity, the cellobiose-based substrate (150 μM in acetate buffer [50 mM, pH 4.5]) was used. Fluorescence was then exited using a 375 nm laser. To assay protease activity, the Enzchek Protease Assay substrate (150 μg/ml in acetate buffer [50 mM, pH 4.0]) was used. Fluorescence was then exited using a 488 nm laser. Droplet fluorescence was measured just after injection (reaction initiation, t 0 ) and after on-chip incubation (10 min at room temperature, t outlet ).

Screening for thermoresistant xylanase Construction of error prone xlnc mutant libraries

The expression cassette from plasmid JME2603 (Additional file 12) is flanked by zeta regions and is composed of a URA3 marker, the pTEF promoter, and the xlnc gene (Additional file 14). We built libraries of error-prone xlnc variants using a two-step PCR amplification according to [START_REF] Bordes | Isolation of a thermostable variant of Lip2 lipase from Yarrowia lipolytica by directed evolution and deeper insight into the denaturation mechanisms involved[END_REF]. First, the PCR fragment composed of Zeta1, the URA3 marker, and the pTEF promoter was amplified using Zeta1for (5'-GCGGCCGCTGTCGGGAACCG-3') and ep-PCRrev (5'-CCGATCCTTCGGGTGTGAGTT-GACAAGGAG-3') as primers and JME2603 as a template under standard conditions (100 pmoles of each primer, 5 ng of DNA template, 0.25 mM dNTP, 1U Pyrobest [Takara], and 1X Pyrobest buffer in a final 50 μl), which produced a 2037-bp fragment (Additional file 14 PCR1). Then, the PCR fragment composed of xlnc and Zeta2 was amplified using epPCRfor (5'-CTCCTTGT-CAACTCACACCCGAAGGATCGG-3') and Zeta2rev

(5'-GCGGCCGCACTGAGGGCTTTG-3') as primers and JME2603 as a template under mutagenic conditions (epPCR), which produced a 1557-bp fragment (Additional file 14; PCR2). A low-fidelity Taq DNA polymerase (NEB) was used, as per standard procedure; we also added either 1 or 2 μl of a dNTP mutation mix (8 mM dTTP, 8 mM dCTP, 96 mM MgCl 2 , and 10 mM MnCl 2 in water) to generate a low mutation rate library A or a medium mutation rate for library B, respectively. Primers epPCRrev and epP-CRfor are reverse and complementary. The PCR fragments were recovered from agarose gels using a QIAquick Gel Extraction Kit (Qiagen). A fusion PCR was then performed under standard conditions using Zeta1for and Zeta2rev as primers and an equimolar proportion of both fragments as templates (Additional file 14; PCR3). The resulting PCR fragments containing the full mutated expression cassette were used to transform Y. lipolytica JMY2566 as described in the section on strain and plasmid.

Library analysis in microfluidics

The JMY4510 (xlnc) wild-type, library A, and library B were encapsulated in 20 pl droplets as described above. After 16 h of incubation at 28 °C, the droplets were loaded into the integrated screening device to assay xylanase activity as described above. To generate the heat shock, the droplets were run through PTFE tubing (0.56 mm × 1.07 mm) immersed in a 90 °C water bath. The flow rate was set such that residence time within the tubing was 30 s. The droplets were then collected in a glass vial and reloaded into the integrated screening device to assay post-heat-shock levels of xylanase activity.

Xylanase activity screening of mutant library in microtiter plate

The microtiter plate cultures were centrifuged, and the supernatants were transferred to new microtiter plates. Xylanase activity was assayed using the EnzChek Ultra Xylanase Assay Kit (Thermo Fisher Scientific, Villebonsur-Yvette, France). Briefly, 5 μl of supernatant was diluted in 45 μl of buffer. 25 μl of substrate (50 μg/ml) was added, and the resulting fluorescence was monitored (Biotek Mx microtiter plate reader, em = 360 nm, em = 460 nm) under constant agitation for 30 min. Vmax was calculated using a five-point window. The supernantants were then exposed to a heat shock event (90 °C, 30 s) and placed on ice. Xylanase activity was assayed as described above.

Fig. 1 FluorescenceFig. 2

 12 Fig. 1 Fungal gene expression SDS-PAGE profiles of producing strains. a Genetic background. Lane 1, crude supernatant from empty strain JMY2810; b producing strains. Lanes 1-5, crude supernatant from respectively strains JMY4407 (aspartic protease; 41 kDa), JMY4510 (endo-β-1,4-xylanase C; 33.5 kDa), JMY4343 (endo-β-1,4-xylanase B; 22 kDa), JMY4346 (endoglucanase A; 26 kDa) and JMY4469 (1,4-β-cellobiohydrolase A; 48 kDa)

Fig. 3

 3 Fig.3Droplet-based microfluidic screening platform. 1. Single cell encapsulation: yeast cells were encapsulated in 20 pl droplets at 1200 droplets/s by flow-focusing of a cell suspension with two streams of Novec7500 oil containing 2.5% (w/w) KryJeffD surfactant. Droplets were collected offchip in a glass vial. 2. Cell growth and enzyme secretion: droplets were incubated for 16 h at 28 °C. 3. Enzymatic screening: droplets were loaded into an integrated device and spaced using Novec7500 oil containing 2.5% (w/w) KryJeffD 900 surfactant. To initiate the enzymatic reaction, a fluorogenic substrate was picoinjected in each droplet at a rate of 300 droplets/s by applying an AC field (20 kHz, 200 V pp ). Droplets were incubated on-chip along a delay line and spaced with Novec7500 oil for fluorescence detection (i.e. enzymatic activity). Droplets could be sorted based on fluorescence at 300 droplets/s by applying AC field pulses (30 kHz; 1200 V pp ; 0.3-0.6 ms). a-f Microscopic images of the different steps of the microfluidic system. The red arrows indicate encapsulated Y. lipolytica cells. Unless specified, scale bars are 30 μm

Fig. 4

 4 Fig. 4 JMY4346 ( xlnb) and JMY4510 ( xlnc) strains activity in droplets 2D histograms showing the blue fluorescence (xylanase activity) and the red fluorescence (barcoding) of a binary 20 pl droplets emulsion containing JMY4346 (xlnb) and JMY4510 (xlnc) strains analyzed in the integrated screening device just after injection of the xylanase substrate (a) and after on-chip incubation (b). On the right are displayed the 1D histograms showing the number of droplets observed as a function of the xylanase activity (blue fluorescence) corresponding to each droplet population (JMY4510 (xlnc) and JMY4346 (xlnb) strains)

Fig. 5

 5 Fig. 5 JMY4346 (xlnb), JMY4469 (cbha) and JMY4407 ( pep1) strains activity in droplets 1D histograms showing the number of droplets observed as a function of the enzymatic activity (blue or green fluorescence) for a ternary emulsion containing JMY4346 (xlnb), JMY4469 (cbha) and JMY4407 (pep1) strains. The emulsion was analyzed in the integrated screening device just after injection of the relevant enzymatic substrate (left) and after on-chip incubation (right) with the xylanase substrate (a), the cellulase substrate (b) and the protease substrate (c)

Fig. 6

 6 Fig. 6 Screening for thermostable variants of endo-β-1,4-xylanase C. a Thermostability of endo-β-1,4-xylanase B and C. Xylanase activity of the crude supernatant for the strains JMY4510 (xlnc; black line) and JMY4346 (xlnb; red line) before (continuous line) and after (dotted line) a heat shock (90 °C, 30 s). The negative control (i.e., growth medium) is shown in gray. Error bars correspond to ±1 standard deviation (N = 3). b Microfluidics library analysis. The two libraries A and B and the parental strain (JMY4510 (xlnc)) were analyzed using the integrated microfluidic screening device.The histograms show the general percentage of variants displaying activity (left) and the proportion of variants with activity levels greater than of the parental strain (right) for the wild-type and each library before (black) and after (white) heat shock (90 °C, 30 s). c 1D histograms showing the number of droplets observed as a function of the xylanase activity before and after heat shock for the wild-type, library A and library B. The mean signal for the wild-type is indicated with a green line, and values σ and >3 σ are to the right of the red lines. Strains were considered to be positives when displaying a xylanase activity higher than (mean wild-type activity + 3 σ)

Acknowledgements

We thank Estelle Mayot for synthesizing the xylobiose-and cellobiose-based substrates as well as the surfactant. This work was supported by Bpifrance and Ets. J. Soufflet (OSIRIS project). This work used microfluidics tools belonging to Ets. J. Soufflet and hosted by ESPCI, Paris. We would also like to thank Jessica Pearce for its language editing services.

Availability of data and materials

Please contact corresponding author for data requests.

Authors' contributions TB, AD, JMN and TR designed the experiments. TB conducted the experiments, collected the data, and analyzed the experiments. ST constructed the strains and the libraries. TR performed the microtiter-based screening of thermostable xylanase. AD, AG, JMN and TR conceived the project and supervised the experiments. TB, TR, and JMN wrote the manuscript. All authors read and approved the final manuscript.

Author details 1 Ecole Supérieure de Physique et de Chimie industrielles de la Ville de Paris (ESPCI Paris), CNRS UMR 8231, 10 rue Vauquelin, 75005 Paris, France. 2 CNRS, University of Bordeaux, CRPP, UPR 8641, 115 Avenue Albert Schweitzer, 33600 Pessac, France. 3 Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, 78352 Jouy-en-Josas, France. 4 Ets J. Soufflet/CRIS-OSIRIS, Quai Sarrail, BP12, 10400 Nogent-sur-Seine, France.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Our manuscript does not contain any individual person's data in any form.

Ethics approval and consent to participate

Our manuscript does not report data collected from humans or animals. S2. Y. lipolytica and E. coli strains used in this study.

Additional file 12: Table

Additional file 13: Table S3. List of primers used to check cassette integration at the specific Zeta locus.

Additional file 14: Figure S6. Construction of xlnc libraries using errorprone PCR. The construction of xlnc mutant libraries was obtained by PCR amplification in two steps. First step, the PCR fragment composed of Zeta1, URA3 marker and pTEF promoter was amplified using primers Zeta-1for and epPCRrev using the JME2603 plasmid as a template (top figure) under normal PCR condition (PCR1). Then, the PCR fragment composed of xlnc and Zeta2 was amplified using primers epPCR-for and Zeta2rev and the JME2603 plasmid as a template (top figure) under epPCR mutagenic conditions (PCR2). Second step, a fusion PCR was performed under standard PCR conditions using an equimolar proportion of both fragments as templates and using primers Zeta1for and Zeta2rev (PCR3).


Seydou Ouedraogo

Tahirou Coulibaly

Rita Meunier-Prest

Mabinty Bayo-Bangoura

Marcel Bouvet
email: marcel.bouvet@u-bourgogne.fr

Mabinty Bayo- Bangoura

p-Type and n-type conductometric behaviors of octachloro-metallophthalocyanine-based heterojunctions, the key role of the metal

Keywords: molecular semiconductor, conductometric transducer, gas sensor, ammonia, devices

The electrical properties of octachloro-phthalocyanines are determined. In combination with LuPc2, they are engaged in heterojunctions used as ammonia sensors. Cobalt, copper and zinc complexes exhibit dramatically different electrical behaviors. Co(Cl8Pc) is a n-type material, Cu(Cl8Pc) a p-type material and Zn(Cl8Pc) is an ambipolar material.

INTRODUCTION

Among porphyrinoid complexes, highly used as sensing materials in chemical sensors [1][2][3][4][5], with acoustic, conductometric and optical transducers, phthalocyanine complexes are mainly associated with conductometric transducers.

In metallophthalocyanine complexes, the metal center plays a key role in their magnetic properties and catalytic activies [6].

On the contrary, the electrical properties do not depend on the nature of the metal center, but rather on the electronic density in the macrocycle. Indeed, the frontier molecular orbitals, HOMO and LUMO, involved in the creation of charge carriers are of -type and localized on the phthalocyanine ring. Thus, in molecular materials, it is well known that the energy gap is directly related to the difference between the first reduction and first oxidation potentials determined for the molecule in solution when they deal with the macrocycle [7]. In addition, even the nature of majority charge carriers depends on the electron-donating, or electron-withdrawing character of the substituents. Thus, perfluorinated phthalocyanines are n-type materials whereas the non substituted phthalocyanines are p-type, as a result of the stabilization of the occupied and empty energy levels, as determined by photoelectron spectroscopy [8]. As a consequence, perfluorinated phthalocyanines lead to organic field-effect transistors with a n-type channel [9] and were used as n-type materials in heterojunctions [10][11][12][13]. On the contrary, chlorinated phthalocyanines were rarely reported, certainly because of their higher sublimation temperature, in relation with their higher molecular weight and stronger van der Waals interactions compared to fluorinated compounds. However, we can cite the work of Pakhomov showing the decrease in conductivity of perchloro-copper phthalocyanine under nitrogen dioxide, which indicates the n-type nature of majority charge carriers in this materials, contrarily to tetrachloro-and unsubstituted analogues [14]. Very thin films of octachloro-zinc phthalocyanine were studied by scanning tunneling microscopy under ultra-high vacuum, showing the molecular packing was driven by C-Cl…H-C hydrogen bonds between adjacent molecules, but their electrical properties were not determined [15]. The electrical properties of tetra, octa and hexadeca-chloro-cobalt phthalocyanines were also reported, but on pressed pellets and without determination of the nature of charge carriers [16].

The metal center plays generally a minor role on the electrical properties of phthalocyanines. Only one study of the sensitivity of metallophthalocyanine-based chemiresistors (MPc, M = Co, Ni, Cu, Zn and 2H) towards a series of vapor phase electron-donating gases was reported [17]. Their responses and recovery times were exponentially correlated with the binding enthalpy of analytes with the sensing materials. Even though their responses to one gas were not very different from each other, the analysis of the relative sensitivities of all studied MPcs allowed to distinguish among the series of analytes, regardless of their concentrations. It is a remarkable example of the metal effect on the sensing properties of phthalocyanines.

In the present paper, we report on the electrical properties of 2,3,9,10,16,17,23,24-octachloro-cobalt phthalocyanine (1) and 2,3,9,10,16,17,23,24-octachloro-copper phthalocyanine (2). Films were deposited by evaporation under secondary vacuum and characterized by Raman spectroscopy and current-voltage (I(V)) responses. Heterojunctions were prepared from these two molecules as sublayers, in association with the lutetium bisphthalocyanine, LuPc2, and used to detect ammonia in the ppm range. This type of conductometric transducer was designed and patented by one of the authors [13,18,19]. EXPERIMENTAL Chemicals 2,3,9,10,16,17,23,24-octachloro-cobalt phthalocyanine (1). A mixture of 4,5-dichlorophthalic anhydride (0.5 g, 2.72 mmol), cobalt (II) acetate tetrahydrate (0.169 g, 0.8 mmol), of urea (1.4 g, 3 mmol) and ammonium heptamolybdate (0.015 g) was refluxed in nitrobenzene for 5 hours. The solid obtained was filtered off, washed successively with ethanol and distilled water, then treated by hot 10% aqueous HCl solution, followed by successive washings with toluene, ethanol, methanol and acetone in a soxhlet. 0.288 g (0.4 mmol) of an intense blue color powder was obtained. Yield = 60%. Anal.

UV-visible, Infra-red and Raman spectroscopies, and mass spectrometry

UV-visible electronic absorption spectra were recorded on a Shimadzu UV-2600 spectrophotometer. FT-IR spectra were recorded on a Bruker Vector 22 spectrometer in transmission mode, using KBr pellets. Raman spectroscopy was conducted employing a Renishaw inVia Raman microscope with a 473 nm laser. Mass spectra were recorded on a MALDI (matrix assisted laser desorption ionization) BRUKER Proflex III spectrometer, using 1,8,9-anthracenetriol as a matrix.

Devices and electrical measurements

Electrical measurements were carried out with Indium Tin Oxide (ITO) interdigitated electrodes (IDE), separated by 75 m, lithographied on a 1 x 1 cm² glass substrate. Thin films of phthalocyanines were prepared by sublimation under secondary vacuum (ca. 10 -6 mbar) in a UNIVEX 250 thermal evaporator (Oerlikon, Germany), by heating in a temperature range of 400-450°C for LuPc2, and in the range 500-550°C for the chlorinated phthalocyanines. The deposited thicknesses were 30 nm for each chlorinated phthalocyanine complex and 50 nm for LuPc2, as determined by a quartz cristal microbalance during the deposition process. The workbench used for NH3 exposure, at different relative humidity (rh) values, was described previously [20]. Synthetic air, ammonia gas, 1000 ppm and 100 ppm in synthetic air were used from standard cylinders, purchased from Air Liquide, France. The total flow was in the range 0.5-0.55 NL.min -1 depending on ammonia concentration and the volume of the test chamber was 8 cm 3 . Gas sensing experiments were carried out in a dynamic fashion, with rest periods alternating with exposure periods, the length of which are precised in the text. In the present study, all the electrical measurements were carried out at the lab temperature (18-22 °C) unless otherwise specified.

RESULTS AND DISCUSSION

Synthesis and characterization of octachloro-metallophthalocyanine complexes

The cobalt and copper octachloro-phthalocyanines 1 and 2 were synthesized from the 4,5-dichlorophthalic anhydride, by reaction with metal acetate, in the presence of urea and ammonium heptamolybdate, by reflux in a solvent, as previously reported for unsubstituted phthalocyanines (Scheme 1) [START_REF] Mckeown | The Synthesis of Symmetrical Phthalocyanines[END_REF]. Even though the 4,5-dichlorophthalonitrile was obtained from the 4,5-dichloroanhydride [START_REF] Wöhrle | [END_REF][START_REF] Sharman | Synthesis of Phthalocyanine Precursors[END_REF], octachloro-metallophthalocyanines were mainly synthesized from the corresponding phthalimide, based on a method firstly reported by Linstead [START_REF] Barrett | [END_REF]25]. MALDI-TOF high resolution mass spectra of compounds 1 and 2 show a parent ion at that corresponds to [M] + . Thus, for compound 1, the most intense peak lies at m/z = 846.6399, for a calculated value of 846.7658. The isotopic profiles of obtained and calculated peaks are given in Supplementary Materials (Fig. S1). For compound 2, the most intense peak lies at m/z = 850.6697, for a calculated value of 850.7545.

Films characterizations

The electronic absorption spectra of bilayers were the superimposition of the spectra of each material, as shown in Figure 1 for the LuPc2/2 bilayer, indicative of no significant charge transfer between the two layers. The maximum wavelength, at 668 nm, belongs to LuPc2 and the shoulders at 635 and 701 nm belong to 2. The weak band at 470 nm corresponds to a transition of a filled orbital towards the semi-occupied orbital of LuPc2 [26,27]. The same type of spectrum was observed for LuPc2/1 (Fig. S2).

Raman spectroscopy analysis was conducted on powders of 1, 2 and LuPc2 and on the devices made from films of 1 and 2 covered by LuPc2. The results are compiled in Table 1. The Raman spectra show peaks that can be directly attributed to the two layers, as shown by the superimposition of the spectra of sublayers, LuPc2 and heterojunctions (Fig. S3 and S4), and examplified in the range 1350-1650 cm -1 for 2 (Fig. 2 right). Thus, for the LuPc2/2 device, peaks can be directly attributed to the LuPc2 layer [28], in particular the peaks at 578 cm -1 corresponding to Pc breathing, at 781 cm -1 to C=N aza breathing and at 1158 cm -1 to pyrrole breathing (Table 1). The one at 1335 cm -1 is attributed to C=C pyrole and benzene stretching and this at 1408 cm -1 to isoindole stretching. The peak at 1513 cm -1 corresponds to C=C pyrrole stretching and coupling of pyrrole and aza stretching. Other peaks can be assigned to the sublayer, at 659, 812, 864, 1051, 1531 and 1581 cm -1 .

Additionally, peaks are common to both materials, such as the peaks at 547 cm -1 (Pc breathing), 1121 cm -1 , 1175 cm 1 (C-H bending), 1206 and 1302 cm -1 (C-H bending) and 1600 cm -1 (benzene stretching). These results confirm that the vacuum evaporation of 1 and 2 has been achieved correctly.

Electrical properties

Resistors made from 1 and from 2 exhibited a very high resistivity, estimated to be ca. 10 9 -10 10 .cm. However, they were engaged as sublayers in heterojunctions, by vacuum evaporation of lutetium bisphthalocyanine, LuPc2, 50 nm in thickness, on films of 1 and 2. The I(V) characteristics of heterojunctions are non linear, but symmetrical, with a current value at +10 V of ca. 6.3 µA for LuPc2/1 and 3 µA for LuPc2/2 (Fig. 3). The conductivity value remained in the same decade for both devices. The observed difference could come from a slight different in morphology. The non linearity clearly shows that an energy barrier exists between the two materials or/and between the electrodes and the sublayers. A plateau exists at low voltages, but above a given potential the I(V) curve is linear. The non linearity of these curves can be characterized by a threshold voltage obtained by the intercept of the linear part of curves with the x-axis. Thus, for LuPc2/1 and LuPc2/2 devices, values of 1.86 V and 2.75 V were obtained, respectively, showing that the energy barrier is slightly higher with 2.

Chemical sensors

We first studied the devices at 90 ppm NH3, with long exposure/recovery cycles, namely 10 min/40 min cycles (Fig. 4). The two devices behave differently; the current increases under NH3 for LuPc2/1 whereas it decreases for LuPc2/2. LuPc2/1 exhibits a n-type behavior whereas LuPc2/2 exhibits a p-type behavior. The relative response, RR, defined as is ca. +50% for LuPc2/1 against -55% for LuPc2/2. For the first time, it appears that the nature of charge carriers changed with the metal in the phthalocyanine complex. It is worth noting that LuPc2, an intrinsic semiconductor, owns positive majority charge carriers in air, with a decrease of the current under NH3 for LuPc2 resistors. However, they are poor ammonia sensors due to a low RR value and an important drift of the baseline [29]. For understanding the particular electrical behavior of octachloro-phthalocyanines, we need to remind us of this of the octafluoro-copper phthalocyanine, Cu(F8Pc), which was reported as an ambipolar material [30]. Depending on the experimental conditions, the Cu(F8Pc) -based conductometric device exhibits a positive or a negative response towards ammonia. Likewise a balance that owns only one equilibrium state but an infinity of unbalanced positions, the ambipolarity can be achieved only if the positive and negative mobile charges participate equally to the electrical current. Obviously, theoretically this condition is attained with intrinsic semiconductors, but its observation can be prevented by trapping effects and specific interactions with environment. In the present case, the change of the metal center can bring the small variation necessary to move the system from a p-type to a n-type behvior.

The effect of the NH3 concentration was studied during short exposure/recovery cycles (1 min/4 min), in the range 10-90 ppm (Fig. 5). For LuPc2/1, except at the first exposure period, the response was stable cycle after cycle and dependent of the NH3 concentration, with a RR value of 84% at 90 ppm and 13% at 10 ppm. The LuPc2/2 heterojunction exhibits also a very stable response, with a lower RR, of -27% and -7% at 90 and 10 ppm, respectively. However, the difference between the RR values at 90 and 60 ppm is very small, showing that a saturation occurs in this concentration range. To evidence this phenomenon, we studied also the devices at lower NH3 concentrations, in the 1-9 ppm range (Fig. 6). For LuPc2/2, at 1 ppm, the limit of detection (LOD) seems attained. Taking into account the baseline current I0 and the noise N at 1 ppm, on the I(t) curve, LOD can be calculated (Eq. 1):

(1) By plotting the RR value as a function of the NH3 concentration (Fig. 6 right), two domains can be defined with different below 10 ppm NH3, with a LOD of 250 ppb. Such a value is of the same order of magnitude as the best ammonia sensors operating at room temperature. Thus, conducting polymer -carbone nanotube based resistors exhibit a LOD of 200 ppb [31,32]. However, other reported heterojunctions, based on the combination of a conducting polymer with LuPc2, exhibited a LOD of 314 ppb [33] and 450 ppb [34], in the same ammonia concentration range, depending on the nature of the polymer and on the geometry of the heterojunction.

CONCLUSION

We showed that the octachloro-phthalocyanines can behave as p-type or n-type materials, depending on the metal center.

The cobalt complex exhibits a n-type behavior whereas the copper complex exhibits a p-type behavior. As expected for monophthalocyanines, their conductivity is low, but their combination with LuPc2 allowed forming a heterojunction capable to detect ammonia in the ppm range. We have to keep in mind that, in the same experimental conditions, LuPc2 resistors exhibit a very bad response to ammonia, in particular with an important drift of the baseline [29]. The sensitivity of the devices comes from the radical nature of lutetium bisphthalocyanine, which makes it easily oxidized and reduced. However, the key role of the octachlorinated phthalocyanine as a sublayer in the heterojunction is clearly established, since the ppm NH3, was obtained with the cobalt complex as a sublayer, with a LOD of 250 ppb, which makes it among the best ammonia sensors operating in ambient air and at room temperature. The copper complex is less sensitive with a LOD of ca.

1 ppm in the same conditions.

2, 3 , 9 ,

 39 10,16,17,23,24-octachloro-copper phthalocyanine (2). A mixture of 4,5-dichlorophthalic anhydride (0.5 g, 2.72 mmol), copper (II) acetate tetrahydrate (0.169 g, 0.8 mmol), urea (1.4 g, 3 mmol) and ammonium heptamolybdate (0.015 g) was refluxed in nitrobenzene for 5 hours. The solid obtained was filtered off, washed successively with ethanol and distilled water, then treated by hot 10% aqueous HCl solution, followed by successive washings with toluene, ethanol, methanol and acetone in a soxhlet. 0.288 g (0.4 mmol) of an intense blue color powder was obtained. Yield = 60%. Anal. calcd. for C32H8Cl8N8Cu: C, 45.13; H, 0.95; N, 13.16; found: C, 44.1; H, 0.87; N, 13.21. MALDI-TOF-MS (matrix DIT) m/z: 850.6697 (M .+ ); calculated for C32H8Cl8N8Cu: 850.7545. FT-IR, (, cm -1 ): 1604, 1512, 1410, 1387, 1341, 1297, 1201, 1140, 1086, 1071, 965, 889, 851, 785, 749, 705, 660, 548, 501, 432.

24 Scheme 1 .

 241 Scheme 1. Synthetic route to obtain octachloro-phthalocyanines 1 (M = Co), 2 (M = Cu) and 3 (M = Zn).

Figure 1 .

 1 Figure 1. UV-vis electronic absorption spectra of a film of LuPc2/2 on glass (black dashed line), compared to this of a film of 2 (red solid line).

Figure 2 .

 2 Figure 2. (left) Raman spectrum of a LuPc2/2 heterojunction. The assignment of peaks characteristic of both materials is given; (right) superimposition of spectra of 2 (dotted line), LuPc2 (solid line) and LuPc2/2 (dashed line) in the range 1350-1650 cm -1 ; the Raman shift value of the most intense peaks of the two components is given.

Figure 3 .

 3 Figure 3. Typical I(V) characteristics of LuPc2/1 (dashed line) and LuPc2/2 (dotted line) heterojunctions; the value of the threshold voltage is given for the two devices. A schematic of the heterojunctions is given; the arrows indicate the main path for charges.

Figure 4 .

 4 Figure 4. Response of LuPc2/1 (left) and LuPc2/2 (right) heterojunctions to 90 ppm ammonia, at 30% relative humidity, both during exposure/recovery cycles (10 min/40 min), at RT, with a bias of 1 V. The x-axis of the right curve has been arbitrary shifted for clarity.

Figure 5 .

 5 Figure 5. Response of LuPc2/1 (left) and LuPc2/2 (right) heterojunctions to NH3, in the 10-90 ppm range, at 30% relative humidity, during exposure/recovery cycles (1 min/4 min), at 20 °C, with a bias of 1 V.

Figure 6 .

 6 Figure 6. Response of LuPc2/2 to NH3, in the 1-9 ppm range, at 30% relative humidity, during exposure/recovery cycles (1 min/4 min), at 20 °C, with a bias of 1 V (left) and relative response as a function of the NH3 concentration for LuPc2/1 () and LuPc2/2 () heterojunctions (right).

  calcd. for C32H8Cl8N8Co: C, 45.38; H, 0.95; N, 13.23; found: C, 44.1; H, 1.01; N, 13.18. MALDI-TOF-MS (matrix DIT) m/z: 846.6399 (M .+ ); calculated for C32H8Cl8N8Co: 846.7658. FT-IR, (, cm -1 ): 1603, 1523, 1416, 1388, 1339, 1292, 1201, 1138, 1086, 1073, 965, 886, 850, 783, 749, 708, 661, 551, 503, 439, 430.

ACKNOWLEGMENTS

The authors acknowledge the Agence Nationale de la Recherche for funding through the ANR project OUTSMART ANR-2015-CE39-0004-03. Financial support from the European Union (FEDER), The Conseil Régional de Bourgogne Franche-Comté and the Conseil Régional de Bourgogne through the FABER and the PARI SMT 08 and CDEA programs is gratefully acknowledged. We also acknowledge the Conseil Régional de Bourgogne through the CPER program. S. O. is indebted to the Science Institute of Burkina-Faso, for supporting missions in France.

16

Table 1. Raman data (in cm -1 ) of LuPc2/1, LuPc2/2, and LuPc2/3 devices compared to these of each product as powder, and their attribution (s: strong, m: medium and w: weak).