Karla Perez-Toralla

Iago Pereiro

Sonia Garrigou

Fahima Di Federico

Charlotte Proudhon

François-Clément Bidard

Jean-Louis Viovy

Valérie Taly
email: valerie.taly@parisdescartes.fr

Stéphanie Descroix
email: stephanie.descroix@curie.fr

Microfluidic extraction and digital quantification of circulating cell-free DNA from serum

Keywords:

Miniaturized devices for the extraction of DNA have been used for assessing genetic material in biological, forensic and environmental samples. However, the ability to isolate trace amounts of highly fragmented DNA from biological fluids remains a challenge. The current work reports a microfluidic approach that combines on line a dynamic magnetic extraction procedure with droplet-based digital PCR (ddPCR). This strategy maximizes the surface area for DNA binding within the chip, in order to capture short DNA fragments, with the possibility of recovering the purified samples into picoliter volumes for high sensitivity mutation detection. The application of this technology to capture circulating cell-free DNA (ccfDNA) from serum samples of cancer patients is demonstrated herein, with efficiencies comparable to standard column-based DNA extraction methods. This technology uses lesser amounts of required material and reagents, and has a higher potential for automation and multiplex DNA analysis. Furthermore, this approach can also be extended for the detection of other circulating biomarkers, such as nucleic acid sequences with aberrant methylation patterns or miRNA.

Introduction

The study of circulating tumor DNA (ctDNA) released in the peripheral blood during tumor cell death has emerged as a promising alternative to invasive tissue biopsies [START_REF] Wan | Liquid biopsies come of age: towards implementation of circulating tumour DNA[END_REF]. It provides genetic and epigenetic information similar to that of the primary tumor [START_REF] Garrigou | A study of hypermethylated circulating tumor DNA as a universal colorectal cancer biomarker[END_REF], and its noninvasive nature, via a simple venal puncture, allows real-time monitoring of tumor burden and treatment response [START_REF] Diaz | Liquid biopsies: genotyping circulating tumor DNA[END_REF][START_REF] Alix-Panabieres | Clinical applications of circulating tumor cells and circulating tumor DNA as liquid biopsy[END_REF][START_REF] Dawson | Analysis of circulating tumor DNA to monitor metastatic breast cancer[END_REF][START_REF] Lebofsky | Circulating tumor DNA as a non-invasive substitute to metastasis biopsy for tumor genotyping and personalized medicine in a prospective trial across all tumor types[END_REF]. Microfluidicbased technologies such as targeted next generation sequencing (NGS) and droplet-based digital PCR (ddPCR) are currently being developed for the highly sensitive detection and quantification of ctDNA [START_REF] Decraene | Multiple hotspot mutations scanning by single droplet digital PCR[END_REF][START_REF] Malapelle | Development of a gene panel for next-generation sequencing of clinically relevant mutations in cell-free DNA from cancer patients[END_REF][START_REF] Garlan | Early evaluation of circulating tumor DNA as marker of therapeutic efficacy in metastatic colorectal cancer patients (PLACOL study)[END_REF][START_REF] Pecuchet | Analysis of base-position error rate of next-generation sequencing to detect tumor mutations in circulating DNA[END_REF]. However, their performance is still limited by pre-analytical steps performed prior to DNA analysis (e.g., sample collection and DNA extraction procedures) [START_REF] Diaz | Liquid biopsies: genotyping circulating tumor DNA[END_REF][START_REF] Messaoudi | Circulating cell free DNA: preanalytical considerations[END_REF][START_REF] Van Ginkel | Preanalytical blood sample workup for cell-free DNA analysis using droplet digital PCR for future molecular cancer diagnostics[END_REF]. Indeed, it remains challenging to isolate efficiently ctDNA from blood, due to the very low concentration (typically few ng/mL), and high fragmentation (in the 50-1000 bp size range) of these molecules, in particular at early stages of cancer [START_REF] Diaz | Liquid biopsies: genotyping circulating tumor DNA[END_REF][START_REF] Messaoudi | Circulating cell free DNA: preanalytical considerations[END_REF]. More efficient isolation methods are thus needed for the use of ctDNA as a non-invasive cancer biomarker.

Sample pre-treatment usually involves time consuming procedures with manual pipetting and numerous centrifugation steps that could increase the potential sources of error, DNA loss and sample contamination [START_REF] Reinholt | Microfluidic isolation of nucleic acids[END_REF]. Developing miniaturized procedures for DNA isolation can help simplifying the process and interfacing with downstream analysis modules. Reducing the volumes of samples and reagents required per test can also be beneficial when only low amounts of samples https://doi.org/10.1016/j.snb.2019.01.159 Received 28 September 2018; Received in revised form 26 January 2019; Accepted 30 January 2019 are available (e.g. fine needle aspirates). A variety of microfluidic devices has been developed for the solid-phase extraction of nucleic acids [START_REF] Reinholt | Microfluidic isolation of nucleic acids[END_REF][START_REF] Christel | Rapid, automated nucleic acid probe assays using silicon microstructures for nucleic acid concentration[END_REF], but only few studies have focused on the purification of cellfree DNA from biological samples [START_REF] Sonnenberg | Dielectrophoretic isolation of DNA and nanoparticles from blood[END_REF][START_REF] Sonnenberg | Rapid electrokinetic isolation of cancer-related circulating cell-free DNA directly from blood[END_REF][START_REF] Hahn | Microsystem for isolation of fetal DNA from maternal plasma by preparative size separation[END_REF]. Microdevices employing silica chemistries (based on previous developments for standard spin column procedures) have been used for the isolation of DNA from prepurified samples or concentrated genomic DNA from blood samples (> 1 ng/μL), showing extraction efficiencies ranging from 45 to 70% [START_REF] Tian | Evaluation of silica resins for direct and efficient extraction of DNA from complex biological matrices in a miniaturized format[END_REF][START_REF] Cady | Nucleic acid purification using microfabricated silicon structures[END_REF][START_REF] Breadmore | Microchip-based purification of DNA from biological samples[END_REF][START_REF] Duarte | Characterization of dynamic solid phase DNA extraction from blood with magnetically controlled silica beads[END_REF]. However, silica-based technologies have been limited by the need of chaotropic salts for efficient DNA binding to the silica surface, and the use of several alcohol washing steps that can inhibit PCR reactions. In contrast, electrophoretic [START_REF] Sonnenberg | Dielectrophoretic isolation of DNA and nanoparticles from blood[END_REF] or electrostatic-based approaches (e.g. those employing surface chemical treatments with a pHinduced DNA binding effect) [START_REF] Cao | Chitosan as a polymer for pHinduced DNA capture in a totally aqueous system[END_REF][START_REF] Nakagawa | Fabrication of amino silane-coated microchip for DNA extraction from whole blood[END_REF][START_REF] Gan | Chitosan-modified filter paper for nucleic acid extraction and "in situ PCR" on a thermoplastic microchip[END_REF] have the capability to operate in an all-aqueous environment, and would thus facilitate the downstream analysis of the extracted molecules. To increase the surface area for DNA interaction, paramagnetic microparticles have been implemented as solid supports for the capture of nucleic acids in microfluidic devices [START_REF] Duarte | Characterization of dynamic solid phase DNA extraction from blood with magnetically controlled silica beads[END_REF][START_REF] Duarte | Dynamic solid phase DNA extraction and PCR amplification in polyester-toner based microchip[END_REF][START_REF] Lien | Extraction of genomic DNA and detection of single nucleotide polymorphism genotyping utilizing an integrated magnetic bead-based microfluidic platform[END_REF][START_REF] Liu | Detection of Mycobacterium tuberculosis using a capillary-array microsystem with integrated DNA extraction, loop-mediated isothermal amplification, and fluorescence detection[END_REF], allowing for the in situ manipulation of particles (suspension and collection) via external magnets [START_REF] Ferraro | Microfluidic platform combining droplets and magnetic tweezers: application to HER2 expression in cancer diagnosis[END_REF][START_REF] Teste | Selective handling of droplets in a microfluidic device using magnetic rails[END_REF]. Their surface can also be functionalized with charge switchable amine-terminated groups to capture negatively charged DNA by electrostatic interaction [START_REF] Hagan | Chitosan-coated silica as a solid phase for RNA purification in a microfluidic device[END_REF][START_REF] Tiwari | Magneto-separation of genomic deoxyribose nucleic acid using pH responsive Fe3O4@silica@chitosan nanoparticles in biological samples[END_REF].

We have previously reported the use of a miniaturized fluidized bed with functionalized paramagnetic particles for the detection of trace amounts of bacteria from complex samples [START_REF] Pereiro | A new microfluidic approach for the one-step capture, amplification and label-free quantification of bacteria from raw samples[END_REF]. We standardized the microfluidic geometry to control the recirculation of microparticles inside a diamond-shaped microfluidic chamber, and maximize the binding affinity to the solid support [START_REF] Pereiro | Magnetic fluidized bed for solid phase extraction in microfluidic systems[END_REF]. Using an external magnetic source and a flow control module we can actively adjust the porosity of the bed of beads, with a global balance between hydrodynamic drag and magnetic forces (Fig. 1). This approach allowed processing important volumes of samples in reduced times (> 1 μL/min) while avoiding clogging artifacts, and providing low backflow pressure, as well as an important surface area for analyte interaction [START_REF] Mai | Single-step immunoassays and microfluidic droplet operation: towards a versatile approach for detection of amyloid-beta peptide-based biomarkers of Alzheimer's disease[END_REF].

Here, we developed a microfluidic Magnetic ExTRactiOn procedure (METRO) for efficient DNA isolation using the fluidized bed, allowing to process raw biological samples, such as undiluted serum, and deliver concentrated and inhibitor-free eluted DNA samples for downstream analysis. Our strategy relies on the combination of a first module for the selective capture of all DNA sequences present in the sample (mutant and wild type) with a second emulsification module for the compartmentalization of extracted molecules into millions of picoliter droplets for high-resolution genetic testing by ddPCR. This digital approach provides high sensitivity and specificity to distinguish the proportion of mutant ctDNA in circulating cell-free DNA (ccfDNA, naturally present in the blood). During the first phase of this study, we used synthetic and genomic DNA samples for the characterization of the different steps of METRO. We then demonstrated the compatibility of our system with DNA amplification for the detection of low concentrated target molecules in serum, using a commercial ddPCR system. We applied these developments to the extraction and detection of circulating DNA from the blood of breast and colon cancer patients, and confronted our results with conventional column-based extraction procedures in a blind protocol. Finally, we demonstrated the direct coupling of the miniaturized DNA extraction procedure with ddPCR on chip.

Materials and methods

Microfluidic device fabrication and experimental setup

The Cyclic olefin copolymer (COC, Topas ® 8007, Topas Advanced Polymers, Extrusion Lab, Frankfurt, Germany) microfluidic devices were fabricated by hot embossing and sealed using a solvent assisted bonding method, as described earlier [START_REF] Perez-Toralla | New non-covalent strategies for stable surface treatment of thermoplastic chips[END_REF][START_REF] Bruchet | Centrifugal microfluidic platform for radiochemistry: potentialities for the chemical analysis of nuclear spent fuels[END_REF]. Channel walls were treated with Pluronic F127 (1 wt% in water, Sigma Aldrich, St. Louis, MO, USA) to prevent nonspecific adsorption of particles or DNA molecules [START_REF] Perez-Toralla | New non-covalent strategies for stable surface treatment of thermoplastic chips[END_REF]. An inverted microscope (Nikon Ti Eclipse) equipped with a cooled CCD camera (CoolSNAP HQ2, Photometrics, Roper scientific-Princeton instruments) was used for optical inspection and fluorescence analysis. Pressure and flow rate were controlled using a pressure controller and a flow sensor (Fluigent, France).

DNA and serum samples

200 bp dsDNA PCR products were produced using fluorescently labeled Forward and Reverse primers (EuroFins, Ebersberg, Germany, Appendix A Supplementary material Section 1.1, Fig. S1 and Table S1). DNA from the HT29 cancer cell line (ATCC, Manassas, VA, US) was fragmented with S220 sonicator (Covaris, Woburn, MA, USA) (Fig. S2). DNA fragment sizes were measured using the LabChip ® GX/GXII Microfluidic system (Perkin-Elmer, MA, USA) with the DNA assay 5 K reagent kit (Perkin-Elmer) and using the 2100 Bioanalyser (Agilent, Santa Clara, CA, USA) with the DNA 12,000 Kit (Agilent). A Qubit 2.0 fluorometer (Invitrogen, ThermoFisher Scientific) with the dsDNA HS Assay (Invitrogen) was used to measure DNA concentrations. Serum samples from 10 healthy male donors (Biological Specialty Corporation, Colmar, US), were pooled, centrifuged at 3000 rpm, and aliquoted for storage at -80 °C. A new aliquot was thawed and centrifuged at 5000 rpm for 15 min prior to each experiment. 7 archived serum samples from cancer patients, were retrospectively chosen based solely upon the identification of a potential target mutation in a biopsy from the primary tumor.

DNA extraction

The procedure for batch extraction of DNA from serum samples was adapted from the protocol provided by the manufacturer (ChargeSwitch ® gDNA Mini Tissue Kit, Life Technologies, Carlsbad, CA), using the minimal volumes and number of particles that preserved the extraction efficiency and reproducibility of the protocol. We miniaturized the batch extraction procedure to implement the METRO protocol on chip (Appendix A Supplementary material Section 1.2 and Table S2). Briefly, we injected binding buffer into the COC device and used a NdFeB12 permanent magnet (30 × 20 × 20 mm, N50, ChenYang Technologies, Germany) for loading and manipulating magnetic microparticles inside the capture chamber (25 μg of particles, 850-900 nm in diameter equivalent to a surface area of 0.1-0.2 mm²), as described earlier [START_REF] Pereiro | A new microfluidic approach for the one-step capture, amplification and label-free quantification of bacteria from raw samples[END_REF]. The porosity of the bed was tuned, from highly packed to disperse particles, by simply controlling the flow rate inside the chip (up to 1 μL/min), while the position of the external magnetic source remained constant (Fig. 1a-i,ii,iii). Samples were incubated with lysis buffer (with RNAse and Proteinase K) for 1 h at room temperature, prior to injection. Following the addition of low pH binding buffer (pH < 6), samples were perfused through the expanded fluidized bed for DNA capture (30-60 μL of sample at 0.4-0.5 μL/min). After a washing step (15-30 μL of wash buffer at 0.5-1 μL/min), DNA was eluted (5-15 μL of elution buffer at 0.2-1 μL/min) and collected for DNA quantification (pH > 8.5).

Picoliter ddPCR analysis

The extracted DNA was quantified using the RainDrop Digital PCR System (Raindance Technologies, Billerica, US) for DNA emulsification and droplet fluorescence read-out [START_REF] Milbury | Determining lower limits of detection of digital PCR assays for cancer-related gene mutations[END_REF], with ddPCR assays developed earlier [START_REF] Zonta | Multiplex detection of rare mutations by picoliter droplet based digital PCR: sensitivity and specificity considerations[END_REF]. Emulsions were thermal cycled on a C1000 Touch Thermal Cycler (Bio-rad, Hercules, CA, USA). For the integrated extraction and emulsification procedure on chip, we measured the end-point fluorescence signal from each droplet using the RainDrop dPCR Sense after DNA amplification. Data were analyzed using the RainDrop Analyst software as described by the manufacturer.

Results and discussion

DNA extraction capabilities on chip

Miniaturized DNA extraction procedure

Extraction using ChargeSwitch ® beads follows three main steps: A) binding of negatively charged nucleic acids to the positively charged surface of the beads, B) washing to remove non-specifically bound molecules, and C) elution of DNA from the beads. We first qualitatively assessed the behavior of the fluidized bed during these steps using fluorescently labeled dsDNA (Fig. 1b). Prior to sample perfusion, the bed of beads was fluidized by flowing binding buffer to equilibrate the surface charge of particles, and homogenize their density in the diamond-shaped chamber (Fig. 1b-i). During step A, the DNA sample was injected, resulting in a rapid increase in the fluorescence intensity at the surface of the beads, verifying that fluorescent DNA molecules were captured. The fluorescence signal was higher in the center of the fluidized bed at the beginning of the capture step, and all particles were covered with fluorescent DNA molecules by the end of the process (Fig. 1b-ii,iii). This redistribution of the fluorescent signal across the chamber was driven by the sideward recirculation of magnetic particles during the fluidization regime (Fig. 1a-iv, Video S1) [START_REF] Pereiro | Magnetic fluidized bed for solid phase extraction in microfluidic systems[END_REF], and was crucial for maximizing the DNA capture capacity of the device. During step B, a continuous recirculation of the bed of beads with no loss of particles was observed, enabling efficient removal of proteins and other inhibitors from the fluidized bed. More importantly, the fluorescence intensity on the beads did not decrease significantly during this step, indicating no loss of captured material. During step C, an immediate decrease in the fluorescence signal at the surface of the particles was observed, to a level comparable to the initial low auto-fluorescence from the beads (Fig. 1b-iv), demonstrating the effective flow-through elution of DNA without the need for an incubation step. We could thus expect that the amount of DNA remaining on the surface of the particles at the end of the procedure was very low (at least not detectable by fluorescence measurements).

We then evaluated the selectivity of our system regarding DNA size, by capturing and eluting fragments with varying lengths (100 bp-3000 bp). We observed through microchip gel electrophoresis analysis that the injected and eluted samples presented the same size distribution with the same relative peak areas (Appendix A Supplementary material Section 1.3, Table S3 and Fig. S3), showing the possibility to isolate fragmented DNA molecules with no preferential selection of a particular size.

DNA binding capacity using model DNA samples

To quantify the capture efficiency of METRO, we perfused different volumes of sample containing fluorescently labeled dsDNA in PBS buffer (200 bp in length, MW = 129,000 g/mol, and [C] = 11 ng/μL). To evaluate the amount of captured DNA in real time, we assessed the level of fluorescence intensity from the sample in the outlet channel (Fig. 1a-i): 1) before injecting the beads, and 2) during the DNA capture process (Appendix A Supplementary material, Section 1.4). We measured a DNA capture rate higher than 97% for samples with up to 200 ng of DNA, with a slight decrease in capture rate for 300 ng of DNA (89 ± 8%), probably due to saturation effects and steric repulsion from DNA molecules captured on the surface of the beads (Fig. 2a). We observed a linear capture rate for DNA inputs in the 0-250 ng range (Fig. S4), demonstrating the capabilities of our system for the efficient extraction of dsDNA fragments in ideal conditions (i.e. using an aqueous matrix). We estimated the DNA binding capacity of our system to be at least in the order of 10 μg of DNA/mg of beads; placing our system in the higher end of the 5-10 μg of DNA/mg range expected from the supplier, using the batch procedure recommended for the ChargeSwitch ® particles, and well above the capacity for other commercially available particles [START_REF] Cao | Chitosan as a polymer for pHinduced DNA capture in a totally aqueous system[END_REF].

DNA extraction from human serum

To evaluate the effect of the sample matrix on DNA binding affinity, we processed human serum samples from healthy donors, spiked with different amounts of fluorescent dsDNA. We measured a capture rate higher than 63% for samples containing up to 100 ng of DNA and a stabilization of the capture efficiency around 50% for higher amounts of DNA (48 ± 6% for 200 ng) (Fig. 2b). As expected, we observed a lower capture rate when using undiluted serum as a matrix, as compared to DNA samples in PBS, which could be related to non-specific adsorption of serum proteins (e.g., albumin) or other nucleic acids (e.g., ccfDNA or miRNA from serum) to the beads surface through electrostatic interactions [START_REF] Messaoudi | Circulating cell free DNA: preanalytical considerations[END_REF][START_REF] Clark | Extraction of DNA by magnetic ionic liquids: tunable solvents for rapid and selective DNA analysis[END_REF]. The high molecular content of complex matrices such as human serum or plasma has indeed limited the implementation of microfluidic DNA isolation procedures. One approach to avoid potential screening effects or clogging artifacts during the isolation process has been to dilute the samples prior to their injection on chip (10-100-fold) [START_REF] Tian | Evaluation of silica resins for direct and efficient extraction of DNA from complex biological matrices in a miniaturized format[END_REF][START_REF] Cao | Chitosan as a polymer for pHinduced DNA capture in a totally aqueous system[END_REF]. However, such approach is not suitable for the analysis of ctDNA, as it would radically limit sample processing capabilities, as well as the sensitive detection of rare events. In this work, the capture rate for DNA samples in serum is still particularly high considering the use of undiluted human serum, the small size of extracted DNA molecules, and the electrostatic-based (thus non-specific) nature of the DNA binding strategy.

To estimate the global extraction efficiency (including elution) for the spiked serum samples on chip, we collected the eluted DNA for offchip quantification (Qubit ® assay, Fig. S5). For an input of 200 ng of DNA using METRO, we obtained an average extraction efficiency of 64 ± 9% (n = 5), in the same range as the capture rate for 200 ng measured above using continuous fluorescence measurements on chip. We thus expect that all DNA captured inside the fluidized bed was efficiently eluted (i.e. no detectable DNA remaining at the surface of the beads). In comparison to the batch ChargeSwitch ® procedure, the elution volume on chip was reduced by at least 15-fold (the volume of the microfluidic chamber is 0.65 μL), while maintaining a similar extraction efficiency (Fig. S5).

Other groups have demonstrated the interest of electrostatic-based approaches for DNA isolation on chip. Nakagawa et al. used an amino silane-coated microchip to obtain 60% capture and 45% elution rates for λ-DNA (2 ng/μL) [START_REF] Nakagawa | Fabrication of amino silane-coated microchip for DNA extraction from whole blood[END_REF]. However, their recovery ratio decreased to 27-40% when extracting human genomic DNA from whole blood, due to the competitive binding of negatively charged proteins, as demonstrated here. More recently, Gan et al. developed an original approach combining a chitosan-modified filter paper inside a microfluidic device for DNA extraction [START_REF] Gan | Chitosan-modified filter paper for nucleic acid extraction and "in situ PCR" on a thermoplastic microchip[END_REF]. Although they obtained a 62-89% capture efficiency using λ-DNA (with concentrations from 0.05 to 1 ng/μL), only 8.9-11.5% of the captured DNA could be eluted, thus limiting the possibility of interfacing with downstream analysis modules. Finally, non-commercial chitosan-coated magnetic particles have also been used for efficient DNA isolation, providing high stability, pH tunability and yield (52 μg from 3 mL human saliva), but their implementation on chip has not been demonstrated [START_REF] Tiwari | Magneto-separation of genomic deoxyribose nucleic acid using pH responsive Fe3O4@silica@chitosan nanoparticles in biological samples[END_REF]. Our technology provides similar or better DNA extraction efficiencies to those reported in the literature with the added advantages of a higher binding capacity, as well as a higher potential for scalability and integration with DNA detection and quantification techniques, such as droplet-based digital PCR, as shown in the following sections.

Processing biological samples

Quantification of DNA extraction efficiency by ddPCR

To evaluate the efficiency of METRO using more realistic samples and demonstrate its compatibility with ddPCR analysis (RainDrop Digital PCR System), we processed undiluted serum from healthy donors spiked with cancer cell line DNA, with fragment size lengths and concentrations relevant to ctDNA analysis (from 50 to 1,100 bp and [C] = 0-357 ng/mL, Fig. S2) [START_REF] Mouliere | High fragmentation characterizes tumour-derived circulating DNA[END_REF]. To differentiate between spiked mutant DNA (MUT) and non-mutated ccfDNA (wild type, WT, initially present in serum samples), we used a mutation specific assay containing allele-specific hydrolysis probes (TaqMan™ probes targeting the R273H mutation in the TP53 gene). We first evaluated the levels of WT DNA for non-spiked samples, and detected an average concentration of 23 ± 11 ng/mL (equivalent to 7045 ± 3315 copies in 1 mL of serum, n = 11), in line with concentrations of ccfDNA previously described for healthy donors [START_REF] Messaoudi | Circulating cell free DNA: preanalytical considerations[END_REF]. We observed no false positives or MUT events in the healthy test samples (Fig. 3a). We then extracted the different spiked samples, and used the digital quantification to compare the number of expected vs. extracted MUT and WT genomes for each sample (Fig. 3b). We established a calibration curve for the MUT genomes (Fig. 3c) and observed a linear capture/elution relationship for the different input mutant DNA with an overall 37% extraction and detection efficiency. In parallel, we performed the extraction of the same spiked samples using the ChargeSwitch ® procedure in batch. However, the analysis of eluted DNA from the bench-top protocol did not generate consistent ddPCR results, probably due to the presence of inhibiting reagents or molecules still present in the eluent (Fig. S6). METRO allowed for a more efficient washing process and buffer exchange inside the microfluidic device, thus delivering inhibitor free samples compatible with ddPCR analysis. These results demonstrated that our system allows analyzing MUT DNA fragments in concentrations as low as 3 ng/mL of serum in a background of WT DNA (leading to a mutational ratio of about 10% when considering the amount of WT ccfDNA in serum samples), using a minute amount of sample (60 μL).

Analysis of serum samples from cancer patients

As a proof of concept, we implemented METRO for the detection of DNA in serum samples from breast (n = 5) and colon (n = 2) cancer patients with a known somatic variant in tumor tissue (Table S4). Specifically, we extracted 60 μL of serum from each of these patients on chip and quantified the number of MUT and WT genomes, using ddPCR assays specific to each patient [START_REF] Garrigou | A study of hypermethylated circulating tumor DNA as a universal colorectal cancer biomarker[END_REF][START_REF] Zonta | Multiplex detection of rare mutations by picoliter droplet based digital PCR: sensitivity and specificity considerations[END_REF][START_REF] Laurent-Puig | Clinical relevance of KRAS-mutated subclones detected with picodroplet digital PCR in advanced colorectal cancer treated with anti-EGFR therapy[END_REF]. We performed two extractions in parallel for each patient sample, using two microfluidic devices with the same operating conditions. The quantification results showed concentrations of total ccfDNA (WT + MUT) ranging from 0 to 342 ng/mL of serum (Table S4), in line with previously reported concentrations for ccfDNA in the serum of cancer patients [START_REF] Messaoudi | Circulating cell free DNA: preanalytical considerations[END_REF]. We detected the somatic variant from patient #6 in the serum samples extracted on chip, with a concentration of 15.45 ng/mL of MUT ctDNA (equivalent of a 47.5% mutational ratio for the G12D mutation in the KRAS gene). We considered this sample as positive for ctDNA, since the concentration was higher than the limit of detection determined above for spiked serum samples (Fig. 4b,c and Table S4). We also noticed that patient #1 presented a high concentration of WT DNA but no detectable MUT ctDNA in serum (Fig. 4a,c and Table S4), which could be related to an important background of DNA from non-tumor cells (e.g., leukocytes). We observed a linear relationship when comparing the total ccfDNA concentrations obtained on chip and through a standard column-based protocol, for values above the limit of detection of the microfluidic METRO procedure (Fig. 4c, Table S5, Appendix A Supplementary material, Section 1.5).

Integrated DNA extraction and detection procedure on chip

Finally, we developed an integrated device combining METRO, with a microfluidic module enabling the direct emulsification of the eluted DNA, together with PCR reagents, on chip (Fig. 5a,b). The generated droplets were collected unto 6 individual tubes and submitted to thermal cycling for DNA amplification (Appendix A Supplementary material, Section 1.6). We evaluated the number of positive events (droplets containing amplified DNA) on each elution fraction through fluorescence detection on chip (Fig. 5c), and estimated an overall DNA extraction efficiency of 64%. We observed that most eluted DNA was contained in elution fractions from 2 to 4 (with occupation ratios ranging from 0.3 to 1.5%). Although the amount of DNA in elution fractions 1 and 6 was very low (< 250 pg), it was readily preserved and amplified using this integrated procedure. Considering the operating conditions, the complete DNA extraction and emulsification of 30 μL of sample was performed in less than 2 h (Table S2).

Although a DNA isolation and amplification procedure using continuous flow microfluidics and microchambers for sample compartmentalization was demonstrated earlier [START_REF] Tian | An integrated temporary negative pressure assisted microfluidic chip for DNA isolation and digital PCR detection[END_REF], we present in this work the first direct eluent emulsification method for picoliter droplet-based dPCR. This approach enables the generation of millions of individual compartments for single molecule detection. It also allows for the efficient elution and amplification of all DNA captured at the beads surface and offers the possibility to rationally modify the PCR amplification environment for each elution fraction (e.g. emulsification with a variety of primers and probes or using different thermal cycling conditions), thus opening the route for multiplex analyses.

Conclusion

We have combined on-chip, for the first time, a miniaturized DNA extraction platform with ddPCR. We applied this method for the detection and quantification of ctDNA from biological samples. Specifically, we isolated fragmented DNA in low concentrations directly from serum samples from cancer patients and demonstrated the compatibility of our system with sensitive DNA amplification methods, such as ddPCR. As compared to conventional column-based methods, this approach has the ability to perform both sample preparation and DNA detection in a single device, providing comparable results but using only a few microliters of sample, which could be of great interest for the implementation of minimally invasive cancer diagnostic procedures. Furthermore, the METRO procedure offers a high degree of flexibility in terms of size, number and surface functionalities of the magnetic particles that can be implemented (e.g. to selectively capture mRNA, microRNA, methylated DNA sequences, as well as bacterial or viral DNA).

Fig. 1 .

 1 Fig. 1. Schematic of the setup for METRO using the microfluidic fluidized bed (a-i). Micrographs of the fluidized bed before (a-ii) and during (a-iii) sample perfusion on chip. Magnified view showing the recirculation of beads. Fluorescence micrographs showing DNA-beads interaction inside the fluidized bed, before (b-i), during (b-ii), at the end of DNA capture step (b-iii), and after DNA elution (b-iv). Scale bars: 500 μm.

Fig. 2 .

 2 Fig. 2. Capture rates for METRO measured for different DNA inputs using fluorescently labeled dsDNA spiked in (a) PBS and (b) human serum samples from healthy donors.

Fig. 3 .

 3 Fig. 3. Detection of R273H TP53 mutation in serum samples spiked with mutant HT29 cell line DNA. (a-b) Two dimensional histograms of the digital PCR assay for the analysis of circulating DNA extracted from healthy donors (a) and from spiked samples (b). Calibration curve (c): Extracted R273H TP53 mutant DNA digitally quantified for varying spike in DNA amounts (0.0033 ng/genome copies). MUT probes: FAM, 6-carboxyfluorescein; WT probes: VIC, a proprietary dye of Life Technologies; A.U., arbitrary units.

Fig. 4 .

 4 Fig. 4. Detection of ccfDNA in the serum of breast and colon cancer patients. (a-b) Two dimensional histograms of the digital PCR assay for the analysis of circulating DNA extracted on chip from two patients bearing TP53 (a) and KRAS mutations (b). (c) Digital quantification for total ccfDNA concentration (MUT + WT) following extraction using the microfluidic METRO procedure and standard column-base protocol. MUT probes: FAM, 6-carboxyfluorescein; WT probes: VIC, a proprietary dye of Life Technologies; A.U., arbitrary units.

Fig. 5 .

 5 Fig. 5. a) Schematic of the integrated procedure for DNA extraction and emulsification for ddPCR analysis. b) Micrograph images from the fluidized bed (bi) and the droplet generator module (bii-iii) during elution and encapsulation of DNA. c) Amount of extracted and amplified DNA by ddPCR for the different elution fractions, collected in PCR tubes (emulsion collection tubes in Fig. 5a-iii).

K. Perez-Toralla et al.

Acknowledgments

This work was supported by the SIRIC CARPEM, the Ministère de l'Enseignement Supérieur et de la Recherche, the Université Paris-Descartes, the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé et de la Recherche Médicale (INSERM), the French Ile-de-France canceropôle, by ANR "Investissements d'Avenir" for Digidiag project (ANR Nanobiotechnologies; no. ANR-10-NANO-0002-09), the ligue nationale contre le cancer (LNCC, Program "Equipe labelisée LIGUE"; no. EL2016.LNCC/VaT) as well as Labex and Equipex IPGG, and by European FP7 programs (LOVEFOOD FP7-ICT-2011-317742, NAPES FP7-NMP-2013-604241, CATCH-U-DNA, FETOPEN H2020 and ERC Advanced Grant CellO (FP7-IDEAS-ERC-321107). The authors acknowledge the Plateforme Technologique of the Institut Universitaire d'Hématologie (IUH, Paris, France) and the biochemistry and molecular biology service (BMB168, Institut Curie, UMR168) for help with DNA analysis.

Appendix A. Supplementary data

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.snb.2019.01.159.


E Zago

M Guglielmin

S Pierno

G Viovy

Mistura

D Ferraro

M Serra

D Filippi

L Zago

E Guglielmin

M Pierno

S Descroix

J.-L Viovy

G Mistura

Controlling the distance of highly confined droplets in a capillary by interfacial tension for merging on-demand †

Controlling the distance of highly confined droplets in a capillary by interfacial tension for merging on-demand

Introduction

Droplet microfluidics is a fast developing subfield of microfluidics, dealing with emulsions of aqueous droplets (or dispersed phase) in oil (or continuous phase). It has been drawing a lot of attention, due to its interesting applications in biomedical and chemical fields. [1][2][3][4] Droplets are generated by specific channel geometries 5 or by pipetting. 6 Surfactants, 7 which reduce the interfacial tension between the continuous and dispersed phases, are usually added to facilitate the droplet generation and, in some cases, to prevent their merging by steric repulsion. 7 Among different implemented platforms, the "plug format", in which droplets are highly confined in a capillary 8,9 are particularly suitable in applications requiring multi-step assays. 9,10 In detail, since they are bigger than the channel diameter, droplets assume an elongated shape and flow as independent wagons in a train, which may contain different reagents, preserving the deterministic order of production and allowing their identification without requiring any complicated barcoding. In this context, circular capillaries are extensively used to transport droplets between the different moduli of platforms devoted to complex protocols, such as ELISA, 11,12 chemical synthesis, 13 incubation 14,15 and purification reactions. 16 Importantly, even if confined, droplets are never in contact with the channel wall because, under suitable conditions, 17 they are surrounded by a thin lubrication film of the continuous phase. 18 This film is essential to reduce crosscontamination risks and it also plays a fundamental role on the droplet motion. In detail, in circular tubings the droplet is always faster than the continuous phase 19,20 and its speed depends on the thickness of the lubrication layer surrounding the droplet: the thicker the film, the faster the droplet. Starting from this observation, recent theoretical studies have investigated in more detail this phenomen, focusing on the role of the viscosity ratio between the continuous and the dispersed phases. [21][22][23] A few experimental works have measured the thickness of the lubrication film, [24][25][26][27] yielding contradictory results. These are typically justified assuming the presence of unwanted molecules in the fluids, 28 which may act as surfactants that modify the interfacial tension and, especially, introduce Marangoni's stresses due to a nonhomogenous distribution at the interfaces. 7 The effect of surfactants on the lubrication film thickness in confined geometries has been addressed, both numerically [29][30][31] experimentally. 25 These investigations show that surfactants cause an elongation of the moving droplet, which leads to a thicker lubrication film and thus to a higher speed. 28 Other works focusing on the speed ratio between the dispersed and continuous phases have identified different regimes depending on the viscosity ratio between the two liquids. 32,33 Although these results suggest that both droplet length and viscosity ratio may affect the droplet speed, a comprehensive understanding of these phenomena is still lacking. More importantly, no study has directly addressed the role played by the interfacial tension. The latter is especially relevant in bioanalytical assays, in which numerous agents contained in the droplets are surface-active. They may involve surfactants 7 or ethanol, 34 used e.g. for cleaning or stabilization purposes, and biomolecules themselves. These surface-active molecules may induce large variations in interfacial tensions between the droplet and the oil. 35 Given such a complex scenario, controlling the separation distance between confined droplets, essential to prevent or promote their contact and the eventual merging, is not a trivial task. Actually, among the different operations that can be performed with droplets, merging (or coalescence) of two (or more) droplets represents a fundamental step for triggering a variety of chemical and biological applications, e.g., kinetic studies, chemical synthesis, screening of biological contents and bio-medical diagnosis. 36 Merging strategies can be conveniently divided into passive and active ones. In the former case, droplets are not sterically stabilized by the surfactant 16 (such as PFD, PFO), therefore they spontaneously coalesce upon contact. Active merging is instead preferred when the carrier fluid contains surfactants, such as Krytox-PEG, that prevent merging by steric interactions at the interface; the fusion must be thus achieved by applying an external perturbation (pressure drop, 37 electric field, 38 mechanical vibration, 39 local heating 40 ) or, in case of non-confined droplets, endowing microchannels with specific geometric features, 41 tuning contact time and surfactant concentration. 42 Regardless of the adopted approach, the merging of two droplets requires the contact between their interfaces. When droplets are flowing in separated channels, this can be achieved by synchronizing them using ladder-like geometries, 43 droplet on demand generators 44 or railroad-like microstructures. 45 Differently, when confined droplets flow in a linear sequence along the same channel, specific geometrical architectures, like channel expansions, must be used to vary the local speed of a droplet and to allow its merging with the subsequent one. [46][47][48] All these methods require the use of exclusive and specifically engineered microfluidic chips, which generally need dedicated and long fabrication procedures in clean room facilities. A simpler alternative is to induce an intrinsic difference in speed between two consecutive droplets. In this work we demonstrate that the speed difference induced by the interfacial tension between two or more confined droplets, transported by a constant flow rate of carrier fluid in a straight circular capillary, can be exploited to precisely trigger their meeting point, and therefore the eventual merging, without the need of any specific microchannel feature or external equipment. As a proof of concept, we apply this strategy to the implementation of a well established enzymatic reaction 8 for the detection of the H 2 O 2 released from biological samples. In order to fully characterize the relative droplets motion, we have systematically investigated the speed of a single confined droplet in a circular capillary having uniform cross-section, and its dependence on i) the viscosity ratio between dispersed and continuous phase, ii) the droplet length, iii) the oil flow rate and, especially, iv) the interfacial tension. The latter is varied by: i) using different hydro-organic solutions and ii) adding a commonly used surfactant (Tween20) in the dispersed phase. As expected, both approaches lead to a decrease in the interfacial tension, however a different behavior is found between the two systems due to the presence of Marangoni's stress in the case of Tween20. The latter case is particularly relevant since surfactants are typically present in most of the commercial available biological reagents, such as PCR mix, cell culture medium or protein buffers. Since in most applications surfactants are added in the continuous phase, 7 we have also investigated the contribution of the oildispersed surfactant to the droplet speed.

Materials and methods

Continuous and dispersed liquid phases

Table 1 lists all the liquids used in this study and their main physical properties (see also Table S1 in ESI †). FC-40 oil (by 3M) is used as continuous phase either pure (labeled as FC40) or with 2% (w/w) of surfactant. Two different surfactants are used: 1H,1H,2H,2H-perfluoro-1-decanol (labeled as FC40 + PFD, by Fluorochem) and PEG-Krytox (labeled as FC40 + PK, by RAN Biotechnologies). The dispersed phase consists of MilliQ-water in different combinations: pure, in solution with various concentrations of glycerol (by Sigma-Aldrich) to modify the viscosity ratio λ between the dispersed and the continuous phases, and with ethanol or Tween20 (by Sigma-Aldrich) to tune the interfacial tension γ. The interfacial tension and the viscosity are measured by the pending drop method 49 and by an Ostwald's viscometer, respectively.

The liquids solutions are chosen to investigate the dependence of the droplet speed for variable surface tensions γ, while keeping λ constant and, vice versa, to study the effect of the viscosity ratio for a fixed interfacial tension. 50 As a matter of fact, as shown in Table 1, the presence of glycerol in water does not strongly affect the value of γ, while it causes a variation of one order of magnitude in λ (from λ ≃ 0.24 to λ ≃ 3.40). Differently, adding ethanol or Tween20 to the aqueous phase does not induce significant variations in λ, while γ drastically changes. Similarly, the introduction of surfactants in the oil phase causes a drop in γ, while λ values are not affected (see Tables 1 andS1 in ESI †). The apparent critical micelle concentration (CMC app ) of the water/Tween20 mixture with the oil phases, evaluated from the measured equilibrium interfacial tensions, 51 are found to be about This journal is © The Royal Society of Chemistry 2019 0.05%, 0.02% and 0.01% in FC40, FC40 + PFD and FC40 + PK cases, respectively (see Fig. S1 in ESI † for more details).

Finally, the Amplex Red Hydrogen Peroxide/Peroxidase Assay kit (by Thermofisher) is used for the enzymatic reaction. Fresh substrate solutions of the supplied H 2 O 2 in reaction buffer are prepared the day of the experiment, in a range between about 3.90 × 10 -1 μM and 7.62 × 10 -2 μM. Then a mixture containing 97% reaction buffer (with 0.01% w/w Tween20 detergent added), 2% of the horseradish peroxidase enzyme (HRP, 0.25 U mL -1 ) and 1% of the Amplex Red probe (10 mM) is prepared as enzymatic solution. All the relative prepa-rations, as well as the experiments, are performed in a dark room to avoid exposure of the Amplex Red to light. 52

Experimental setups

Two customized experimental setups have been developed with the aim of: i) measuring the speed of a single confined droplet in a circular capillary (or tube) (see setup 1) and ii) demonstrating the feasibility of passive droplet merging-ondemand and its capability in the implementation of enzymatic reactions (setup 2).

Table 1 List of the liquids used as continuous and dispersed phases during the experiments, with the corresponding interfacial tension γ and viscosity ratio λ between the dispersed and the continuous phases (FC40 and FC40 + PFD). All dilutions are measured as % w/w Setup 1 is schematically shown in Fig. 1a. A single water-phase droplet is generated in a circular PTFE tube (inner nominal diameter 0.3 mm, outer nominal diameter 0.6 mm) by pipetting 16 (at a flow rate of 0.2 μL s -1 ), using a 100 μL syringe (by SGE) mounted on a syringe pump (PHD 22/2000, by Harvard apparatus) and pre-filled with the oil phase. Negligible oscillations in the generated flow are observed during the measurements (see Fig. S2 and Table S2 in ESI †). One extremity of the tube is connected to the syringe, while the other is moved between two conventional Eppendorf tubes containing the oil and the waterphase solutions to be investigated. By this approach, droplets having volumes between 15 nL and 300 nL are generated with a dispersion of about 2%. 16 The length L of each droplet (Fig. 1b) is measured at rest by transmission microscopy (Nikon Eclipse Ci, with 4× and 10× objectives, MiKrotron camera) before and after every run of measurements. The acquired images are analyzed by ImageJ software. The estimated droplet volume 53 is compared to the expected pipetted volumes in order to verify the reliability of both the pipetting and the measured droplet length. The tube is held in a 3D printed structure (see Fig. S3 in ESI †), designed to integrate six optical detectors, placed at fixed positions along the tube length to record the droplet passage. Each detector is composed of two optical fibres facing each other across the tube. These fibres are respectively coupled with a LED (L-7113GC by Kingbright) and a photodiode (OPT101, by Texas Instruments), used to emit and collect the light passing through the tube, respectively (see Fig. 1c). The droplet crossing the detection region defocuses the optical beam, causing a typical decrease in the transmitted intensity (see Fig. 1d). This signal is processed by an Arduino board controlled by a customized LabVIEW (National Instruments) program, which allows to record the crossing time of the droplet at each photodetector. The crossing time is defined as the time at which the signal decreases below a defined threshold (dashed line in Fig. 1d). Then, the detector positions are plotted in terms of the crossing times and the droplet speed is evaluated as the slope of the linear fit of these data points (see Fig. 1e). During a typical run, a single droplet is flown at least 6 times back and forth along the tube, then the oil phase flow rate ϕ is changed and the operation is repeated. The normalized speed β is calculated as the speed of the droplet V divided by the average speed of the oil phase U = ϕ/πr 2 , where r is the inner tube radius. In order to estimate the accuracy of the syringe pump, selected values of ϕ are compared with those deduced by weighing the mass of the liquid flown out the capillary in fixed amounts of time. 33 The discrepancy is found to be systematic and less than 1% (see Table S3 in ESI †). Additionally, for a more accurate estimate of U, the inner radius (r) of the tube is evaluated by repeated direct measurements with a transmission microscope (Nikon Eclipse Ti) (see Fig. S4 in ESI †), yielding r = 145 ± 1 μm. Finally, all measurements are performed in a constantly monitored air-conditioned room (RT = 22 °C with a maximum oscillation of 1.5 °C during different days).

A second experimental setup (setup 2) is designed to demonstrate the passive droplet merging approach based on precise surface tension tuning. The same setup is also exploited for the implementation of an enzymatic reaction, as a proofof-concept application. It is composed by a microfluidic PDMS device produced by conventional double replica molding 54,55 of a master fabricated by micromilling 37 (see Fig. S5 in ESI †). As sketched in Fig. 2, the chip presents two consecutive T-junctions to generate pairs of droplets containing two different solutions. The inlets of the device are also connected to a pressure controller (MFCS, by Fluigent) and the flows are regulated by 3 customized micro-valves, described in ref. 56, for droplet-on-demand generation. 57 The pressure used for the droplets generation is 200 mbar, while for the oil flow is between 150 and 300 mbar.

This approach allows the independent control of the droplet volumes, their frequency, the oil gap separating two consecutive droplets and the flow rate of the continuous phase. Droplets pairs are then flown out of the device in the circular PTFE tube, which is fixed above a meter to measure the merging position by a movable camera (Nikon D3300). Finally, in the case of the enzymatic reaction experiments (dashed square in Fig. 2), the tube passes through a customized fluorescence detector. 12

Results and discussion

In this work we present a novel method for achieving the passive merging of droplets flowing in a circular channel. It relies on the tuning of the droplet speed via the control of several parameters of the system: i) the normalized droplet length  = L/w (where L is the droplet length and w the tube diameter), ii) the viscosity ratio λ between the dispersed and the continuous phase, iii) the interfacial tension γ between the dispersed and the continuous phase, iv) the flow rate ϕ of the continuous phase. In the following, the influence of these physical quantities will be presented and discussed in separated sections for the sake of clarity.

Variation of the droplet speed with the continuous phase flow rate ϕ and the viscosity ratio λ

At first, the influence of the normalized droplet length  and of the carrier oil flow rate ϕ on the speed of a pure water droplet is evaluated, keeping constant both γ and λ. In detail, Fig. 3a shows the normalized droplet speed (or droplet mobility) β as a function of , for different ϕ. In agreement with earlier results, 28 the droplet speed V is always larger than the average oil speed U, i.e. β = V/U > 1. Furthermore, the data corresponding to different ϕ exhibit the same trend: upon increasing the droplet length, β decreases up to a critical length (about  = 5), then increases reaching a plateau. Similar trends have been observed in numerical simulations, 21,58 although at higher capillary numbers (Ca) than those used in our work (Ca = 10 -4 ÷ 10 -3 ). Considering that the speed This journal is © The Royal Society Chemistry 2019 depends on the thickness of the lubrication film and that the droplet is deformed during the flow, 21,58 we suggest that the plateau is due to the fact that, above a certain length, the -dependence of the average value of the film thickness saturates, while for shorter droplet, yet still confined, the average film thickness is significantly affected by the deformation of the droplet frontal and rear meniscii. Furthermore, the data show that an increase in the flow rate of the continuous phase ϕ leads to an increase in the droplet mobility ratio β (see inset Fig. 3a). This was expected considering that the lubrication film around the droplet increases with ϕ, 22 leading to higher values of β. Finally, similar trends, slightly enhanced by about 1-2%, can be observed by adding PFD to the continuous phase (see Fig. S6 in ESI † for more details).

We have also evaluated the effect of the viscosity ratio λ between the dispersed and the continuous phase on the droplet speed. In detail, to vary the viscosity without affecting the surface tension, we have used three glycerol/water mixtures 50 at different concentrations as the dispersed phase, achieving a varia-tion of more than one order of magnitude in λ (from 0.23 to 3.6, see Table 1). Fig. 3b shows the droplet mobility β plotted as a function of the droplet length  for the three λ values considered and for a specific oil flow rate (ϕ = 40 μL min -1 ). Despite the large variation of λ, the graph presents a maximum oscillation in β of about 1.5%, barely above the sensitivity of our experiment. We can then safely conclude that for  > 1, β is very weakly affected by the viscosity ratio.

Variation of the droplet speed with γ by modifying the hydroorganic composition of the dispersed phase

The effect of the surface tension on the droplet speed is studied by considering different hydro-organic (water/ethanol mix) solutions as the dispersed phase. An increase in ethanol concentration leads to a decrease of γ, while keeping almost constant the viscosity ratio 50 (see Table 1).

Considering that β slightly varies with the normalized droplet length  (see Fig. 3a), we first evaluate the influence of γ for various . Fig. 4a displays the normalized Fig. 2 Scheme of the experimental setup for the droplet merging study. Pairs of droplets containing pure water (blue) and Tween20 aqueous solution (yellow) are generated in a PDMS chip by a sequence of T-junctions using the droplet-on-demand approach. The three inlets of the junctions are connected to a pressure controller. The distance between the droplets and the oil phase flow can be easily tuned by acting on the valves actuation time and applied pressure. After generation, the droplets flow in the capillary that is placed in correspondence of a meter. A camera is used to acquire the position of the merging event. In the case of the enzymatic reaction (dashed rectangle), the emitted fluorescence intensity is measured by a detector connected to a computer. droplet speed β as function of  for various γ (between 51 and 17mN), using FC40 oil as the continuous phase. At first, it is clearly shown that the droplet speed increases as the interfacial tension γ decreases. Furthermore, independently from γ, the droplet mobility assumes a value of about β = 1.05 for  close to 1. Then, β increases for longer droplets and reaches a plateau above a critical length ( ≃ 5). Moreover, the final constant values increase with ethanol concentration and thus, with decreasing interfacial tensions. This behaviour may be explained considering that β increases with the average thickness of the lubrication film, 19 which is affected by the local droplet deformation. In fact, the drainage of the oil surrounding the droplet, which tends to get thinner the film, is expected to be easier for shorter droplets. This results in an increase of the droplet mobilities β with  (up to  ≃ 5). Then, for sufficiently long droplets (about  > 5), the results indicate that β, thus the film thickness, is constant with , as found in ref. 21 and 58. Since in most applications, surfactants are dispersed in the continuous phase, we evaluate their influence by repeating the same study in FC40 + PFD conditions (see Materials and methods). The results, reported in Fig. 4b, show similar trends as in the previous case (pure FC40); nevertheless, data are distributed on a wider range of β, meaning that a smaller variation in γ leads to a more important variation in the droplet mobility. This represents an interesting effect from the application point of view: the merging of two consecutive droplets can be passively achieved acting on their difference in interfacial tensions.

Remarkably, these observations hold not only in the presence and absence of surfactants in the continuous phase, but also for different flow rates (see Fig. S7 in ESI †). To better evaluate the influence of the interfacial tension on droplet speed differences, in Fig. 4c, β is plotted against γ for a fixed droplet length in the plateau region ( = 8), with both FC40 (full symbols) and FC40 + PFD (empty symbols) carrier phases, and for different flow rates (ϕ = 40, 70 100 μL min -1 ). As previously observed, the graph shows that the decrease of the interfacial tension leads to an increase in the droplet speed β (see dashed line). Overall, the graph indicates that decreasing γ by one order of magnitude causes a variation in the droplet speed between pure water and ethanol solution as high as 16%. This variation increases with the flow rate and it reaches 25% when γ = 5 mN m -1 and ϕ = 100 μL min -1 . Similar trends have been observed in the case of FC40 + PK (see Fig. S9a in ESI †). Finally, the effect of viscosity ratio on the droplet speed (see Fig. 3b) is found to be significantly smaller than that due to changes in the interfacial tension (Fig. 4a). of the droplet speed with γ by adding surfactant to the dispersed phase Differently from the case discussed above, in which the interfacial tension is modified acting on the hydro-organic composition of the dispersed phase, we further investigate the dependence on γ by considering the addition of various concentration of a surfactant (Tween20) to the dispersed phase.

In particular, the Tween20 concentrations in water are chosen to obtain values of γ similar to those of the hydroorganic solutions above discussed. As in the previous case, solutions of FC40 oil, both pure and in presence of surfactant (FC40 + PFD or FC40 + PK), are used as continuous phases.

Fig. 5a shows β as a function of , for a fixed flow rate of the continuous phase (ϕ = 70 μL min -1 ). The results present a strong dependence on the Tween20 concentration. Below the critical micellar concentration, CMC app (≃0.05%), β decreases with , reaching a plateau for long droplets ( > 5); above the CMC app , β initially decreases with  and then, for longer droplets ( > 3), increases, reaching a plateau as well for  > 5. By adding the surfactant to the continuous phase (FC40 + PFD, CMC app ≃ 0.02%), β exhibits the same behaviour as with pure FC40 however, as for the hydro-organic mixtures, the values of β are now distributed on a wider range of values (see Fig. 5b). As in the previous case, this represents an interesting feature for the final merging applications. Finally, the same general trends are also found for different oil flow rates (see Fig. S8 in ESI †) and in the case of FC40 + PK (see Fig. S9b andc in ESI †).

To better clarify the influence of the Tween20 in the dispersed phase, Fig. 6a shows the normalized speed β as a function of γ, for different ϕ and for a fixed droplet length  = 8, which guarantees to work in the asymptotic region. In both FC40 and FC40 + PFD, β increases by decreasing γ. However, there is no overlap between the two sets of data because γ ≈ 15 mN m -1 corresponds to the smallest (for FC40) and the highest (for FC40 + PFD) obtainable values, respectively. For a given ϕ, the maximum β variation, corresponding to a decrease in γ of one order of magnitude, is about 14%. In Fig. 6b, the same data are plotted versus Tween20 concentration, showing that β initially increases with the surfactant concentration even beyond the CMC app point, and eventually reaches an asymptotic value that depends on the applied ϕ. Similar behaviors for the FC40 + PK case are discussed in ESI † (see point 8). Therefore, the addition of Tween20 to the dispersed phase leads to a more complex behaviour than the one observed for the hydro-organic solutions. In order to discuss the experimental results, different effects must be considered. On the one side, it is known that an increasing concentration of surfactants makes the droplet interface less compressible (or more incompressible) 59 due to the compact arrangement of the molecules, thus the droplet tends to be elongated instead of being deformed in the rear part. This leads to a thicker lubrication film, thus to a higher speed. 29 On the other side, due to the internal recirculation flows associated with the droplet motion, the surfactant distribution at the interface is not homogenous: molecules are more concentrated at the rear meniscus than at the front one, and almost absent in the central region. 7 This generates an interfacial tension gradient (∇γ) that creates a Marangoni's stress acting against the droplet motion. 29 However, the inhomogeneous distribution of the surfactants decreases with their concentration, 29 leading to a reduction in Marangoni's stress for high concentrations. We expect that the resulting droplet speed is a combination of these different contributions. In fact, for short droplets ( < 3), ∇γ may be neglected with respect to the incompressibility 59 of the interface, leading to a droplet speed which mainly depends on the surfactant concentration: higher concentration leads to higher incompressibility, thus higher elongation and speed. Differently, for longer droplets ( > 3), β is influenced by both ∇γ and concentration. In fact, as shown in Fig. 5, for low concentrations of surfactant (<CMC app ), β decreases with , suggesting that the contribution of ∇γ is dominant. Differently, for high concentrations (>CMC app ), ∇γ is less effective than the surface incompressibility, which increases with the surfactant concentration leading to higher β.

Passive on demand droplet merging and enzymatic reaction

We show here that the observed variation in droplet speed with interfacial tension can be exploited to induce the merging between droplets in a channel of constant circular crosssection. By using the experimental setup 2, pairs of droplets of about 130 nL ( ≃ 7), separated by a tunable initial distance (ΔS i ), are generated in FC40 + PFD. The first droplet containing pure water moves slower than the second one, containing a water phase solution with 1% of Tween20. They eventually merge after a certain distance (ΔS m ), as shown in the sequence of images of Fig. 7 and in the Movie S1. † Additionally, the graph in Fig. 7 shows that ΔS m is linearly proportional to ΔS i , and that higher flow rates lead to faster merging events. Both results are expected considering the previously presented results: i) droplets show uniform motions and ii) higher flow rate of the continuous phase leads to higher droplet speed. Thus, the merging of two droplets based on their difference in interfacial tension can be easily achieved, and for a given composition of droplets, the position of the merging along the capillary can be precisely tuned in space and time without the need of any specific geometrical features or external equipment, by simply varying ΔS i or/ and the flow rate of the continuous phase, as shown in the Movie S2-4. † Therefore, the merging point can be easily predicted, facilitating the design and the integration of this ondemand merging step in a microfluidic platform.

This innovative on-demand merging approach is validated by implementing, in a droplet microfluidic device, an enzymatic reaction aimed to quantify the hydrogen peroxide content in a solution. 60 In conventional format, these assays, characterized by a time-dependent readout, are affected by operator-dependent variability due to the multiple steps required. 12,61 Integration and are, therefore, fundamental to improve processing time, reproducibility and sensitivity. 62,63 In order to measure the enzymatic activity or detect a target analyte, precise triggering of the fusion step between the sample and the reagent droplet, as well as a reproducible and reliable monitoring of the product formation are mandatory, in both continuous and stopped assay methods. 64 Using the same experimental setup 2 as above, two droplets (200 nL volume) are generated containing i) different concentrations of hydrogen peroxide (from 3.90 × 10 -1 μM to 7.62 × 10 -2 μM) and ii) the enzymatic solution including 0.01% Tween20 (see Material and methods). The initial separation between the two droplets (ΔS i ) is fixed at a constant distance and each pair is transported at a constant flow rate of 15 μL min -1 . In these conditions, the merging point is expected at about 14 cm from the detector. Upon coalescence, the enzymatic reaction starts and the HRP enzyme catalyses the reduction of hydrogen peroxide to convert the non-fluorescent Amplex Red molecules into resorufin, a redfluorescent oxidation product, proportionally to the content of hydrogen peroxide. 60,65 The fluorescence signal is monitored by flowing the merged droplet back and forth several times across the detector during the reaction period, 12 as performed in a continuous assay. As commonly carried out in batch, the resulting signal from a negative assay, obtained by using a sample droplet containing only the buffer solution (without H 2 O 2 ), is subtracted to the one corresponding to the droplet containing hydrogen peroxide. Fig. 8a reports the time evolution of the fluorescent intensity during the reaction for different hydrogen peroxide concentrations of interest. Each point of the graph represents the average of three independent measurements and the error bars represent standard deviations. As expected, the amount of formed products increases with time and is dependent on the H 2 O 2 concentration. From these curves, the enzyme kinetics can be investigated by plotting the slope of the curve at the beginning of the reaction against the H 2 O 2 concentration (see inset of Fig. 8a). The enzymatic activity follows the Michaelis-Menten model, 66 a typical description of the kinetic properties of a wide class of enzymes, characterized by an initial linear increase with the substrate concentration and by a levelling off at higher substrate concentrations. Additionally, the Michaelis constant K M obtained as fitting parameter results 1.17 ± 0.17 μM in good agreement with the value obtained for the same substrate/enzyme combination in batch. 67 This assures that the efficiency of the enzymatic reaction under investigation is not affected by the fact of being performed in small volumes (nL ranges) and in presence of a small amount of surfactants. Indeed, surfactants are often added in conventional reactions buffers, to prevent non-specific binding in immunoassays, to promote cell lysis, to solubilize the released intracellular materials or to denature proteins and other macromolecules. [68][69][START_REF] Arkin | Inhibition of Protein-Protein Interactions: Non-Cellular Assay Formats, Eli Lilly & Company and the National Center for Advancing Translational Sciences[END_REF][START_REF] Lever | [END_REF] Alternatively, the enzymatic reaction product can be evaluated by measuring the fluorescent readout after a fixed incubation time from the beginning of the reaction. This method, known as stopped assay, provides one single point and typically requires the introduction of an additional solution to quench the reaction. 64 Differently, our approach allows to precisely define the space between the merging point and the detector, thus the incubation time (t m-d ) for the reaction, without any quenching solution. With this aim, a fixed H 2 O 2 concentration of 6.09 × 10 -1 μM is chosen and several pairs of droplets with the same composition as above described are generated varying their initial distance ΔS i from 1.0 cm to This journal is © The Royal Society of Chemistry 51.5 cm to define five different incubation times, between about 61 s and 427 s. In this way, as shown in Fig. 8b, the temporal enzymatic reaction curve previously obtained (solid purple curve) is successfully recovered within the experimental errors. This result confirms the robustness of the ondemand merging approach, and provides a simplification in the detection protocol of a common enzymatic reaction, allowing a reliable observation time without additional steps to quench the reaction, as needed in conventional batch methods.

Conclusion

In this paper, we present an innovative microfluidic method for tuning the separation distance of pairs of confined droplets (or plugs) flowing in capillaries, by suitably varying the interfacial tension. The method requires standard equipment and circular tubings without the need of any microfabrication steps. It has been achieved after an extensive investigation of the droplet speed in a capillary as a function of several physico-chemical parameters, notably the droplet interfacial tension. Interestingly, different trends are observed by varying the droplet length () and by adding surfactants to either the continuous or dispersed phases. Correspondingly, the variation of the relative droplet speed can be as high as 25%. Thanks to this, we prove that the merging of droplets transported by the same carrier fluid can be easily and precisely triggered in space and time, in an on-demand fashion, by selecting the initial droplet distance and the carrier phase flow rate. As a proof of concept, we successfully applied this method to perform a standard enzymatic reaction for the detection of hydrogen peroxide in a solution, in both continuous and stopped assay configurations. More generally, our method can be easily extended to any protocol in which a reliable and/or time-resolved merging is required to start or stop a reaction. In particular, differently from a previous work, 8 it allows triggering several different types of reactions in a row after a predefined incubation time. Furthermore, it can be exploited to bring into contact two droplets stabilized by surfactants that guarantee a steric stabilization of the droplet interfaces (e.g.: PEG-Krytox 72 ), which can be merged by an external stimulus. 38 Finally, we point out that the investigated dependence of droplet speed on the interfacial tension can also be exploited to increase or maintain the distance between consecutive droplets of different contents. This represents an interesting alternative to the introduction of air bubbles 12,73 to prevent undesired coalescence in trains of droplets.

Fig. 1

 1 Fig.1Scheme of the experimental setup used to measure the droplet speed. (a) A single droplet is generated by pipetting alternatively the water and the oil phase solutions. The droplet is moved in front of the objective to evaluate its length (b) and then is flowed along the tube. Six pairs of optical fibers (c) are used to detect the droplet crossing signal, recording the time of this event (red lines in d). The droplet speed is evaluated as the slope of linear fits of its travelled distance as a function of time (e).

Fig. 3

 3 Fig. 3 (a) Normalized droplet speed β as a function of the normalized droplet length  using pure water as the dispersed phase and pure FC40 as the continuous phase, for different flow rates ϕ. The inset shows β as a function of ϕ for two different values of . (b) Dependence of β on  at different viscosity ratios λ between the dispersed and the continuous phase, for a fixed flow rate ϕ = 40 μL min -1 . Dashed lines are only a guide to the eyes.

Fig. 4

 4 Fig. 4 Normalized droplet mobility β as a function of the droplet length  for droplets of ethanol-water solutions of different concentrations carried out by pure FC40 oil (a) or by FC40 oil added with PFD surfactant (b) at a fixed flow rate (ϕ = 70 μL min -1 ). The vertical scales are different in the two graphs for a better data visualization. (c) β as a function of the static interfacial tension γ obtained by varying the ethanol concentration in the droplet phase. The data refer to droplets having  = 8 and different flow rates. Dashed lines are reported as a guide to the eyes.

Fig. 5

 5 Fig. 5 Normalized droplet mobility β as a function of the droplet length  varying the Tween20 concentration in the dispersed phase using different liquids as continuous phase (a) FC40 and (b) FC40 + PFD. The flow rate is the same: ϕ = 70 μL min -1 .

Fig. 6

 6 Fig. 6 Droplet mobility β as a function of (a) the static surface tension γ and (b) the Tween20 concentration, in both FC40 and FC40 + PFD case. The inset represents a magnification of the same graph at small concentrations, indicating the CMC app for FC40 (solid line) and FC40 + PFD (dashed line) condition.

Fig. 7

 7 Fig. 7 Droplet merging experiments. (left) Sequence of images showing two droplets (130 nL) containing 1) pure water and 2) Tween20 solution (at 1%) flowing in the capillary; even if they are initially separated (a and b) of about 2.5 mm, they progressively get closer (c-e) and finally merge (f). (right) Graph of the distance travelled by the droplet before the merging (ΔS m ) as function of the initial generation space (ΔS i ), for three different flow rate ϕ of the continuous phase (FC40 + PFD). The solid lines are the linear fits of the data.

Fig. 8

 8 Fig. 8 Implementation of an enzymatic reaction for the detection of hydrogen peroxide in a solution based on the passive merging between a sample droplet and an enzymatic solution one. a) Continuous assay: temporal evolution of the resulting fluorescence intensity for different peroxidase concentrations (ranging from about 7.62 × 10 -2 μM to 3.90 × 10 -1 μM). The initial slopes of these curves are plotted in the inset as a function of the respective hydrogen peroxide concentrations to determine the enzyme activity. The experimental points (in black) follow the Michaelis-Menten model for enzymes kinetics (red fit curve). b) Stopped assay: fixing the hydrogen peroxide concentration (6.09 × 10 -1 μM) and varying the initial spacing ΔS i between the droplets, it is possible to define different incubation times by delaying the merging. The enzymatic reaction curve (full purple line) is successfully recovered (black points). Each point is an average of three independent measurements and standard deviations are taken as error bars.

  FC40 (viscosity: 4.14 ± 0.02 mPa s) FC40 + PFD (viscosity: 4.25 ± 0.03 mPa s)

	Dispersed phase	γ (mN m -1 )	λ	Dispersed phase	γ (mN m -1 )	λ
	MilliQ water	51.2 ± 0.3	0.243 ± 0.003	MilliQ water	14.9 ± 0.2	0.243 ± 0.003
	Glycerol 49%	48.3 ± 0.5	1.230 ± 0.004	Glycerol 49%	14.3 ± 0.3	1.201 ± 0.004
	Glycerol 67%	46.2 ± 0.6	3.402 ± 0.009	Glycerol 67%	13.9 ± 0.3	3.310 ± 0.008
	EtOH 4%	40.4 ± 0.5	0.252 ± 0.003	EtOH 12%	13.1 ± 0.1	0.279 ± 0.003
	EtOH 8%	33.1 ± 0.5	0.256 ± 0.004	EtOH 25%	7.4 ± 0.1	0.414 ± 0.003
	EtOH 15%	27.3 ± 0.2	0.329 ± 0.003	EtOH 50%	4.0 ± 0.1	0.514 ± 0.003
	EtOH 31%	17.6 ± 0.2	0.500 ± 0.003	Tween20-0.001%	13.1 ± 0.4	0.241 ± 0.003
	Tween20-0.00125%	40.9 ± 0.1	0.241 ± 0.003	Tween20-0.01%	7.3 ± 0.2	0.241 ± 0.003
	Tween20-0.01%	26.7 ± 0.2	0.242 ± 0.003	Tween20-0.1%	5.6 ± 0.1	0.242 ± 0.003
	Tween20-0.1%	18.8 ± 0.2	0.244 ± 0.003	Tween20-1%	5.2 ± 0.1	0.249 ± 0.003
	Tween20-0.5%	17.9 ± 0.1	0.246 ± 0.003	Tween20-6%	4.7 ± 0.1	0.251 ± 0.003
	Tween20-1%	17.6 ± 0.2	0.249 ± 0.003
	Tween20-6%	17.3 ± 0.1	0.251 ± 0.003
	Tween20-12%	16.5 ± 0.2	0.252 ± 0.003

This journal is © The Royal Society of Chemistry 2019

Acknowledgements

We thank Marie-Caroline Jullien for the scientific discussion, Vélan Taniga and INOREVIA for support in the optical detec-tors development, Giogio Delfitto for developing the electronic components. This work was supported by the French National Research Agency (ANR) as part of the "Investissements d'Avenir" program (reference: ANR 10-NANO-0207), by the Labex and Equipex IPGG (references: 10-LABX-0031 and 10-EQPX-0034), by ERC "CellO" (FP7-IDEAS-ERC-321107) and by the ARC foundation for young researcher fellowship (DF). MS was supported by a Curie Institute International PhD fellowship. DF, MS, J-LV and SD are coinventors of a patent application filed by Institut Curie and CNRS, encompassing some of the technologies described in this article.

Conflicts of interest

There are no conflicts to declare.