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Monitoring Single Cell Bioenergetics via the Coarsening of Emulsion Droplets
L. Boitard, D. Cottinet, C. Kleinschmitt, N. Bremond, J. Baudry, G. Yvert, J. Bibette
Proc. Nat. Acad. Sci. USA - 109(19) :7181-6 - DOI:10.1073/pnas.1200894109 - 2012
Microorganisms are widely used to generate valuable products, and their efficiency is a major industrial focus. Bioreactors are typically composed of billions of cells, and available measurements only reflect the overall performance of the population. However, cells do not equally contribute, and process optimization would therefore benefit from monitoring this intrapopulation diversity. Such monitoring has so far remained difficult because of the inability to probe concentration changes at the single-cell level. Here, we unlock this limitation by taking advantage of the osmotically driven water flux between a droplet containing a living cell toward surrounding empty droplets, within a concentrated inverse emulsion. With proper formulation, excreted products are far more soluble within the continuous hydrophobic phase compared to initial nutrients (carbohydrates and salts). Fast diffusion of products induces an osmotic mismatch, which further relaxes due to slower diffusion of water through hydrophobic interfaces. By measuring droplet volume variations, we can deduce the metabolic activity down to isolated single cells. As a proof of concept, we present the first direct measurement of the maintenance energy of individual yeast cells. This method does not require any added probes and can in principle apply to any osmotically sensitive bioactivity, opening new routes for screening, and sorting large libraries of microorganisms and biomolecules.
UV-Induced Bursting of Cell-Sized Multicomponent Lipid Vesicles in a Photosensitive Surfactant Solution
A. Diguet, M. Yanagisawa, Y. J. Liu, E. Brun, S. Abadie, S. Rudiuk and D. Baigl
JACS - 134(10) :4898-904 - DOI:10.1021/ja211664f - 2012
We study the behavior of multicomponent giant unilamellar vesicles (GUVs) in the presence of AzoTAB, a photosensitive surfactant. GUVs are made of an equimolar ratio of dioleoylphosphatidylcholine (DOPC) and dipalmitoylphosphatidylcholine (DPPC) and various amounts of cholesterol (Chol), where the lipid membrane shows a phase separation into a DPPC-rich liquid-ordered (Lo) phase and a DOPC-rich liquid-disordered (Ld) phase. We find that UV illumination at 365 nm for 1 s induces the bursting of a significant fraction of the GUV population. The percentage of UV-induced disrupted vesicles, called bursting rate (Yburst), increases with an increase in [AzoTAB] and depends on [Chol] in a non-monotonous manner. Yburst decreases when [Chol] increases from 0 to 10 mol % and then increases with a further increase in [Chol], which can be correlated with the phase composition of the membrane. We show that Yburst increases with the appearance of solid domains ([Chol] = 0) or with an increase in area fraction of Lo phase (with increasing [Chol] = 10 mol %). Under our conditions (UV illumination at 365 nm for 1 s), maximal bursting efficiency (Yburst = 53%) is obtained for [AzoTAB] = 1 mM and [Chol] = 40 mol %. Finally, by restricting the illumination area, we demonstrate the first selective UV-induced bursting of individual target GUVs. These results show a new method to probe biomembrane mechanical properties using light as well as pave the way for novel strategies of light-induced drug delivery.
A completely in vitro ultrahigh-throughput droplet-based microfluidic screening system for protein engineering and directed evolution
Fallah-Araghi, J.-C. Baret, M. Ryckelynck, and A.D. Griffiths
Lab. Chip - 12(5) :882–91 - DOI: 10.1039/c2lc21035e - 2012
In vitro screening systems based on the coupled transcription and translation of genes using cell-free systems have a number of attractive features for protein engineering and directed evolution. We present a completely in vitro ultrahigh-throughput screening platform using droplet-based microfluidics. Single genes are compartmentalized in aqueous droplets, dispersed in inert carrier oil, and amplified using the polymerase chain reaction (PCR). After amplification, the droplets, now containing 30,000 copies of each gene, are fused one-to-one with droplets containing a cell-free coupled transcription-translation (IVTT) system and the reagents for a fluorogenic assay. Fluorescence-activated electrocoalescence with an aqueous stream is then used to selectively recover genes from droplets containing the desired activity. We demonstrate, by selecting mixtures of lacZ genes encoding the enzyme β-galactosidase and lacZmut genes encoding an inactive variant, that this system can sort at 2000 droplets s(-1): lacZ genes were enriched 502-fold from a 1 : 100 molar ratio of lacZ : lacZmut genes. Indeed, the false positive and false negative error rates were both <0.004 and the results indicate that enrichment is not limited by the sorting efficiency, but by the co-encapsulation of multiple genes in droplets, which is described by the Poisson distribution. Compared to screening using microtiter plate-based systems, the volume and cost of PCR and IVTT reagents are reduced by almost 10(5)-fold, allowing the screening of 10(6) genes using only 150 μL of reagents.
Functional single-cell hybridoma screening using droplet-based microfluidics
B. El Debs, R. Utharala, I.V. Balyasnikova, A.D. Griffiths and C.A. Merten
Proc. Nat. Acad. Sci. USA - 109(29) :11570-5 - DOI: 10.1073/pnas.1204514109 - 2012
Monoclonal antibodies can specifically bind or even inhibit drug targets and have hence become the fastest growing class of human therapeutics. Although they can be screened for binding affinities at very high throughput using systems such as phage display, screening for functional properties (e.g., the inhibition of a drug target) is much more challenging. Typically these screens require the generation of immortalized hybridoma cells, as well as clonal expansion in microtiter plates over several weeks, and the number of clones that can be assayed is typically no more than a few thousand. We present here a microfluidic platform allowing the functional screening of up to 300,000 individual hybridoma cell clones within less than a day. This approach should also be applicable to nonimmortalized primary B-cells, as no cell proliferation is required: Individual cells are encapsulated into aqueous microdroplets and assayed directly for the release of antibodies inhibiting a drug target based on fluorescence. We used this system to perform a model screen for antibodies that inhibit angiotensin converting enzyme 1, a target for hypertension and congestive heart failure drugs. When cells expressing these antibodies were spiked into an unrelated hybridoma cell population in a ratio of 1:10,000 we observed a 9,400-fold enrichment after fluorescence activated droplet sorting. A wide variance in antibody expression levels at the single-cell level within a single hybridoma line was observed and high expressors could be successfully sorted and recultivated.
Membraneless glucose/O2 microfluidic biofuel cells using covalently bound enzymes
T. Beneyton, I Putu Mahendra Wijaya,a C. Ben Salem, A. D. Griffiths and V. Taly
Chem. Comm. - 49 1094–1096 - - 2012
Obstructed breakup of slender drops in a microfluidic T-junction
A.M. Leshansky, S. Afkhami, M.C Jullien, P. Tabeling
Phys. Rev. Lett. - 108(26) :4502 - DOI:10.1103/PhysRevLett.108.264502 - 2012
In this Letter we present a theoretical analysis of the droplet breakup with “permanent obstruction” in a microfluidic T junction [M.-C. Jullien et al., Phys. Fluids 21 072001 (2009)]. The proposed theory is based on a simple geometric construction for the interface shape combined with Tanner’s law for the local contact angle. The resulting scaling of the droplet deformation with time and capillary number is in excellent agreement with the results of direct numerical simulations and prior experiments. More rigorous analysis based on the lubrication approximation reveals a self-similar behavior analogous to the classical problem of a droplet spreading over a preexisting liquid film.
A low-cost, label-free DNA detection method in lab-on-chip format based on electrohydrodynamic instabilities, with application to long-range PCR
M. Lemine Youba Diakité, J. Champ, S. Descroix, L. Malaquin, F. Amblard and J.-L. Viovy
Lab. Chip - 12(22) :4738-47 - DOI:10.1039/C2LC40372B - 2012
In order to evolve from a "chip in the lab" to a "lab on a chip" paradigm, there is still a strong demand for low-cost, portable detection technologies, notably for analytes at low concentrations. Here we report a new label-free DNA detection method with direct electronic read, and apply it to long-range PCR. This method uses a nonlinear electrohydrodynamic phenomenon: when subjected to high electric fields (typically above 100 V cm(-1)), suspensions of large polyelectrolytes, such as long DNA molecules, create "giant" dynamic concentration fluctuations. These fluctuations are associated with large conductivity inhomogeneities, and we use here a contact-mode local conductivity detector to detect these fluctuations. In order to decouple the detection electronics from the high voltage excitation one, an original "doubly symmetric" floating mode battery-operated detection scheme was developed. A wavelet analysis was then applied, to unravel from the chaotic character of the electohydrodynamic instabilities a scalar signal robustly reflecting the amplification of DNA. As a first proof of concept, we measured the products of the off-chip amplification of 10 kbp DNA from lambda phage DNA, achieving a sensitivity better than 100 fg DNA in the original 50 µl sample. This corresponds to the amplification products of less than 100 initial copies of target DNA. The companion enabling technologies developed to implement this new concept, i.e. the doubly symmetric contact conductivity detection and wavelet analysis, may also find various other applications in lab-on-chips.
A programmable magnetic tweezers and droplet microfluidic platform for high throughput nanoliter multi-steps assays
Ali-Cherif, S. Begolo, S. Descroix, J.L. Viovy, L.Malaquin
Angew Chem Int Ed Engl. - 51(43) :10765-9 - DOI:10.1002/anie.201203862 - 2012
Tweezing out the answer: A microfluidic device combining droplets (less than 100 nL) and magnetic particles was implemented for fast heterogeneous multiplexed assays. Magnetic tweezers can perform the manipulations required in an immunoassay (capture, extraction, mixing, and rinsing). This method was applied to the diagnosis of congenital hypothyroidism with 14 pM sensitivity. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Carbamazepine removal from water by dielectric barrier discharge : comparison of ex situ and in situ discharge on water
Y. Liu, S. Mei, D. Iya-Sou, S. Cavadias, S. Ognier
Chemical Engineering And Processing - 56 :10-18 - DOI:10.1016/j.cep.2012.03.003 - 2012
Dielectric barrier discharges (DBD) were used for the degradation of carbamazepine (CBZ) in aqueous solution. The electric discharge was generated either ex situ or in situ directly on the water surface. To maintain the same ozone concentration of 40 ppm in both instances, the power injected was 0.7 W in the ex situ discharge and 12 W in the in situ discharge. The results showed 100% CBZ removal after 3 min of treatment with the ex situ discharge, while the in situ discharge only removed 81% of the CBZ after 60 min. According to measurements of UV absorbance at 285 nm and 254 nm, and of total organic carbon, the ex situ discharge system also proved to be more effective than the in situ system. The measurement of nitrogen oxides in both gaseous and liquid phases indicated that high energy in situ discharges produced a large amount of NOx. These species contributed to decreased pH and significantly slowed the CBZ oxidation rate, due to their competition with ozone. Production of NOx should be avoided when using the DBD technique for wastewater treatment.
Atmospheric Pressure Deposition of Thin Functional Coatings: Polymer Surface Patterning by DBD and Post-Discharge Polymerization of Liquid Vinyl Monomer from Surface Radicals
J.P. Borra, A. Valt, F. Arefi-Khonsari, M. Tatoulian
Plasma Process - 9(11-12) :1104-15 - DOI:10.1002/ppap.201100210 - 2012
We present a route for grafting polyacid and polyether coatings on polymers by post-discharge polymerization of liquid vinyl monomer. Surface modifications of polymer films by Micro-Discharges in air Dielectric Barrier Discharge (MD, DBD) are depicted with sub-micrometer craters homogeneously distributed. Both the energy per MD and the power density are critical to avoid thermal film deformation. Homogeneous surface composition is related to the DBD energy density. The polymerization mechanism is depicted from yields versus DBD energy density and time of exposure to air between DBD and monomer deposition. Both parameters control the surface density of radicals and peroxides, triggering the post-DBD polymerization with 80 and 73% of monomer functionality remaining in acid and ether coatings, respectively. The effect of deposition conditions on coatings properties is shown as well as the stability of coatings upon washing.


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579 publications.