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Quantitative and sensitive detection of rare mutations using droplet microfluidics
D. Pekin, Y. Skhiri, J.-C. Baret, D. Le Corre, L. Mazutis, C. Ben Salem, F. Millot, A. El Harrak, J.B. Hutchison, J.W. Larson, D.R. Link, P. Laurent-Puig, A.D. Griffiths and V. Taly
Lab. Chip - 11(13) :2156-66 - DOI: 10.1039/c1lc20128j - 2011
Somatic mutations within tumoral DNA can be used as highly specific biomarkers to distinguish cancer cells from their normal counterparts. These DNA biomarkers are potentially useful for the diagnosis, prognosis, treatment and follow-up of patients. In order to have the required sensitivity and specificity to detect rare tumoral DNA in stool, blood, lymph and other patient samples, a simple, sensitive and quantitative procedure to measure the ratio of mutant to wild-type genes is required. However, techniques such as dual probe TaqMan(®) assays and pyrosequencing, while quantitative, cannot detect less than ∼1% mutant genes in a background of non-mutated DNA from normal cells. Here we describe a procedure allowing the highly sensitive detection of mutated DNA in a quantitative manner within complex mixtures of DNA. The method is based on using a droplet-based microfluidic system to perform digital PCR in millions of picolitre droplets. Genomic DNA (gDNA) is compartmentalized in droplets at a concentration of less than one genome equivalent per droplet together with two TaqMan(®) probes, one specific for the mutant and the other for the wild-type DNA, which generate green and red fluorescent signals, respectively. After thermocycling, the ratio of mutant to wild-type genes is determined by counting the ratio of green to red droplets. We demonstrate the accurate and sensitive quantification of mutated KRAS oncogene in gDNA. The technique enabled the determination of mutant allelic specific imbalance (MASI) in several cancer cell-lines and the precise quantification of a mutated KRAS gene in the presence of a 200,000-fold excess of unmutated KRAS genes. The sensitivity is only limited by the number of droplets analyzed. Furthermore, by one-to-one fusion of drops containing gDNA with any one of seven different types of droplets, each containing a TaqMan(®) probe specific for a different KRAS mutation, or wild-type KRAS, and an optical code, it was possible to screen the six common mutations in KRAS codon 12 in parallel in a single experiment.
High-resolution dose-response screening using droplet-based microfluidics
O.J. Miller, A.E. Harrak, T. Mangeat, J.-C. Baret, L. Frenz, B. El Debs, E. Mayot, M.L. Samuels, E.K. Rooney, P. Dieu, M. Galvan, D.R. Link and A.D. Griffiths
Proc. Nat. Acad. Sci. USA - 109(2) :378–83 - DOI:10.1073/pnas.1113324109 - 2011
A critical early step in drug discovery is the screening of a chemical library. Typically, promising compounds are identified in a primary screen and then more fully characterized in a dose–response analysis with 7–10 data points per compound. Here, we describe a robust microfluidic approach that increases the number of data points to approximately 10,000 per compound. The system exploits Taylor–Aris dispersion to create concentration gradients, which are then segmented into picoliter microreactors by droplet-based microfluidics. The large number of data points results in IC50 values that are highly precise (± 2.40% at 95% confidence) and highly reproducible (CV = 2.45%, n = 16). In addition, the high resolution of the data reveals complex dose–response relationships unambiguously. We used this system to screen a chemical library of 704 compounds against protein tyrosine phosphatase 1B, a diabetes, obesity, and cancer target. We identified a number of novel inhibitors, the most potent being sodium cefsulodine, which has an IC50 of 27 ± 0.83 μM.
Analysis of gene expression at a single cell level in microdroplets
P. Mary, L. Dauphinot, N. Bois, M.C Potier, V. Studer, P. Tabeling
Biomicrofluidics - 5(2) :24109 - DOI:10.1063/1.3596394 - 2011
In the present work, we have measured the messenger RNA expression of specific genes both from total RNA and cells encapsulated in droplets. The microfluidic chip introduced includes the following functionalities: RNA/cell encapsulation, lysis, reverse transcription and real-time polymerase chain reaction. We have shown that simplex and duplex gene expression measurements can be carried out over a population of 100 purified RNA samples encapsulated simultaneously in 2 nl droplets in less than 2 h. An analysis of 100 samples containing one to three cells has shown excellent consistency with standard techniques regarding average values. The cell-to-cell distributions of the E-cadherin expression suggest fluctuations on the order of 80% in the number of transcripts, which is highly consistent with the general findings from the literature. A mathematical model has also been introduced to strengthen the interpretation of our results. The present work paves the way for the systematic acquisition of such information in biological and biomedical studies.
Ultrasound Internal Tattooing
O. Couture, M. Faivre, N. Pannacci, A. Babataheri, V. Servois, P. Tabeling, M. Tanter
Med. Phys - 38(2) :1116-23 - PMID:21452748 - 2011
The ability of remotely tagging tissues in a controlled and three-dimensional manner during preoperative imaging could greatly help surgeons to identify targets for resection. The authors' objective is to selectively and noninvasively deposit markers under image guidance for such internal tattooing.
This study describes the production of new ultrasound-inducible droplets carrying large payloads of fluorescent markers and the in vivo proof of concept of their remote and controlled deposition via focused ultrasound. The droplets are monodispersed multiple emulsions produced in a microfluidic system, consisting of aqueous fluorescein in perfluorocarbon in water. Their conversion (either by vaporization or cavitation) is performed remotely using a clinical ultrasonic imaging probe.
When submitted to 5 MHz imaging pulses, the droplets vaporize in vitro at 1.4 MPa peak-negative pressure and eject their content. After several seconds, a brightly fluorescent spot (0.5 mm diameter) is observed at the focus of the transducer. Experiments in the chorioallantoique membrane of chicken eggs and chicken embryo demonstrate that the spot is stable and is easily seen by naked eye.
These ultrasound-inducible multiple emulsions could be used to deliver large amounts of contrast agents, chemotherapy, and genetic materials in vivo using a conventional ultrasound scanner.
New family of fluorinated polymer chips for droplet and organic solvent microfluidics
Begolo S, Colas G, Viovy JL, Malaquin L.
Lab. Chip - 11(3) :508-12 - DOI:10.1039/c0lc00356e - 2011
We present a new family of microfluidic chips hot embossed from a commercial fluorinated thermoplastic polymer (Dyneon THV). This material shares most of the properties of fluoro polymers (very low surface energy and resistance to chemicals), but is easier to process due to its relatively low melting point. Finally, as an elastic material it also allows easy world to chip connections. Fluoropolymer films can be imprinted by hot embossing from PDMS molds prepared by soft lithography. Chips are then sealed by an original technique (termed Monolithic-Adhesive-Bonding), using two different grades of fluoropolymer to obtain uniform mechanical, chemical and surface properties. This fabrication process is well adapted to rapid prototyping, but it also has potential for low cost industrial production, since it does not require any curing or etching step. We prepared microfluidic devices with micrometre resolution features, that are optically transparent, and that provide good resistance to pressure (up to 50 kPa). We demonstrated the transport of water droplets in fluorinated oil, and fluorescence detection of DNA within the droplets. No measurable interaction of the droplets with the channels wall was observed, alleviating the need for surface treatment previously necessary for droplet applications in microfluidic chips. These chips can also handle harsh organic solvents. For instance, we demonstrated the formation of chloroform droplets in fluorinated oil, expanding the potential for on chip microchemistry.
Microchip integrating magnetic nanoparticles for allergy diagnosis
Teste B, Malloggi F, Siaugue JM, Varenne A, Kanoufi F, Descroix S.
Lab. Chip - 11(24) :4207-13 - DOI:10.1039/C1LC20809H - 2011
We report on the development of a simple and easy to use microchip dedicated to allergy diagnosis. This microchip combines both the advantages of homogeneous immunoassays i.e. species diffusion and heterogeneous immunoassays i.e. easy separation and preconcentration steps. In vitro allergy diagnosis is based on specific Immunoglobulin E (IgE) quantitation, in that way we have developed and integrated magnetic core-shell nanoparticles (MCSNPs) as an IgE capture nanoplatform in a microdevice taking benefit from both their magnetic and colloidal properties. Integrating such immunosupport allows to perform the target analyte (IgE) capture in the colloidal phase thus increasing the analyte capture kinetics since both immunological partners are diffusing during the immune reaction. This colloidal approach improves 1000 times the analyte capture kinetics compared to conventional methods. Moreover, based on the MCSNPs' magnetic properties and on the magnetic chamber we have previously developed the MCSNPs and therefore the target can be confined and preconcentrated within the microdevice prior to the detection step. The MCSNPs preconcentration factor achieved was about 35?000 and allows to reach high sensitivity thus avoiding catalytic amplification during the detection step. The developed microchip offers many advantages: the analytical procedure was fully integrated on-chip, analyses were performed in short assay time (20 min), the sample and reagents consumption was reduced to few microlitres (5 µL) while a low limit of detection can be achieved (about 1 ng mL(-1)).
Stress Clamp Experiments on Multicellular Tumor Spheroids
Montel F, Delarue M, Elgeti J, Malaquin L, Basan M, Risler T, Cabane B, Vignjevic D, Prost J, Cappello G, Joanny JF.
Phys. Rev. Lett. - 107(18) :188102 - PMID:22107677 - 2011
Pour croître une tumeur doit « faire sa place » au sein d’un tissu : c’est tout un jeu de forces mécaniques qui s’exercent alors entre la tumeur et le tissu sain et dont le perdant pourrait être la tumeur. L’application d’une pression même faible sur un tissu tumoral bloque son expansion et lorsque l’on cesse d’exercer cette pression, la croissance tumorale repart. La pression stoppe la prolifération des cellules, mais pas de façon homogène : les cellules au centre de leur amas cellulaire ne se divisent plus, alors que celles en périphérie continuent. Dans l’organisme, seules les tumeurs capables de soutenir cette pression pourraient continuer à proliférer. L’environnement mécanique pourrait un jour devenir un outil à prendre en compte dans le diagnostic.
Probing ribosomal protein–RNA interactions with an external force
Pierre Mangeol, Thierry Bizebard, Claude Chiaruttini, Marc Dreyfus, Mathias Springer, and Ulrich Bockelmann
Proc. Nat. Acad. Sci. USA - 108(45) :18272-6 - DOI:10.1073/pnas.1107121108 - 2011
Ribosomal (r-) RNA adopts a well-defined structure within the ribosome, but the role of r-proteins in stabilizing this structure is poorly understood. To address this issue, we use optical tweezers to unfold RNA fragments in the presence or absence of r-proteins. Here, we focus on Escherichia coli r-protein L20, whose globular C-terminal domain (L20C) recognizes an irregular stem in domain II of 23S rRNA. L20C also binds its own mRNA and represses its translation; binding occurs at two different sites—i.e., a pseudoknot and an irregular stem. We find that L20C makes rRNA and mRNA fragments encompassing its binding sites more resistant to mechanical unfolding. The regions of increased resistance correspond within two base pairs to the binding sites identified by conventional methods. While stabilizing specific RNA structures, L20C does not accelerate their formation from alternate conformations—i.e., it acts as a clamp but not as a chaperone. In the ribosome, L20C contacts only one side of its target stem but interacts with both strands, explaining its clamping effect. Other r-proteins bind rRNA similarly, suggesting that several rRNA structures are stabilized by “one-side” clamping.
Quantifying how DNA stretches, melts and changes twist under tension
Peter Gross, Niels Laurens, Lene B. Oddershede, Ulrich Bockelmann, Erwin J.G. Peterman, and Gijs J. L. Wuite
Nature Physics - 7 :731-6 - DOI:10.1038/nphys2002 - 2011
In cells, DNA is constantly twisted, bent and stretched by numerous proteins mediating genome transactions. Understanding these essential biological processes requires in-depth knowledge of how DNA complies to mechanical stress. Two important physical features of DNA, helical structure and sequence, are not incorporated in current descriptions of DNA elasticity. Here we connect well-defined force–extension measurements with a new model for DNA elasticity: the twistable worm-like chain, in which DNA is considered a helical, elastic entity that complies to tension by extending and twisting. In addition, we reveal hitherto unnoticed stick–slip dynamics during DNA overstretching at ~65?pN, caused by the loss of base-pairing interactions. An equilibrium thermodynamic model solely based on DNA sequence and elasticity is presented, which captures the full complexity of this transition. These results offer deep quantitative insight in the physical properties of DNA and present a new standard description of DNA mechanics.
Rectification of the current in alpha-hemolysin pore depends on the cation type : the alkali series probed by MD simulations and experiments
S. Bhattacharya, L. Muzard, L. Payet, J. Mathe, U. Bockelmann, A. Aksimentiev, and V. Viasnoff
J Phys Chem C - 115(10) :4255-64 - PMID:21860669 - 2011
A striking feature of the alpha-hemolysin channel-a prime candidate for biotechnological applications-is the dependence of its ionic conductance on the magnitude and direction of the applied bias. Through a combination of lipid bilayer single-channel recording and molecular dynamics (MD) simulations, we characterized the current-voltage relationship of alpha-hemolysin for all alkali chloride salts at neutral pH. The rectification of the ionic current was found to depend on the type of cations and increase from Li(+) to Cs(+). Analysis of the MD trajectories yielded a simple quantitative model that related the ionic current to the electrostatic potential, the concentration and effective mobility of ions in the channel. MD simulations reveal that the major contribution to the current asymmetry and rectification properties originates from the cationic contribution to the current that is significantly reduced in a cationic dependent way when the membrane polarity is reversed. The variation of chloride current was found to be less important. We report that the differential affinity of cations for the charged residues positioned at the channel's end modulates the number of ions inside the channel stem thus affecting the current properties. Through direct comparison of simulation and experiment, this study evaluates the accuracy of the MD method for prediction of the asymmetric, voltage dependent conductances of a membrane channel.


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579 publications.