Fragmentation of DNA in a sub-microliter microfluidic sonication device.
Tseng, Q., Lomonosov, A.M., Furlong, E.E. & Merten, C.A.
Lab Chip. 2012 Nov 21;12(22):4677-82. doi: 10.1039/c2lc40595d.
Fragmentation of DNA is an essential step for many biological applications including the preparation of next-generation sequencing (NGS) libraries. As sequencing technologies push the limits towards single cell and single molecule resolution, it is of great interest to reduce the scale of this upstream fragmentation step. Here we describe a miniaturized DNA shearing device capable of processing sub-microliter samples based on acoustic shearing within a microfluidic chip. A strong acoustic field was generated by a Langevin-type piezo transducer and coupled into the microfluidic channel via the flexural lamb wave mode. Purified genomic DNA, as well as covalently cross-linked chromatin were sheared into various fragment sizes ranging from approximately 180 bp to 4 kb. With the use of standard PDMS soft lithography, our approach should facilitate the integration of additional microfluidic modules and ultimately allow miniaturized NGS workflows.
Functional single-cell hybridoma screening using droplet-based microfluidics.
El Debs, B., Utharala, R., Balyasnikova, I.V., Griffiths, A.D. & Merten, C.A.
Proc Natl Acad Sci U S A. 2012 Jul 2. Research Highlight in Nature Methods
9(9): 866. 2012. Epub 2012 Jul 2.
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.
Micro segmented-flow in biochemical and cell-based assays.
Clausell-Tormos, J. & Merten, C.A.
Front Biosci (Elite Ed). 2012 Jan 1;4:1768-79.
Micro-segmented flow (e.g. in microfluidic channels, capillaries or a length of tubing) has become a promising technique in modern biology. Compared to conventional formats such as microtiter plates, sample volumes can be reduced about 1000-fold, thus allowing a massive reduction of assay costs and the use of samples available in low quantities, only (e.g. primary cells). Furthermore, assays can be highly parallelized and performed at superb spatio-temporal resolution. Here, we review the state-of-the-art in micro-segmented flow as applied in biochemical, cell- and multicellular organisms-based assays. We discuss likely future applications such as single cell / single organism proteomics and transcriptomics and point out the specific advantages and limitations compared to emulsion-based (droplet-based) approaches.
A competitive co-cultivation assay for cancer drug specificity evaluation.
El Debs, B.W., Tschulena, U., Griffiths, A.D. & Merten, C.A.
J Biomol Screen. 2011 Sep;16(8):818-24. Epub 2011 Jul 25.
The identification of compounds that specifically inhibit or kill cancer cells without affecting cells from healthy tissues is very challenging but very important for reducing the side effects of current cancer therapies. Hence, there is an urgent need for improved assays allowing the selectivity of a given compound to be monitored directly. The authors present an assay system based on the competitive co-cultivation of an excess of cancer cells with a small fraction of noncancer human indicator cells generating a fluorescence signal. In the absence of a specific anticancer compound, the cancer cells outgrow the indicator cells and abolish the fluorescence signal. In contrast, the presence of specific anticancer drugs (such as Tyrphostin-AG1478 or PLX4720) results in the selective growth of the indicator cells, giving rise to a strong fluorescence signal. Furthermore, the authors show that the nonspecific cytotoxic compound sodium azide kills both cancer and noncancer cells, and no fluorescence signal is obtained. Hence, this assay system favors the selection of compounds that specifically target cancer cells and decreases the probability of selecting nonspecific cytotoxic molecules. Z factors of up to 0.85 were obtained, indicating an excellent assay that can be used for high-throughput screening.
Microfluidic devices for diagnostic applications.
Eicher, D. & Merten, C.A.
Expert Rev Mol Diagn. 2011 Jun;11(5):505-19. doi: 10.1586/ERM.11.25.
Microfluidic systems have become an attractive platform for diagnostic applications. The technology not only allows drastically decreased assay volumes (down to picoliters), but also accelerates sample processing and readout. In this article we give an overview of the different kinds of systems, ranging from non- or minimally instrumented point-of-care devices for the detection of pathogens to fully-instrumented cutting-edge technologies such as next-generation sequencing and droplet-based microfluidics. We expect that microfluidic systems will have a major impact on future diagnostic (e.g., disposable devices) and therapeutic approaches (e.g., personalized medicine). Here we summarize current trends and critically review the limitations.
Retroviral display in gene therapy, protein engineering, and vaccine development.
Urban, J.H. & Merten, C.A.
ACS Chem Biol. 2011 Jan 21;6(1):61-74. Epub 2010 Dec 20.
The display and analysis of proteins expressed on biological surfaces has become an attractive tool for the study of molecular interactions in enzymology, protein engineering, and high-throughput screening. Among the growing number of established display systems, retroviruses offer a unique and fully mammalian platform for the expression of correctly folded and post-translationally modified proteins in the context of cell plasma membrane-derived particles. This is of special interest for therapeutic applications such as gene therapy and vaccine development and also offers advantages for the engineering of mammalian proteins toward customized binding affinities and catalytic activities. This review critically summarizes the basic concepts and applications of retroviral display and analyses its benefits in comparison to other display techniques.
Miniaturization and Parallelization of Biological and Chemical Assays in Microfluidic Devices.
Vyawahare, S., Griffiths, A.D. & Merten, C.A.
Chem Biol. 2010 Oct 29;17(10):1052-65.
Microfluidic systems are an attractive solution for the miniaturization of biological and chemical assays. The typical sample volume can be reduced up to 1 million-fold, and a superb level of spatiotemporal control is possible, facilitating highly parallelized assays with drastically increased throughput and reduced cost. In this review, we focus on systems in which multiple reactions are spatially separated by immobilization of reagents on two-dimensional arrays, or by compartmentalization in microfabricated reaction chambers or droplets. These systems have manifold applications, and some, such as next-generation sequencing are already starting to transform biology. This is likely the first step in a biotechnological transformation comparable to that already brought about by the microprocessor in electronics. We discuss both current applications and likely future impacts in areas such as the study of single cells/single organisms and high-throughput screening.
Screening Europe 2010: an update about the latest technologies and applications in high-throughput screening.
Expert Rev Mol Diagn. 2010 Jul;10(5):559-63.
This article reviews important presentations from the 7th Screening Europe Conference, and extracts general trends and developments. Technological advances, as well as novel applications are discussed, thus providing an overview on state-of-the-art high-throughput screening. Among other topics, cell-based assays were highly popular, since they reflect the clinical situation much more closely than screens based on purified drug targets. A further approach to reduce the costly attrition of drug candidates subsequent to initial screens is the use of label-free technology. By measuring desired properties directly, without the use of any reporter (e.g., calorimetric measurements of binding affinities), the selection of false positives can be decreased drastically. Additional improvements in high-throughput screening are resulting from novel technology platforms utilizing sophisticated imaging systems and/or miniaturization. These and other important topics from the Screening Europe 2010 Conference are discussed in this article, thus providing a current snapshot of the field.
An automated two-phase microfluidic system for kinetic analyses and the screening of compound libraries.
Clausell-Tormos, J., Griffiths, A.D. & Merten, C.A.
Lab Chip. 2010 May 21;10(10):1302-7. Epub 2010 Mar 3.
Droplet-based microfluidic systems allow biological and chemical reactions to be performed on a drastically decreased scale. However, interfacing the outside world with such systems and generating high numbers of microdroplets of distinct chemical composition remain challenging. We describe here an automated system in which arrays of chemically distinct plugs are generated from microtiter plates. Each array can be split into multiple small-volume copies, thus allowing several screens of the same library. The system is fully compatible with further on-chip manipulation(s) and allows monitoring of individual plugs over time (e.g. for recording reaction kinetics). Hence the technology eliminates several bottlenecks of current droplet-based microfluidic systems and should open the way for (bio-)chemical and cell-based screens.
Coupling the inhibition of viral transduction with a positive fluorescence signal.
Clausell-Tormos, J., Griffiths, A.D. & Merten, C.A.
Comb Chem High Throughput Screen. 2010 May;13(4):352-7.
Cell-based assays for the inhibition of viral infections most commonly couple a positive signal (e.g., an increase in fluorescence) to the infection itself and not to its inhibition. When performing drug screens, compounds decreasing the signal are therefore considered as putative inhibitors. However, this approach can cause the selection of many false positives, since, for example, both killing of the host cell and inhibiting viral cell-entry results in the same signal. Using a model system based on murine leukemia virus (MLV) particles pseudotyped with the G-protein of vesicular stomatitis virus (VSV-G), we have developed generic assays coupling a positive readout to the inhibition of viral transduction. Consequently, the system favors drug candidates (and concentrations thereof) that do not harm human cells and significantly decreases the probability for selecting false positives. The assay allows Z-factors of approximately 0.9, takes cytotoxic side effects into account and could in theory be adapted for high-throughput screening of inhibitors against further viral species.
Nanoliter plates--versatile tools for the screening of split-and-mix libraries on-bead and off-bead.
Upert, G., Merten, C.A. & Wennemers, H.
Chem Commun (Camb). 2010 Apr 7;46(13):2209-11. Epub 2010 Feb 23.
Nanoliter plates are versatile tools for the screening of split-and-mix libraries both on-bead and off-bead. The method was evaluated for the identification of hydrolytically active compounds and enzyme inhibitors.
High-throughput screening of enzymes by retroviral display using droplet-based microfluidics.
Granieri, L., Baret, J.C., Griffiths, A.D. & Merten, C.A.
Chem Biol. 2010 Mar 26;17(3):229-35.
During the last 25 years, display techniques such as phage display have become very powerful tools for protein engineering, especially for the selection of monoclonal antibodies. However, while this method is extremely efficient for affinity-based selections, its use for the selection and directed evolution of enzymes is still very restricted. Furthermore, phage display is not suited for the engineering of mammalian proteins that require posttranslational modifications such as glycosylation or membrane anchoring. To circumvent these limitations, we have developed a system in which structurally complex mammalian enzymes are displayed on the surface of retroviruses and encapsulated into droplets of a water-in-oil emulsion. These droplets are made and manipulated using microfluidic devices and each droplet serves as an independent reaction vessel. Compartmentalization of single retroviral particles in droplets allows efficient coupling of genotype and phenotype. Using tissue plasminogen activator (tPA) as a model enzyme, we show that, by monitoring the enzymatic reaction in each droplet (by fluorescence), quantitative measurement of tPA activity in the presence of different concentrations of the endogenous inhibitor PAI-1 can be made on-chip. On-chip fluorescence-activated droplet sorting allowed the processing of 500 samples per second and the specific collection of retroviruses displaying active wild-type tPA from a model library with a 1000-fold excess of retroviruses displaying a non-active control enzyme. During a single selection cycle, a more than 1300-fold enrichment of the active wild-type enzyme was demonstrated.
A competition-based assay for the screening of species-specific antibiotics.
Granieri, L., Miller, O.J., Griffiths, A.D. & Merten, C.A.
J Antimicrob Chemother. 2009 Jul;64(1):62-8. Epub 2009 Apr 28.
OBJECTIVES: To develop a high throughput screening-compatible assay for the selection of species-specific antibiotics that do not harm human cells. METHODS: Staphylococcus aureus and human reporter cells continuously generating a fluorescence signal were competitively co-cultivated. The fluorescence signals were determined in the presence and absence of the specific antibiotic streptomycin and the toxic compound sodium azide. The results were compared with a standard cfu assay. RESULTS: In the absence of an effective antibiotic, S. aureus outgrew the human reporter cells and thus abolished the fluorescence signal. Conversely, the addition of streptomycin resulted in the growth of the reporter cells and a strong fluorescence signal. When sodium azide was added instead of streptomycin, only a very low background signal was obtained indicating toxicity and damage to the human reporter cells. The assay proved to be highly reliable (Z-factor >0.9) and high fluorescence signals correctly correlated with the efficient inhibition of S. aureus, as determined in comparative cfu assays. CONCLUSIONS: In contrast to conventional cfu assays, the co-cultivation system allows the effects of a drug candidate on pathogens and human cells to be monitored simultaneously. Cytotoxic compounds can, therefore, be quickly ruled out during a primary screen. The nature of the screen also enables effective antibiotics to be identified without engineering the target pathogen to yield a fluorescence signal.
Droplets and emulsions: very high-throughput screening in biology
Baret, J.C., Taly, V., Ryckelynck, M., Merten, C.A. & Griffiths, A.D.
Med Sci (Paris). 2009 Jun-Jul;25(6-7):627-32.
The combination of microfluidic manipulation of emulsion droplets and in vitro compartmentalization offers a means to parallelize biological and chemical assays in droplets. These droplets behave as independent microreactors that are produced, actuated and analyzed at rates of the order of 1000 droplets per seconds providing tools to parallelize assays on small volumes (pL to nL range) for high-throughput -screening: the throughput of the assays performed in droplets is 1000 times larger than the throughput of existing technologies, based on micromanipulation of liquid in microtitre plates by robotic devices. The droplet-based microfluidic technology enables controlled manipulation, analysis and sorting of cells, genes and macromolecules based on their enzymatic activities, as well as chemical compounds based on their activity on biological targets. The integration of microfluidic systems for a series of complex individual operations on droplets could offer a solution to the miniaturization and automation of biological assays, combined with a decrease of the assay volumes and an increase of throughput, going beyond the capacities of conventional screening systems.
Imaging of mRNA in live cells using nucleic acid-templated reduction of azidorhodamine probes.
Pianowski, Z., Gorska, K., Oswald, L., Merten, C.A. & Winssinger, N.
J Am Chem Soc. 2009 May 13;131(18):6492-7.
Nucleic acid-templated reactions leading to a fluorescent product represent an attractive strategy for the detection and imaging of cellular nucleic acids. Herein we report the use of a Staudinger reaction to promote the reduction of profluorescent azidorhodamine. The use of two cell-permeable GPNA probes, one labeled with the profluorescent azidorhodamine and the other with trialkylphosphine, enabled the detection of the mRNA encoding O-6-methylguanine-DNA methyltransferase in intact cells.
Drop-based microfluidic devices for encapsulation of single cells.
Koster, S., Angile, F.E., Duan, H., Agresti, J.J., Wintner, A., Schmitz, C., Rowat, A.C., Merten, C.A., Pisignano, D., Griffiths, A.D. & Weitz, D.A.
Lab Chip. 2008 Jul;8(7):1110-5. Epub 2008 May 23.
We use microfluidic devices to encapsulate, incubate, and manipulate individual cells in picoliter aqueous drops in a carrier fluid at rates of up to several hundred Hz. We use a modular approach with individual devices for each function, thereby significantly increasing the robustness of our system and making it highly flexible and adaptable to a variety of cell-based assays. The small volumes of the drops enables the concentrations of secreted molecules to rapidly attain detectable levels. We show that single hybridoma cells in 33 pL drops secrete detectable concentrations of antibodies in only 6 h and remain fully viable. These devices hold the promise of developing microfluidic cell cytometers and cell sorters with much greater functionality, allowing assays to be performed on individual cells in their own microenvironment prior to analysis and sorting.
Droplet-based microfluidic platforms for the encapsulation and screening of Mammalian cells and multicellular organisms.
Clausell-Tormos, J., Lieber, D., Baret, J.C., El-Harrak, A., Miller, O.J., Frenz, L., Blouwolff, J., Humphry, K.J., Koster, S., Duan, H., Holtze, C., Weitz, D.A., Griffiths, A.D. & Merten, C.A.
Chem Biol. 2008 May;15(5):427-37. Research Highlight in Nature Methods 5(7): 580-581. 2008.
High-throughput, cell-based assays require small sample volumes to reduce assay costs and to allow for rapid sample manipulation. However, further miniaturization of conventional microtiter plate technology is problematic due to evaporation and capillary action. To overcome these limitations, we describe droplet-based microfluidic platforms in which cells are grown in aqueous microcompartments separated by an inert perfluorocarbon carrier oil. Synthesis of biocompatible surfactants and identification of gas-permeable storage systems allowed human cells, and even a multicellular organism (C. elegans), to survive and proliferate within the microcompartments for several days. Microcompartments containing single cells could be reinjected into a microfluidic device after incubation to measure expression of a reporter gene. This should open the way for high-throughput, cell-based screening that can use >1000-fold smaller assay volumes and has approximately 500x higher throughput than conventional microtiter plate assays.
Fusoselect: cell-cell fusion activity engineered by directed evolution of a retroviral glycoprotein.
Merten, C.A., Stitz, J., Braun, G., Medvedovska, J., Cichutek, K. & Buchholz, C.J.
Nucleic Acids Res. 2006 Mar 15;34(5):e41. Print 2006.
Membrane fusion plays a key role in many biological processes including vesicle trafficking, synaptic transmission, fertilization or cell entry of enveloped viruses. As a common feature the fusion process is mediated by distinct membrane proteins. We describe here 'Fusoselect', a universal procedure allowing the identification and engineering of molecular determinants for cell-cell fusion-activity by directed evolution. The system couples cell-cell fusion with the release of retroviral particles, but can principally be applied to membrane proteins of non-viral origin as well. As a model system, we chose a gamma-retroviral envelope protein, which naturally becomes fusion-active through proteolytic processing by the viral protease. The selection process evolved variants that, in contrast to the parental protein, mediated cell-cell fusion in absence of the viral protease. Detailed analysis of the variants revealed molecular determinants for fusion competence in the cytoplasmic tail (CT) of retroviral Env proteins and demonstrated the power of Fusoselect.
Circumventing tolerance to the prion protein (PrP): vaccination with PrP-displaying retrovirus particles induces humoral immune responses against the native form of cellular PrP.
Nikles, D., Bach, P., Boller, K., Merten, C.A., Montrasio, F., Heppner, F.L., Aguzzi, A., Cichutek, K., Kalinke, U. & Buchholz, C.J.
J Virol. 2005 Apr;79(7):4033-42.
Passive immunization with antibodies directed against the cellular form of the prion protein (PrPC) can protect against prion disease. However, active immunization with recombinant prion protein has so far failed to induce antibodies directed against native PrPC expressed on the cell surface. To develop an antiprion vaccine, a retroviral display system presenting either the full-length mouse PrP (PrP209) or the C-terminal 111 amino acids (PrP111) fused to the transmembrane domain of the platelet-derived growth factor receptor was established. Western blot analysis and immunogold electron microscopy of the retroviral display particles revealed successful incorporation of the fusion proteins into the particle membrane. Interestingly, retroviral particles displaying PrP111 (PrPD111 retroparticles) showed higher incorporation efficiencies than those displaying PrP209. Already 7 days after intravenous injection of PrPD111 retroparticles, PrPC-deficient mice (Prnp(o/o)) showed high immunoglobulin M (IgM) and IgG titers specifically binding the native PrPC molecule as expressed on the surface of T cells isolated from PrPC-overexpressing transgenic mice. More importantly, heterozygous Prnp(+/o) mice and also wild-type mice showed PrPC-specific IgM and IgG antibodies upon vaccination with PrPD111 retroparticles, albeit at considerably lower levels. Bacterially expressed recombinant PrP, in contrast, was unable to evoke IgG antibodies recognizing native PrPC in wild-type mice. Thus, our data show that PrP or parts thereof can be functionally displayed on retroviral particles and that immunization with PrP retroparticles may serve as a novel promising strategy for vaccination against transmissible spongiform encephalitis.
Directed evolution of retrovirus envelope protein cytoplasmic tails guided by functional incorporation into lentivirus particles.
Merten, C.A., Stitz, J., Braun, G., Poeschla, E.M., Cichutek, K. & Buchholz, C.J.
J Virol. 2005 Jan;79(2):834-40.
In contrast to most gammaretrovirus envelope proteins (Env), the Gibbon ape leukemia virus (GaLV) Env protein does not mediate the infectivity of human immunodeficiency virus type 1 (HIV-1) particles. We made use of this observation to set up a directed evolution system by creating a library of GaLV Env variants diversified at three critical amino acids, all located around the R-peptide cleavage site within the cytoplasmic tail. This library was screened for variants that were able to functionally pseudotype HIV-1 vector particles. All selected Env variants mediated the infectivity of HIV-1 vector particles and encoded novel cytoplasmic tail motifs. They were efficiently incorporated into HIV particles, and the R peptide was processed by the HIV protease. Interestingly, in some of the selected variants, the R-peptide cleavage site had shifted closer to the C terminus. These data demonstrate a valuable approach for the engineering of chimeric viruses and vector particles.
Displaying epidermal growth factor on spleen necrosis virus-derived targeting vectors.
Merten, C.A., Engelstaedter, M., Buchholz, C.J. & Cichutek, K.
Virology. 2003 Jan 5;305(1):106-14.
Targeted gene transfer into human cells has previously been achieved with spleen necrosis virus (SNV)-derived vector particles harboring envelope (Env) proteins which carry single chain Fv (scFv) domains derived from antibodies. Such cell targeting vectors have been found to directly transduce human cells expressing the cell surface molecules recognized by the respective scFv. In an attempt to achieve targeted gene transfer into epidermal growth factor receptor (EGFR)-positive human cells, SNV vector particles carrying a surface (SU) envelope protein N-terminally modified with the EGF domain and the wildtype transmembrane protein were generated. However, direct transduction of EGFR-positive cells was not detected. Canine D17 cells, which can be infected by wildtype SNV, were also not transduced. Infectivity of D17 cells was restored by removal of the EGF modification via cleavage of a factor Xa site located between the EGF domain and the SU protein or by blocking the EGFRs on the cell surface by EGF treatment. The properties of SNV-EGF vector particles as described here are similar to those of murine leukemia virus-derived vector particles harboring envelope proteins modified with a growth factor-derived domain. It seems therefore that, although scFv-modified SNV allows direct cell targeting, EGF-modified SNV allows only indirect cell targeting.
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