Manuel Marquez

Ultrahigh-throughput screening in drop-based microfluidics for directed evolution

The explosive growth in our knowledge of genomes, proteomes, and metabolomes is driving ever-increasing fundamental understanding of the biochemistry of life, enabling qualitatively new studies of complex biological systems and their evolution. This knowledge also drives modern biotechnologies, such as molecular engineering and synthetic biology, which have enormous potential to address urgent problems, including developing potent new drugs and providing environmentally friendly energy. Many of these studies, however, are ultimately limited by their need for even-higher-throughput measurements of biochemical reactions. We present a general ultrahigh-throughput screening platform using drop-based microfluidics that overcomes these limitations and revolutionizes both the scale and speed of screening. We use aqueous drops dispersed in oil as picoliter-volume reaction vessels and screen them at rates of thousands per second. To demonstrate its power, we apply the system to directed evolution, identifying new mutants of the enzyme horseradish peroxidase exhibiting catalytic rates more than 10 times faster than their parent, which is already a very efficient enzyme. We exploit the ultrahigh throughput to use an initial purifying selection that removes inactive mutants; we identify ∼100 variants comparable in activity to the parent from an initial population of ∼107. After a second generation of mutagenesis and high-stringency screening, we identify several significantly improved mutants, some approaching diffusion-limited efficiency. In total, we screen ∼108 individual enzyme reactions in only 10 h, using < 150 μL of total reagent volume; compared to state-of-the-art robotic screening systems, we perform the entire assay with a 1,000-fold increase in speed and a 1-million-fold reduction in cost.

Glass-like dynamics of collective cell migration

Collective cell migration in tissues occurs throughout embryonic development, during wound healing, and in cancerous tumor invasion, yet most detailed knowledge of cell migration comes from single-cell studies. As single cells migrate, the shape of the cell body fluctuates dramatically through cyclic processes of extension, adhesion, and retraction, accompanied by erratic changes in migration direction. Within confluent cell layers, such subcellular motions must be coupled between neighbors, yet the influence of these subcellular motions on collective migration is not known. Here we study motion within a confluent epithelial cell sheet, simultaneously measuring collective migration and subcellular motions, covering a broad range of length scales, time scales, and cell densities. At large length scales and time scales collective migration slows as cell density rises, yet the fastest cells move in large, multicell groups whose scale grows with increasing cell density. This behavior has an intriguing analogy to dynamic heterogeneities found in particulate systems as they become more crowded and approach a glass transition. In addition we find a diminishing self-diffusivity of short-wavelength motions within the cell layer, and growing peaks in the vibrational density of states associated with cooperative cell-shape fluctuations. Both of these observations are also intriguingly reminiscent of a glass transition. Thus, these results provide a broad and suggestive analogy between cell motion within a confluent layer and the dynamics of supercooled colloidal and molecular fluids approaching a glass transition.

Coaxial jets generated from electrified Taylor cones. Scaling laws

Abstract An experimental investigation on the electrified co-axial jets of two immiscible liquids issuing from a structured Taylor cone (Science 295 (5560) (2002) 1695) has been carried out. The structure of these almost conical electrified menisci consists of an outer meniscus surrounding an inner one. The liquid threads which issue from the vertex of each one of the menisci give rise to a two-concentric layered jet whose eventual breakup results in an aerosol of relatively monodisperse compound droplets with the outer liquid encapsulating the inner one. The effect of the flow rates of both liquids on the current transported by these coaxial jets and on the size of the compound droplets has been investigated. Several couples of liquids have been used to explore the influence on the spraying process of the properties of the liquids: i.e. the electrical conductivity K , dielectric constant β , interfacial tension of the liquid couple γ , viscosity μ , etc. We have found that the measurements of the current emitted through the coaxial jet when they are made dimensionless fit satisfactorily the current scaling law of regular electrosprays. Data of the mean diameter of the compound droplets have been obtained using a non-intrusive laser system. As expected the breakup process and therefore the droplet size are strongly dependent on the liquid viscosities and on the ratio of the liquid flow rates.

A new device for the generation of microbubbles

In this paper we present a new method for the production of bubble-liquid suspensions (from now on BLS) composed of micron-sized bubbles and with gas to liquid volume ratios larger than unity. We show that the BLS gas fraction λ=Qg/Ql, being Qg and Ql the flow rates of gas and liquid, respectively, is controlled by a dimensionless parameter which accounts for the ratio of the gas pressure inside the device to the liquid viscous pressure drop from the orifices where the liquid is injected to the exit, where the BLS is obtained. This parameter permits the correct scaling of the BLS gas volume fraction of all the experiments presented.

Confined assembly of asymmetric block-copolymer nanofibers via multiaxial jet electrospinning.

Multiaxial (triaxial/coaxial) electrospinning is utilized to fabricate block copolymer (poly(styrene-b-isoprene), PS-b-PI) nanofibers covered with a silica shell. The thermally stable silica shell allows post-fabrication annealing of the fibers to obtain equilibrium self-assembly. For the case of coaxial nanofibers, block copolymers with different isoprene volume fractions are studied to understand the effect of physical confinement and interfacial interaction on self-assembled structures. Various confined assemblies such as co-existing cylinders and concentric lamellar rings are obtained with the styrene domain next to the silica shell. This confined assembly is then utilized as a template to guide the placement of functional nanoparticles such as magnetite selectively into the PI domain in self-assembled nanofibers. To further investigate the effect of interfacial interaction and frustration due to the physically confined environment, triaxial configuration is used where the middle layer of the self-assembling material is sandwiched between the innermost and outermost silica layers. The results reveal that confined block-copolymer assembly is significantly altered by the presence and interaction with both inner and outer silica layers. When nanoparticles are incorporated into PS-b-PI and placed as the middle layer, the PI phase with magnetite nanoparticles migrates next to the silica layers. The migration of the PI phase to the silica layers is also observed for the blend of PS and PS-b-PI as the middle layer. These materials not only provide a platform to further study the effect of confinement and wall interactions on self-assembly but can also help develop an approach to fabricate multilayered, multistructured nanofibers for high-end applications such as drug delivery.

Microwave, Photo- and Thermally Responsive PNIPAm−Gold Nanoparticle Microgels

Microwave-, photo- and thermo-responsive polymer microgels that range in size from 500 to 800 microm and are swollen with water were prepared by a novel microarray technique. We used a liquid-liquid dispersion technique in a system of three immiscible liquids to prepare hybrid PNIPAm- co-AM core-shell capsules loaded with AuNPs. The spontaneous encapsulation is a result of the formation of double oil-in-water-in-oil (o/w/o) emulsion. It is facilitated by adjusting the balance of the interfacial tensions between the aqueous phase (in which a water-soluble drug may be dissolved), the monomer phase and the continuous phase. The water-in-oil (w/o) droplets containing 26 wt% NIPAm and Am monomers, 0.1 wt% Tween-80 surfactant, FITC fluorescent dye and colloidal gold nanoparticles spontaneously developed a core-shell morphology that was fixed by in situ photopolymerization. The results demonstrate new reversibly swelling and deswelling AuNP/PNIPAm hybrid core-shell microcapsules and microgels that can be actuated by visible light and/or microwave radiation (<or=1,250 nm) and/or temperature. This is the first study to demonstrate that incorporating AuNPs speeds up the response kinetics of PNIPAm, and hence enhances the sensitivity to external stimuli of PNIPAm. These microgels can have potential applications for microfluidic switches or microactuators, photosensors, and various nanomedicine applications in controlled delivery and release.

A microfluidic approach to encapsulate living cells in uniform alginate hydrogel microparticles.

A microfluidic technique is described to encapsulate living cells in alginate hydrogel microparticles generated from monodisperse double-emulsion templates. A microcapillary device is used to fabricate double emulsion templates composed of an alginate drop surrounded by a mineral oil shell. Hydrogel formation begins when the alginate drop separates from the mineral oil shell and comes into contact with Ca(2+) ions in the continuous phase. Alginate hydrogel microparticles with diameters ranging from 60 to 230 µm are obtained. 65% of the cells encapsulated in the alginate microparticles were viable after one week. The technique provides a useful means to encapsulate the living cells in monodisperse hydrogel microparticles.

Materials: Spontaneous formation of inorganic helices

Attempts to generate films, wires, helices, rings and regular patterns out of inorganic materials have been going on for the past 100 years. We show here that stable helices of porous manganese oxide materials can be formed spontaneously from uniform sols and that they are excellent semiconductors. These inorganic helices contain micropores, can be converted into other structures, and their composition can be varied.

Monodisperse structured multi-vesicle microencapsulation using flow-focusing and controlled disturbance

A method to produce monodisperse structured microcapsules in the diameter range from 10–100 µm is here presented. Flow-focusing is a well known technique whereby a steady capillary micro-jet is generated by the action of a highly accelerated co-flowing stream forced through a small orifice. The micro-jet breaks up owing to capillary instability, giving rise to droplets with a narrow size distribution. In the present study, flow-focusing gives rise not to simple but to compound capillary jets. At break-up, under suitable control parameters, such jets give rise to microcapsules where an outer liquid (shell liquid) surrounds a core liquid integrated by one or more vesicles. Furthermore, under adequate stimulation combining a sinusoidal signal with intermitent pulses, the jet break-up can be controlled. Highly monodisperse microcapsules are produced; fundamental geometric parameters (main diameter, shell thickness or number of cores) are reliably controlled. Rather than using a gas flow to focus the concentric stream of two immiscible liquids, this study has investigated in some detail the evolution of a concentric stream of three immiscible liquids forced through a small orifice. The selection of the surface tension coefficients between the three phases ensures the robust production of a microcapsule structure involving a plurality of vesicles homogeneously distributed in the capsule bulk, the number of cores being a freely chosen parameter. Such composite microcapsules find a broad field of technological applications in the pharmaceutical, food or biotechnology industries.

Selective recognition of bacterial membranes by zinc(II)-coordination complexes

Two fluorophore-dipicolylamine-Zn2+ conjugates are shown by epifluorescence microscopy to stain the membranes of bacterial cells in preference to mammalian cells.