Laurent Malaquin

Microfluidic sorting and multimodal typing of cancer cells in self-assembled magnetic arrays

We propose a unique method for cell sorting, “Ephesia,” using columns of biofunctionalized superparamagnetic beads self-assembled in a microfluidic channel onto an array of magnetic traps prepared by microcontact printing. It combines the advantages of microfluidic cell sorting, notably the application of a well controlled, flow-activated interaction between cells and beads, and those of immunomagnetic sorting, notably the use of batch-prepared, well characterized antibody-bearing beads. On cell lines mixtures, we demonstrated a capture yield better than 94%, and the possibility to cultivate in situ the captured cells. A second series of experiments involved clinical samples—blood, pleural effusion, and fine needle aspirates— issued from healthy donors and patients with B-cell hematological malignant tumors (leukemia and lymphoma). The immunophenotype and morphology of B-lymphocytes were analyzed directly in the microfluidic chamber, and compared with conventional flow cytometry and visual cytology data, in a blind test. Immunophenotyping results using Ephesia were fully consistent with those obtained by flow cytometry. We obtained in situ high resolution confocal three-dimensional images of the cell nuclei, showing intranuclear details consistent with conventional cytological staining. Ephesia thus provides a powerful approach to cell capture and typing allowing fully automated high resolution and quantitative immunophenotyping and morphological analysis. It requires at least 10 times smaller sample volume and cell numbers than cytometry, potentially increasing the range of indications and the success rate of microbiopsy-based diagnosis, and reducing analysis time and cost.

Microfluidic: an innovative tool for efficient cell sorting.

At first mostly dedicated to molecular analysis, microfluidic systems are rapidly expanding their range of applications towards cell biology, thanks to their ability to control the mechanical, biological and fluidic environment at the scale of the cells. A number of new concepts based on microfluidics were indeed proposed in the last ten years for cell sorting. For many of these concepts, progress remains to be done regarding automation, standardization, or throughput, but it is now clear that microfluidics will have a major contribution to the field, from fundamental research to point-of-care diagnosis. We present here an overview of cells sorting in microfluidics, with an emphasis on circulating tumor cells. Sorting principles are classified in two main categories, methods based on physical properties of the cells, such as size, deformability, electric or optical properties, and methods based on biomolecular properties, notably specific surface antigens. We document potential applications, discuss the main advantages and limitations of different approaches, and tentatively outline the main remaining challenges in this fast evolving field.

Programmable Magnetic Tweezers and Droplet Microfluidic Device for High‐Throughput Nanoliter Multi‐Step Assays

Droplet microfluidics offer unique capabilities for the development of high-throughput analytical systems. It allows fluids to be aliquoted into volumes in the nanoliter or picoliter range, and then transported over arbitrary distances without dispersion or cross-contamination. Several droplet-based functions, such as merging, 3] splitting, sorting, or cell encapsulation, have already been demonstrated. Moreover, thanks to their “pipeline” architecture, in which different samples follow the same track in a row, droplet microfluidics enable the implementation of high-throughput assays with relatively simple microfluidic designs, as compared for example, to systems in which the aliquoting is performed by valves integrated in the microfluidic system itself. Unfortunately, the ability to efficiently purify or extract molecules of interest from a complex matrix, a key component of most biochemical methods, is still missing from the functions currently available for droplet microfluidics. In macroscopic methods, the use of superparamagnetic beads as a solid state support has become very popular: they can bind an analyte of interest, be retained with a magnet while the supernatant fluid is removed, and release the analyte in an elution buffer. This process can be multiplexed, for example, using multiple magnets at the bottom of microtiter plates, but it still suffers from significant constraints, notably owing to 1) mass transfer limitations, 2) the need for relatively large volumes, and 3) poor mixing and washing efficiencies. We already proposed an alternative approach, using selfassembled magnetic microcolumns in microfluidic format. A large reduction in analysis time and increased automation was achieved in this way, but as with other microcolumnbased systems, this device does not allow for a high level of multiplexing and is not adapted to low-volume sample handling (that is less than 10 mL). In this respect, the combination of magnetic solid-phase extraction with droplet microfluidics is appealing. Magnetic-bead transfer between two droplets of several microliters was first introduced by Shikida et al. Results were achieved by the displacement of a permanent magnet along a millimeter-sized fluidic channel, interconnecting different reservoirs. A similar method, using multiple wetting valves to allow for more complex designs, was recently presented, but it still requires rather large volumes and incubation times. To increase the flexibility of these assays, Sista et al. combined this strategy with droplets manipulated on an array of electrodes by electrowetting (EWOD). 13] This device, generally named a digital microfluidic device, is very flexible, but it requires complex microfabrication steps to integrate the array of electrodes onto the device surface. A variant of this method, in which drops are immobilized on hydrophilic patches and droplets containing magnetic particles are magnetically actuated through microfabricated coils, was also proposed. The drop size, however, is large (approximately 10 mL) and microfabrication of the system is almost as demanding as that of the EWOD systems. Another strategy was proposed recently, in which droplets containing magnetic particles are hydrodynamically split in the presence of an asymmetric field. 16] This approach shares with conventional droplet microfluidic devices the possibility of processing droplets at a high throughput of tens to hundreds of drops per second. However, even if effective particle separation in a single daughter droplet is achieved, using this method for a purification process is not efficient, since the removal of the supernatant fluid is inefficient. More recently, Gu et al. attempted to induce droplet splitting in sub-nanoliter droplets by direct magnetic extraction of particles. Unfortunately, in such small droplets, ferromagnetic particles were needed to reach a high enough magnetic trapping force. Because of the permanent magnetization induced in large ferromagnetic particles, resuspention of the magnetic particles after trapping was impossible, and the magnetic “plug” had to be recollected in a macroscopic tube to perform the next step (in this case PCR). Thus, on the one hand, a good separation of the magnetic particles was acheived, but on the other hand the ability to perform multiple steps was lost. Herein, we create a device that combines these advantages. It involves a new design for magnetic capture based on programmable “magnetic tweezers”. Because of multiple trapping–release sequences, the particles can be exchanged with an extremely low carryover of supernatant (less than 2%) between droplets with volumes of 80 nL or lower. Starting from reagents contained in a microtiter plate, full automation of the method, involving several exchanges of reagents and rinsing solutions, is acheived by a combination of [*] Dr. A. Ali-Cherif, Dr. S. Begolo, [+] Dr. S. Descroix, Dr. J.-L. Viovy, Dr. L. Malaquin Institut Curie UMR 168, Research Center, CNRS, UMR168 11 rue Pierre et Marie Curie, 75005 Paris (France) E-mail: laurent.malaquin@curie.fr [] Current address: California Institute of technology 1200 E. California Blvd. MC 101-20, Pasadena, CA 91125 (USA) [] These authors contributed equally to this work.

Highly parallel mix-and-match fabrication of nanopillar arrays integrated in microfluidic channels for long DNA molecule separation

We report on a mix-and-match method based on a combination of soft UV nanoimprint lithography, contact optical lithography, and reactive-ion-etch techniques, which is applicable for high throughput manufacturing of nanostructure integrated microfluidic devices. We demonstrate the integration of high density and high aspect ratio nanopillars into microfluidic channels as electrophoresis sieving matrices. As a result, λ DNA and T4 DNA can be separated within a few minutes. By changing the pattern design, the device could be used for separation of other types of molecules.

Using polydimethylsiloxane as a thermocurable resist for a soft imprint lithography process

Nanoimprint lithography has been investigated using polydimethylsiloxane as a thermocurable resist. This novel process allowed us to reduce both pressure (<10 bars) and temperature (80 °C) when compared to a conventional imprinting process with a thermoplastic polymer resist such as polymethylmethacrylate. Using a new formulation of the elastomeric material, we have demonstrated high quality imprinting of both micronic and nanometric structures with no evidence of any viscous flow problems. The excellent etching resistance of the polydimethylsiloxane structures to a reactive ion etching silicon process and the compatibility with a lift-off procedure for pattern transfer are also presented.

Combining Microfluidics, Optogenetics and Calcium Imaging to Study Neuronal Communication In Vitro

In this paper we report the combination of microfluidics, optogenetics and calcium imaging as a cheap and convenient platform to study synaptic communication between neuronal populations in vitro. We first show that Calcium Orange indicator is compatible in vitro with a commonly used Channelrhodopsine-2 (ChR2) variant, as standard calcium imaging conditions did not alter significantly the activity of transduced cultures of rodent primary neurons. A fast, robust and scalable process for micro-chip fabrication was developed in parallel to build micro-compartmented cultures. Coupling optical fibers to each micro-compartment allowed for the independent control of ChR2 activation in the different populations without crosstalk. By analyzing the post-stimuli activity across the different populations, we finally show how this platform can be used to evaluate quantitatively the effective connectivity between connected neuronal populations.

Collective beating of artificial microcilia.

We combine technical, experimental, and theoretical efforts to investigate the collective dynamics of artificial microcilia in a viscous fluid. We take advantage of soft lithography and colloidal self-assembly to devise microcarpets made of hundreds of slender magnetic rods. This novel experimental setup is used to investigate the dynamics of extended cilia arrays driven by a precessing magnetic field. Whereas the dynamics of an isolated cilium is a rigid body rotation, collective beating results in a symmetry breaking of the precession patterns. The trajectories of the cilia are anisotropic and experience a significant structural evolution as the actuation frequency increases. We present a minimal model to account for our experimental findings and demonstrate how the global geometry of the array imposes the shape of the trajectories via long-range hydrodynamic interactions.

Fabrication of multiple nano-electrodes for molecular addressing using high-resolution electron beam lithography and their replication using soft imprint lithography

Abstract We investigate the ability for High Resolution Electron Beam Lithography (HREBL) to fabricate multiple nano-electrodes with the smallest gap in the smallest area. By using both standard PolyMethylMethAcrylate (PMMA) as resist and standard MIBK/IPA development, we show that up to 10 nano-electrodes can be realized in an area as small as 65 nm. The obtained structures have been used either for the realization of embedded electrodes in SiO 2 by wet etching followed by lift-off, or for the fabrication of molds for Nano-Imprint Lithography using PMMA as a mask for Reactive Ion Etching of silicon. Preliminary results on the replication of these molds using a soft nano-imprint process are also presented.

Microfluidic platform combining droplets and magnetic tweezers: application to HER2 expression in cancer diagnosis

The development of precision medicine, together with the multiplication of targeted therapies and associated molecular biomarkers, call for major progress in genetic analysis methods, allowing increased multiplexing and the implementation of more complex decision trees, without cost increase or loss of robustness. We present a platform combining droplet microfluidics and magnetic tweezers, performing RNA purification, reverse transcription and amplification in a fully automated and programmable way, in droplets of 250nL directly sampled from a microtiter-plate. This platform decreases sample consumption about 100 fold as compared to current robotized platforms and it reduces human manipulations and contamination risk. The platform’s performance was first evaluated on cell lines, showing robust operation on RNA quantities corresponding to less than one cell, and then clinically validated with a cohort of 21 breast cancer samples, for the determination of their HER2 expression status, in a blind comparison with an established routine clinical analysis.

A three dimensional thermoplastic microfluidic chip for robust cell capture and high resolution imaging

We present a low cost microfluidic chip integrating 3D micro-chambers for the capture and the analysis of cells. This device has a simple design and a small footprint. It allows the implementation of standard biological protocols in a chip format with low volume consumption. The manufacturing process relies on hot-embossing of cyclo olefin copolymer, allowing the development of a low cost and robust device. A 3D design of microchannels was used to induce high flow velocity contrasts in the device and provide a selective immobilization. In narrow distribution channels, the liquid velocity induces a shear stress that overcomes adhesion forces and prevents cell immobilization or clogging. In large 3D chambers, the liquid velocity drops down below the threshold for cell attachment. The devices can be operated in a large range of input pressures and can even be handled manually using simple syringe or micropipette. Even at high flow injection rates, the 3D structures protect the captured cell from shear stress. To validate the performances of our device, we implemented immuno-fluorescence labeling and Fluorescence in Situ Hybridization (FISH) analysis on cancer cell lines and on a patient pleural effusion sample. FISH is a Food and Drug Administration approved cancer diagnostic technique that provides quantitative information about gene and chromosome aberration at the single cell level. It is usually considered as a long and fastidious test in medical diagnosis. This process can be easily implanted in our platform, and high resolution fluorescence imaging can be performed with reduced time and computer intensiveness. These results demonstrate the potential of this chip as a low cost, robust, and versatile tool adapted to complex and demanding protocols for medical diagnosis.