Emmanuel Delamarche

Microfluidic Chips for Point‐of‐Care Immunodiagnostics

We might be at the turning point where research in microfluidics undertaken in academia and industrial research laboratories, and substantially sponsored by public grants, may provide a range of portable and networked diagnostic devices. In this Progress Report, an overview on microfluidic devices that may become the next generation of point-of-care (POC) diagnostics is provided. First, we describe gaps and opportunities in medical diagnostics and how microfluidics can address these gaps using the example of immunodiagnostics. Next, we conceptualize how different technologies are converging into working microfluidic POC diagnostics devices. Technologies are explained from the perspective of sample interaction with components of a device. Specifically, we detail materials, surface treatment, sample processing, microfluidic elements (such as valves, pumps, and mixers), receptors, and analytes in the light of various biosensing concepts. Finally, we discuss the integration of components into accurate and reliable devices.

Capillary pumps for autonomous capillary systems

Autonomous capillary systems (CSs), where liquids are displaced by means of capillarity, are efficient, fast and convenient platforms for many bioanalytical applications. The proper functioning of these microfluidic devices requires displacing accurate volumes of liquids with precise flow rates. In this work, we show how to design capillary pumps for controlling the flow properties of CSs. The capillary pumps comprise microstructures of various shapes with dimensions from 15-250 microm, which are positioned in the capillary pumps to encode a desired capillary pressure. The capillary pumps are designed to have a small flow resistance and are preceded by a constricted microchannel, which acts as a flow resistance. Therefore, both the capillary pump and the flow resistance define the flow rate in the CS, and flow rates from 0.2-3.7 nL s(-1) were achieved. The placement and the shape of the microstructures in the capillary pumps are used to tailor the filling front of liquids in the capillary pumps to obtain a reliable filling behaviour and to minimize the risk of entrapping air. The filling front can, for example, be oriented vertically or tilted to the main axis of the capillary pump. We also show how capillary pumps having different hydrodynamic properties can be connected to program a sequence of slow and fast flow rates in a CS.

Lithography beyond light: microcontact printing with monolayer resists

We describe high-resolution lithography based on transfer of a pattern from an elastomeric stamp to a solid substrate by conformal contact : a nanoscale interaction between substrate and stamp on macroscopic scales that allows transport of material from stamp to substrate. The stamp is first formed by curing poly(dimethyl siloxane) (PDMS) on a master with the negative of the desired surface, resulting in an elastomeric solid with a pattern of reliefs, typically a few microns deep, on its surface. The stamp provides an ink that forms a self-assembled monolayer (SAM) on a solid surface by a covalent, chemical reaction. Because SAMs act as highly localized and efficient barriers to some wet etches, microcontact printing forms part of a convenient lithographic system not subject to diffraction or depth of focus limitations while still providing simultaneous transfer of patterned features. Our study helps to define the strengths and limitations of microcontact printing with SAMs, a process that is necessary to assess its worth to technology. We used lithography based on scanning tunneling microscopy (STM) to demonstrate that disruption of SAMs on gold allowed the formation of etched features as small as 20 nm using a CN - /O 2 etch. This result implied that etching occurred where damage of a few molecules in the ordered SAM allowed passage of cyanide, whereas adjacent molecules in the SAM remained unperturbed at this scale. Features as small as 30 nm 2 etched in gold over areas greater than 1 cm 2 resulted from microcontact printing with replicas of electron-beam-formed masters, with the transfer of these printed SAMs requiring only 1 s. STM studies of these transferred SAMs revealed an achievable order indistinguishable from that found for SAMs prepared from solution. Facile alignment of printing steps at submicron scales may result from new designs of stamps that exploit their limited deformability and lock-and-key-type approaches to mate stamp and substrate.

High-sensitivity miniaturized immunoassays for tumor necrosis factor α using microfluidic systems

We use microfluidic chips to detect the biologically important cytokine tumor necrosis factor alpha (TNF- alpha) with picomolar sensitivity using sub-microliter volumes of samples and reagents. The chips comprise a number of independent capillary systems (CSs), each of which is composed of a filling port, an appended microchannel, and a capillary pump. Each CS fills spontaneously by capillary forces and includes a self-regulating mechanism that prevents adventitious drainage of the microchannels. Thus, interactive control of the flow in each CS is easily achieved via collective control of the evaporation in all CSs by means of two Peltier elements that can independently heat and cool. Long incubation times are crucial for high sensitivity assays and can be conveniently obtained by adjusting the evaporation rate to have low flow rates of approximately 30 nL min(-1). The assay is a sandwich fluorescence immunoassay and takes place on the surface of a poly(dimethylsiloxane)(PDMS) slab placed across the microchannels. We precoat PDMS with capture antibodies (Abs), localize the capture of analyte molecules using a chip, then bind the captured analyte molecules with fluorescently-tagged detection Abs using a second chip. The assay results in a mosaic of fluorescence signals on the PDMS surface which are measured using a fluorescence scanner. We show that PDMS is a compatible material for high sensitivity fluorescence assays, provided that detection antibodies with long excitation wavelength fluorophores ( > or =580 nm) are employed. The chip design, long incubation times, proper choice of fluorophores, and optimization of the detection Ab concentration all combine to achieve high-sensitivity assays. This is exemplified by an experiment with 170 assay sites, occupying an area of approximately 0.6 mm(2) on PDMS to detect TNF-alpha in 600 nL of a dendritic cell (DC) culture medium with a sensitivity of approximately 20 pg mL(-1)(1.14 pM).

Fabricating Microarrays of Functional Proteins Using Affinity Contact Printing

Phenomena involving the binding between biomolecules are ubiquitous in biology and are essential for cell growth, signal transmission, and immune defense. In the latter system, the binding between antibody and antigen has already been exploited technologically to perform affinity purifications on columns and immunoassays on surfaces.[1] Recently, the fabrication of microarrays of proteins which require the immobilization of a large number of receptors on a surface have fueled the invention of novel patterning techniques such as pin-spotting and drop-on-demand.[2] Microarrays of proteins may find utility in proteomics, immunoassays, or for screening libraries of (bio)chemicals. It is at present not clear which patterning method will be the one best suited to pattern proteins on surfaces, but classical lithography does not seem capable of fabricating microarrays of proteins. Soft lithography[3] offers the possibility of manipulating proteins and other biomolecules by printing them from a micropatterned stamp to a surface[4] or by depositing them from a liquid using microfluidic networks ( FNs).[5] Affinity microcontact printing ( CP)[6] is a refined soft-lithographic technique that uses an elastomeric stamp made of polydimethylsiloxane (PDMS) and derivatized with binding biomolecules to extract corresponding binding partners from an impure, dilute source for placing them on a surface with spatial control. Herein, we describe, by using one particular example, how specific binding between biomolecules provides a unique opportunity to make use of self-assembly processes in technology: we propose different variants of CP to pattern surfaces with ensembles of biomolecules where the pattern on the affinity stamp ( -stamp) is not determined by its topography but by the position of various proteins covalently linked to a planar -stamp (Figure 1). This modified surface enables the simultaneous capture of different target proteins on the -stamp from a complex solution (Figure 1A). Thus, the capture step (Figure 1B) directs the assembly of an array of target molecules on the stamp (Figure 1C), which can be Figure 1. Microarrays of proteins on surfaces can be fabricated using an stamp derivatized with various capture sites that can extract target biomolecules from a complex solution and release them on a surface in a single microcontact-printing step. The -stamp can be reused for several inking and printing cycles.

Affinity capture of proteins from solution and their dissociation by contact printing

Biological experiments at the solid/liquid interface, in general, require surfaces with a thin layer of purified molecules, which often represent precious material. Here, we have devised a method to extract proteins with high selectivity from crude biological sample solutions and place them on a surface in a functional, arbitrary pattern. This method, called affinity-contact printing (αCP), uses a structured elastomer derivatized with ligands against the target molecules. After the target molecules have been captured, they are printed from the elastomer onto a variety of surfaces. The ligand remains on the stamp for reuse. In contrast with conventional affinity chromatography, here dissociation and release of captured molecules to the substrate are achieved mechanically. We demonstrate this technique by extracting the cell adhesion molecule neuron-glia cell adhesion molecule (NgCAM) from tissue homogenates and cell culture lysates and patterning affinity-purified NgCAM on polystyrene to stimulate the attachment of neuronal cells and guide axon outgrowth.

Nanopatterning Reveals an ECM Area Threshold for Focal Adhesion Assembly and Force Transmission that is regulated by Integrin Activation and Cytoskeleton Tension

Summary Integrin-based focal adhesions (FA) transmit anchorage and traction forces between the cell and the extracellular matrix (ECM). To gain further insight into the physical parameters of the ECM that control FA assembly and force transduction in non-migrating cells, we used fibronectin (FN) nanopatterning within a cell adhesion-resistant background to establish the threshold area of ECM ligand required for stable FA assembly and force transduction. Integrin–FN clustering and adhesive force were strongly modulated by the geometry of the nanoscale adhesive area. Individual nanoisland area, not the number of nanoislands or total adhesive area, controlled integrin–FN clustering and adhesion strength. Importantly, below an area threshold (0.11 µm2), very few integrin–FN clusters and negligible adhesive forces were generated. We then asked whether this adhesive area threshold could be modulated by intracellular pathways known to influence either adhesive force, cytoskeletal tension, or the structural link between the two. Expression of talin- or vinculin-head domains that increase integrin activation or clustering overcame this nanolimit for stable integrin–FN clustering and increased adhesive force. Inhibition of myosin contractility in cells expressing a vinculin mutant that enhances cytoskeleton–integrin coupling also restored integrin–FN clustering below the nanolimit. We conclude that the minimum area of integrin–FN clusters required for stable assembly of nanoscale FA and adhesive force transduction is not a constant; rather it has a dynamic threshold that results from an equilibrium between pathways controlling adhesive force, cytoskeletal tension, and the structural linkage that transmits these forces, allowing the balance to be tipped by factors that regulate these mechanical parameters.

A Vertical Microfluidic Probe

Performing localized chemical events on surfaces is critical for numerous applications. We earlier invented the microfluidic probe (MFP), which circumvented the need to process samples in closed microchannels by hydrodynamically confining liquids that performed chemistries on surfaces (Juncker et al. Nat. Mater. 2005, 4, 622-628). Here we present a new and versatile probe, the vertical MFP (vMFP), which operates in the scanning mode while overcoming earlier challenges that limited the practical implementation of the MFP technology. The key component of the vMFP is the head, a microfluidic device (∼1 cm(2) in area) consisting of glass and Si and having microfluidic features fabricated in-plane in the Si layer. The base configuration of the head has two micrometer-size channels that inject/aspirate liquids and terminate at the apex which is ∼1 mm(2). In scanning mode, the head is oriented vertically with the apex parallel to the surface with typical spacing of 1-30 μm. Such length scales and using flow rates from nanoliters/second to microliters/second allow chemical events to be performed on surfaces with tens of picoliter quantities of reagents. Before scanning, the head is clipped on a holder for leak-free, low dead volume interface assembly, providing a simple world-to-chip interface. Surfaces are scanned by mounting the holder on a computer-controlled stage having ∼0.1 μm resolution in positioning. We present detailed steps to fabricate vMFP heads having channels with dimensions from 1 μm × 1 μm to 50 μm × 50 μm for liquid localization over areas of 10-10,000 μm(2). Additionally, advanced design strategies are described to achieve high yield in fabrication and to support a broad range of applications. These include particulate filters, redundant aperture architectures, inclined flow-paths that service apertures, and multiple channels to enable symmetric flow confinement. We also present a method to characterize flow confinement and estimate the distance between the head and the surface by monitoring the evolution of a solution of fluorescently labeled antibody on an activated glass surface. This flow characterization reveals regimes of operation suitable for different surface topographies. We further integrate the dispensing of immersion liquid to the vMFP head for processing surfaces for extended periods of time (∼60 min). The versatility of the vMFP is exemplified by patterning fluorescently labeled proteins, inactivation of cells using sodium hypochlorite, and staining living NIH fibroblasts with Cellomics. These applications are enabled by the compact design of the head, which provides easy access to the surface, simplifies alignment, and enables processing surfaces having dimensions from the micrometer to the centimeter scale and with large topographical variations. We therefore believe that ease-of-operation, reconfigurability, and conservative use of chemicals by the vMFP will lead to its widespread use by microtechnologists and the chemical and biomedical communities.

Continuous flow in open microfluidics using controlled evaporation

This paper presents a method for programming the flow rate of liquids inside open microfluidic networks (MFNs). A MFN comprises a number of independent flow paths, each of which starts with an open filling port, has a sealed microchannel in which assays can be performed, and an open capillary pump (CP). The MFN is placed over Peltier elements and its flow paths initially fill owing to capillary forces when liquids are added to the filling ports. A cooling Peltier element underneath the filling ports dynamically prevents evaporation in all filling ports using the ambient temperature and relative humidity as inputs. Another Peltier element underneath the CPs heats the pumps thereby inducing evaporation in the CPs and setting the flow rate in the microchannels. This method achieves flow rates in the microchannels ranging from approximately 1.2 nL s(-1) to approximately 30 pL s(-1), and is able to keep 90% of a 0.6 microL solution placed in an open filling port for 60 min. This simple and efficient method should be applicable to numerous assays or chemical reactions that require small and precise flow of liquids and reagents inside microfluidics.

High-Performance Immunoassays Based on Through-Stencil Patterned Antibodies and Capillary Systems

We present a simple method to pattern capture antibodies (cAbs) on poly(dimethylsiloxane) (PDMS), with high accuracy and in a manner compatible with mass fabrication for use with capillary systems (CSs), using stencils microfabricated in Si. Capture antibodies are patterned as 60-270 microm wide and 2 mm long lines on PDMS and used with CSs that have been optimized for convenient handling, pipetting of solutions, pumping of liquids, such as human blood serum, and visualization of signals for fluorescence immunoassays. With the use of this method, C-reactive protein (CRP) is detected with a sensitivity of 0.9 ng mL(-1) (7.8 pM) in 1 microL of CRP-spiked human serum, within 11 min and using only four pipetting steps and a total volume of sample and reagents of 1.35 microL. This exemplifies the high performances that can be achieved using this approach and an otherwise conventional surface sandwich fluorescence immunoassay. This method is simple and flexible and should therefore be applicable to a large number of demanding immunoassays.