Emmanuel Roy

Serial siphon valving for centrifugal microfluidic platforms

Today, the focus in microfluidic platforms for diagnostics is on the integration of several analysis steps toward sample-to-answer systems. One of the main challenges to integration is the requirement for serial valving to allow the sequential release of fluids in a temporally and spatially controlled manner. The advantages offered by centrifugal microfluidic platforms make them excellent candidates for integration of biological analysis steps, yet they are limited by the lack of robust serial valving technologies. This is especially true for the majority of centrifugal microfluidic devices that rely on hydrophilic surfaces, where few passive serial valving techniques function reliably. Building on the useful functionality of centrifugal microfluidic siphoning previously shown, a novel serial siphon valve is introduced that relies on multiple, inline siphons to provide for a better controlled, sequential release of fluids. The introduction of this novel concept is followed by an analytical analysis of the device. Proof-of-concept is also demonstrated, and examples are provided to illustrate the range of functionality of the serial siphon valve. The serial siphon is shown to be robust and reproducible, with variability caused by the dependence on contact angle, rotation velocity, and fluidic properties (viz., surface tension) significantly reduced compared to current microfluidic, centrifugal serial valving technologies.

Surface modification of thermoplastics—towards the plastic biochip for high throughput screening devices

Microarrays have become one of the most convenient tools for high throughput screening, supporting major advances in genomics and proteomics. Other important applications can be found in medical diagnostics, detection of biothreats, drug discovery, etc. Integration of microarrays with microfluidic devices can be highly advantageous in terms of portability, shorter analysis time and lower consumption of expensive biological analytes. Since fabrication of microfluidic devices using traditional materials such as glass is rather expensive, there is great interest in employing polymeric materials as a low cost alternative that is suitable for mass production. A number of commercially available plastic materials were reviewed for this purpose and poly(methylmethacrylate) Zeonor 1060R and Zeonex E48R were identified as promising candidates, for which methods for surface modification and covalent immobilization of DNA oligonucleotides were developed. In addition, we present proof-of-concept plastic-based microarrays with and without integration with microfluidics.

Surface topography induces 3D self-orientation of cells and extracellular matrix resulting in improved tissue function

The organization of cells and extracellular matrix (ECM) in native tissues plays a crucial role in their functionality. However, in tissue engineering, cells and ECM are randomly distributed within a scaffold. Thus, the production of engineered-tissue with complex 3D organization remains a challenge. In the present study, we used contact guidance to control the interactions between the material topography, the cells and the ECM for three different tissues, namely vascular media, corneal stroma and dermal tissue. Using a specific surface topography on an elastomeric material, we observed the orientation of a first cell layer along the patterns in the material. Orientation of the first cell layer translates into a physical cue that induces the second cell layer to follow a physiologically consistent orientation mimicking the structure of the native tissue. Furthermore, secreted ECM followed cell orientation in every layer, resulting in an oriented self-assembled tissue sheet. These self-assembled tissue sheets were then used to create 3 different structured engineered-tissue: cornea, vascular media and dermis. We showed that functionality of such structured engineered-tissue was increased when compared to the same non-structured tissue. Dermal tissues were used as a negative control in response to surface topography since native dermal fibroblasts are not preferentially oriented in vivo. Non-structured surfaces were also used to produce randomly oriented tissue sheets to evaluate the impact of tissue orientation on functional output. This novel approach for the production of more complex 3D tissues would be useful for clinical purposes and for in vitro physiological tissue model to better understand long standing questions in biology.

Microfluidic ELISA on non-passivated PDMS chip using magnetic bead transfer inside dual networks of channels

Achieving efficient passivation of micro-channels against non-specific adsorption of biomolecules is a critical aspect in the development of microfluidic ELISA systems. Usual surface treatments such as pre-coating of the channels with serum albumin, exposure to oxygen plasma, polyethylene glycol grafting however exhibit a lack of long-term stability, with procedures that can be time-consuming, complex or associated with costly materials and instruments. In this paper, we present a new fluidic design combined with an original strategy of manipulating magnetic beads in order to reduce assay noise in bead-based microfluidic ELISA without the need for prior channel pre-treatment. The novelty of the system relies on the physical separation of the immune complex formation phase and the enzymatic reaction phase into two independent networks of channels. These networks are linked by fluidic bridges, whose openings are controlled by pressure valves, and through which the beads are magnetically transferred. A standard curve for the quantification of a model antibody was obtained within 30 minutes. A detection limit of 100 pg mL(-1) (660 fM) and good linearity of the signal up to 4 ng mL(-1) were observed.

Microfluidic patterning of miniaturized DNA arrays on plastic substrates.

This paper describes the patterning of DNA arrays on plastic surfaces using an elastomeric, two-dimensional microcapillary system (muCS). Fluidic structures were realized through hot-embossing lithography using Versaflex CL30. Like elastomers based on poly(dimethylsiloxane), this thermoplastic block copolymer is able to seal a surface in a reversible manner, making it possible to confine DNA probes with a level of control that is unparalleled using standard microspotting techniques. We focus on muCSs that support arrays comprising up to 2 x 48 spots, each being 45 mum in diameter. Substrates were fabricated from two hard thermoplastic materials, poly(methylmethacrylate) and a polycyclic olefin (e.g., Zeonor 1060R), which were both activated with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride and N-hydroxysuccinimide to mediate covalent attachment of DNA molecules. The approach was exemplified by using 0.25-32 muM solutions of amino-modified oligonucleotides labeled with either Cy3 or Cy5 fluorescent dye in phosphate-buffered saline, allowing for a direct and sensitive characterization of the printed arrays. Solutions were incubated for durations of 1 to >48 h at 22, 30, and 40 degrees C to probe the conditions for obtaining uniform spots of high fluorescence intensity. The length (l) and depth (d) of microfluidic supply channels were both important with respect to depletion as well as evaporation of the solvent. While selective activation of the substrate proved helpful to limit unproductive loss of oligonucleotides along trajectories, incubation of solution in a humid environment was necessary to prevent uncontrolled drying of the liquid, keeping the immobilization process intact over extended periods of time. When combined, these strategies effectively promoted the formation of high-quality DNA arrays, making it possible to arrange multiple probes in parallel with a high degree of uniformity. Moreover, we show that resultant arrays are compatible with standard hybridization protocols, which allowed for reliable discrimination of individual strands when exposed to a specific ssDNA target molecule.

3D thermoplastic elastomer microfluidic devices for biological probe immobilization

Microfluidics has emerged as a valuable tool for the high-resolution patterning of biological probes on solid supports. Yet, its widespread adoption as a universal biological immobilization tool is still limited by several technical challenges, particularly for the patterning of isolated spots using three-dimensional (3D) channel networks. A key limitation arises from the difficulties to adapt the techniques and materials typically used in prototyping to low-cost mass-production. In this paper, we present the fabrication of thin thermoplastic elastomer membranes with microscopic through-holes using a hot-embossing process that is compatible with high-throughput manufacturing. The membranes provide the basis for the fabrication of highly integrated 3D microfluidic devices with a footprint of only 1 × 1 cm(2). When placed on a solid support, the device allows for the immobilization of up to 96 different probes in the form of a 10 × 10 array comprising isolated spots of 50 × 50 μm(2). The design of the channel network is optimized using 3D simulations based on the Lattice-Boltzmann method to promote capillary action as the sole force distributing the liquid in the device. Finally, we demonstrate the patterning of DNA and protein arrays on hard thermoplastic substrates yielding spots of excellent definition that prove to be highly specific in subsequent hybridization experiments.

Rapid isothermal substrate microfabrication of a biocompatible thermoplastic elastomer for cellular contact guidance

The use of microstructured substrates to study and influence cell orientation, which plays an important role in tissue functionality, has been of great interest lately. Silicon and poly(dimethylsiloxane) substrates have typically been used, but long processing times and exogenous protein surface coating, required to enhance cell viability, limit their use as large-scale platforms. There is thus a need for a non-biodegradable biocompatible substrate that allows rapid and low cost microfabrication. In this paper a styrene-(ethylene/butylene)-styrene block co-polymer (SEBS) microstructured by a rapid replication technique using low pressure an isothermal hot embossing approach has been demonstrated. SEBS substrates were treated with oxygen plasma to enhance cell adhesion and sterilized using ethylene oxide gas. While cell adhesion to and proliferation on these substrates was as good as on tissue culture polystyrene, cellular alignment on microstructured SEBS was also very high (97.7±0.5%) when calculated within a 10° angle variation from the longitudinal axis. Furthermore, tissue sheets on microstructured SEBS have been produced and exhibited cellular alignment within the engineered tissue. In addition, these results were obtained without coating the material with exogenous proteins. Such substrates should be helpful in the culture of tissue engineered substitutes with an intrinsic orientation and to elucidate questions in cell biology.

Stretching the Stamp: A Flexible Approach to the Fabrication of Miniaturized DNA Arrays

DNA arrays have emerged as key tools in genomic research to measure relative expression levels of genes among different samples in parallel andwithminimal sample consumption. In addition, they are also becoming popular in other areas such as biomedical diagnostics, cellular analysis, and drug development. DNA arrays typically comprise a set of synthesized gene sequences immobilized on a solid support, which are subjected to hybridization with specific target genes. In most cases, a fluorescence marker such as Cy3 (lex1⁄4 550 nm, lem1⁄4 570 nm) or Cy5 (lex1⁄4 650 nm, lem1⁄4 670 nm) is linked to the target DNA, which allows for the detection of hybridization events throughfluorescence readoutof thearray.ThenumberofDNA probes per surface area is an important criterion for their efficacy: the higher their density, the more information can be extracted from a single experiment. DNA microarrays are commonly fabricated by spotting minute amounts of DNA solutiononto aproper substrate byusing eithermetal pins or microactuated dispensing systems. To this end, the resolution of most commercial spotting techniques is limited to arrays containing 100 to 5000 spots per cm depending on the specificationsof the tools, the conditionsof spotting, and the characteristics of the substrate. Arrays of higher density (e.g., spots 100 nm in diameter or below) have been realized by using cantilever probes, yet reliability and throughput of this technology need to be improved before it may become suitable for production purposes. An alternative fabrication scheme is in situ synthesis, which employs light-sensitive chemistry in conjunction with photolithographic techniques to construct a

Towards Low Cost Disposable High Throughput Screening Devices

Microarrays have become one of the most convenient tools for high throughput screening, supporting major advances in genomics and proteomics. Other important applications can be found in medical diagnostics, detection of biothreats, drug discovery, etc. Integration of microarrays with microfluidic devices can be highly advantageous in terms of portability, shorter analysis time and lower consumption of expensive biological analytes. Since fabrication of microfluidic devices using traditional materials such as glass is rather expensive, there is a high interest in employing polymeric materials as a low cost alternative that is suitable for mass production. A number of commercially available plastic materials were reviewed for this purpose and poly(methylmethacrylate) and Zeonor™ 1060R were identified as promising candidates, for which methods for surface modification and covalent immobilization of DNA oligonucleotide were developed. In addition, we present proof-of-concept plastic-based microarrays with and without integration with microfluidics.

Fabrication of Microfluidic Devices in Thermoplastic Elastomeric Materials for DNA Detection on Thermal Plastic Substrate

Thermoplastic elastomer (TPE) based microfluidic devices integrated with a microfluidic pumping manifold which consists of 4 electromagnetic valves (EMV) were fabricated. The back and forth shuttling flow and its application in the DNA hybridization process were validated on a thermal plastic Zeonor 1060R substrate. The flow rate can be as fast as 23μl/min when the channel width and the channel height are in 100μm, and 25μm, respectively. The DNA hybridization process is detected by using a fluorescence microscopy. Remarkable DNA hybridization is achieved with the continuous flow of the target DNA at a concentration of 10 nM within the first 1 min by using this device.