A "dry and wet hybrid" lithography technique for multilevel replication templates: Applications to microfluidic neuron culture and two-phase global mixing.

A broad range of microfluidic applications, ranging from cell culture to protein crystallization, requires multilevel devices with different heights and feature sizes (from micrometers to millimeters). While state-of-the-art direct-writing techniques have been developed for creating complex three-dimensional shapes, replication molding from a multilevel template is still the preferred method for fast prototyping of microfluidic devices in the laboratory. Here, we report on a "dry and wet hybrid" technique to fabricate multilevel replication molds by combining SU-8 lithography with a dry film resist (Ordyl). We show that the two lithography protocols are chemically compatible with each other. Finally, we demonstrate the hybrid technique in two different microfluidic applications: (1) a neuron culture device with compartmentalization of different elements of a neuron and (2) a two-phase (gas-liquid) global micromixer for fast mixing of a small amount of a viscous liquid into a larger volume of a less viscous liquid.

Block copolymer-oligonucleotide conjugates for genotyping on microarrays.

Polymer-oligonucleotide conjugates were synthesized from the amphiphilic block copolymer poly(tert-butylacrylamide-b-(N-acryloylmorpholine-co-N-acryloxysuccinimide)) using an original solid-phase DNA synthesis strategy. This method provided conjugates highly functionalized with oligonucleotides throughout the polymer chain. After purification, block copolymer-oligonucleotide conjugates were spotted on a multidetection microarray system developed by Apibio using a standard nanodroplet piezo inkjet spotting technique to develop the oligosorbent assay (OLISA). Two genotyping models (HLA-DQB1 and platelet glycoproteins [GPs]), which are particularly difficult to study with standard systems, were evaluated. For both models, block copolymer-oligonucleotide conjugates used as capture probes amplified the responses of in vitro diagnostic assays. The detection limit reached by using conjugates was estimated at 15 pM for a 219-bp DNA target (HLA-DQB1 model). Moreover, single nucleotide polymorphism was detected in the platelet GPs genotyping model. The use of polymer conjugates led to a significant improvement in both sensitivity and specificity of standard hybridization assays even when applied to complex biological models.

Surface treatment and characterization: perspectives to electrophoresis and lab-on-chips.

The control and modification of surface state is a major challenge in bioanalytical sciences, and in particular in electrokinetic separation methods, due to the importance of electroosmosis. This topic has gained recently a renewed interest, associated with the development of "lab-on-chips" systems that extend the range of materials in which separation channels are fabricated. The surface science community has developed through the years a large toolbox of characterization tools and surface modification protocols, which is not yet fully exploited in the bioanalytical world. In this paper, we try and present an overview of these tools, in order to stimulate new ideas for improved and more controlled surface treatment strategies for separations in capillaries and microchannels. We briefly describe some physical and chemical aspects of electroosmosis (global and spatially resolved), streaming current, and streaming potential. We also review the main strategies for surface coating, and compare the advantages of physisorption, well-organized thin self-assembled monolayers, or conversely thick polymer "brushes". Examples of existing applications to electrophoresis in microchannel are also given.

Polymer-oligonucleotide conjugate synthesis from an amphiphilic block copolymer. Applications to DNA detection on microarray.

An amphiphilic block copolymer poly(tert-butylacrylamide-b-(N-acryloylmorpholine-N-acryloxysuccinimide)) (poly(TBAm-b-(NAM/NAS)) and a random copolymer poly(NAM/NAS), synthesized by the reversible addition-fragmentation chain transfer (RAFT) polymerization process, have been used as support for oligonucleotide (ODN) synthesis, to elaborate polymer-oligonucleotide conjugates. In a first step, starters of ODN solid-phase synthesis were coupled to activated ester functions of polymers, and second, resulting functionalized polymers were covalently grafted onto hydroxylated controlled pore glass (CPG) support to further accomplish ODN synthesis. An efficient capping of residual hydroxyl functions of CPG was performed before synthesis, with both acetic anhydride and diethoxy-N,N-diisopropyl-phosphoramidite reagents, to suppress parasite-free ODN population present in conjugate crude material and resulting from syntheses directly initiated on silica beads. After purification, conjugates were evaluated in a DNA hybridization assay on a microarray, as macromolecules being able to favor capture of the target. Conjugate coating conditions were studied on the dT25/dA25 model. The role of the hydrophobic part (poly(TBAm)) of the conjugate synthesized with the block copolymer in the orientation of the conjugate after coating was revealed by spotting experiments achieved in a mixed solvent (DMF/H(2)O). The use of block copolymer-dT25 conjugate afforded a significant sensitivity improvement of the hybridization assay.