Integration of a zero dead-volume PDMS rotary switch valve in a miniaturised (bio)electroanalytical system

This work features the design, fabrication and characterisation of a miniaturised electroanalytical lab on a chip that allows the performance of a complete bioassay, from the capture of magnetic particles through their functionalisation and sample incubation to the detection of electroactive reaction products. The system is built using mainly polymeric materials such as PMMA and PDMS and fast prototyping techniques such as milling and moulding. The system also includes a set of microelectrodes, photo-lithographed on a silicon chip. The novelty lies in the design of the rotary microvalve, which contains a microreactor so that various reaction and incubation steps can be carried out in isolation from the detection event with zero dead volume. This avoids contamination and fouling of the electrodes by proteins or other organic matter, and extends the useful lifetime of the detector. The system operation is demonstrated by a model example, consisting in the functionalisation of streptavidin-coated magnetic particles with biotinylated β-galactosidase over periods ranging from 5 to 15 minutes, at which point the particles saturate. Although the system is intended for the development of enzyme-based electrochemical bioassays, the concept of its rotary microreactor can be applied more broadly.Peer reviewe

Multi-stringency wash of partially hybridized 60-mer probes reveals that the stringency along the probe decreases with distance from the microarray surface

Here, we describe a multi-parametric study of DNA hybridization to probes with 20–70% G + C content. Probes were designed towards 71 different sites/mutations in the phenylalanine hydroxylase gene. Seven probe lengths, three spacer lengths and six stringencies were systematically varied. The three spacer lengths were obtained by placing the gene-specific sequence in discrete steps along the 60-mer probes. The study was performed using Agilent 8 × 15 000 probes custom-made arrays and a home-built array washer providing different stringencies to each of the eight sub-arrays on the slides. Investigation of hybridization signals, specificity and dissociation curves indicated that probes close to the surface were influenced by an additional stringency provided by the microarray surface. Consistent with this, probes close to the surface required 4 × SSC, while probes placed away from the surface required 0.35 × SSC wash buffers in order to give accurate genotyping results. Multiple step dissociation was frequently observed for probes placed furthest away from surface, but not for probes placed proximal to the surface, which is consistent with the hypothesis that there is different stringency along the 60-mer. The results have impact on design of probes for genotyping, gene expression and comparative genome hybridization analysis