Detlef Snakenborg

Measurements of scattered light on a microchip flow cytometer with integrated polymer based optical elements.

Flow cytometry is widely used for analyzing microparticles, such as cells and bacteria. In this paper, we report an innovative microsystem, in which several different optical elements (waveguides, lens and fiber-to-waveguide couplers) are integrated with microfluidic channels to form a complete microchip flow cytometer. All the optical elements, the microfluidic system, and the fiber-to-waveguide couplers were defined in one layer of polymer (SU-8, negative photoresist) by standard photolithography. With only a single mask procedure required, all the fabrication and packaging processes can be finished in one day. Polystyrene beads were measured in the microchip flow cytometer, and three signals (forward scattering, large angle scattering and extinction) were measured simultaneously for each bead. To our knowledge this is the first time forward scattered light and incident light extinction were measured in a microsystem using integrated optics. The microsystem can be applied for analyzing different kinds of particles and cells, and can easily be integrated with other microfluidic components.

Lab-on-a-chip with integrated optical transducers

Taking the next step from individual functional components to higher integrated devices, we present a feasibility study of a lab-on-a-chip system with five different components monolithically integrated on one substrate. These five components represent three main domains of microchip technology: optics, fluidics and electronics. In particular, this device includes an on-chip optically pumped liquid dye laser, waveguides and fluidic channels with passive diffusive mixers, all defined in one layer of SU-8 polymer, as well as embedded photodiodes in the silicon substrate. The dye laser emits light at 576 nm, which is directly coupled into five waveguides that bring the light to five different locations along a fluidic channel for absorbance measurements. The transmitted portion of the light is collected at the other side of this cuvette, again by waveguides, and finally detected by the photodiodes. Electrical read-out is accomplished by integrated metal connectors. To our knowledge, this is the first time that integration of all these components has been demonstrated.

BioXTAS RAW, a software program for high- throughput automated small-angle X-ray scattering data reduction and preliminary analysis

A fully open source software program for automated two-dimensional and one-dimensional data reduction and preliminary analysis of isotropic small-angle X-ray scattering (SAXS) data is presented. The program is freely distributed, following the open-source philosophy, and does not rely on any commercial software packages. BioXTAS RAW is a fully automated program that, via an online feature, reads raw two-dimensional SAXS detector output files and processes and plots data as the data files are created during measurement sessions. The software handles all steps in the data reduction. This includes mask creation, radial averaging, error bar calculation, artifact removal, normalization and q calibration. Further data reduction such as background subtraction and absolute intensity scaling is fast and easy via the graphical user interface. BioXTAS RAW also provides preliminary analysis of one-dimensional data in terms of the indirect Fourier transform using the objective Bayesian approach to obtain the pair-distance distribution function, PDDF, and is thereby a free and open-source alternative to existing PDDF estimation software. Apart from the TIFF input format, the program also accepts ASCII-format input files and is currently compatible with one-dimensional data files from SAXS beamlines at a number of synchrotron facilities. BioXTAS RAW is written in Python with C++ extensions.

Microstructure fabrication with a CO2 laser system

In this paper, we investigate the use of a commercial CO2 laser system for fabrication of microfluidic systems in polymers. We discuss the cutting process with the laser system and present a straightforward model for the channel depth of microchannels dependent on the fabrication parameters. In particular, we examine the influence of the cutting sequence, the number of cut passes, the laser beam velocity and the laser radiant flux. The model allows the prediction of microchannel depths within a maximum deviation of 8 µm for channels that are up to 210 µm in depths. It was shown that, at constant channel depth, the channel width could be varied by 27% by using different cutting parameters. The optimum cutting sequence for the production of a channel -junction is also presented in the paper. The laser system is shown to be a flexible and rapid tool for the production of polymer microfluidic prototypes.

On-chip electro membrane extraction with online ultraviolet and mass spectrometric detection.

Electro membrane extraction was demonstrated in a microfluidic device. The device was composed of a 25 μm thick porous polypropylene membrane bonded between two poly(methyl methacrylate) (PMMA) substrates, each having 50 μm deep channel structures facing the membrane. The supported liquid membrane (SLM) consisted of 2-nitrophenyl octyl ether (NPOE) immobilized in the pores of the membrane. The driving force for the extraction was a 15 V direct current (DC) electrical potential applied across the SLM. Samples containing the basic drugs pethidine, nortriptyline, methadone, haloperidol, loperamide, and amitriptyline were used to characterize the system. Extraction recoveries were typically in the range of 65-86% for the different analytes when the device was operated with a sample flow of 2.0 μL/min and an acceptor flow of 1.0 μL/min. With the sample flow at 9.0 μL/min and the acceptor flow at 0.0 μL/min, enrichment factors exceeding 75 were obtained during 12 min of operation from a total sample volume of only 108 μL. The on-chip electro membrane system was coupled online to electrospray ionization mass spectrometry and used to monitor online and real-time metabolism of amitriptyline by rat liver microsomes.

Improved bacteria detection by coupling magneto-immunocapture and amperometry at flow-channel microband electrodes

This paper describes the first immunosensing system reported for the detection of bacteria combining immunomagnetic capture and amperometric detection in a one-step sandwich format, and in a microfluidic environment. Detection is based on the electrochemical monitoring of the activity of horseradish peroxidase (HRP), an enzyme label, through its catalysis of hydrogen peroxide (H(2)O(2)) in the presence of the mediator hydroquinone (HQ). The enzymatic reaction takes place in an incubation micro-chamber where the magnetic particles (MPs) are confined, upstream from the working electrode. The enzyme product is then pumped along a microchannel, where it is amperometrically detected by a set of microelectrodes. This design avoids direct contact of the biocomponents with the electrode, which lowers the risk of electrode fouling. The whole assay can be completed in 1h. The experiments performed with Escherichia coli evidenced a linear response for concentrations ranging 10(2)-10(8) cell ml(-1), with a limit of detection of 55 cells ml(-1) in PBS, without pre-enrichment steps. Furthermore, 100 cells ml(-1) could be detected in milk, and with negligible interference by non-target bacteria such as Pseudomonas.

A cyclo olefin polymer microfluidic chip with integrated gold microelectrodes for aqueous and non-aqueous electrochemistry

This paper presents an entirely polymeric microfluidic system, made of cyclo olefin polymer (COP), with integrated gold microband electrodes for electrochemical applications in organic media. In the present work, we take advantage of the COPs high chemical stability to polar organic solvents in two different ways: (i) to fabricate gold microelectrodes using COP as a substrate by standard lithographic and lift-off techniques; and (ii) to perform electrochemical experiments in organic media. In particular, fourteen parallel gold microelectrodes with a width of 14 microm and separated from their closest neighbour by 16 microm were fabricated by lithographic and lift-off techniques on a 188 microm thick COP sheet. A closed channel configuration was obtained by pressure-assisted thermal bonding between the COP sheet containing the microelectrodes and a microstructured COP sheet, where a 3 cm long, 50 microm wide and 24 microm deep channel was fabricated via hot embossing. Cyclic voltammetric measurements were carried out in aqueous and organic media, using a solution consisting of 5 mM ferrocyanide/ferricyanide in 0.5 M KNO(3) and 5 mM ferrocene in 0.1 M TBAP/acetonitrile, respectively. Experimental currents obtained for different flow rates ranging from 1 to 10 microL min(-1) were compared to the theoretical steady state currents calculated by the Levich equation for a band electrode (R. G. Compton, A. C. Fisher, R. G. Wellington, P. J. Dobson and P. A. Leigh, J. Phys. Chem., 1993, 97, 10410-10415). In both cases, the difference between the experimental and the predicted data is less than 5%, thus validating the behaviour of the fabricated device. This result opens the possibility to use a microfluidic system made entirely from COP with integrated microband electrodes in organic electroanalysis and in electrosynthesis.

Multi-channel peristaltic pump for microfluidic applications featuring monolithic PDMS inlay

The design, fabrication and characterization of a miniaturized, mechanically-actuated 12-channel peristaltic pump for microfluidic applications and built from simple, low-cost materials and fabrication methods is presented. Two pump configurations are tested, including one which reduces pulsating flow. Both use a monolithic PDMS pumping inlay featuring three-dimensional geometries favourable to pumping applications and 12 wholly integrated circular channels. Flow rates in the sub-microL min(-1) to microL min(-1) range were obtained. Channel-to-channel flow rate variability was comparable to a commercial pumping system at lower flow rates. The small footprint, 40 mm by 80 mm, of the micropump renders it portable, and allows its use on microscope stages adjacent to microfluidic devices, thus reducing system dead volumes. The micropumps design allows potential use in remote and resource-limited locations.

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.

A fast and reliable way to establish fluidic connections to planar microchips

In this work, we present a non-permanent method to connect microfluidic devices. The approach uses short flexible tubes that are plugged into bottom-flat holes and ensure fast and reliable interconnections. The small available dimensions allow the tube to be directly attached to the side of planar microchips. A theoretical model to estimate the maximum applicable pressure was developed, and verified with experimental data. Furthermore, the tube connections were compared to other non-permanent interconnection types.