Pieter Telleman

Microsystem engineering of lab-on-a-chip devices

Introduction Clean rooms Microfluidics - theoretical aspects Microfluidics - components Simulations in microfluidics Silicon and cleanroom processing Glass micromachining Polymer micromachining Packaging of microsystems Analytical chemistry on microsystems Index

Development of a sensitive DNA microarray suitable for rapid detection of Campylobacter spp.

Campylobacter is the most common cause of human acute bacterial gastroenteritis worldwide, widely distributed and isolated from human clinical samples as well as from many other different sources. To comply with the demands of consumers for food safety, there is a need for development of a rapid, sensitive and specific detection method for Campylobacter. In this study, we present the development of a novel sensitive DNA-microarray based detection method, evaluated on Campylobacter and non-Campylobacter reference strains, to detect Campylobacter directly from the faecal cloacal swabs. The DNA-microarray method consists of two steps: first, both universal bacterial sequences and specific Campylobacter sequences (size range: 149-307 bp) are amplified and fluorescently labeled using multiplex-PCR, targeting the 16S rRNA, the 16S-23S rRNA intergenic region and specific Campylobacter genes. Secondly, the Cy5 labeled PCR-amplicons are hybridised to immobilised capture probes on the microarray. The method allows detection of three to thirty genome equivalents (6-60 fg DNA) of Campylobacter within 3 h, with a hands on time of only 15 min. Using the DNA-microarrays, two closely related Campylobacter species, Campylobacter jejuni and Campylobacter coli could be detected and differentiated directly from chicken faeces. The DNA-microarray method has a high potential for automation and incorporation into a dedicated mass screening microsystem.

Integrated optical measurement system for fluorescence spectroscopy in microfluidic channels

A transportable miniaturized fiber-pigtailed measurement system is presented which allows quantitative fluorescence detection in microliquid handling systems. The microliquid handling chips are made in silica on silicon technology and the optical functionality is monolithically integrated with the microfluidic channel system. This results in inherent stability and photolithographic alignment precision. Permanently attached optical fibers provide a rugged connection to the light source, detection, and data processing unit, which potentially allows field use of such systems. Fluorescence measurements with two dyes, fluorescein, and Bodipy 650/665 X, showed good linear behavior over a wide range of concentrations. Minimally detected concentrations were 250 pM for fluorescein and 100 nM for Bodipy.

Polymer microfluidic chip for online monitoring of microarray hybridizations

A disposable single use polymer microfluidics chip has been developed and manufactured by micro injection molding. The chip has the same outer dimensions as a standard microscope slide (25 x 76 x 1.1 mm) and is designed to be compatible with existing microscope slide handling equipment like microarray scanners. The chip contains an inlet, a 10 microL hybridization chamber capable of holding a 1000 spot array, a waste chamber and a vent to allow air to escape when sample is injected. The hybridization chamber ensures highly homogeneous hybridization conditions across the microarray. We describe the use of this chip in a flexible setup with fluorescence based detection, temperature control and liquid handling by computer controlled syringe pumps. The chip and the setup presented in this article provide a powerful tool for highly parallel studies of kinetics and thermodynamics of duplex formation in DNA microarrays. The experimental setup presented in this article enables the on-chip microarray to be hybridized and monitored at several different stringency conditions during a single assay. The performance of the chip and the setup is demonstrated by on-line measurements of a hybridization of a DNA target solution to a microarray. A presented numerical model indicates that the hybridization process in microfluidic hybridization assays is diffusion limited, due to the low values of the diffusion coefficients D of the DNA and RNA molecules involved.

One-step immobilization of aminated and thiolated DNA onto poly(methylmethacrylate) (PMMA) substrates

Direct immobilisation of modified DNA oligonucleotides (aminated or thiolated) onto a plastic substrate, poly(methylmethacrylate), (PMMA) is described. Using the methyl esters present on non-modified PMMA, it was possible to establish a covalent bond between the electron donor of a DNA probe and the C terminal ester of the PMMA substrate. Since the procedure consists of a single brief wash in isopropanol or ethanol, the procedure is simple and environmentally friendly. The new immobilization strategy was characterized by analysing DNA microarray performance. The new procedure resulted in probe- and hybridization densities that were greater or equivalent to those obtained with commercially available surfaces and other procedures to immobilize DNA onto PMMA. The described chemistry selectively immobilized the DNA via terminal thiol or amine groups indicating that probe orientation could be controlled. Furthermore, the chemical bond between the immobilized DNA and the PMMA could endure repeated heat cycling with only 50% probe loss after 20 cycles, indicating that the chemistry could be used in integrated PCR/microarray devices.

Use of Culture, PCR Analysis, and DNA Microarrays for Detection of Campylobacter jejuni and Campylobacter coli from Chicken Feces

ABSTRACT A DNA microarray for detection of Campylobacter spp. was recently developed and applied to detect Campylobacter spp. directly from chicken feces. Sixty-five pooled chicken cloacal swab samples from 650 individual broiler chickens were included in the study. The results of Campylobacter sp. detection obtained with DNA microarrays were compared to those obtained by conventional culture and gel electrophoresis. By conventional culture, 60% of the samples were positive for either Campylobacter jejuni or Campylobacter coli. By PCR and capillary electrophoresis, 95% of the samples were positive for Campylobacter spp., whereas with DNA microarrays all samples were positive for Campylobacter spp. By application of DNA microarray analysis, the isolates in 4 samples (6%) could not be identified to the species level, whereas by PCR-capillary electrophoresis, the isolates in 12 samples (19%) remained unidentified. Interestingly, PCR-capillary electrophoresis analysis revealed that two (3%) of the samples were positive for both C. jejuni and C. coli, while DNA microarray analysis revealed that nine (14%) of the samples were positive for both species. Of 65 samples, 2 samples were identified to contain C. coli by conventional culture but were positive for C. jejuni by both PCR-capillary electrophoresis and DNA microarray analysis. The discrepancy between the methods is discussed.

Ultraviolet transparent silicon oxynitride waveguides for biochemical microsystems

The UV wavelength region is of great interest in absorption spectroscopy, which is employed for chemical analysis, since many organic compounds absorb in only this region. Germanium-doped silica, which is often preferred as the waveguide core material in optical devices for telecommunication, cannot accommodate guidance below 400 nm, owing to the presence of UV-absorbing centers. We show that silicon oxynitride (SiO(x) N(y)) waveguides exhibit very good UV performance. The propagation loss for 24-microm -wide SiO(x)N (y) waveguides was found to be ~1.0dB/cm in the wavelength range 220-550 nm. The applicability of these waveguides was demonstrated in a biochemical microsystem consisting of multimode buried-channel SiO(x)N (y) waveguides that were monolithically integrated with microfluidic channels. Absorption measurements of a beta -blocking agent, propranolol, at 212-215 nm were performed. The detection limit was reached at a concentration of 13microM , with an optical path length of 500microm (signal/noise ratio, 2).

Cell Sorting in Microfluidic Systems

Cell sorting has become increasingly important in basic research and medical diagnostics. We demonstrate here that magnetic activated sorting and fluorescent activated sorting can also be envisaged in microfluidic silicon structures. In these structures we take advantage of the laminar flow in microstructures. Paramagnetic particles sheeted by two buffer streams are separated from non-magnetic particles by deflection in a magnetic field gradient. Particles labelled with a fluorochrome are sheeted by two buffer streams, excited and detected by a photo multiplier tube. The photo multiplier tube switches a valve on one of the outlets of the sorter microstructure and selects a particle by forcing it to the collecting outlet. Sorting in these structures is now being optimised with paramagnetic and fluorescent beads, but ultimately it is our intention to sort living cells. One of the major advantages of microfluidic structures is the capability of integrating various functional modules. Simultaneously with optimising our sorter structures, we are investigating various levels of system integration that may improve the performance of the structure. For many cell sorting applications involving biohazardous materials or requiring absolute sterile techniques, a single use microstructure would be advantages. Using silicon processing techniques, metal plating and injection moulding we have produced polymer microfluidic structures. Laminar flow and flow switching have been demonstrated in these polymer microstructures and we are currently investigating their performance in sorting.

Rare Event Cell Surging in a Microfluidic System for Application in Prenatal Diagnosis

A recently developed prototype of a microfluidic cell sorting system fabricated on silicon wafers using semiconductor technology is presented. Preliminary results show that we can successfully sort fluorescent particles from non-fluorescent particles. When the performance of the structure has been optimised we will initiate sorting of fluorescently labelled foetal cells. Sorting foetal cells from mother’s blood offers a safer alternative to currently used methods of prenatal genetic diagnostic screening that carry a substantial risk of harming the foetus and may even lead to pregnancy loss.

Somatic Cell Counting with Silicon Apertures

Counting and sizing of biological cells has been used for half a century as a tool in health care and food quality control. This paper describes the employment of a method known as “Coulter sizing” which is based on a small aperture in a membrane. The possibility of microfabrication of silicon provides a cheap and better alternative to current day technologies of aperture manufacture. Silicon technology allows for the aperture to become cheap enough so it can be used as a disposable unit in a Coulter counter. We have manufactured and investigated silicon apertures with different sizes for sizing and counting of particles. One of the places where a simple particle counter would be applicable is on diary farms, where somatic cell counts (SCC) are used for quality control.