Henk Stapert

Quality control of inkjet technology for DNA microarray fabrication

A robust manufacturing process is essential to make high-quality DNA microarrays, especially for use in diagnostic tests. We investigated different failure modes of the inkjet printing process used to manufacture low-density microarrays. A single nozzle inkjet spotter was provided with two optical imaging systems, monitoring in real time the flight path of every droplet. If a droplet emission failure is detected, the printing process is automatically stopped. We analyzed over 1.3 million droplets. This information was used to investigate the performance of the inkjet system and to obtain detailed insight into the frequency and causes of jetting failures. Of all the substrates investigated, 96.2% were produced without any system or jetting failures. In 1.6% of the substrates, droplet emission failed and was correctly identified. Appropriate measures could then be taken to get the process back on track. In 2.2%, the imaging systems failed while droplet emission occurred correctly. In 0.1% of the substrates, droplet emission failure that was not timely detected occurred. Thus, the overall yield of the microarray manufacturing process was 99.9%, which is highly acceptable for prototyping.

Rapid Genotyping of Human Papillomavirus by Post-PCR Array-Based Hybridization Techniques

ABSTRACT Kinetic hybridization measurements on a microarray are expected to become a valuable tool for genotyping applications. A method has been developed that enables kinetic hybridization measurements of PCR products on a low-density microarray. This is accomplished by pumping a solution containing PCR products up and down through a porous microarray substrate. After every pumping cycle, the fluorescently labeled PCR products hybridized to capture probes immobilized on the solid surface of the porous microarray substrate are measured. By this method, both binding curves and high-resolution melting curves are obtained instead of the single endpoint hybridization intensities as with commonly used post-PCR array-based hybridization techniques. We used 20 subtypes of the human papillomavirus (HPV) as a model system to test our detection method and blindly analyzed 216 clinical samples. We compared our microarray flowthrough method with a reference method, PCR followed by a reverse line blot (RLB). Real-time hybridization measurements followed by high-resolution melting curves of low concentrations of fluorescently labeled HPV targets on a microarray were successfully carried out without any additional chemical signal amplification. The results of our new method were in good agreement (93%, with a kappa coefficient of κ = 0.88 [95% CI, 0.81 to 0.94]) with the RLB results. All discrepant samples were analyzed by a third method, enzyme immunoassay (EIA). Furthermore, in a number of cases, we were able to identify false-positive samples by making use of the information contained in the kinetic binding and melting curves. This clearly demonstrates the added value of the use of kinetic measurements and high-resolution melting curves, especially for highly homologous targets.

Mass transfer effects on DNA hybridization in a flow-through microarray

The goal of this study is to assess the influence of mass transfer phenomena on DNA hybridization kinetics in a flow-through, porous microarray for fast molecular testing. We present a scaled mathematical model of coupled convection, diffusion and reaction in porous media, which was used to simulate hybridization kinetics and to analyze the influence of convective transport on the reaction rate. In addition to computer simulations, we also present experimental data of hybridization collected on our microarray system for different flow rates. The results reported in this paper provide for a better understanding of the interaction between reaction and mass transfer processes during flow-through hybridization and suggest criteria for system design and optimization.

Liquid crystal aberration compensation devices

The application of aberration compensation devices can support the trend to higher information densities in optical data storage. The information content of a disc may be increased while maintaining acceptable tolerance levels. For example, disc tilt dependence may require coma compensation, whereas double layer formats require spherical aberration compensation. Liquid crystals (LCs) can be successfully applied in such devices. The design of two practical implementations is discussed; a coma compensating LC-cell with special electrode-design for optimized tolerances w.r.t. decentering, and a lens made of photopolymerizable LC, which can be used to compensate for spherical aberration.

Immobilization of oligonucleotides with homo-oligomer tails onto amine-functionalized solid substrates and the effects on hybridization.

Microarrays have become important tools for the detection and analysis of nucleic acid sequences. Photochemical (254 nm UV) DNA immobilization onto amine-functionalized substrates is often used in microarray fabrication and Southern blots, although details of this process and their effects on DNA functionality are not well understood. By using Cy5-labeled model oligonucleotides for UV immobilization and Cy3-labeled complementary sequences for hybridization, we measured independently the number of immobilized and hybridized oligonucleotides on the microarray surface. By using a two-color fluorescence LED setup and a novel method to compile the data, a full analysis has been made of the effects of oligonucleotide composition (length and sequence) on both immobilization and hybridization. Short homo-oligomer sequences (tails) of uracils, thymines, and, to a limited extent, guanines attached to a hybridization sequence improve immobilization. We propose a possible mechanism explaining the grafting of these nucleotides to amine-functionalized substrates, and we found evidence that the DNA backbone is possibly involved in the immobilization process. Hybridization, on the other hand, greatly improves as a function of tail length regardless of tail composition. On the basis of statistical arguments, the probes increasingly bind via their tail, with the hybridization sequence becoming more accessible to its complement. We conclude that all tails, sequence independent, improve hybridization signals, which is caused by either improved immobilization (especially thymine and uracil) or improved hybridization (most pronounced with guanine tails).

A novel nano-photonics biosensor concept for rapid molecular diagnostics

We present a novel nano-photonics biosensor concept that offers an ultra-high surface specificity and excellent suppression of background signals due to the sample fluid on top of the biosensor. In our contribution, we will briefly discuss the operation principle and fabrication of the biosensor, followed by a more detailed discussion on the experimentally determined performance parameters. Recent results on detection of fluorescently labeled molecules in a highly fluorescent background will be shown, and we will give an outlook on real-time detection of bio-molecules such as proteins and nucleic acids.