Oligonucleotide microarrays are tools used to analyze samples for the presence of specific DNA sequences. In the system as presented here, specific DNA sequences are first amplified by a polymerase chain reaction (PCR) during which process they are labeled with fluorophores. The amplicons are subsequently hybridized onto an oligonucleotide microarray, which in our case is a porous nylon membrane with microscopic spots. Each spot on the membrane contains oligonucleotides with a sequence complementary to part of one specific target sequence. The solution containing the amplicons flows by external agitation many times up and down through the porous substrate, thereby reducing the time delaying effect of diffusion. By excitation of the fluorophores the emitted pattern of fluorophores can be detected by a charge-coupled device camera. The recorded pattern is a characteristic of the composition of the sample. The oligonucleotide capture probes have been deposited on the substrate by using noncontact piezo ink jet printing, which is the focus of our study. The objective of this study is to understand the mechanisms that determine the distribution of the ink jet printed capture probes inside the membrane. The membrane is a porous medium: the droplets placed on the membrane penetrate in the microstructure of it. The three-dimensional (3D) distribution of the capture probes inside the membrane determines the distribution of the hybridized fluorescent PCR products inside the membrane and thus the emission of light when exposed to the light source. As the 3D distribution of the capture probes inside the membrane eventually determines the detection efficiency, this parameter can be controlled for optimization of the sensitivity of the assay. The main issues addressed here are how are the capture probes distributed inside the membrane and how does this distribution depend on the printing parameters. We will use two model systems to study the influences of different parameters: a single nozzle print head jetting large droplets at a low frequency and a multinozzle print head emitting small droplets at a high frequency. In particular, we have investigated the effects when we change from usage of the first system to the second system. Furthermore, we will go into detail how we can obtain smaller spot sizes in order to increase the spot density without having overlapping spots, leading eventually to lower manufacturing costs of microarrays. By controlling the main print parameters influencing the 3D distribution inside the porous medium, the overall batch-to-batch variations can possibly be reduced.
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