Department Healthcare Devices and Instrumentation, Philips Research Europe, High Tech Campus 4, 5656 AE Eindhoven, The Netherlands.
Biomicrofluidics. 2008 Oct 14;2(4):44101. doi: 10.1063/1.2994715.
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.
寡核苷酸微阵列是用于分析样品中特定 DNA 序列存在的工具。在本系统中，首先通过聚合酶链反应 (PCR) 扩增特定的 DNA 序列，在此过程中，它们被荧光标记。随后，扩增子与寡核苷酸微阵列杂交，在我们的情况下，寡核苷酸微阵列是带有微观斑点的多孔尼龙膜。膜上的每个斑点都包含与一个特定靶序列的一部分互补的寡核苷酸序列。含有扩增子的溶液通过外部搅拌多次流过多孔基质，从而减少了扩散的时滞效应。通过激发荧光团，可以通过电荷耦合器件 (CCD) 相机检测到荧光团的发射模式。记录的模式是样品组成的特征。寡核苷酸捕获探针已通过非接触式压电喷墨打印沉积在基底上，这是我们研究的重点。本研究的目的是了解决定喷墨打印捕获探针在膜内分布的机制。膜是一种多孔介质：放置在膜上的液滴会渗透到其微结构中。膜内捕获探针的三维 (3D) 分布决定了膜内杂交荧光 PCR 产物的分布，从而决定了暴露在光源下的发光情况。由于膜内捕获探针的 3D 分布最终决定了检测效率，因此可以控制该参数以优化分析的灵敏度。这里主要解决的问题是捕获探针在膜内是如何分布的，以及这种分布如何依赖于打印参数。我们将使用两个模型系统来研究不同参数的影响：一个喷嘴打印头以低频喷射大液滴，以及一个多喷嘴打印头以高频发射小液滴。特别是，我们研究了从使用第一个系统改为使用第二个系统时的影响。此外，我们将详细研究如何获得更小的斑点尺寸，以增加斑点密度而不产生重叠斑点，最终降低微阵列的制造成本。通过控制影响多孔介质内 3D 分布的主要打印参数，可以可能降低批次间的整体变化。
