Laboratory for MEMS Applications, Department of Microsystems Engineering (IMTEK), University of Freiburg, Georges-Koehler-Allee 106, 79110 Freiburg, Germany.
Lab Chip. 2010 Oct 7;10(19):2519-26. doi: 10.1039/c004954a. Epub 2010 Jul 7.
We present a novel process flow enabling prototyping of microfluidic cartridges made out of polymer films. Its high performance is proven by implementation of a microfluidic genotyping assay testing 22 DNA samples including clinical isolates from patients infected by methicilin-resistant Staphylococcus aureus (MRSA). The microfluidic cartridges (disks) are fabricated by a novel process called microthermoforming by soft lithography (microTSL). Positive moulds are applied allowing for higher moulding precision and very easy demoulding when compared to conventional microthermoforming. High replication accuracies with geometric disk-to-disk variations of less than 1% are typical. We describe and characterise fabrication and application of microfluidic cartridges with wall thicknesses <188 microm thus enabling efficient thermocycling during real-time polymerase chain reaction (PCR). The microfluidic cartridges are designed for operation in a slightly modified commercial thermocycling instrument. This approach demonstrates new opportunities for both microfluidic developments and well-established laboratory instruments. The microfluidic protocol is controlled by centrifugal forces and divides the liquid sample parallely into independent aliquots of 9.8 microl (CV 3.4%, N = 32 wells). The genotyping assays are performed with pre-stored primers and probes for real-time PCR showing a limit of detection well below 10 copies of DNA per reaction well (N = 24 wells in 3 independent disks). The system was evaluated by 44 genotyping assays comprising 22 DNA samples plus duplicates in a total of 11 disks. The samples contained clinical samples of seven different genotypes of MRSA as well as positive and negative controls. The results are in excellent agreement with the reference in microtubes.
我们提出了一种新颖的工艺流程，能够对聚合物薄膜制成的微流控芯片进行原型制作。通过实施一种微流控基因分型检测试验，对 22 个 DNA 样本（包括耐甲氧西林金黄色葡萄球菌（MRSA）感染患者的临床分离株）进行了测试，证明了其高性能。微流控芯片（圆盘）是通过一种称为软光刻微热成型（microTSL）的新工艺制造的。使用正模具，与传统的微热成型相比，可以实现更高的模具精度和非常容易的脱模。典型的几何圆盘对圆盘变化小于 1%，复制精度非常高。我们描述并表征了制造和应用微流控芯片的过程，其壁厚度<188 微米，从而能够在实时聚合酶链反应（PCR）过程中实现高效的热循环。微流控芯片专为在稍微修改的商业热循环仪器中运行而设计。这种方法为微流控开发和成熟的实验室仪器都提供了新的机会。微流控协议由离心力控制，并将液体样本平行地分为 9.8 微升的独立等分试样（CV 为 3.4%，N=32 个孔）。基因分型检测使用预先存储的引物和探针进行实时 PCR 检测，每个反应孔的检测下限低于 10 个拷贝的 DNA（N=24 个孔，3 个独立的圆盘）。该系统通过 44 个基因分型检测进行了评估，其中包括 22 个 DNA 样本，加上 11 个圆盘中的重复样本。这些样本包含七种不同基因型的耐甲氧西林金黄色葡萄球菌的临床样本，以及阳性和阴性对照。结果与微管中的参考值非常吻合。
