Fraunhofer USA Center for Manufacturing Innovation, Boston, MA, USA.
Lab Chip. 2009 Oct 7;9(19):2803-10. doi: 10.1039/b904854e. Epub 2009 Jun 29.
In this paper, we present a fully integrated lab-on-a-chip and associated instrument for the detection of bacteria from liquid samples. The system conducts bacterial lysis, nucleic acid isolation and concentration, polymerase chain reaction (PCR), and end-point fluorescent detection. To enable truly low-cost manufacture of the single-use disposable chip, we designed the plastic chip in a planar format without any active components to be amenable to injection molding and utilized a novel porous polymer monolith (PPM) embedded with silica that has been shown to lyse bacteria and isolate the nucleic acids from clinical samples (M. D. Kulinski, M. Mahalanabis, S. Gillers, J. Y. Zhang, S. Singh and C. M. Klapperich, Biomed. Microdevices, 2009, 11, 671-678).(1) The chip is made of Zeonex(R), a thermoplastic with a high melting temperature to allow PCR, good UV transmissibility for UV-curing of the PPM, and low auto-fluorescence for fluorescence detection of the amplicon. We have built a prototype instrument to automate control of the fluids, temperature cycling, and optical detection with the capability of accommodating various chip designs. To enable fluid control without including valves or pumps on the chip, we utilized a remote valve switching technique. To allow fluid flow rate changes on the valveless chip, we incorporated speed changing fluid reservoirs. The PCR thermal cycling was achieved with a ceramic heater and air cooling, while end-point fluorescence detection was accomplished with an optical spectrometer; all integrated in the instrument. The chip seamlessly and automatically is mated to the instrument through an interface block that presses against the chip. The interface block aligns and ensures good contact of the chip to the temperature controlled region and the optics. The integrated functionality of the chip was demonstrated using Bacillus subtilis as a model bacterial target. A Taqman assay was employed on-chip to detect the isolated bacterial DNA.
在本文中，我们提出了一种完全集成的微流控芯片系统及其相关仪器，用于从液体样本中检测细菌。该系统进行细菌裂解、核酸分离和浓缩、聚合酶链反应（PCR）以及终点荧光检测。为了实现真正的低成本制造一次性使用的芯片，我们设计了平面格式的塑料芯片，没有任何有源组件，便于注塑成型，并利用了一种新型多孔聚合物整体（PPM），其中嵌入了二氧化硅，已被证明能够裂解细菌并从临床样本中分离核酸（M.D.Kulinski、M.Mahalanabis、S.Gillers、J.Y.Zhang、S.Singh 和 C.M.Klapperich，Biomed.Microdevices，2009，11，671-678）。（1）芯片由 Zeonex（R）制成，这种热塑性材料具有较高的熔点，可用于 PCR，具有良好的紫外线透过率，可用于 PPM 的紫外线固化，并且荧光检测时的自发荧光较低。我们已经构建了一个原型仪器，可自动控制流体、温度循环和光学检测，并能够适应各种芯片设计。为了在芯片上不包含阀门或泵的情况下实现流体控制，我们利用了远程阀门切换技术。为了允许无阀芯片上的流量变化，我们结合了变速流体储液器。PCR 热循环通过陶瓷加热器和空气冷却来实现，而终点荧光检测则通过光学光谱仪来完成；所有这些都集成在仪器中。芯片通过一个接口块与仪器自动对接，该接口块压在芯片上。接口块对准并确保芯片与温度控制区域和光学元件良好接触。通过使用枯草芽孢杆菌作为模型细菌靶标，展示了芯片的集成功能。在芯片上进行了 Taqman 检测，以检测分离的细菌 DNA。
