Schuler Friedrich, Trotter Martin, Geltman Marcel, Schwemmer Frank, Wadle Simon, Domínguez-Garrido Elena, López María, Cervera-Acedo Cristina, Santibáñez Paula, von Stetten Felix, Zengerle Roland, Paust Nils
Hahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, Germany and Laboratory for MEMS Applications, IMTEK - Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.
Hahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.
Lab Chip. 2016 Jan 7;16(1):208-16. doi: 10.1039/c5lc01068c.
Existing systems for digital droplet PCR (ddPCR) either suffer from low integration or are difficult to introduce to mass fabrication. Here we present an integrated system that is compatible to mass fabrication and combines emulsification, PCR, and fluorescence readout in a single chamber within a disposable cartridge (disk). Droplets are generated by injecting the sample into fluorinated oil via centrifugal step emulsification. The resulting emulsion is aligned in the PCR and readout zone by capillary action. During thermocycling, gas bubbles generated by degassing are removed by capillary driven transport through tapered regions in the PCR chamber. Thereby, the positioning of the emulsion within the readout zone of the PCR chamber is ensured at any time and no bubbles are present during readout. Manual handling of the disk solely requires pipetting of oil and PCR mix into the inlet structures, placing the disk into the thermocycler and subsequently into a microarray scanner. The functionality of the ddPCR process chain is demonstrated by quantitative detection of the cystic fibrosis causing mutation p.Phe508del, which is of interest for non-invasive prenatal testing (NIPT). The mutation was detected in a concentration range spanning four orders of magnitude. We envision that this work will lay the base for the development of highly integrated sample-to-digital-answer PCR systems that can be employed in routine clinical diagnosis.
现有的数字液滴聚合酶链式反应（ddPCR）系统要么集成度低，要么难以引入大规模制造。在此，我们展示了一种与大规模制造兼容的集成系统，该系统在一次性试剂盒（盘）的单个腔室内结合了乳化、聚合酶链式反应（PCR）和荧光读数功能。通过离心分步乳化将样品注入氟化油中来产生液滴。所得乳液通过毛细作用在PCR和读数区域中排列。在热循环过程中，脱气产生的气泡通过毛细驱动运输穿过PCR腔室中的锥形区域而被去除。由此，可随时确保乳液在PCR腔室读数区域内的位置，并且在读数期间不存在气泡。对盘的手动操作仅需将油和PCR混合物吸移到入口结构中，将盘放入热循环仪中，随后再放入微阵列扫描仪中。通过对导致囊性纤维化的p.Phe508del突变进行定量检测，证明了ddPCR工艺链的功能，该突变在无创产前检测（NIPT）中具有重要意义。在跨越四个数量级的浓度范围内检测到了该突变。我们设想这项工作将为开发可用于常规临床诊断的高度集成的从样品到数字答案的PCR系统奠定基础。
