Department of Biomedical Engineering, School of Medicine, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Tsinghua University, Beijing, 100084, China.
Lab Chip. 2018 Sep 11;18(18):2854-2864. doi: 10.1039/c8lc00543e.
A pipette-actuated capillary array comb (PAAC) system operated on a smartphone-based hand-held device has been successfully developed for the multiplex detection of bacteria in a "sample-to-answer" manner. The PAAC consists of eight open capillaries inserted into a cylindrical plastic base with a piece of chitosan-modified glass filter paper embedded in each capillary. During the sample preparation, a PAAC was mounted into a 1 mL pipette tip with an enlarged opening and was operated with a 1 mL pipette for liquid handling. The cell lysate was drawn and expelled through the capillaries three times to facilitate the DNA capture on the embedded filter discs. Following washes with water, the loop-mediated isothermal amplification (LAMP) reagents were aspirated into the capillaries, in which the primers were pre-fixed with chitosan. After that, the PAAC was loaded into the smartphone-based device for a one-hour amplification at 65 °C and end-point detection of calcein fluorescence in the capillaries. The DNA capture efficiency of a 1.1 mm-diameter filter disc was determined to be 97% of λ-DNA and the coefficient of variation among the eight capillaries in the PAAC was only 2.2%. The multiplex detection of genomic DNA extracted from Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus provided limits of detection of 200, 500, and 500 copies, respectively, without any cross-contamination and cross reactions. "Sample-to-answer" detection of E. coli samples was successfully completed in 85 minutes, demonstrating a sensitivity of 200 cfu per capillary. The multiplex "sample-to-answer" detection, the streamlined operation, and the compact device should facilitate a broad range of applications of our PAAC system in point-of-care testing.
一种基于智能手机的手持设备上的移液管驱动毛细管阵列梳（PAAC）系统已成功开发用于以“样本到答案”的方式对细菌进行多重检测。PAAC 由八个插入圆柱形塑料底座的开口毛细管组成，每个毛细管中嵌入一块壳聚糖改性的玻璃滤纸片。在样品制备过程中，将 PAAC 安装到带有扩大开口的 1 毫升移液管吸头中，并使用 1 毫升移液管进行液体处理。将细胞裂解物通过毛细管抽吸和排出三次，以促进嵌入滤纸片上的 DNA 捕获。用水洗涤后，将环介导等温扩增（LAMP）试剂吸入毛细管中，其中引物预先用壳聚糖固定。之后，将 PAAC 装入基于智能手机的设备中，在 65°C 下进行一小时的扩增，并在毛细管中检测钙黄绿素荧光的终点。直径为 1.1 毫米的滤纸片的 DNA 捕获效率被确定为 λ-DNA 的 97%，PAAC 中的八个毛细管之间的变异系数仅为 2.2%。从大肠杆菌、肺炎克雷伯菌和金黄色葡萄球菌提取的基因组 DNA 的多重检测提供了分别为 200、500 和 500 拷贝的检测限，没有任何交叉污染和交叉反应。成功在 85 分钟内完成了对大肠杆菌样品的“样本到答案”检测，每个毛细管的灵敏度为 200 cfu。多重“样本到答案”检测、简化的操作和紧凑的设备应该有助于我们的 PAAC 系统在即时检测中广泛应用。
