Chemical and Biomolecular Engineering Department, University of California, Los Angeles, Los Angeles, CA 90095, USA.
Lab Chip. 2018 Jul 24;18(15):2291-2299. doi: 10.1039/c8lc00452h.
A nucleic acid amplification-free, optics-free platform has been demonstrated for sequence-specific detection of Escherichia coli (E. coli) 16S rRNA at 1 aM (10-18 M) against a 106-fold (1 pM) background of Pseudomonas putida (P. putida) RNA. This work was driven by the need for simple, rapid, and low cost means for species-specific bacterial detection at low concentration. Our simple, conductometric sensing device functioned by detecting blockage of a nanopore fabricated in a sub-micron-thick glass membrane. Upon sequence-specific binding of target 16S rRNA, otherwise charge-neutral, PNA oligonucleotide probe-polystyrene bead conjugates become electrophoretically mobile and are driven to the glass nanopore of lesser diameter, which is blocked, thereby generating a large, sustained and readily observable step decrease in ionic current. No false positive signals were observed with P. putida RNA when this device was configured to detect E. coli 16S rRNA. Also, when a universal PNA probe complementary to the 16S rRNA of both E. coli and P. putida was conjugated to beads, a positive response to rRNA of both bacterial species was observed. Finally, the device readily detected E. coli at 10 CFU mL-1 in a 1 mL sample, also against a million-fold background of viable P. putida. These results suggest that this new device may serve as the basis for small, portable, low power, and low-cost systems for rapid detection of specific bacterial species in clinical samples, food, and water.
一种无需核酸扩增、无需光学元件的平台已被证明可用于在 10^-18 M（1 aM）的浓度下，针对 10^6 倍（1 pM）假单胞菌 RNA 背景，对大肠杆菌（E. coli）16S rRNA 进行序列特异性检测。这项工作的驱动力是需要一种简单、快速且低成本的方法，以便在低浓度下对特定细菌进行特异性检测。我们的简单、电导传感装置通过检测亚微米厚玻璃膜中制造的纳米孔的阻塞来发挥作用。在目标 16S rRNA 特异性结合后，原本电荷中性的 PNA 寡核苷酸探针-聚苯乙烯珠缀合物变得具有电泳迁移性，并被驱动到直径较小的玻璃纳米孔中，从而被阻塞，从而产生大的、持续的且易于观察到的离子电流急剧下降。当该装置被配置为检测大肠杆菌 16S rRNA 时，用假单胞菌 RNA 不会观察到假阳性信号。此外，当与两种细菌的 16S rRNA 互补的通用 PNA 探针与珠粒缀合时，观察到两种细菌 rRNA 的阳性反应。最后，该装置可在 1 毫升样品中以 10 CFU mL-1 的浓度轻松检测到大肠杆菌，同时还能抵抗百万倍活假单胞菌的背景。这些结果表明，这种新装置可能成为用于在临床样本、食品和水中快速检测特定细菌的小型、便携式、低功率和低成本系统的基础。
