Department of Electrical and Biomedical Engineering, University of Nevada, Reno, NV, 89557, USA.
J Nanobiotechnology. 2017 Nov 9;15(1):78. doi: 10.1186/s12951-017-0315-0.
Rapid identification of bacteria can play an important role at the point of care, evaluating the health of the ecosystem, and discovering spatiotemporal distributions of a bacterial community. We introduce a method for rapid identification of bacteria in live cell assays based on cargo delivery of a nucleic acid sequence and demonstrate how a mixed culture can be differentiated using a simple microfluidic system.
C60 Buckyballs are functionalized with nucleic acid sequences and a fluorescent reporter to show that a diversity of microorganisms can be detected and identified in live cell assays. The nucleic acid complexes include an RNA detector, targeting a species-specific sequence in the 16S rRNA, and a complementary DNA with an attached fluorescent reporter. As a result, each bacterium can be detected and visualized at a specific emission frequency through fluorescence microscopy.
The C60 probe complexes can detect and identify a diversity of microorganisms that include gram-position and negative bacteria, yeast, and fungi. More specifically, nucleic-acid probes are designed to identify mixed cultures of Bacillus subtilis and Streptococcus sanguinis, or Bacillus subtilis and Pseudomonas aeruginosa. The efficiency, cross talk, and accuracy for the C60 probe complexes are reported. Finally, to demonstrate that mixed cultures can be separated, a microfluidic system is designed that connects a single source-well to multiple sinks wells, where chemo-attractants are placed in the sink wells. The microfluidic system allows for differentiating a mixed culture.
The technology allows profiling of bacteria composition, at a very low cost, for field studies and point of care.
快速识别细菌可以在医疗点发挥重要作用，评估生态系统的健康状况，并发现细菌群落的时空分布。我们介绍了一种基于核酸序列货物传递的活细胞分析中快速鉴定细菌的方法，并展示了如何使用简单的微流控系统来区分混合培养物。
C60 富勒烯用核酸序列和荧光报告物进行功能化，以表明可以在活细胞分析中检测和鉴定多种微生物。核酸复合物包括 RNA 探测器，针对 16S rRNA 中的物种特异性序列，以及带有附着荧光报告物的互补 DNA。因此，每个细菌都可以通过荧光显微镜在特定发射频率下进行检测和可视化。
C60 探针复合物可以检测和鉴定包括革兰氏阳性和阴性细菌、酵母和真菌在内的多种微生物。更具体地说，设计了核酸探针来识别枯草芽孢杆菌和链球菌或枯草芽孢杆菌和铜绿假单胞菌的混合培养物。报告了 C60 探针复合物的效率、串扰和准确性。最后，为了证明混合培养物可以分离，设计了一种微流控系统，将单个源井连接到多个汇井，在汇井中放置化学引诱剂。微流控系统允许区分混合培养物。
该技术允许以非常低的成本对现场研究和医疗点的细菌组成进行分析。
