Centrifugal microfluidic lab-on-a-chip system with automated sample lysis, DNA amplification and microarray hybridization for identification of enterohemorrhagic Escherichia coli culture isolates
Centrifugal microfluidic lab-on-a-chip system with automated sample lysis, DNA amplification and microarray hybridization for identification of enterohemorrhagic Escherichia coli culture isolates
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DOI：
10.1039/d0an01232g
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发表时间：
2020-11-07
期刊：
影响因子：
4.2
通讯作者：
Veres, Teodor
中科院分区：
文献类型：
--
作者：
Geissler, Matthias;Brassard, Daniel;Veres, Teodor
细菌培养分离物快速、自动鉴定技术的发展可以帮助监管机构缩短食品安全监督、合规和执法以及疫情调查的响应时间。虽然聚合酶链反应（PCR）等分子方法能够以高灵敏度和特异性鉴定微生物，但它们通常依赖于复杂的仪器和复杂的工作流程来进行样品制备，并且需要高水平的动手参与。在这里，我们描述了一个集成热裂解、PCR扩增和微阵列杂交在同一个药筒上的芯片实验室系统的设计、操作和性能。该分析在离心微流控平台上进行，该平台允许在旋转过程中气动驱动液体，从而可以以全自动方式执行所有流体操作，而无需在微流控盒上集成主动控制元件。该药筒由硬和软热塑性聚合物制成，与大批量制造（例如注射成型）兼容。芯片设计和热接口都进行了优化，以确保有效的传热，并允许在PCR过程中快速热循环。集成的工作流包括14个步骤，只需不到2小时即可完成。唯一的手动步骤与样品和试剂的装载有关，以及微阵列的荧光成像。片上裂解和PCR扩增都提供了与台式仪器获得的结果相当的结果。该微阵列包含一组寡核苷酸探针，用于多重检测七种肠出血性大肠杆菌优先血清型，在环烯烃共聚物底物上使用一种新的激活方案，该方案涉及使用溴化氰将羟基（来自氧等离子体处理）转化为活性氰酸酯。通过多重PCR扩增步骤获得的大肠杆菌O157:H7 （rfbO157, eae， vt1和vt2）的荧光标记基因标记，以非定量方式证明了芯片上杂交。
The development of technology for the rapid, automated identification of bacterial culture isolates can help regulatory agencies to shorten response times in food safety surveillance, compliance, and enforcement as well as outbreak investigations. While molecular methods such as polymerase chain reaction (PCR) enable the identification of microbial organisms with high sensitivity and specificity, they generally rely on sophisticated instrumentation and elaborate workflows for sample preparation with an undesirably high level of hands-on engagement. Herein, we describe the design, operation and performance of a lab-on-a-chip system integrating thermal lysis, PCR amplification and microarray hybridization on the same cartridge. The assay is performed on a centrifugal microfluidic platform that allows for pneumatic actuation of liquids during rotation, making it possible to perform all fluidic operations in a fully-automated fashion without the need for integrating active control elements on the microfluidic cartridge. The cartridge, which is fabricated from hard and soft thermoplastic polymers, is compatible with high-volume manufacturing (e.g., injection molding). Chip design and thermal interface were both optimized to ensure efficient heat transfer and allow for fast thermal cycling during the PCR process. The integrated workflow comprises 14 steps and takes less than 2 h to complete. The only manual steps are related to loading of the sample and reagents on the cartridge as well as fluorescence imaging of the microarray. On-chip lysis and PCR amplification both provided results comparable to those obtained by bench-top instrumentation. The microarray, incorporating a panel of oligonucleotide probes for multiplexed detection of seven enterohemorrhagic E. coli priority serotypes, was implemented on a cyclic olefin copolymer substrate using a novel activation scheme that involves the conversion of hydroxyl groups (derived from oxygen plasma treatment) into reactive cyanate ester using cyanogen bromide. On-chip hybridization was demonstrated in a non-quantitative fashion using fluorescently-labelled gene markers for E. coli O157:H7 (rfbO157, eae, vt1, and vt2) obtained through a multiplexed PCR amplification step.