基于创新微流控技术的快速低成本高通量细菌耐药性检测方法

Rapid, Low-cost, High-Throughput Test of Antimicrobial Susceptibility Based on Novel Microfluidic Technology..Antimicrobial resistance (AMR) is a natural phenomenon developed in microorganisms, and is accelerated by misuse of antibiotics. AMR makes the treatment difficult, costly, or even impossible, thus is causing major health threat as well as enormous financial loss worldwide. The issues of AMR in China are especially severe...Two strategies are effective in addressing AMR issues: (1) use narrow-spectrum antibiotics based on accurate diagnosis, preferentially, using antimicrobial susceptibility test (AST); (2) develop new antibiotics. However, both strategies are challenged by the lack of effective and affordable method to test the response of target microorganisms to drug administration. An AST involves a large number of tests, especially when the minimum inhibitory concentration (MIC) of each drug is desired. Current ASTs are based on observing cell-growth, which is costly and time-consuming. Similar challenge exists in the drug development which requires large-scale screening. Tremendous efforts have been made to improve AST technology; however, those new strategies, based on PCR, nanoparticles, or dielectrophoresis, are still impractical to be used for real specimens. ..Herein we propose a microfluidic platform which could have major contribution to addressing the aforementioned challenges. We are developing a new strategy to detect the response of microorganisms to drug treatment, using a microfluidic on-chip culture and counting platform which could detect the response of drug-affected bugs quicker than conventional methods. The device will be made of a new type of material, perfluoropolymers, which addresses major challenges associated with channel fouling in drug-loaded on-chip culture. Also, the system supports high-throughput operation without massive manipulation such as pipetting, thereby improving the reliability of the assay. Moreover, a special design of the microchannel will incorporate a concentration effect to further reduce the time need for incubation therefore accelerating the test. ..Through this project, we expect an inexpensive, user-friendly but quick and reliable method to benefit the control of AMR. As our method is designed to allow maximum accessibility, the broad range of clinics currently could not afford AST would be able to conduct routine test. At the mean time, the unique platform will allow us to obtain important new knowledge about the mechanism of AMR development. Finally, the platform could be implemented to drug discovery and other applications where similar tool is sought for.

致病微生物的产生耐药性会使已有的药物失效，耐药性的产生因广谱抗生素滥用而加快。耐药性已成为世界重大危机，且形势在中国尤为严峻。利用可靠的药效筛选手段指导选用特异性抗生素，可显著减缓耐药性产生。然而，目前药效筛选基于细胞培养，费时且昂贵，无法指导用药。基于基因或纳米科技的新尝试尚有明显缺陷难以用于常规检测。本研究致力于建立一个基于创新的微流控技术的快速药效筛选方法。我们通过在特制芯片上进行细胞培养，并在单细胞尺度进行观测，更快地检测到致病微生物对药物的反应。此外，我们用具超强抗污性能的全氟化合物制作芯片，以解决通道沾污造成药物浓度不可控的难点问题；此设计支持高通量操作，且芯片的特殊设计具有浓缩功能，减少培养所需时间，进一步加快检测速度，且提高对体外难增殖细胞的响应。通过本项目，我们希望建立一套经济、便捷和可靠的方法，有助于有效控制微生物抗药性。除临床应用前景，这一平台还有望用于药物开发等。

致病微生物产生耐药性会使已有的药物失效，耐药性的产生因广谱抗生素滥用而加快。耐药性已成为世界重大危机，且形势在中国尤为严峻。利用可靠的药效筛选手段指导选用特异性抗生素，可显著减缓耐药性产生。然而，目前药效筛选基于细胞培养，费时且昂贵，无法指导用药。基于基因或纳米科技的新尝试尚有明显缺陷难以用于常规检测。本研究建立了一个基于创新的微流控技术的快速药效筛选方法。我们通过在特制芯片上进行细胞培养，并在单细胞尺度进行观测，更快地检测到致病微生物对药物的反应，将传统24-48小时完成的测试缩短至2-3小时。我们系统研究了芯片内流体剪切力对细菌耐药性测试的影响，并结合计算机模拟分析设计了特别的芯片结构，解决了剪切力带来的挑战。我们用具出色抗污性能的化合物制作芯片，以解决通道沾污造成药物浓度不可控的难点问题，实现了比传统PDMS芯片更好的测试可靠性，测试结果与国际临床标准完全吻合，同时芯片成本也低于PDMS芯片，利于大规模推广使用。此外，我们实现了支持高通量操作的芯片设计，且通过特别的加工方法低成本实现了一种纳米细胞筛结构，该结构具有细菌浓缩功能，减少培养所需时间，进一步加快检测速度，且提高对体外难增殖细胞的响应。基于上述创新的集成系统实现了快速自动化的细菌抗药性测试，在数小时内可靠完成测试结果，通过普通的手机配合一个转换镜头即可拍照获取测试结果。通过本项目，我们建立了一套经济、便捷和可靠的方法，有助于有效控制微生物抗药性。除临床应用前景，这一平台还有望用于药物开发等。此外在开展本项目的过程中我们还创立了多种基于非传统材料的芯片微加工方法。