Implementation of Microfluidics for Antimicrobial Susceptibility Assays: Issues and Optimization Requirements.
Implementation of Microfluidics for Antimicrobial Susceptibility Assays: Issues and Optimization Requirements.
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DOI：
10.3389/fcimb.2020.547177
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发表时间：
2020
影响因子：
5.7
通讯作者：
Hicks LM
中科院分区：
文献类型：
--
作者：
Parsley NC;Smythers AL;Hicks LM
尽管多重耐药病原体不断出现，但进入市场的新型抗菌药物数量却极少。天然产物肽是生物活性化合物的丰富来源，质谱技术的进步在发现和表征新分子种类方面取得了前所未有的能力。然而，传统的生物活性测定形式阻碍了天然产物抗菌肽（AMP）的发现和生化表征，需要大量样品和显着优化实验参数才能实现准确/一致的活性测量。微流体装置为批量分析系统提供了一种有前景的替代方案。在此，将基于微流体的生物测定与各自市售硬件中的传统 96 孔板格式进行了比较。使用针对大肠杆菌 ATCC 25922 筛选的 Viola inconspicua 肽库来比较每种测定类型的生物活性。明场显微镜用于确定微流体通道中的生物活性，同时在基于板的测定中严格评估基于常见光学和荧光的细胞活力测量。所有基于板的测定在光密度和基于荧光的测量中表现出一些变化，都赋予后期洗脱的 V. inconspicua 文库级分的生物活性。然而，发现基于板的测定和微流体测定的生物活性特征存在显着差异，并且可能源自构成每种测定装置的材料或每种形式中使用的生长/测定条件。虽然需要新技术来克服传统生物活性测定的局限性，但我们证明，现成的微流控装置的实施并非易事，需要进行重大的方法开发/优化，才能实现传统用途，以灵敏、快速地检测天然产物基质中的 AMP。
Despite the continuous emergence of multi-drug resistant pathogens, the number of new antimicrobials reaching the market is critically low. Natural product peptides are a rich source of bioactive compounds, and advances in mass spectrometry have achieved unprecedented capabilities for the discovery and characterization of novel molecular species. However, traditional bioactivity assay formats hinder the discovery and biochemical characterization of natural product antimicrobial peptides (AMPs), necessitating large sample quantities and significant optimization of experimental parameters to achieve accurate/consistent activity measurements. Microfluidic devices offer a promising alternative to bulk assay systems. Herein, a microfluidics-based bioassay was compared to the traditional 96-well plate format in respective commercially-available hardware. Bioactivity in each assay type was compared using a Viola inconspicua peptide library screened against E. coli ATCC 25922. Brightfield microcopy was used to determine bioactivity in microfluidic channels while both common optical and fluorescence-based measurements of cell viability were critically assessed in plate-based assays. Exhibiting some variation in optical density and fluorescence-based measurements, all plate-based assays conferred bioactivity in late eluting V. inconspicua library fractions. However, significant differences in the bioactivity profiles of plate-based and microfluidic assays were found, and may be derived from the materials comprising each assay device or the growth/assay conditions utilized in each format. While new technologies are necessary to overcome the limitations of traditional bioactivity assays, we demonstrate that off-the-shelf implementation of microfluidic devices is non-trivial and significant method development/optimization is required before conventional use can be realized for sensitive and rapid detection of AMPs in natural product matrices.
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影响因子：
5.7
作者：
Kirkpatrick CL;Parsley NC;Bartges TE;Wing CE;Kommineni S;Kristich CJ;Salzman NH;Patrie SM;Hicks LM
通讯作者：
Hicks LM
DOI：
10.1038/nrm4041
发表时间：
2015-09
期刊：
Nature reviews. Molecular cell biology
影响因子：
--
作者：
Duncombe TA;Tentori AM;Herr AE
通讯作者：
Herr AE
影响因子：
9.4
作者：
Gomez-Lopez, A;Aberkane, A;Cuenca-Estrella, M
通讯作者：
Cuenca-Estrella, M
DOI：
10.1073/pnas.0707879105
发表时间：
2008-02-26
影响因子：
11.1
作者：
Horinek, D.;Serr, A.;Netz, R. R.
通讯作者：
Netz, R. R.
影响因子：
7.4
作者：
Kirkpatrick CL;Broberg CA;McCool EN;Lee WJ;Chao A;McConnell EW;Pritchard DA;Hebert M;Fleeman R;Adams J;Jamil A;Madera L;Strömstedt AA;Göransson U;Liu Y;Hoskin DW;Shaw LN;Hicks LM
通讯作者：
Hicks LM