Facile syringe filter-enabled bacteria separation, enrichment, and buffer exchange for clinical isolation-free digital detection and characterization of bacterial pathogens in urine.
Facile syringe filter-enabled bacteria separation, enrichment, and buffer exchange for clinical isolation-free digital detection and characterization of bacterial pathogens in urine.
复制标题
DOI：
10.1039/d1an00039j
复制
发表时间：
2021-04-26
期刊：
影响因子：
--
通讯作者：
中科院分区：
文献类型：
--
作者：
开发针对传染病的病原体鉴定（ID）和抗菌药物敏感性测试（AST）的加速方法对于促进循证抗生素治疗并减少临床对广谱抗生素的过度依赖是必要的。为此，基于液滴的微流控技术已经实现了具有单细胞和单分子分辨率的非常快速的诊断测定。然而，液滴平台总是依赖于测试经过长时间（> 16 小时）铺板后临床分离的纯化细菌样品。虽然基于平板的临床分离对于从临床样本中的背景中富集和分离细菌以及促进缓冲液交换非常重要，但它产生了诊断瓶颈，最终阻碍了基于液滴的方法显着加快获得结果的时间。为了缓解这一瓶颈，我们开发了方便的注射器过滤器策略，用于从尿液样本中分离、富集和缓冲液交换。通过选择适当尺寸的滤膜，我们将细菌细胞与尿液样本中的背景颗粒分离，并在这一步骤过滤后实现高达 91% 的细菌回收率。当与基于液滴的细菌细胞检测结合使用时，一步过滤将细菌 ID 和定量的检测限提高了一个数量级以上。我们还开发了一种简便的缓冲液交换策略，用于制备尿液样本中的细菌，用于基于液滴的 AST，在缓冲液交换过程中实现了高达 10 倍的细菌富集。我们的过滤策略可以轻松集成到液滴工作流程中，实现无需临床隔离的样品到答案 ID 和 AST，并显着加快标准传染病诊断工作流程的周转速度。我们设计了一种基于过滤的简单细菌回收和缓冲液交换过程，并将其与液滴微流体连接起来，用于尿液中细菌病原体的临床免隔离数字检测和表征。
The development of accelerated methods for pathogen identification (ID) and antimicrobial susceptibility testing (AST) for infectious diseases is necessary to facilitate evidence-based antibiotic therapy and reduce clinical overreliance on broad-spectrum antibiotics. Towards this end, droplet-based microfluidics has unlocked remarkably rapid diagnostic assays with single-cell and single-molecule resolution. Yet, droplet platforms invariably rely on testing purified bacterial samples that have been clinically isolated after lengthy (>16 h) plating. While plating-based clinical isolation is important for enriching and separating out bacteria from background in clinical samples and also facilitating buffer exchange, it creates a diagnostic bottleneck that ultimately precludes droplet-based methods from achieving significantly accelerated times-to-result. To alleviate this bottleneck, we have developed facile syringe filter-enabled strategies for bacterial separation, enrichment, and buffer exchange from urine samples. By selecting appropriately sized filter membranes, we separated bacterial cells from background particulates in urine samples and achieve up to 91% bacterial recovery after such 1-step filtration. When interfaced with droplet-based detection of bacterial cells, 1-step filtration improved the limit of detection for bacterial ID and quantification by over an order of magnitude. We also developed a facile buffer exchange strategy to prepare bacteria in urine samples for droplet-based AST that achieved up to 10-fold bacterial enrichment during buffer exchange. Our filtration strategies, can be easily integrated into droplet workflows, enable clinical isolation-free sample-to-answer ID and AST, and significantly accelerate the turnaround of standard infectious disease diagnostic workflows. We devised and interfaced a facile filtration-based bacteria recovery and buffer exchange process with droplet microfluidics for clinical isolation-free digital detection and characterization of bacteria pathogens in urine.
登录
查看更多内容
影响因子：
6.1
作者：
Rane TD;Chen L;Zec HC;Wang TH
通讯作者：
Wang TH
影响因子：
5.7
作者：
Andrade, Patricia Fernanda;de Faria, Andreia Fonseca;Goncalves, Maria do Carmo
通讯作者：
Goncalves, Maria do Carmo
影响因子：
2.7
作者：
Li Y;Yang X;Zhao W
通讯作者：
Zhao W
影响因子：
2.9
作者：
Ku, Seockmo;Kreke, Thomas;Ladisch, Michael R.
通讯作者：
Ladisch, Michael R.
影响因子：
4.4
作者：
Francy, Donna S.;Stelzer, Erin A.;Lindquist, H. D. Alan
通讯作者：
Lindquist, H. D. Alan