基于微液滴阵列芯片的病原细菌数字化分析

Rapid and accurate detection of pathogenic bacteria plays an important role in the diagnosis and treatment of infectious diseases. Current clinical bacteria analysis methods have obvious defections in detection time, throughput and quantitative accuracy, which make it difficult to meet clinical needs. Droplets microfluidics based digital analysis, because of its advantages for example fast detection, high throughput and accurate quantitate, is suitable for rapid quantitative detection of pathogenic bacteria. This project proposed a droplet-array microfluidics based digital assay for pathogenic bacteria analysis, in which several key steps of bacteria analysis are integrated in the microfluidic chip, high-throughput nano-liter droplets encapsulated single bacterium are used as reaction units and floatage based trapping method is used to captured droplets in different domains and form the droplet array. In experiment, bacterial samples and various chromogenic medium / antibiotic are mixed in pairs to generate droplets. After bacteria culture, the bacterial species, quantity and characteristics of antibiotic susceptibility in original sample are calculated via digital method by Poisson distribution theory according to the color results in-droplet. Series of standard strains and a batch of clinical samples will be used to verify the system performance and clinical feasibility of the proposed method, and the goal of rapid, automated, quantitative detection of a variety of pathogenic bacteria within the body fluid sample will be accomplished ultimately. The applicant expects to make useful attempt for the development of new technologies of clinical bacterial analysis through this study.

快速、准确的病原细菌检测对感染性疾病的诊断和治疗意义重大。现有细菌分析手段在分析时间、检测通量和定量准确性等方面存在欠缺，难以满足临床需求。基于液滴微流控芯片的数字化分析技术具有分析快速、通量高和易于实现定量测定等优点，特别适合于进行病原细菌的快速定量检测。本研究拟发展一种基于微液滴阵列芯片的病原细菌数字化分析方法，在芯片上集成细菌分析的多个关键步骤，以大规模纳升级微液滴包裹单个细菌作为反应单元，利用浮力实现液滴的分区捕获以构建微液滴阵列；将细菌样本与多种显色培养基/抗生素配对混合以生成液滴并培养得到显色结果，根据显色结果利用泊松分布理论进行数字化分析，进而推算出原始样本中的细菌种类、数量及其抗生素敏感性。实验将使用系列标准菌株和批量临床样本验证系统性能及其临床可行性，最终实现对病人体液样本内多种病原细菌的快速、自动化、定量化检测。申请人期望藉此研究为临床细菌检验新技术的开发进行有益尝试。

本项目的主要研究内容为：利用微流控芯片技术和数字化分析技术，建立一种快速、简便的病原细菌抗生素敏感性检测方法。目前主要研究结果包括：（1）搭建了一种多功能液滴操作微流控阵列芯片，并验证了其针对单细胞的捕获和检测能力；（2）研制了一种新型抗生素浓度梯度（2倍稀释）生成芯片，并将其于阵列化液滴芯片结合；（3）初步检测了几种临床获得的耐甲氧西林金黄色葡萄球菌（MRSA）的抗生素敏感性，并将其用于几种新型咪唑类低聚物抗菌材料的抗菌性研究。该项目的科学意义主要在于：一旦成功，将实现对病人体液样品内多种病原细菌的抗生素敏感性的快速、自动化、定量化检测，直接有效指导用药，极大降低对细菌感染性疾病的治疗时间与成本；此外，该项目对新型抗菌药物的开发与筛选也具有重要作用。