Unidad Monterrey , Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional , Vía del Conocimiento 201 , Parque PIIT, Apodaca , Nuevo León CP 66628 , México.
Mayo Clinic , Rochester , Minnesota 55905 , United States.
Anal Chem. 2019 Apr 16;91(8):5133-5141. doi: 10.1021/acs.analchem.8b05689. Epub 2019 Mar 27.
The ability to detect multiple analytes in a small sample volume has significance for numerous areas of research, including organs-on-chip, small animal experiments, and neonatology. The objective of this study was to develop an automated microfluidics platform for multiplexed detection of analytes in microliter sample volumes. This platform employed computer-controlled microvalves to create laminar co-flows of sample and assay reagent solutions. It also contained valve-regulated cross-junction for discretizing sample/reagent mixtures into water-in-oil droplets. Microfluidic automation allowed us to control parameters related to frequency of droplet generation and the number of droplets of the same composition, as well as the size of droplets. Each droplet represented an individual enzymatic assay carried out in a sub-nanoliter (0.8 nL) volume reactor. An enzymatic reaction involving target analyte and assay reagents produced colorimetric or fluorescent signals in droplets. Importantly, intensity of optical signal was proportional to the concentration of analyte in question. This microfluidic bioanalysis platform was used in conjunction with commercial "mix-detect" assays for glucose, total bile acids, and lactate dehydrogenase (LDH). After characterizing these assays individually, we demonstrated sensitive multiplexed detection of three analytes from as little as 3 μL. In fact, this volume was sufficient to generate multiple repeat droplets for each of the three biochemical assays as well as positive control droplets, confirming the quality of assay reagents and negative control droplets to help with background subtraction. One potential application for this microfluidic bioanalysis platform involves sampling cell-conditioned media in organ-on-chip devices. To highlight this application, hepatocyte spheroids were established in microfluidic devices, injured on-chip by exposure to lipotoxic agent (palmitate), and then connected to the bioanalysis module for daily monitoring of changes in cytotoxicity (LDH), energy metabolism (glucose), and liver function (total bile acids). Microfluidic in-droplet assays revealed increased levels of LDH as well as reduction in bile acid synthesis-results that were consistent with hepatic injury. Importantly, these experiments highlighted the fact that in-droplet assays were sufficiently sensitive to detect changes in functional output of a relatively small (∼100) number of hepatocyte spheroids cultured in a microfluidic device. Moving forward, we foresee increasing the multiplexing capability of this technology and applying this platform to other biological/medical scenarios where detection of multiple analytes from a small sample volume is desired.
该能力在小样本量中检测多种分析物对于包括器官芯片、小动物实验和新生儿学在内的众多研究领域具有重要意义。本研究的目的是开发一种用于微升样本量中分析物的多重检测的自动化微流控平台。该平台采用计算机控制的微阀来创建样品和分析试剂溶液的层流共流。它还包含阀控十字结，用于将样品/试剂混合物离散化成油包水液滴。微流控自动化使我们能够控制与液滴生成频率和相同组成的液滴数量以及液滴大小相关的参数。每个液滴代表在亚纳升级（0.8 nL）体积反应器中进行的单个酶促分析。涉及目标分析物和分析试剂的酶反应在液滴中产生比色或荧光信号。重要的是，光学信号的强度与所研究分析物的浓度成正比。该微流控生物分析平台与葡萄糖、总胆汁酸和乳酸脱氢酶 (LDH) 的商业“混合检测”分析试剂盒一起使用。在单独表征这些分析试剂盒后，我们证明了从少至 3 μL 的三种分析物的灵敏多重检测。事实上，该体积足以生成每个三种生化分析的多个重复液滴以及阳性对照液滴，从而确认了分析试剂的质量和阴性对照液滴有助于背景扣除。这种微流控生物分析平台的一个潜在应用涉及在器官芯片设备中采样细胞条件培养基。为了突出这一应用，肝细胞球状体在微流控设备中建立，通过暴露于脂毒性剂（棕榈酸）在芯片上受伤，然后连接到生物分析模块，以每日监测细胞毒性（LDH）、能量代谢（葡萄糖）和肝功能（总胆汁酸）的变化。微流控液滴内分析揭示了 LDH 水平的升高以及胆汁酸合成的减少-这些结果与肝损伤一致。重要的是，这些实验强调了这样一个事实，即液滴内分析足够灵敏，能够检测到在微流控设备中培养的相对较少（约 100 个）肝细胞球状体的功能输出变化。展望未来，我们预计将提高该技术的多重检测能力，并将该平台应用于其他需要从小样本量中检测多种分析物的生物/医学场景。
