纳米孔—液滴微流控联用方法在单分子检测和癌症诊断中的应用

Owing to ultra-high sensitivity and ultra-low detection limitation, label-free electrochemical nanopore sensing method has been a rapidly growing tool for single molecule analysis in applications such as biological detection and medical diagnostics. Although promising, nanopore sensing has drawback being the lack of molcule capture efficiency. This stems from the fact that nanopore can only sense the molecules which emerge several microns around the pore centre. In highly diluted solution, for example nM or pM, this results in long waing time for the detection. Recently, droplet based microfluidics has attracted substantial intrests in single molecule analysis. Droplet encapsulates and enriches molecules inside small volume, which significantly increases its concentration. However, this technology usually requires complex flow control to handle and label molecules, for example fluorescent or magnetic label. It even needs to stop the flow and detach the chip for the next step, leading to incomplete and discontinuous on-chip detection. Here, we report a nanopore integrated droplet-microfluidic method for single molecule detection. This method fully takes use of combined advantages of both droplet and nanopore by firstly confining the molecules near the pore centre using droplet to increase capture efficiency. Secondly, it avoids complex and time-consuming process exhausted in sample label and pretreatment. Last, the combined method has the potential to realize ultra-low conentration detection (fM), which provides a new pathway for rare cancer biomarker detection.

纳米孔技术是一种具有超高灵敏度、超低检测限和免荧光标记的单分子电化学检测方法，在生物检测和医学诊断领域有着独特的应用。但是纳米孔技术局限于其分子捕获率，因为纳米孔只能捕捉到孔周围几个微米范围内的分子，当溶液的浓度极低时（纳摩尔或皮摩尔量级），往往需要很长的检测时间。液滴微流控技术近年来在分子检测领域受到极大关注。液滴可以将分子包裹在有限的体积内，能有效提高待测分子的浓度，但是该技术通常需要复杂的流体控制对分子进行各种标记（如荧光标记、磁性标记等）和预处理，甚至需要中断流体流动，拆开芯片等，往往不能完整、连续地在一个芯片内实现分子检测全过程。本项目提出纳米孔—液滴微流控联用方法，通过微米大小的液滴将分子有效地限制在纳米孔的周围，提高分子捕获率，纳米孔技术则避免了繁琐的样品标记和预处理过程，能大大缩短检测时间，实现极低浓度分子的检测（费摩尔），为稀有的癌症生物标志物检测提供了新的思路。

纳米孔技术是一种具有超高灵敏度、超低检测限和免荧光标记的单分子电化学检测方法，在生物检测和医学诊断领域有着独特的应用。但是纳米孔技术局限于其分子捕获率，因为纳米孔只能捕捉到孔周围几个微米范围内的分子，当溶液的浓度极低时（纳摩尔或皮摩尔量级），往往需要很长的检测时间。液滴微流控技术近年来在分子检测领域受到极大关注。液滴可以将分子包裹在有限的体积内，能有效提高待测分子的浓度，但是该技术通常需要复杂的流体控制对分子进行各种标记（如荧光标记、磁性标记等）和预处理，甚至需要中断流体流动，拆开芯片等，往往不能完整、连续地在一个芯片内实现分子检测全过程。本项目提出纳米孔—液滴微流控联用方法，通过微米大小的液滴将分子有效地限制在纳米孔的周围，提高分子捕获率，纳米孔技术则避免了繁琐的样品标记和预处理过程，能大大缩短检测时间，实现极低浓度分子的检测（费摩尔），为稀有的癌症生物标志物检测提供了新的思路。创新性提出通过廉价易得的石英毛细管作为制备固态纳米孔的新材料，通过可在普通实验室装配的激光加热拉制技术作为新工艺，加工出孔径在20-60nm范围内的新型纳米孔结构，通过在纳米孔两端构建电化学检测系统获取电流信号来定量分析待测分子，通过对纳米孔内壁进行抗体修饰来定性的分析待测抗原分子，完成对pM至fM量级极低浓度分子的检测。