基于微纳界面浓度极化和分子穿孔效应的生物分析新方法研究

Low concentrated molecules, such as some protein and nucleic acid molecules, play essentials roles in a body. One typical example is micro-RAN. Even the powerful polymerase chain reaction amplification is difficult to apply due to the limited bases of the short single nucleotides. New analytical methods for obtaining information about these biomolecules are of great interest for the development of life science. The analysis of low concentration solute in a limited amount of sample itself is a great challenge in analytical chemistry. Chip analytical system is one of the technical platforms that could be used for the solution of the above challenge due to its potential advantages, such as small consumption of reagent and sample, speediness, portability and integration. Chip system is especially suitable for bioanalysis where the sample amount is limited, and the reagents are expensive. However, the feature of small sample consumption could also be a limitation in term of full use of the limited and rare samples. Meanwhile, the detection of low concentration solute is even tougher to the available detection methods. One solution is to drive the target molecules from the introduced limited sample into a given space online in the chip till the concentration of the target molecule increased above the detection limits. Electric filed induced stacking has shown its great pre-concentration powers. Concentration polarization occurs when an electric filed is applied across the charged nanopore(s). Million times concentration enhancement comparable to that of PCR amplification for DNA has been demonstrated with microfluidic chip system with a nanofluidic interface. Another important application of the interface is discriminating single molecular events by using interfaces with single nanopore of molecular size. Translocation of big molecules through such pores causes conductance changes, providing information about the concentration, size, or even molecular interactions such as hybridization, immunoassay etc. Chip analytical platform together with well introduced micro-nanofluidic interfaces exhibit unique and great potential to confront the above challenges. In this proposal, ion concentration polarization and translocation effect will be systematically investigated aiming to analysis of typical biomolecules. The contents include establishment of various types of nanofluidic interface with obvious concentration polarization and translocation effects, investigation of typical phenomena and process by numerical simulation and applications of the system by local reaction/detection at the interface and by online interfacing with electrophoresis separation and detection. This study will provide solid scientific and technical basis for the challenging problem in analytical chemistry, especially for the analysis of extremely low concentration biomolecules that are difficult to implement with conventional signal amplification methods.

从微量样品中获得低浓度生物分子信息对生命科学研究具有重要意义。芯片分析系统具有样品和试剂消耗少、速度快、便携性好等特点，特别适合生物分析的需要。然而，芯片分析存在样品利用低和对检测灵敏度要求苛刻的问题。如何在微尺度条件下提高现有检测手段的检出能力成为解决问题的关键。微纳界面上的浓度极化效应和和分子穿孔效应在如上问题的解决中表现出独特的优势和潜力。本项目拟基于微纳流控技术，研究在微分析平台上引入具有微纳效应界面的方法，研究典型生物分子在不同界面和介质条件下的传输行为和浓度极化规律，研究利用浓度极化效应和纳米孔界面分子穿孔效应进行超低浓度生物分子的浓集，反应及检测的方法，包括芯片上定位浓集，电泳分离和检测的方法，用建模和数值模拟的方法对典型现象及过程进行研究，通过对典型生物分子（蛋白，核酸）的浓集、分离和检测展示微纳流控效应在提高芯片分析系统检测性能中的作用。

微流控芯片分析系统的高通量、分析速度、节省试剂和样品的特点已经得到广泛认可，在复杂生物样品分析中有很大的应用潜力。然而系统尺度的减小和有限量的样品输入对现有检测方法提出了很大的挑战：超微量样品中可供检测的分子数极其有限，甚至少至单分子水平。提高微尺度下的检测灵敏度的方法很多，在微分析系统中表现为将有限量样品中的目标分子进行定位浓集。除了传统的样品浓缩方法，还可借助微纳界面上的离子浓度极化效应或者单个纳米孔上的分子计数的方法进行。本项目针对如上问题进行了深入研究。在自行建立的固态单个纳米孔界面和生物纳米孔传感界面上，以核酸为典型被测物，在单分子水平上对其传输及分子相互作用进行了检测，同时对单个纳米孔界面上的离子浓度极化现象进行了探讨。在微通道微纳界面研究的基础上还在纸基流体通道上借助离子交换膜建立了具有显著浓度极化效应的界面，对各种操作模式进行了探讨，展示了在尿液样品现场快速分析的应用潜力。借助数字仿真方法对电场驱动条件下微通道中的混合效应进行了探讨。研究还发现，在电场驱动下，还可利用动态中和反应在开放的纸基流体通道上建立与浓度极化效应类似的电场梯度和pH梯度界面，在此界面上不仅可以实现快速高效的电堆积，还可实现无胶和无两性电解质条件的快速等电聚焦分离。基于这种效应在纸基微流控系统上展示了高盐生物样品中的蛋白及环境污染离子的快速堆积和检测。在纸基流通通道上还展示了正负离子组分的同时点堆积。研究还展示，基于纸基微流控系统的快速电动堆积和分离还可能成为光谱及质谱等大型实验室分析的样品预处理方法，具有高效，价廉和快速的特点。该研究表明，基于电场驱动下的电动堆积效应的电堆积可有效提高微量样品中目标检测物的检测灵敏度。纸基微流控系统与传统微通道加工系统相比，具有成本低，容易加工和容易推广的特点。