蛋白分子粒径谱的多角度光纤动态光散射测量方法研究

Characterizing the particle size spectrum (PSS) of protein molecules has profound significance for monitoring the agglomerate state of blood protein molecules and accurate medical intervention. Among many currently available techniques of macromolecule PSS analysis, the dynamic light scattering performs outstanding in analyzing PSS of liquid biopsy, for its non-interfering, simplicity of operation and rapid analysis. However, the existing dynamic light scattering technique has poor accuracy in measuring the samples with high concentration and multimodal particle size distribution. This project proposed a dynamic light scattering method combining the microfluidic chip and fiber and built an inversion model for the multimodal PSS, to study the relationship between the protein macromolecule PSS and volume scattering function of protein molecules. Furthermore, the work of synchronous measurement of multiangle scattering light, coupling of weak optical signal by fiber, elimination of multiple scattering noise, the noise analysis of multiangle scattering light and inversion method of multimodal PSS have to be solved and much study have to be done. This method has excellent application scalability that it is not only appropriate for monitoring the aggregation process of the protein molecule, but also has well performance of PSS analysis for particles, whose diameters are in the nanometer to the micron level. Finally it has the perspective to achieve a PSS analysis technology for liquid biopsy and in situ measurement with non-interfering and small sensor size.

表征蛋白分子的粒径谱，对于监测血液蛋白分子聚集状态，进行准确的医学干预具有重要意义。在现有的粒径谱分析方法中，动态光散射技术对样品无干扰、操作简单、分析速度快，是实现液体活检粒径谱分析的最佳技术。然而现有的动态光散射技术在测量高浓度样品、多峰值分布粒径谱样品时，测量误差较大。本项目拟研究一种在多个角度测量蛋白分子群动态光散射的微流控光纤传感技术，并建立利用多角度动态散射光反演多峰分布粒径谱的数学模型，进而提出一种可直接检测高浓度样品、可准确分析多峰值蛋白分子粒径谱的多角度光纤动态光散射（MFDLS）测量方法。解决其中所涉及的微小散射体积控制、低噪声散射光的产生、利用光纤采集微弱散射光、多角度散射光的有效利用等问题。该方法不仅适用于蛋白分子粒径谱分析，也可拓展应用于其它领域中纳米-亚微米颗粒物的粒径谱分析，并最终有望于形成一种传感器尺寸小、对样品无干扰的液体活检、原位测量粒径谱分析技术。

在生物医学中，表征分子的粒径谱，对于监测血液蛋白分子聚集状态，进行准确的医学干预具有重要意义。在现有的粒径谱分析方法中，动态光散射技术对样品无干扰、操作简单、分析速度快，是实现液体活检粒径谱分析的最佳技术。然而现有的动态光散射技术在测量高浓度样品、多峰值分布粒径谱样品时，测量误差较大。为了解决这个问题，本课题结合微流控技术和光纤技术，实现了一种双角度动态光散射系统，提出了一种具有峰值识别和自适应选择功能的高精度多角度粒度分布反演算法。所设计和实现的微流控芯片中的微流道宽度仅为500μm，样品体积仅为30μl。通过超短的散射光程和超小的样品体积有效的降低多重散射现象的干扰。光纤与微流控芯片技术的结合使得整套系统便携、对实验环境要求比较简单。实验结果表明，借助于线性回归工具，该仪器可以对浓度在8~22mg/ml范围内的亚微米颗粒悬浮液实现高精度测量（相对误差约为10%）。此外，我们首次在基于微流控芯片的光纤DLS系统中实现了双角度在线测量。实验结果表明，与单角度DLS测量相比，双角度DLS测量具有更高的重复性。本项目对研究蛋白质聚集状态的表征进行了调研，以血红蛋白为应用实例，研究了血红蛋白与红细胞形态学的关系，建立了血红细胞特别是镰状血红细胞和聚集血红细胞的光散射模型。验证了本项目所建立的动态光散射技术粒径测量技术在血红细胞粒径测量的可行性。