基于适配体识别-刺激响应介孔材料信号放大的体内蛋白多肽类药物定量分析新方法的研究

Biopharmaceutical analysis has become an important part of the development and evaluation of protein and polypeptide drugs (PPD). LC-MS is the main method to determine drugs in vivo. However, the determination of PPD in biological samples by LC-MS directly exists some problems, such as difficulties in sample pretreatment process and low sensitivity. The methods currently used for determination of PPD in vivo,such as the immunosorbent assay methods also present some disadvantages, like narrow linear range, bad precision and low sensitivity. In view of the above problems, this project intends to combine “aptamer-recognition” and “signal transduction of stimulus-responsive mesopores materials” to develop a novel method for quantification of PPD. The aptamers on the surface of the “magnetic nanoparticles probes” and “stimulus-responsive Micro-/Nanocontainers probes” can recognize specifically the same PPD in the biological samples and form complexes. Then the complexes can be purified by magnetic field. Lastly, the pore-capping units should be unlocked with external triggers, allowing the controlled release of the signal molecules from the stimulus-responsive Micro-/Nanocontainers probes. The “signal molecules” can be detected instead of the targeted PPD, which can achieve the conversion of the target to detect and the transduction of signal simultaneously. Thus the method could achieve high sensitivity, as the PPDs can be enriched and extracted specifically from the biological samples and the detectable amounts of the targeted PPD would be amplified with more signal molecules. In addition, the “signal molecules” with fluorophore can also achieve the “visual” detection. This study will provide a novel method for the quantification of PPD in biological samples.

蛋白多肽类药物（PPD）的体内定量分析是其研发评价的重要环节。液质联用作为体内药物分析的主要方法，直接用于体内PPD分析时存在样品处理困难、灵敏度低等问题；常用的免疫学分析方法也存在线性范围窄、重现性差等缺陷。为解决上述问题，本项目提出基于“适配体识别”和“刺激响应介孔材料信号转导”的PPD定量分析新方法。即采用“磁响应探针”和“刺激响应微纳米囊探针”表面的适配体分别特异地识别生物样品中同一PPD，形成复合物，外加磁场使该复合物分离纯化，通过外界刺激触发闭孔剂使孔解锁，以释放微纳米囊中负载的“信号分子”代替目标PPD进行检测，完成检测目标转换和信号转导。该方法可以专属地分离PPD，同时对检测信号的数量和响应进行放大，从而实现生物样品中PPD的高灵敏度检测。此外，选择具有荧光响应的“信号分子”还可达到“可视化”检测的效果。本研究将为PPD体内定量测定及研发提供新方法和新思路。

与传统化学药物相比，蛋白多肽类药物给药剂量小、血药浓度低、结构特殊且不稳定，因而对检测方法的灵敏度和准确度要求较高。为确保药物的安全性和有效性，结合蛋白多肽类药物自身的特点，建立全面的蛋白多肽类药物分析方法和药代动力学评价体系具有非常重要的意义。目前蛋白多肽类药物采用的酶联免疫法等技术存在线性范围窄、测定误差大等缺陷。本项目针对上述多肽类药物体内分析遇到的困难和现存分析方法的局限性，探索了一种“基于适配体识别-刺激响应介孔材料信号放大的体内蛋白多肽类药物定量分析”的新方法，设计了显著增强质谱响应的标签分子，可大量修饰在纳米材料表面，实现信号转化及信号放大。以灵敏度、准确度、选择性、可靠性为考量，建立了几种效果显著且灵活多变、普适性好的信号转化及信号放大方法，实现了复杂生物基质中凝血酶的高灵敏度LC-MS检测，为蛋白多肽类大分子的检测提供了新的思路。顺利完成了项目计划书中的研究内容，成功解决了本项目提出的关键问题。同时，针对该项目所提出的“双适配体（Aptamers，Apt）识别-标签增敏”方法使用中的一些问题（例如：1）要求待检测的PPD需同时具有两个不同识别位点的适配体；2）Apt与靶标结合易受到缓冲溶液离子强度和pH的影响，因此适用范围非常有限；3）纳米粒探针装载的质谱标签分子数量不足等）进行了一些新的探索。