摘要
超顺磁性的复合粒子在生物医学中的应用日趋深入、成熟。但在免疫检测中的应用,目前只集中在利用磁性复合粒子的磁性分离、富集、浓缩的功能上。而其本身具备的具有穿透性、高灵敏度的磁信号,却没有在生物检测中得到广泛深入的应用。原因之一就是现有的合成方式所制备的磁性复合粒子在尺寸大小、生物相容性、饱和磁化强度、胶体稳定性和标记效率等方面没有办法很好的兼顾,而且,作为较新的标记材料,在与原有的反应体系复合的时候需要进行相应的调整,才能形成有效的技术路线。
本文通过制备新型Fe3O4/poly(St-co-MPS)/SiO2复合粒子,提高了粒子的稳定性,得到了尺寸合适、高磁化强度、高稳定性的复合粒子;随后,根据免疫检测的特点,对其表面进行了可控数量的羧基修饰,提高了生物相容性,通过系统的比对工作,确定了抗体偶联的调整条件,并针对免疫检测的两大类型:“夹心法”和“竞争抑制法”,进行了整体的匹配调整,改进了增速剂、封闭剂等条件,确定了针对不同样本(血液和尿液)检测的解决方案。
最终制备的新型磁性复合粒子应用在对乙肝表面抗原和盐酸克伦特罗(瘦肉精)的检测中,达到了快速、高灵敏度的检测效果。并在国家指定的验证机构进行了严格的比对实验,分别达到了现有两个检测项目ELISA方法的灵敏度,而检测时间缩短至20分钟。从而确认了粒子制备和整个反应体系的有效性。
总的来看,由此得到的技术路线,将快速免疫检测的灵敏度和可靠性大幅提高,也为目前这一领域所面临的技术瓶颈问题找到了解决办法,为其他纳米材料在生物检测方面的应用和相关基础研究提供了参考。
With the development of nanotechnology and biotechnology, many novel nano- materials are playing important roles in bio-assays as new labels. Because of their special characters, superparamagnetic composite particles are more and more widely used in immunoassays. However, most of these applications focus in separation, concentration and purification of analytes in specimens, while their penetrating and high-sensitive magnetic signal is seldom involved.
As a rapid and simple detection method, the technology of lateral flow immunoassays (LFIAs) has gained wide acceptance in a variety of fields since its initial development in the 1990s. But because of the limits of the signal particles used in it, LFIAs can not be performed as a high sensitive assay. Thus, it limits the development of LFIAs in the past ten years. Recently, superparamagnetic LFIAs have suggested us a new way to approach more sensitive immunoassays. The signal can be detected“in”nitrocellulose membrane, not like optical absorbance labels, can just be detected“on”the membrane, because of the penetrability of magnetism. It can reduce the influence by membrane surface nonuniformity, and makes the assay more sensitive and reliable. Moreover, magnetism signal itself can’t be interfered by the color of test specimens, for example, whole blood and urine. Thus, some routine sample treatment courses, such as centrifugation of blood, extraction of urine, etc. can be avoided. And the test procedure is simplified.
However, when we follow the routine procedures to prepare superparamagnetic composite particles, there are always some difficulties to give simultaneous considerations for the sizes, magnetism, stability, biocompatibility and labeling efficiency of the particles. These problems limit their applications on LFIAs. Furthermore, as a new signal labeling system, it has to be optimized thoroughly.
Our studies took a different route to synthesize superparamagnetic composite particles to make them more suitable for LFIAs. First, Fe3O4/poly(St-co-MPS) composite particles were prepared by miniemulsion copolymerization of 3-trimethoxysilylpropylmeth- acrylate (MPS) and styrene(St). Subsequent growth of SiO2 layer by the conventional St?ber method allows the preparation of core-Shell superparamagnetic Fe3O4/poly(St-co-MPS)/SiO2 composite particles with controllable thickness of SiO2 shell. And then we made them carboxyl-functionalized on the surface with defined carboxyl density (MPs). The particles were highly magnetizable. And their saturation magnetization was as high as 45 emu/g at room temperature. Generally, they are sized-controlled, and has better biological compatibility, higher magnetism saturation, better condition tolerance, and more suitable for high sensitive magnetic LFIAs. The test signal can be read by superparamagnetic resonance reader (MAR, Quantum Design). Almost 100% of labeled analytes can be quantified through the whole thickness of membrane that makes the assay much more sensitive. Then, contents-controlled carboxyl groups were synthesized on particle surface. Finally, two surface-functionalized particles with high magnetism saturation and different carboxyl contents were achieved to study the improvement of labeling efficiency and detection sensitivity in next step.
In chapter 3, we take human chorionic gonadotropin (hCG) detection as a model to study the application of magnetic composite particles we made and the optimization of the new system. The proper reaction time was determined as 20 minutes. Secondly, we regulated the surfactant and its working concentration for the system; Thirdly, the comparison on labeling efficiency and influence on sensitivity between two surface-functionalized particles with different carboxyl contents was done. The proper carboxyl content was determined. At last, the stability of the system was demonstrated by“aging process”.
In chapter 4, we take human hepatitis surface antigen (HBsAg) detection as a sample to evaluate our novel particles for“sandwich”magnetic LFIAs. PreS1 antigen was introduced into the preparation of monoclonal antibody against HBsAg to improve the specificity of the assay. Secondly, we added EDTA into the system to reduce the non-specific reaction during the assay. Finally we prepared our new magnetic LFIAs strips for HBsAg. And we assessed them by national standard specimens, the sensitivity was equal to ELISA kit, but with shortened test time of 20 minutes. Then, we performed clinical assessment at officially certificated sites, Beijing 302 hospital and Beijing Ditan hospital. Totally 849 serum samples were tested by our strips, and simultaneously compared with ELISA kits from Abbott, US and Kehua, China. Eventually, our strips reached the sensitivity of 100%, specificity of 99.2% and 99.9% of accuracy. These results are much better than other rapid LFIAs.
In chapter 5, we take Clentuterol detection as a sample to develop“competitive inhibition”magnetic LFIAs strips by our novel particles. First, we synthesized BSA-clenbuterol antigen to improve the specificity of the assay. Second, we used new blockers to reduce the interference of related compounds in urine specimens. And the stability of the system was enhanced. For the final magnetic LFIAs strips for clenbuterol we made after the optimizations, we tested them by national standard sample. The sensitivity was 0.5g/L, equal to ELISA kit, but with shortened test time of 20 minutes. The assessment on testing swine urine was performed at Shenzhen Entry-Exit Inspection and Quarantine Bureau. Totally 107 samples were tested, and compared with colloidal gold LFIAs strips from Zhongde Biotech. Randox ELISA kit was taken as the judgment . The sensitivity of our strips was 100%, better than colloidal gold LFIAs system.
After all the works we did, the novel superparamagnetic composite particles we made were demonstrated as a suitable material for magnetic LFIAs, both in“sandwich”and“competitive inhibition”types. And all the related optimizations can also be a useful reference for the development of other magnetic LFIAs. Moreover, some results and phenomenon we got in the study are good points for theoretical researches in the relationship between nano-materials and bio-molecules.
引文
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