基于金纳米棒光学免疫传感检测日本血吸虫循环抗原的研究

Circulating antigen of Schistosoma japonicum(SjCAg) are excreted by worm living in host. The existence of SjCAg can reflect active infection and worm burden. However，the lack of effective detection method, results the dilemma in identifying whether the schistosomiasis patient is cured. Application of basic research on new method with high sensitivity to detect SjCAg is direly needed. Recently, it appears that the detection sensitivity can be remarkably improved through our gold nanorods(GNRs) optical immune-sensing method. SjCAg can be raised by separation and purification followed by highly special antibody chosed as capture antibody. Gold nanorods(GNRs) were routinely acquired in seed-mediated growth method. The aspect ratios of GNRs can be adjusting with variational amount of AgNO3. The GNRs detection film is developed based on the immobilization of the capture antibody onto GNRs-adsorbed conducting the matrix with free hydroxy group. During the whole progress, Mercaptoundecanoic acid (MUA) facilitated is important and layer-by-layer assembly technology is applied. When the detection film was exposed in the solution with SjCAg, antigen-antibody binding occurred followed by the changes in the optical properties of the films, which were observed in UV-vis spectroscopy. A novel label-free optical immuno-sensing method based on gold nanorods for detecting of SjCAg can be set up,which is helpful to differentiate between the current infection and patient with shistosomiasis after therapy. And also it lays the foundation for monitoring the light infected patients with schistosomiasis in transmission controlled area.

国内外研究证明，血吸虫循环抗原可作为血吸虫病现症感染的重要标志。但由于缺少高灵敏检测循环抗原标志物的方法，导致了临床上血吸虫病人治愈与否难以鉴别。因此，开展高灵敏检测方法的应用基础研究是血吸虫病现症病人诊断急需解决的课题。在近年的研究中，我们发现采用基于金纳米棒光学免疫传感的方法可以显著提高检测的灵敏度。我们拟在此基础上，通过对血吸虫循环抗原的分离纯化，制备高特异性的捕获抗体，借助金纳米棒和静电沉积技术制备金纳米棒光学传感膜。采用化学修饰，分别将巯基与金、羧基与生物分子的氨基共价结合，实现抗体与金纳米棒的连接。循环抗原与抗体结合的金纳米棒因表面结构发生变化而导致光谱位移，借此显示抗原与抗体特异性的分子识别过程，从而创建基于金纳米棒的光学免疫传感检测日本血吸虫循环抗原的方法，以期满足血吸虫病现症感染鉴别诊断的需要，为血吸虫病疫情与传播控制地区低感染度人群传染源的监测奠定基础。

近年来，我国血吸虫病防治工作已经进入传播控制阶段，并将在2025年达到消除目标。目前，流行区域的血吸虫病感染率和感染强度逐渐降低，常规的血吸虫病检测方法因灵敏度的不足而导致时常出现漏检和假阳性的结果。因此，开展高灵敏度检测方法的研制以及寻找高特异性检测靶标成为我国血吸虫病防治工作的重点。国内外研究已证明，血吸虫循环抗原可作为血吸虫病现症感染的重要标志，但由于缺少高灵敏检测循环抗原标志物的方法，导致了临床上血吸虫病人治愈与否难以鉴别。.我们研究发现采用基于金纳米棒光学免疫传感的方法可以显著提高检测的灵敏度。通过制备血吸虫循环抗原的高特异性单链抗体，借助金纳米棒和静电沉积技术研制金纳米棒光学传感膜。采用化学修饰，分别将巯基与金、羧基与生物分子的氨基共价结合，实现抗体与金纳米棒的连接。循环抗原与抗体结合的金纳米棒因表面结构发生变化而导致光谱位移，借此显示抗原与抗体特异性的分子识别过程，从而建立了金纳米棒光学免疫传感检测日本血吸虫循环抗原的新方法。.采用种子生长法制备金纳米棒，获得长径比为3.2的金纳米棒。通过对传感基底材料的筛选，获得具有较好导电性能和吸附能力的ITO基底，并实现了与金纳米棒传感器的组装。为了检测日本血吸虫循环抗原，选用来源于血吸虫未成熟卵单链抗体库的未成熟卵单链抗体SIEA26-28kDa-SjscFc为捕捉抗体，利用静电吸附作用实现了单链抗体与金纳米棒光学免疫传感器的连接。选取IHA、ELISA法和金纳米棒光学免疫传感器对感染日本血吸虫兔血清和临床病人血清的检测，发现金纳米棒光学免疫传感器能够识别各个周期的日本血吸虫感染兔血清，而IHA和ELISA法无法实现更早期感染的检测。此外，确诊的血吸虫病病人血清也能够通过金纳米棒光学免疫传感器识别，获得了初步临床检测数据。.金纳米棒光学免疫传感器检测日本血吸虫循环抗原的方法为血吸虫病低感染度的检测提供了一个新的技术平台，也为低流行区血吸虫病感染的筛查和诊断提供快速便捷的检测工具。