科研动态

Simoa技术:飞克级蛋白质高灵敏检测服务——华盈生物

2019/03/27

        蛋白质是生命功能的最终执行者,也是临床诊断、疾病分型、药物筛选等生物医学相关领域重要、应用广的检测对象。酶联免疫吸附实验(ELISA)技术一直以来在蛋白质检测中占据着主导地位。ELISA的灵敏度一般只能达到pg/mL水平,对于超低丰度蛋白的检测往往力不从心。然而,在疾病早期,精确检测这些超低丰度蛋白又对预判疾病的发生与转归有着重大意义,伴随着大健康产业和精准医学的发展,传统方法的技术瓶颈与巨大市场需求之间的矛盾日趋凸显。

        解决矛盾就意味着获得海量市场,各生物技术公司试图从各个角度入手改进现有的蛋白检测技术,以突破超低丰度蛋白质检测的瓶颈问题。它们普遍分为两个技术流派:

        1)以增强检测信号强度为出发点,结合电化学发光而开发的技术,如MSD。

        2)以降低背景噪音为出发点,结合毛细管电泳而开发的技术,如Ella和Erenna等。

        然而这些技术只能称为高灵敏,不能称为超灵敏,它们依然无法满足NfL、Tau等神经因子在外周血清中的检测,也无法在疾病初期近健康状态下检测IL-1β这些低丰度蛋白,应用范围仍然受到限制。

        Simoa(Single-molecule Array)技术灵敏度比ELISA高1000倍以上,它的出现将蛋白质检测技术直接带入到单分子、数字化检测时代,成为fg级超低丰度蛋白质检测领域的优势技术

 

Simoa是如何解决超低丰度蛋白检测问题的呢?

 

        Simoa核心技术环节有两个:
        1. 超低的反应体系,在提高灵敏度的同时,指数级降低背景噪音和信号扩散;

        2. 数字化检测设计与定量方法,实现单分子信号的独立识别与计算,不放过任何一个检测信号。


        Simoa检测的生物学原理仍然是经典的免疫反应-双抗夹心法,所不同的是,Simoa技术将约250,000个捕获抗体包被在2.7 μm的小磁珠上,检测时加入生物素标记的检测抗体及亲和素偶联的酶和底物,通过一层油将单个磁珠分别封闭在238,000个4.5 μm的反应孔(Well)中进行反应。由于每个小孔的反应体系仅仅为50飞升,比传统ELISA小20亿倍,这时小孔中即使只有一个分子,其催化底物就可产生3000个荧光分子,通过CCD摄像头即可捕获到信号,利用泊松分布理论可计算出阳性荧光小孔(On Well)对应的蛋白浓度值,实现数字化单分子检测的愿望。

 

对比.png

图1 Simoa与ELISA对比


        ELISA检测如同墨水滴入2000个鸟巢大小的泳池中,信号会无限地扩散和稀释,只有样品浓度达到一定阈值(比如pg/mL以上),才能被ELISA检测到,再根据所有信号的平均值来计算浓度。而Simoa的检测就好比将一滴墨水滴入矿泉水瓶中,很容易就被观察到。之后通过对238,000个孔中每一个阳性信号孔(On Well)独立纳入浓度计算公式,即Simoa是将前沿的“数字化”检测原理在蛋白检测中进行运用的技术

David Yeung [1]等研究人员对比了目前世界上几乎所有超敏蛋白质检测技术对于IL-2、IL-6、IL-17A、TNF-α等低丰度炎症因子的检测情况,发现Sioma技术无论在灵敏度还是在数据重现性上,相比其他技术均具有显著优势(见表1)。

表1 Simoa与其它高敏蛋白检测技术对比
表1.png

FEAD: Frequency of endogenous analyte detection

 图2.png

 

        综上,我们可以看出Simoa技术是低丰度蛋白检测领域真正的王者,它的检测能力超乎我们的想象,可以满足其它所有技术无法完成的检测需求。

 

        1. Simoa可以在血清/血浆中检测NfL、Tau、pTau、Aβ40、Aβ42等超低丰度神经因子;

        2. Simoa可以通过加大稀释倍数,检测房水、玻璃体、眼泪等微量样品中的炎症因子;

        3. Simoa可以在单个细胞中定量蛋白,实现单个胚胎细胞培养上清中的蛋白检测;

        4. Simoa可以在外泌体等稀少样品类型中检测PD1、PD-L1等蛋白;

        5. Simoa可以在阿尔兹海默症初期(提前16年),在接近正常人的患者血清中检测到蛋白标志物NfL[2]

 
        目前Simoa的商品化试剂盒已经广泛覆盖肿瘤、神经、心血管、免疫等,华盈生物可以为多种类型的样品(如:血清、血浆、脑脊液、细胞培养上清、呼出冷凝气、外泌体、房水、玻璃体、眼泪、汗液、尿液、唾液、微量细胞、单个囊胚培养基、血拓片、凝血块等)提供这些指标的检测服务。

检测目录
商业化试剂盒-01.png


Simoa实测灵敏度范围

检测下限.png



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