Enhancing Protein Capture Using a Combination of Nanoyeast Single-Chain Fragment Affinity Reagents and Alternating Current Electrohydrodynamic Forces.
Enhancing Protein Capture Using a Combination of Nanoyeast Single-Chain Fragment Affinity Reagents and Alternating Current Electrohydrodynamic Forces.
复制标题
结合使用纳米酵母单链片段亲和试剂和交流电流体动力来增强蛋白质捕获。
DOI：
10.1021/acs.analchem.5b02490
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发表时间：
2015
影响因子：
7.4
通讯作者：
Trau,Matt
中科院分区：
文献类型：
--
作者：
Vaidyanathan,Ramanathan;Rauf,Sakandar;Grewal,YadveerS;Spadafora,LaurenJ;Shiddiky,MuhammadJA;Cangelosi,GerardA;Trau,Matt
新的高性能检测技术和更强大的蛋白质捕获剂可以结合起来，快速和特异性地捕获和检测复杂生物样品中与疾病相关的蛋白质生物标志物。在这里，我们展示了使用最近开发的重组亲和试剂，即纳米酵母-scFv，与交流电流体动力学（ac-EHD）诱导的剪切力相结合，以提高蛋白质生物标志物分析过程中的捕获性能。ac-EHD的使用显著改善了穿过捕获域的流体运输，从而增强了传感器-靶相互作用，并同时从电极表面置换非特异性分子。我们证明了这种简单的概念验证方法，用于捕获和检测ofEntamoeba histolytica抗原从消毒粪便，在5分钟的跨度内使用ac-EHD微流体装置。在ac-EHD场下，将抗原捕获在纳米酵母-scFv固定化装置上，随后使用量子点缀合的抗体进行检测。该免疫传感器在消毒粪便中特异性检测抗原，具有低背景噪声，浓度低至58.8 fM，批间重现性（%RSD，n = 3）<17.2%，在缓冲液中检测抗原，浓度低至5.88 fM，批间重现性（% RSD，n= 3）为8.4%。此外，使用该免疫传感器的抗原检测比先前在缓冲液中采用表面增强拉曼散射（Sers）检测的微流体装置中用相同的纳米酵母-scFv试剂获得的灵敏度高10倍，并且比在消毒粪便中使用丝网印刷金电极的方法灵敏至少200倍。我们预测，使用这些稳定的亲和试剂的这种快速和灵敏的方法可能提供一种新的方法来检测生物基质中的蛋白质疾病生物标志物。
New high-performance detection technologies and more robust protein capture agents can be combined to both rapidly and specifically capture and detect protein biomarkers associated with disease in complex biological samples. Here we demonstrate the use of recently developed recombinant affinity reagents, namely nanoyeast-scFv, in combination with alternating current electrohydrodynamic (ac-EHD)-induced shear forces, to enhance capture performance during protein biomarker analysis. The use of ac-EHD significantly improves fluid transport across the capture domain, resulting in enhanced sensor-target interaction and simultaneous displacement of nonspecific molecules from the electrode surface. We demonstrate this simple proof-of-concept approach for the capture and detection ofEntamoeba histolyticaantigens from disinfected stool, within a span of 5 min using an ac-EHD microfluidic device. Under an ac-EHD field, antigens were captured on a nanoyeast-scFv immobilized device and subsequently detected using a quantum dot conjugated antibody. This immunosensor specifically detected antigen in disinfected stool with low background noise at concentrations down to 58.8 fM with an interassay reproducibility (%RSD ofn= 3) < 17.2%, and in buffer down to 5.88 fM with an interassay reproducibility (% RSD,n= 3) of 8.4%. Furthermore, antigen detection using this immunosensor was 10 times more sensitive than previously obtained with the same nanoyeast-scFv reagents in a microfluidic device employing surface-enhanced Raman scattering (SERS) detection in buffer and at least 200 times more sensitive than methods using screen printed gold electrodes in disinfected stool. We predict this rapid and sensitive approach using these stable affinity reagents may offer a new methodology to detect protein disease biomarkers from biological matrices.