Enhanced colorimetric detection of norovirus using in-situ growth of Ag shell on Au NPs
Enhanced colorimetric detection of norovirus using in-situ growth of Ag shell on Au NPs
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DOI：
10.1016/j.bios.2018.10.067
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发表时间：
2019-02-01
影响因子：
12.6
通讯作者：
Park, Enoch Y.
中科院分区：
文献类型：
--
作者：
Khoris, Indra Memdi;Takemura, Kenshin;Park, Enoch Y.
诺如病毒（NoV）是急性胃肠炎的主要病因。NoV的低感染剂量和环境稳定性有利于其通过食物、水和人与人等多种方式有效传播。用于NoV检测的可用酶联免疫吸附测定（ELISA）由于所用过氧化物酶的低催化活性而具有低灵敏度，因此需要可靠的超灵敏生物测定。在这项研究中，我们将掺入银离子的金纳米颗粒（Au/Ag NPs）增强的过氧化物酶样活性应用于NoV检测的比色生物测定中。将抗NoV基因组II抗体固定在96孔微量滴定板的表面，并在抗NoV Ab、NoV和Ab-Au NP之间形成夹心结构。然后，加入含Ag离子的对苯二酚以形成Au/Ag核/壳NP。当H2 O2/3，3 '，5，5'-四甲基联苯胺（TMB）溶液加入到威尔斯孔中时，Ag离子从Au/Ag纳米颗粒表面释放出来，并增强了TMB的氧化。这些反应增强了TMB的氧化，并产生强烈的蓝色。与Au NPs相比，Au/Ag NPs显示出对H2 O2更高的亲和性和催化效率以及基于k（cat）的更高的催化速度，高达7倍。然后优化生物测定以使用NoV样颗粒（NoV-LP）检测临床分离的NoV。检测NoV-LP的检测限为10.8 pg/mL，与金免疫测定和基于辣根过氧化物酶的ELISA相比，灵敏度分别高出1000倍和100倍。检测到临床分离的NoV GII.4和NoV GII.3的病毒RNA拷贝数范围为10（2）-10（6）拷贝/mL粪便溶液，NoV GII.4的检测限为13.2拷贝/mL粪便溶液，相当于132拷贝病毒RNA/g粪便，表明灵敏度显著高于商业免疫测定试剂盒。该生物测定代表了适用于公共卫生早期诊断的低浓度NoV的可行检测测定。
Norovirus (NoV) is a leading cause of acute gastroenteritis. The low infectious dose and environmental stability of NoV facilitate its effective transmission through a variety of modes such as food, water and person-to-person. The available enzyme-linked immunosorbent assay (ELISA) for NoV detection has low sensitivity due to the low catalytic activity of the peroxidase used, and thus, a reliable ultrasensitive bioassay is needed. In this study, we apply the enhanced peroxidase-like activity of silver ion-incorporated gold nanoparticles (Au/Ag NPs) in a colorimetric bioassay for NoV detection. NoV was captured by anti-NoV genogroup II antibodies, which were immobilized on the surface of a 96-well microtiter plate and formed a sandwich structure among anti-NoV Ab, NoV and Ab-Au NP. Then, Ag ion-containing hydroquinone was added to form Au/Ag core/shell NPs. When H2O2/3,3',5,5'-tetramethylbenzidine (TMB) solution was added to the wells, Ag ions were liberated from the surface of Au/Ag NPs and enhanced the oxidation of TMB. These reactions enhanced the oxidation of TMB and developed an intense blue color. The Au/Ag NPs were shown to exhibit higher affinity and catalytic efficiency for H2O2 and higher catalytic velocity based on the k(cat), of up to 7-fold compared with Au NPs. The bioassay was then optimized to detect clinically isolated NoV using NoV-like particles (NoV-LPs). NoV-LPs were detected with a limit of detection of 10.8 pg/mL, corresponding to 1000- and 100-fold higher sensitivity compared to the gold-immunoassay and horseradish peroxidase-based ELISA, respectively. Clinically isolated NoV GII.4 and NoV GII.3 were detected in the range of 10(2)-10(6) copies of viral RNA/mL fecal solution with a detection limit of 13.2 copies/mL fecal solution for NoV GII.4, equivalent to 132 copies of viral RNA/g feces and indicating significantly higher sensitivity compared to commercial immunoassay kits., This bioassay represents a workable detection assay for low concentrations of NoV that is applicable for early-stage diagnosis for public hygiene.