College of Veterinary Medicine, Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Joint International Research Laboratory of Agriculture and Agri-Product Safety, The Ministry of Education of China, Yangzhou University, Yangzhou 225009, China.
Future Industries Institute and ARC Centre of Excellence in Convergent Bio and Nano Science and Technology, University of South Australia, Mawson Lakes Campus, Mawson Lakes, South Australia 5095, Australia.
ACS Sens. 2020 Apr 24;5(4):1140-1148. doi: 10.1021/acssensors.0c00150. Epub 2020 Apr 1.
is a spherical zoonotic pathogen with a diameter of ∼200 nm, which can lead to a wide range of acute and chronic diseases in human body. Early and reliable on-site detection of is the key step to control the spread of the pathogen. However, the lack of a current technology with advantages of rapidity, ultrasensitivity, and convenience limits the implementation of traditional techniques for on-site detection of . Herein, we developed a naked-eye counting of based on the light scattering properties of gold nanoparticle (GNP) under dark-field microscopy (termed "GNP-labeled dark-field counting strategy"). The recognition of single by anti- antibodies-functionalized GNP probes with size of 15 nm leads to the formation of wreath-like structure due to the strong scattered light resulted from hundreds of GNP probes binding on one under dark-field microscopy. Hundreds of GNP probes can bind to the surface of due to the high stability and specificity of the nucleic acid immuno-GNP probes, which generates by the hybridization of DNA-modified GNP with DNA-functionalized antibodies. The limit of detection (LOD) of the GNP-labeled dark-field counting strategy for detection in spiked samples or real samples is down to four per microliter, which is about 4 times more sensitive than that of quantitative polymerase chain reaction (qPCR). Together with the advantages of the strong light scattering characteristic of aggregated GNPs under dark-field microscopy and the specific identification of functionalized GNP probes, we can detect in less than 30 min using a cheap and portable microscope even if the sample contains only a few targets of interest and other species at high concentration. The GNP-labeled dark-field counting strategy meets the demands of rapid detection, low cost, easy to operate, and on-site detection, which paves the way for early and on-site detection of infectious pathogens.
是一种直径约 200nm 的球形人畜共患病原体，可导致人体多种急性和慢性疾病。早期、可靠的现场检测是控制病原体传播的关键步骤。然而，目前缺乏一种具有快速、超灵敏和方便优势的技术，限制了传统现场检测技术的应用。在此，我们开发了一种基于金纳米粒子（GNP）在暗场显微镜下的光散射特性的裸眼计数方法（称为“GNP 标记暗场计数策略”）。通过尺寸为 15nm 的抗抗体功能化 GNP 探针对单个进行识别，由于数百个 GNP 探针在暗场显微镜下结合到一个上而导致强散射光的形成，导致出现环形结构。由于核酸免疫 GNP 探针具有高稳定性和特异性，数百个 GNP 探针可以结合到上，这些探针是由 DNA 修饰的 GNP 与 DNA 功能化抗体杂交生成的。该 GNP 标记暗场计数策略用于检测加标样品或实际样品中的检测限（LOD）低至 4 个/微升，比定量聚合酶链反应（qPCR）灵敏 4 倍。结合暗场显微镜下聚集 GNP 强光散射特性和功能化 GNP 探针的特异性识别优势，我们可以使用便宜且便携的显微镜在 30 分钟内检测到，即使样品中仅含有少量感兴趣的靶标和其他高浓度的物种。GNP 标记暗场计数策略满足快速检测、低成本、易于操作和现场检测的需求，为传染病原体的早期和现场检测铺平了道路。
