基于量子点纳米珠的荧光侧向层析免疫测定
Summary
在这里，我们描述了一种制备量子点纳米珠 （QDNB） 和使用基于 QDNB 的侧流免疫测定条检测疾病生物标志物的方案。可以在紫外光照射下对测试结果进行定性评估，并使用荧光条读数器在 15 分钟内进行定量测量。
Abstract
量子点，也称为半导体纳米晶体，是用于生物成像和传感的新型荧光标记物。然而，通过费力的纯化程序制备的小尺寸 （~10 nm） 量子点抗体偶联物在使用侧向层析免疫测定试纸条检测某些痕量疾病标志物时表现出有限的灵敏度。在此，我们提出了一种使用一步乳液蒸发法制备量子点纳米珠 （QDNB） 的方法。使用制备的 QDNB，以 C 反应蛋白 （CRP） 为例，构建荧光侧流免疫测定法来检测疾病生物标志物。与单个量子点纳米颗粒不同，量子点纳米珠-抗体偶联物作为免疫测定标记物更敏感，因为通过将数百个量子点封装在一个聚合物复合纳米珠中来放大信号。此外，QDNB 的较大尺寸有助于在将 QDNB 与抗体偶联时更容易离心分离。制备基于 QDNBs 的荧光侧流免疫分析，并在 15 min 内测量样品中的 CRP 浓度。测试结果可以在紫外光照射下进行定性评估，并在 15 分钟内使用荧光读数仪进行定量测量。
Introduction
侧向层析免疫测定 （LFIA） 试纸条是床旁的重要快速检测工具 1,2，特别是在流行病期间的疾病筛查中。然而，传统的胶体金基 LFIA 试纸检测灵敏度低，仅提供定性结果3。为了提高 LFIA 的检测灵敏度，出现了各种新的纳米颗粒，包括彩色乳胶 4,5、上转换荧光纳米颗粒6、时间分辨荧光微球 7,8 和量子点 9,10,11。量子点 （QD）12,13，也称为半导体纳米晶体，具有可调谐的发射波长、宽激发范围和高发光效率，使其成为生物成像的理想标记物。
然而，单个量子点发射的荧光信号仍然很弱，导致免疫测定中的检测灵敏度相对较低。在微球中封装大量量子点可以放大信号并提高基于量子点的免疫测定的灵敏度。已采用各种方法，例如逐层自组装 14,15,16,17,18、溶胀法 19,20 和二氧化硅微球 21,22,23,24 封装，将量子点封装在微球内。例如，可以通过增加每个夹心免疫反应的 QD 负载来实现量子点功能化二氧化硅纳米球标记25。配备超声波雾化器的喷雾干燥器也用于制备纳米级 QD-BSA 纳米球26。然而，上述方法存在复杂的多步骤、荧光猝灭和低生产率的问题。
在我们之前的工作27 中，报道了一种将量子点封装在聚合物纳米珠内的乳液溶剂蒸发方法。这种制备技术简单，可保持 QD 的荧光效率，确保高包埋效率，并允许轻松进行放大生产。几个研究小组已经使用通过这种方法制备的 QDNB 成功开发了 LFIA 试纸条，用于食品毒素检测 28,29,30、传染病生物标志物检测31,32 和环境监测33。
该方案介绍了量子点纳米珠 （QDNB）、QDNB 和抗体偶联、基于 QDNB 的 LFIA 的制备以及人血浆样品中 C 反应蛋白 （CRP） 测量的具体制备步骤。
Protocol
该研究获得上海市皮肤病医院机构审查委员会 （No. 2020-15） 批准。所有涉及人类血液样本的实验程序均在生物安全 II 级实验室中进行。本研究中使用的试剂和设备的详细信息列在 材料表中。 1. QDs 纳米珠的制备 注：对于 QD 纳米珠合成，使用乳液溶剂蒸发技术合成 QD 纳米珠，油相与水相的比例为 1：5。 通过 超声处理生成?…
Representative Results
QDNB 制备过程如图 1A 所示。将含有 QD 和氯仿中聚合物的油相与水相混合，通过超声处理获得微型乳液。乳剂通过氯仿的逐渐蒸发而凝固。QDNB 的透射电子显微镜照片 （TEM） 如图 2A 所示。QDNB 具有球形形态，平均直径为 96 nm，在 TEM 图像中测量超过 50 个 QDNB。在纳米珠内清楚地观察到 QD 纳米颗粒，表明封装成功。 图 2B 和 <strong cla…
Discussion
在这里，我们描述了制备量子点纳米珠 （QDNB）27 的方案以及使用 QDNB 制备荧光侧流免疫测定 （LFIA）。证明了样品中 CRP 的定性和定量测量。这种基于 QDNB 的 LFIA 还可以应用于其他疾病生物标志物25,32、食物毒素29,30、病毒16,27 等。
<p class="jove_c…Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了上海市科学技术委员会项目 （STCSM） （22S31902000） 和上海市皮肤病医院临床研究孵化计划 （NO. lcfy2021-10） 的支持。
Materials
| (dimethylamino)propyl)-N’-ethylcarbodiimide hydrochloride | Sigma-Aldrich | 03450 | |
| Absorbance paper | Kinbio Biotech | CH37K | |
| Bovine serum albumin | Sigma-Aldrich | B2064 | |
| Casein | Sigma-Aldrich | C8654 | |
| CdSe/ZnS quantum dot | Suzhou Mesolight Inc. | CdSe/ZnS-625 | |
| Choloroform | Sino Pharm | 10006818 | |
| CRP antibody | Hytest Biotech | 4C28 | |
| Fluorescent lateral flow assay reader | Suzhou Helmence Precision Instrument | FIC-H1 | |
| Glass fiber pad | Kinbio Biotech | SB06 | |
| Goat anti-rabbit IgG | Sangon Biotech | D111018 | |
| Nitrocellulose membrane | Satorious | CN140 | |
| Poly(styrene-maleic anhydride) copolymer | Sigma-Aldrich | S458066 | |
| Rabbit IgG | Sangon Biotech | D110502 | |
| Sodium dodecyl sulfate | Sino Pharm | 30166428 | |
| Sodium hydroxide | Sino Pharm | 10019718 |
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Citar este artigo
Fan, L., Luo, Y., Yan, W., Han, H., Zhang, P. Fluorescent Lateral Flow Immunoassay Based on Quantum Dots Nanobeads. J. Vis. Exp. (208), e67000, doi:10.3791/67000 (2024).