发表机构
TU Braunschweig; Helmholtz Centre for Infection Research（布伦瑞克工业大学; 亥姆霍兹感染研究中心）
机构由 AI 辅助整理，请以论文原文为准。AI 中文总结
本研究用scFv功能化80nm磁性纳米颗粒，结合磁性颗粒光谱检测SARS-CoV-2病毒样颗粒和N蛋白，检出限分别达490fM和1.7nM，分析了灵敏度与颗粒浓度的关系。
AI 中文摘要
磁性纳米颗粒（MNP）可通过功能化修饰抗体应用于磁性免疫分析（MIA）。在遵循“混合即测量”策略的均相分析中，只需加入待测样本，再用磁性测量方法评估MNP的响应即可。磁性颗粒光谱（MPS）是一种快速且灵敏的方法，可通过测量布朗弛豫的变化来确定结合状态。交流 susceptometry（ACS）也可用于进一步分析，但其耗时显著更长，因此不适用于即时检测应用。本研究旨在进一步优化由MPS评估的MIA方法。为此，我们在功能化过程中使用自制的单链可变片段（scFv）替代完整的免疫球蛋白G（IgG）抗体，以保持BNF-葡聚糖（Dextran）MNP的标称直径为80 nm，尽可能减小其尺寸，这使得MNP在结合分析物时能产生更大的相对尺寸变化。我们使用自制的病毒样颗粒（VLP）和SARS-CoV-2的N蛋白作为分析物。由于存在多个结合位点，这些分析物会形成聚类结构，对于VLP，我们通过ACS进行了更深入的研究，以评估其场依赖性。此外，我们还分析了检测灵敏度随MNP浓度的变化关系，并估算了两种分析物的检测限。我们发现，使用scFv功能化的MNP，能够检测到低至490 fM的SARS-CoV-2 VLP和1.7 nM的N蛋白。不过，灵敏度随MNP浓度降低而呈略超比例的提升，并不自动意味着能获得更好的检测限。
英文摘要
Magnetic nanoparticles (MNP) can be used in magnetic immunoassays (MIA) by functionalizing them with antibodies. In homogeneous assays that follow a "mix-and-measure" approach, it is then sufficient to add the sample in question and evaluate the response of the MNPs using a magnetic measurement method. Magnetic Particle Spectroscopy (MPS) is a fast and sensitive method for this purpose, capable of determining the binding state by measuring changes in Brownian relaxation. For further analysis, alternating current susceptometry (ACS) is considered, although it is significantly more time-consuming and therefore no option for applications interested in point of care detection. In this study, we aim to further improve the approach of MIA evaluated by MPS. To this end, we use self-synthesized scFv fragments instead of whole IgG antibodies during functionalization in order to keep the size of the BNF-Dextran MNP with 80 nm nominal diameter as small as possible, which allows for greater relative size changes upon binding to an analyte. Virus-like particles (VLP) and the N protein of SARS-CoV-2, which were also produced in-house, are used as analytes. Due to multiple binding sites, these form cluster structures, which, in the case of VLP, were investigated in greater depth ACS to assess their field dependence. In addition, the sensitivity of the assays was analyzed as a function of MNP concentration, and the detection limit for both analytes was estimated. We found that, using scFv-functionalized MNPs, we were able to detect low concentrations of just 490 fM of SARS-CoV-2 VLP and 1.7 nM of the N protein. However, the slightly above-proportional increase in sensitivity as the MNP concentration decreases does not automatically imply a better detection limit.
