Laboratory for Life Sciences and Technology (LiST), Danube Private University, Viktor-Kaplan-Straße 2, 2700 Wiener, Neustadt, Austria.
Institute of Macromolecular Chemistry, Czech Academy of Sciences, Heyrovského nám. 2, Prague 162 00, Czech Republic.
ACS Appl Mater Interfaces. 2024 Apr 10;16(14):17109-17119. doi: 10.1021/acsami.3c18304. Epub 2024 Mar 26.
The analysis of low-abundance protein molecules in human serum is reported based on counting of the individual affinity-captured analyte on a solid sensor surface, yielding a readout format similar to digital assays. In this approach, a sandwich immunoassay with rolling circle amplification (RCA) is used for single molecule detection (SMD) through associating the target analyte with spatially distinct bright spots observed by fluorescence microscopy. The unspecific interaction of the target analyte and other immunoassay constituents with the sensor surface is of particular interest in this work, as it ultimately limits the performance of this assay. It is minimized by the design of the respective biointerface and thiol self-assembled monolayer with oligoethylene (OEG) head groups, and a poly[oligo(ethylene glycol) methacrylate] (pHOEGMA) antifouling polymer brush was used for the immobilization of the capture antibody (cAb) on the sensor surface. The assay relying on fluorescent postlabeling of long single-stranded DNA that are grafted from the detection antibody (dAb) by RCA was established with the help of combined surface plasmon resonance and surface plasmon-enhanced fluorescence monitoring of reaction kinetics. These techniques were employed for in situ measurements of conjugating of cAb to the sensor surface, tagging of short single-stranded DNA to dAb, affinity capture of the target analyte from the analyzed liquid sample, and the fluorescence readout of the RCA product. Through mitigation of adsorption of nontarget molecules on the sensor surface by tailoring of the antifouling biointerface, optimizing conjugation chemistry, and by implementing weak Coulombic repelling between dAb and the sensor surface, the limit of detection (LOD) of the assay was substantially improved. For the chosen interleukin-6 biomarker, SMD assay with LOD at a concentration of 4.3 fM was achieved for model (spiked) samples, and validation of the ability of detection of standard human serum samples is demonstrated.
本文报道了一种基于在固体传感器表面上对单个亲和捕获分析物进行计数的方法，对人血清中低丰度蛋白质分子进行分析，该方法提供了类似于数字分析的读取格式。在这种方法中，通过将目标分析物与荧光显微镜观察到的空间上不同的亮斑相关联，使用带有滚环扩增（RCA）的三明治免疫测定法进行单分子检测（SMD）。在这项工作中，目标分析物与其他免疫测定成分与传感器表面的非特异性相互作用特别感兴趣，因为它最终限制了该测定的性能。通过设计相应的生物界面和带有聚乙二醇（OEG）头基的硫醇自组装单层，可以最小化这种相互作用，并且使用聚[聚（乙二醇）甲基丙烯酸酯]（pHOEGMA）抗污聚合物刷将捕获抗体（cAb）固定在传感器表面上。该测定法依赖于通过 RCA 从检测抗体（dAb）接枝的长单链 DNA 的荧光后标记，并借助表面等离子体共振和表面等离子体增强荧光监测反应动力学建立。这些技术用于原位测量 cAb 与传感器表面的结合、短单链 DNA 与 dAb 的标记、目标分析物从分析液体样品中的亲和捕获以及 RCA 产物的荧光读出。通过调整抗污生物界面、优化缀合化学和在 dAb 和传感器表面之间引入弱库仑排斥，减少了传感器表面上非目标分子的吸附，从而大大提高了测定的检测限（LOD）。对于所选的白细胞介素-6 生物标志物，对于模型（加标）样品，SMD 测定的检测限达到 4.3 fM，并且证明了检测标准人血清样品的能力。
