Wang Yuling, Rauf Sakandar, Grewal Yadveer S, Spadafora Lauren J, Shiddiky Muhammad J A, Cangelosi Gerard A, Schlücker Sebastian, Trau Matt
Centre for Biomarker Research and Development, Australian Institute for Bioengineering and Nanotechnology (AIBN) and ‡School of Chemistry and Molecular Biosciences, The University of Queensland , Brisbane, Queensland 4072, Australia.
Anal Chem. 2014 Oct 7;86(19):9930-8. doi: 10.1021/ac5027012. Epub 2014 Sep 18.
Quantitative and accurate detection of multiple biomarkers would allow for the rapid diagnosis and treatment of diseases induced by pathogens. Monoclonal antibodies are standard affinity reagents applied for biomarkers detection; however, their production is expensive and labor-intensive. Herein, we report on newly developed nanoyeast single-chain variable fragments (NYscFv) as an attractive alternative to monoclonal antibodies, which offers the unique advantage of a cost-effective production, stability in solution, and target-specificity. By combination of surface-enhanced Raman scattering (SERS) microspectroscopy using glass-coated, highly purified SERS nanoparticle clusters as labels, with a microfluidic device comprising multiple channels, a robust platform for the sensitive duplex detection of pathogen antigens has been developed. Highly sensitive detection for individual Entamoeba histolytica antigen EHI_115350 (limit of detection = 1 pg/mL, corresponding to 58.8 fM) and EHI_182030 (10 pg/mL, corresponding 453 fM) with high specificity has been achieved, employing the newly developed corresponding NYscFv as probe in combination with SERS microspectroscopy at a single laser excitation wavelength. Our first report on SERS-based immunoassays using the novel NYscFv affinity reagent demonstrates the flexibility of NYscFv fragments as viable alternatives to monoclonal antibodies in a range of bioassay platforms and paves the way for further applications.
对多种生物标志物进行定量准确检测，将有助于对病原体引发的疾病进行快速诊断和治疗。单克隆抗体是用于生物标志物检测的标准亲和试剂；然而，其生产成本高昂且 labor-intensive。在此，我们报告新开发的纳米酵母单链可变片段（NYscFv），它是单克隆抗体的一种有吸引力的替代物，具有生产成本效益高、在溶液中稳定性好以及靶向特异性强等独特优势。通过将使用玻璃包覆、高度纯化的表面增强拉曼散射（SERS）纳米颗粒簇作为标记的SERS显微光谱与包含多个通道的微流控装置相结合，开发出了一种用于灵敏双工检测病原体抗原的强大平台。使用新开发的相应NYscFv作为探针，在单一激光激发波长下结合SERS显微光谱，已实现对单个溶组织内阿米巴抗原EHI_115350（检测限 = 1 pg/mL，相当于58.8 fM）和EHI_182030（10 pg/mL，相当于453 fM）的高灵敏度检测，且具有高特异性。我们首次报道使用新型NYscFv亲和试剂的基于SERS的免疫分析，证明了NYscFv片段在一系列生物分析平台中作为单克隆抗体可行替代物的灵活性，并为进一步应用铺平了道路。
