Department of Chemistry, Virginia Commonwealth University, Richmond, Virginia 23284, United States.
Department of Chemical Engineering, University of Tennessee, Chattanooga, Tennessee 37403, United States.
Anal Chem. 2023 Jul 4;95(26):9839-9846. doi: 10.1021/acs.analchem.3c00717. Epub 2023 Jun 16.
Iron-regulated surface determinant protein A (IsdA) is a key surface protein found in the foodborne bacteria─ ()─which is known to be critical for bacterial survival and colonization. is pathogenic and has been linked to foodborne diseases; thus, early detection is critical to prevent diseases caused by this bacterium. Despite IsdA being a specific marker for and several detection methods have been developed for sensitive detection of this bacteria such as cell culture, nucleic acids amplification, and other colorimetric and electrochemical methods, the detection of through IsdA is underdeveloped. Here, by combining computational generation of target-guided aptamers and fluorescence resonance energy transfer (FRET)-based single-molecule analysis, we presented a widely applicable and robust detection method for IsdA. Three different RNA aptamers specific to the IsdA protein were identified and their ability to switch a FRET construct to a high-FRET state in the presence of protein was verified. The presented approach demonstrated the detection of IsdA down to picomolar levels (×10 M, equivalent to ∼1.1 femtomoles IsdA) with a dynamic range extending to ∼40 nM. The FRET-based single-molecule technique that we reported here is capable of detecting the foodborne pathogen protein IsdA with high sensitivity and specificity and has a broader application in the food industry and aptamer-based sensing field by enabling quantitative detection of a wide range of pathogen proteins.
铁调节表面决定蛋白 A（IsdA）是一种关键的表面蛋白，存在于食源性病原体——（）中，这种蛋白对于细菌的生存和定植至关重要。（）是一种致病细菌，与食源性疾病有关；因此，早期检测对于预防由这种细菌引起的疾病至关重要。尽管 IsdA 是（）的特异性标志物，并且已经开发了几种用于该细菌的灵敏检测的方法，如细胞培养、核酸扩增以及其他比色法和电化学方法，但通过 IsdA 检测（）的方法尚未得到充分发展。在这里，通过结合靶向引导适体的计算生成和基于荧光共振能量转移（FRET）的单分子分析，我们提出了一种广泛适用且强大的 IsdA 检测方法。鉴定了三个针对 IsdA 蛋白的不同 RNA 适体，并验证了它们在存在蛋白质的情况下将 FRET 构建体切换到高 FRET 状态的能力。所提出的方法能够以皮摩尔级别的灵敏度（×10 M，相当于约 1.1 飞摩尔 IsdA）检测到 IsdA，动态范围扩展至约 40 nM。我们报道的基于 FRET 的单分子技术能够以高灵敏度和特异性检测食源性病原体蛋白 IsdA，并且通过能够定量检测广泛的病原体蛋白，在食品工业和适体传感领域具有更广泛的应用。
