Department of Chemistry and Biochemistry, Florida International University , 11200 SW 8th Street, Miami, Florida 33199, United States.
ACS Appl Mater Interfaces. 2018 Jan 31;10(4):4233-4242. doi: 10.1021/acsami.7b16914. Epub 2018 Jan 18.
DNA-modified gold nanoparticles (AuNPs) are useful signal-reporters for detecting diverse molecules through various hybridization- and enzyme-based assays. However, their performance is heavily dependent on the probe DNA surface coverage, which can influence both target binding and enzymatic processing of the bound probes. Current methods used to adjust the surface coverage of DNA-modified AuNPs require the production of multiple batches of AuNPs under different conditions, which is costly and laborious. We here develop a single-step assay utilizing dithiothreitol (DTT) to fine-tune the surface coverage of DNA-modified AuNPs. DTT is superior to the commonly used surface diluent, mercaptohexanol, as it is less volatile, allowing for the rapid and reproducible controlling of surface coverage on AuNPs with only micromolar concentrations of DTT. Upon adsorption, DTT forms a dense monolayer on gold surfaces, which provides antifouling capabilities. Furthermore, surface-bound DTT adopts a cyclic conformation, which reorients DNA probes into an upright position and provides ample space to promote DNA hybridization, aptamer assembly, and nuclease digestion. We demonstrate the effects of surface coverage on AuNP-based sensors using DTT-regulated DNA-modified AuNPs. We then use these AuNPs to visually detect DNA and cocaine in colorimetric assays based on enzyme-mediated AuNP aggregation. We determine that DTT-regulated AuNPs with lower surface coverage achieve shorter reaction times and lower detection limits relative to those for assays using untreated AuNPs or DTT-regulated AuNPs with high surface coverage. Additionally, we demonstrate that our DTT-regulated AuNPs can perform cocaine detection in 50% urine without any significant matrix effects. We believe that DTT regulation of surface coverage can be broadly employed for optimizing DNA-modified AuNP performance for use in biosensors as well as drug delivery and therapeutic applications.
DNA 修饰的金纳米粒子（AuNPs）是通过各种杂交和基于酶的测定法检测多种分子的有用信号报告器。然而，它们的性能严重依赖于探针 DNA 的表面覆盖率，这会影响靶标结合和结合探针的酶处理。当前用于调整 DNA 修饰的 AuNPs 表面覆盖率的方法需要在不同条件下生产多批 AuNPs，这既昂贵又费力。我们在这里开发了一种利用二硫苏糖醇（DTT）来微调 DNA 修饰的 AuNPs 表面覆盖率的单步测定法。DTT 优于常用的表面稀释剂巯基己醇，因为它挥发性较低，仅需使用微摩尔浓度的 DTT 即可快速且可重复地控制 AuNPs 表面覆盖率。在吸附后，DTT 在金表面形成致密的单层，提供防污能力。此外，表面结合的 DTT 采用循环构象，将 DNA 探针定向为垂直位置，并提供充足的空间以促进 DNA 杂交、适体组装和核酸酶消化。我们使用 DTT 调节的 DNA 修饰的 AuNPs 来证明表面覆盖率对 AuNP 基传感器的影响。然后，我们使用这些 AuNPs 在基于酶介导的 AuNP 聚集的比色测定法中可视化检测 DNA 和可卡因。我们确定，与使用未经处理的 AuNPs 或表面覆盖率高的 DTT 调节的 AuNPs 的测定法相比，表面覆盖率较低的 DTT 调节的 AuNPs 实现了更短的反应时间和更低的检测限。此外，我们证明我们的 DTT 调节的 AuNPs 可以在没有任何显著基质效应的情况下在 50%尿液中进行可卡因检测。我们相信，DTT 对表面覆盖率的调节可以广泛用于优化 DNA 修饰的 AuNP 性能，以用于生物传感器以及药物输送和治疗应用。
