Department of Materials Science and Engineering , Rutgers University , 607 Taylor Road , Piscataway , New Jersey 08854 , United States.
Department of Chemistry and Chemical Biology , Rutgers University , 123 Bevier Road , Piscataway , New Jersey 08854 , United States.
Bioconjug Chem. 2019 Oct 16;30(10):2555-2562. doi: 10.1021/acs.bioconjchem.9b00495. Epub 2019 Sep 13.
Selective detection and precise quantification of biomolecules in intracellular settings play a pivotal role in the diagnostics and therapeutics of diseases, including various cancers and infectious epidemics. Because of this clinical relevance, nanoprobes with high sensitivity, wide tunability, and excellent biological stability have become of high demand. In particular, nanoflares based on gold nanoparticles have emerged as an attractive candidate for intracellular detection due to their efficient cellular uptake, enhanced binding affinity with complementary targets, and improved biological compatibility. However, nanoprobes, including these nanoflares, are known to be susceptible to the adsorption of proteins present in the biological environment, which leads to the formation of a so-called protein corona layer on their surface, leading to an altered targeting efficiency and cellular uptake. In this work, we leverage the nanoflares platform to demonstrate the effect of protein corona on biomolecular detection, quantification, as well as biological stability against enzymatic degradation. Nanoflares incubated in a biologically relevant concentration of serum albumin proteins (0.50 wt %) were shown to result in more than 20% signal reduction in target detection, with a decrease varying proportionally with the protein concentrations. In addition, similar signal reduction was observed for different serum proteins, and PEG backfilling was found to be ineffective in mitigating the negative impact induced by the corona formation. Furthermore, nuclease resistance in nanoflares was also severely compromised by the presence of the corona shell (∼2-fold increase in hydrolysis activity). This work demonstrates the consequences of an in situ formed protein corona layer on molecular detection/quantification and biological stability of nanoflares in the presence of nuclease enzymes, highlighting the importance of calibrating similar nanoprobes in proper biological media to improve the accuracy of molecular detection and quantification.
在疾病的诊断和治疗中，包括各种癌症和传染病在内，细胞内生物分子的选择性检测和精确定量起着关键作用。由于这种临床相关性，对具有高灵敏度、宽可调性和优异生物稳定性的纳米探针的需求也变得很高。特别是基于金纳米粒子的纳米耀斑由于其高效的细胞摄取、增强与互补靶标的结合亲和力以及提高的生物相容性，已成为细胞内检测的有吸引力的候选物。然而，众所周知，包括这些纳米耀斑在内的纳米探针容易受到生物环境中存在的蛋白质的吸附，这导致在其表面形成所谓的蛋白质冠层，从而改变靶向效率和细胞摄取。在这项工作中，我们利用纳米耀斑平台来证明蛋白质冠对生物分子检测、定量以及对酶降解的生物稳定性的影响。在具有生物相关性的血清白蛋白浓度（0.50wt%）下孵育的纳米耀斑导致靶标检测的信号降低超过 20%，并且信号降低与蛋白质浓度成比例变化。此外，还观察到不同血清蛋白的类似信号降低，并且发现 PEG 回填在减轻冠形成引起的负面影响方面无效。此外，纳米耀斑的核酸酶抗性也因冠层壳的存在而严重受损（水解活性增加约 2 倍）。这项工作证明了在存在核酸酶的情况下，原位形成的蛋白质冠层对纳米耀斑的分子检测/定量和生物稳定性的影响，突出了在适当的生物介质中校准类似纳米探针以提高分子检测和定量准确性的重要性。
