Gold Nanoparticle Interference Study during the Isolation, Quantification, Purity and Integrity Analysis of RNA

Investigations have been conducted regarding the interference of nanoparticles (NPs) with different toxicological assay systems, but there is a lack of validation when conducting routine tests for nucleic acid isolation, quantification, integrity, and purity analyses. The interference of citrate-capped gold nanoparticles (AuNPs) was investigated herein. The AuNPs were added to either BEAS-2B bronchial human cells for 24 h, the isolated pure RNA, or added during the isolation procedure, and the resultant interaction was assessed. Total RNA that was isolated from untreated BEAS-2B cells was spiked with various concentrations (v/v%) of AuNPs and quantified. A decrease in the absorbance spectrum (220–340 nm) was observed in a concentration-dependent manner. The 260 and 280 nm absorbance ratios that traditionally infer RNA purity were also altered. Electrophoresis was performed to determine RNA integrity, but could not differentiate between AuNP-exposed samples. However, the spiked post-isolation samples did produce differences in spectra (190–220 nm), where shifts were observed at a shorter wavelength. These shifts could be due to alterations to chromophores found in nucleic acids. The co-isolation samples, spiked with 100 µL AuNP during the isolation procedure, displayed a peak shift to a longer wavelength and were similar to the results obtained from a 24 h AuNP treatment, under non-cytotoxic test conditions. Moreover, hyperspectral imaging using CytoViva dark field microscopy did not detect AuNP spectral signatures in the RNA isolated from treated cells. However, despite the lack of AuNPs in the final RNA product, structural changes in RNA could still be observed between 190–220 nm. Consequently, full spectral analyses should replace the traditional ratios based on readings at 230, 260, and 280 nm. These are critical points of analyses, validation, and optimization for RNA-based techniques used to assess AuNPs effects.已有研究针对纳米颗粒（nanoparticles, NPs）对各类毒理学检测体系的干扰效应展开探索，但在开展核酸分离、定量、完整性及纯度分析的常规检测时，相关干扰的验证工作仍存在缺失。本研究针对柠檬酸包被金纳米颗粒（citrate-capped gold nanoparticles, AuNPs）的干扰效应展开探究：将AuNPs分别以三种方式引入体系——与人类支气管上皮细胞系BEAS-2B共培养24小时、加入已分离得到的纯RNA中，或在RNA分离过程中添加，随后对所产生的相互作用进行评估。研究人员将不同体积分数（v/v%）的AuNPs加入到从未经处理的BEAS-2B细胞中分离得到的总RNA样本中并进行定量检测，结果观察到220~340 nm波长范围内的吸光度光谱随AuNPs浓度升高呈现浓度依赖性降低，传统用于推断RNA纯度的260 nm与280 nm吸光度比值也发生了改变。通过电泳实验检测RNA完整性时，无法区分经AuNPs处理的样本与对照组；不过，分离后加入AuNPs的样本在190~220 nm波长范围内的光谱出现差异，表现为波长向短波段偏移，此类偏移可能源于核酸中所含发色团的结构改变。在非细胞毒性实验条件下，于RNA分离过程中加入100 μL AuNPs的共分离样本，其光谱峰向长波段偏移，该结果与经24小时AuNPs处理的细胞样本所得结果一致。此外，通过CytoViva暗场显微镜进行高光谱成像，未在经处理细胞分离得到的RNA中检测到AuNPs的光谱特征信号；尽管最终RNA产物中未检测到AuNPs，但在190~220 nm波长范围内仍可观察到RNA的结构变化。因此，完整光谱分析应当取代传统的基于230 nm、260 nm及280 nm吸光度读数的比值分析法。对于用于评估AuNPs生物效应的RNA相关技术而言，上述发现为其分析、验证及优化提供了关键参考依据。