Yang Qin, Keerthisinghe Tharushi Prabha, Tan Tiffany Rou Jie, Cao Xiaoqiong, Setyawati Magdiel Inggrid, DeLoid Glen, Ng Kee Woei, Loo Say Chye Joachim, Demokritou Philip, Fang Mingliang
School of Civil and Environmental Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798, Singapore.
Nanyang Environment and Water Research Institute, Nanyang Technological University, 637141, Singapore.
Environ Sci Nano. 2020 Oct 1;7(10):3155-3166. doi: 10.1039/d0en00568a. Epub 2020 Aug 20.
Human are increasingly exposed to various types of engineered nanomaterials (ENMs) via dietary ingestion of nano-enabled food products, but these ENMs' impact on the gut bacteria health is still poorly understood. Current efforts in understanding the impact of these ENMs are hampered by their optical interferences in conventional quantification and viability assays, such as optical density and whole cell fluorescence staining assays. Therefore, there is a need to develop a more reliable bacteria quantification method in the presence of ENMs to effectively screen the potential adverse effects arising from the exposure of increasing ENMs on human gut microbiome. In this study, we developed a DNA-based quantification (DBQ) method in a 96-well plate format. Post-spiking method was used to correct the interference from ENMs on the reading. We showed the applicability of this method for several types of ENMs, i.e., cellulose nanofiber (CNF), graphene oxide (GO), silicon dioxide (SiO2), and chitosan, both in pure bacterial culture and human gut microbiome community. The detection limit for the highest dosing of CNF, GO, SiO2, and chitosan ENMs was approximately 0.18, 0.19, 0.05, and 0.24 as OD600, respectively. The method was also validated by a dose response experiment of with chitosan in the course of 8 hr. We believe that this method has great potential to be used in screening the effect of ENMs on the growth of gut bacteria or any other models and normalization for metabolites or proteins analysis.
人类通过食用含纳米材料的食品而越来越多地接触到各种类型的工程纳米材料（ENM），但这些ENM对肠道细菌健康的影响仍知之甚少。目前在理解这些ENM影响方面的努力受到它们在传统定量和活力测定（如光密度和全细胞荧光染色测定）中的光学干扰的阻碍。因此，需要开发一种在存在ENM的情况下更可靠的细菌定量方法，以有效筛选因接触越来越多的ENM而对人类肠道微生物群产生的潜在不利影响。在本研究中，我们开发了一种基于DNA的96孔板定量（DBQ）方法。采用加标后方法校正ENM对读数的干扰。我们展示了该方法对几种类型的ENM（即纤维素纳米纤维（CNF）、氧化石墨烯（GO）、二氧化硅（SiO2）和壳聚糖）在纯细菌培养和人类肠道微生物群中的适用性。CNF、GO、SiO2和壳聚糖ENM最高剂量的检测限分别约为0.18、0.19、0.05和0.24（OD600）。该方法还通过壳聚糖在8小时内的剂量反应实验进行了验证。我们相信，该方法在筛选ENM对肠道细菌生长或任何其他模型的影响以及代谢物或蛋白质分析的归一化方面具有巨大潜力。
