基于rGO和荧光探针的RNase A活性的灵敏检测和协同药物筛选,Analytical Chemistry

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基于rGO和荧光探针的RNase A活性的灵敏检测和协同药物筛选
Analytical Chemistry ( IF 6.7 ) Pub Date : 2018-02-02 00:00:00 , DOI: 10.1021/acs.analchem.7b04429
Chunyi Tong ₁ , Chuan Zhao ₁ , Bin Liu ₁ , Bin Li ₂ , Zhaoyang Ai ₃ , Jialong Fan ₁ , Wei Wang ₂

Affiliation

RNase A除了是酶学理论和实验研究的重要对象外，还通过调节各种生理或病理过程，包括细胞生长，增殖，分化和侵袭，在多种疾病的发展中也起着重要作用。因此，它可用作疾病治疗学的有用生物标记。在这里，通过将荧光底物与还原氧化石墨烯（rGO）结合，构建了一种简单，灵敏且低成本的RNase A检测方法。首先将检测限为0.05 ng / mL的方法用于RNase A靶向药物筛选，并鉴定出14种天然化合物作为该酶的激活剂。然后，将其用于检测药物治疗和乙型肝炎病毒（HBV）感染对RNase A活性的影响。结果表明，G-10和Chikusetsusaponin V以浓度依赖的方式上调了肿瘤细胞中RNase A的水平，而与对照组相比，HBV感染组中RNase A的平均水平受到了显着抑制。此外，使用该方法观察到重金属离子对RNase A的浓度依赖性抑制作用，结果表明Ba²⁺，Co 2 ⁺，Pb 2 ⁺，As ³⁺和Cu ²⁺抑制RNase A活性，IC ₅₀值分别为93.7μM（Ba 2 ⁺），90.9μM（Co 2 ⁺），110.6μM（Pb ²⁺），171.5μM（As ³⁺）和165.1μM（Cu ²⁺）。总而言之，考虑到快速性和高灵敏度的好处，该方法可用于生物样品中的RNase A分析以及体外和体内天然化合物的筛选。

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Sensitive Detection of RNase A Activity and Collaborative Drug Screening Based on rGO and Fluorescence Probe

In addition to being an important object in theoretical and experimental studies in enzymology, RNase A also plays an important role in the development of many kinds of diseases by regulating various physiological or pathological processes, including cell growth, proliferation, differentiation, and invasion. Thus, it can be used as a useful biomarker for disease theranostics. Here, a simple, sensitive, and low-cost assay for RNase A was constructed by combining a fluorogenic substrate with reduced graphene oxide (rGO). The method with detection limit of 0.05 ng/mL was first applied for RNase A targeted drug screening, and 14 natural compounds were identified as activators of this enzyme. Then, it was applied to detect the effect of drug treatment and Hepatitis B virus (HBV) infection on RNase A activity. The results indicated that RNase A level in tumor cells was upregulated by G-10 and Chikusetsusaponin V in a concentration-dependent manner, while the average level of RNase A in the HBV infection group was significantly inhibited compared with that in the control group. Furthermore, the concentration-dependent inhibitory effect of heavy metal ions on RNase A was observed using the method and the results indicated that Ba²⁺, Co²⁺, Pb²⁺, As³⁺, and Cu²⁺ inhibited RNase A activity with IC₅₀ values of 93.7 μM (Ba²⁺), 90.9 μM (Co²⁺), 110.6 μM (Pb²⁺), 171.5 μM (As³⁺), and 165.1 μM (Cu²⁺), respectively. In summary, considering the benefits of rapidity and high sensitivity, the method is practicable for RNase A assay in biosamples and natural compounds screening in vitro and in vivo.

更新日期：2018-02-02

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