A novel graphene-based label-free fluorescence 'turn-on' nanosensor for selective and sensitive detection of phosphorylated species in biological samples and living cells
A novel graphene-based label-free fluorescence 'turn-on' nanosensor for selective and sensitive detection of phosphorylated species in biological samples and living cells
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DOI：
10.1039/c5nr07261a
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发表时间：
2016-01-01
期刊：
影响因子：
6.7
通讯作者：
Ling, Yong-Chien
中科院分区：
文献类型：
--
作者：
Ke, Yaotang;Garg, Bhaskar;Ling, Yong-Chien
开发了一种新型的无标记荧光“开启”纳米传感器，用于高选择性和灵敏地检测生物样品和活细胞中的磷酸化物种(Ps)。该设计策略依赖于利用Ti4+固定的聚多巴胺(PDA)包覆的还原石墨烯氧化物(rGO@PDA-Ti4+)作为一个有吸引力的平台，通过磷酸基团与Ti4+之间的离子对相互作用来结合核黄素5‘-单磷酸分子(FMN)。所制备的rGO@PDA-T-I4+-FMN(纳米传感器)，由于FMN与rGO@Pda-Ti4+之间的Forster共振能量传递无效而产生微弱的荧光。实验结果表明，微波辅助纳米传感器与α、β-酪蛋白、卵清蛋白、人血清、脱脂牛奶、蛋清和活细胞(都含有Ps)相互作用，释放FMN(由于磷酸基团与Ti4+之间的高形成常数)，导致良好的荧光‘开启’响应。利用荧光光谱、共聚焦显微镜和MALDI-TOF MS光谱对Ps进行了定性和定量检测。在优化的条件下，该纳米传感器对α-、β-酪蛋白和卵清蛋白的检测下限分别为118.5、28.9和54.8nM。此外，还将标准加入法作为蛋清样品中磷肽定量的标准依据。我们推测，目前的Ps定量检测具有巨大的潜力，并可能为不久的将来的疾病诊断铺平道路。
A novel label-free fluorescence 'turn-on' nanosensor has been developed for highly selective and sensitive detection of phosphorylated species (Ps) in biological samples and living cells. The design strategy relies on the use of Ti4+-immobilized polydopamine (PDA) coated reduced graphene oxide (rGO@PDA-Ti4+) that serves as an attractive platform to bind riboflavin 5'-monophosphate molecules (FMNs) through ion-pair interactions between phosphate groups and Ti4+. The as-prepared rGO@PDA-T-i4+-FMNs (nanosensor), fluoresce only weakly due to the ineffective Forster resonance energy transfer between the FMNs and rGO@PDA-Ti4+. The experimental findings revealed that the microwave-assisted interaction of the nanosensor with alpha-, beta-casein, ovalbumin, human serum, non-fat milk, egg white, and living cells (all containing Ps) releases FMNs (due to the high formation constant between phosphate groups and Ti4+), leading to an excellent fluorescence 'turn-on' response. The fluorescence spectroscopy, confocal microscopy, and MALDI-TOF MS spectrometry were used to detect Ps both qualitatively and quantitatively. Under the optimized conditions, the nanosensor showed a detection limit of ca. 118.5, 28.9, and 54.8 nM for the tryptic digests of alpha-, beta-casein and ovalbumin, respectively. Furthermore, the standard addition method was used as a bench-mark proof for phosphopeptide quantification in egg white samples. We postulate that the present quantitative assay for Ps holds tremendous potential and may pave the way to disease diagnostics in the near future.