Morpholine Derivative-Functionalized Carbon Dots-Based Fluorescent Probe for Highly Selective Lysosomal Imaging in Living Cells

Abstract

The development of a suitable fluorescent probe for the specific labeling and imaging of lysosomes through the direct visual fluorescent signal is extremely important for understanding the dysfunction of lysosomes, which might induce various pathologies, including neurodegenerative diseases, cancer, and Alzheimer's disease. Herein, a new carbon dot-based fluorescent probe (CDs-PEI-ML) was designed and synthesized for highly selective imaging of lysosomes in live cells. In this probe, PEI (polyethylenimine) is introduced to improve water solubility and provide abundant amine groups for the as-prepared CDs-PEI, and the morpholine group (ML) serves as a targeting unit for lysosomes. More importantly, passivation with PEI could dramatically increase the fluorescence quantum yield of CDs-PEI-ML as well as their stability in fluorescence emission under different excitation wavelength. Consequently, experimental data demonstrated that the target probe CDs-PEI-ML has low cytotoxicity and excellent photostability. Additionally, further live cell imaging experiment indicated that CDs-PEI-ML is a highly selective fluorescent probe for lysosomes. We speculate the mechanism for selective staining of lysosomes that CDs-PEI-ML was initially taken up by lysosomes through the endocytic pathway and then accumulated in acidic lysosomes. It is notable that there was less diffusion of CDs-PEI-ML into cytoplasm, which could be ascribed to the presence of lysosome target group morpholine on surface of CDs-PEI-ML. The blue emission wavelength combined with the high photo stability and ability of long-lasting cell imaging makes CDs-PEI-ML become an alternative fluorescent probe for multicolor labeling and long-term tracking of lysosomes in live cells and the potential application in super-resolution imaging. To best of our knowledge, there are still limited carbon dots-based fluorescent probes that have been studied for specific lysosomal imaging in live cells. The concept of surface functionality of carbon dots will also pave a new avenue for developing carbon dots-based fluorescent probes for subcellular labeling.

Fluorescent Probes for Super-Resolution Microscopy of Lysosomes

“…Hence, abnormalities and malfunctioning of these organelles might induce numerous pathologies. 2 Therefore, it becomes highly imperative to develop a technique that enables visualization and tracking of lysosomes and eventually would aid in understanding the lysosomal working principle. Additionally, recording the dynamics of lysosomal movement in real time would facilitate the in-depth understanding of the working principle of lysosomes and the kind of organelles it could interact with.…”

Fluorescent carbon dots as intracellular imaging probes

“…They usually have carbonaceous graphitic core of <10 nm with varying degrees of oxidation (Shi, Li, & Ma, ). These fluorescent carbon nanoparticles can be synthesized from various carbon precursors such as citric acid (He et al, ; Khan, Verma, Chethana, & Nandi, ; Lan et al, ; Liu, Tian, Tian, Wang, & Yang, ; Shangguan et al, ; Shi et al, ; Shu et al, ; Wu, Li, Ling, Huang, & Jia, ; Yang et al, ; Zhang et al, ), activated carbon powder (Chai et al, ), hyaluronic acid (Zhang et al, ), phenylene diamine derivative (Cheng et al, ; Hua, Bao, & Wu, ; Liu, Y., Duan, W., et al, 2017; Song, W., Duan, W., et al, 2017; Xia, Chen, Zou, Yu, & Wang, ), flour (Zhang et al, ), 2‐azidoimidazole (Tang, Lin, Li, & Hu, ), thiomalic acid (Safavi, Ahmadi, Mohammadpour, & Zhou, ), aminosalicylic acid (Song, Y., Zhu, C., et al, 2017), sodium alginate‐tryptophan (Zhu et al, ), neutral red‐triethyl amine (Jiao et al, ), capsicum (Chen et al, ), ethanol (Gao, Ding, Zhu, & Tian, ; Qu, Zhu, Shao, Shi, & Tian, ; Zhu, Qu, Shao, Kong, & Tian, ), (3‐aminopropyl) triethoxysilane (Zou et al, ), malic acid (Zhi et al, ), folic acid (Liu et al, ), and so on. Furthermore, various cost‐effective and simple procedures have been established for their preparation that include solvothermal treatment (Chen et al, ; Zhu et al, ), thermal degradation (Shi et al, ), high temperature reflux (Lan et al, ), microwave‐assisted synthesis (He et al, ; Tang et al, ), pyrolysis (Zhang et al, , ), and oxidative acid treatment (Safavi et al, ).…”

“…Carbon dot‐based imaging of intracellular organelles. (i) Carbon dot‐4‐(2‐aminoethyl) morpholine‐based lysosome imaging in HeLa cells (Reprinted with permission from Wu et al, ). Copyright © 2017, American Chemical Society.…”

Nanostructure and Corresponding Quenching Efficiency of Fluorescent DNA Probes

“…The field of fluorescent DNA probes improved considerably with the introduction of inorganic nanoparticles, such as quantum dots (QDs) [ 4 , 5 ] and gold (Au) nanoparticles (AuNPs) [ 6 , 7 , 8 ] as energy donors and acceptors [ 9 ], respectively. As a new kind of luminescent inorganic fluorophores, QDs are being widely used in chemical sensors [ 10 ], DNA detection [ 11 ], cell labeling [ 12 ], and imaging [ 13 ] because they have a broad and continuous excitation spectrum, a narrow size-tunable symmetric emission spectrum, and a high fluorescence quantum yield. AuNPs are widely used in gene delivery [ 14 ] and cell labeling [ 15 ] because they are bioinert, nontoxic, and readily synthesized and functionalized.…”