Limin Yang

Real-Time Imaging of Mitochondrial Hydrogen Peroxide and pH Fluctuations in Living Cells Using a Fluorescent Nanosensor

Mitochondrial reactive oxygen species (ROS) and pH fluctuations are closely correlated with mitochondrial dysfunctions, which are implicated in various human diseases including neurodegenerative disorders and cancers. Simultaneously monitoring the changes of ROS and pH of mitochondria remains a major challenge in the mitochondrial biology. In this study, we develop a novel mitochondria-targeted fluorescent nanosensor for real-time imaging of the fluctuations of hydrogen peroxide (H2O2) and pH in living cells. The fluorescence probes for detecting pH and H2O2 were loaded in the small-sized mesoporous silica nanoparticles (MSN). Then the polyethylenimine was attached to cap the pores of MSN, the triphenylphosphonium was further modified to target mitochondria in living cells. Confocal fluorescence imaging indicated that the nanosensor could effectively target mitochondria and successfully achieved real-time imaging of mitochondrial H2O2 and pH fluctuations in living cells. Notably, this is a single nanosensing system that is capable of visualizing multiple subcellular analytes at the same time and position by multicolor fluorescence imaging. The current approach can provide a promising tool to investigate the interplaying roles of various subcellular analytes in living cells.

Ratiometric Fluorescence Nanoprobes for Subcellular pH Imaging with a Single-Wavelength Excitation in Living Cells

Abnormal pH values in the organelles are closely associated with inappropriate cellular functions and many diseases. Monitoring subcellular pH values and their variations is significant in biological processes occurring in living cells and tissues. Herein, we develop a series of ratiometric fluorescence nanoprobes for quantification and imaging of pH values with a single-wavelength excitation in cytoplasm, lysosomes, and mitochondria. The nanoprobes consist of mesoporous silica nanoparticles assembled with aminofluorescein as the recognition unit for pH measurement and ethidium bromide as reference fluorophore. Further conjugation of subcellular targeting moiety enables the nanoprobes to specifically target lysosome and mitochondria. Confocal fluorescence imaging demonstrated that the nanoprobes could effectively monitor the pH fluctuations from 5.0 to 8.3 in living cells by ratio imaging with 488 nm excitation. Subcellular pH determination and imaging in lysosome and mitochondria could also be achieved in different conditions. The current method can offer a general strategy to determine subcellular analytes and investigate the interactions in biological samples.

Hollow Mesoporous Silica Nanoparticles with Tunable Structures for Controlled Drug Delivery

A size-controllable and facile synthetic strategy has been developed to fabricate a series of hollow mesoporous silica nanoparticles (HMSNs) with tunable hollow cores or shell thicknesses by employing gold nanoparticles (Au NPs) and cetyltrimethylammonium bromide (CTAB) as dual templates. Various sizes of Au NPs and different amounts of tetraethyl orthosilicate contributed to structure-tailored mesoporous silica-coated Au NPs. After calcination, CTAB molecules were completely removed, and Au NPs could still support the silica shell due to the high melting point. HMSNs were ultimately obtained by etching Au NPs. Applications of HMSNs as nanocarriers for delivering drugs were investigated. Significantly, it was flexible and convenient to control drug-loading/releasing behavior of HMSNs just by tuning the hollow cores or shell thicknesses. Intracellular experiments have proven that HMSNs are suitable for delivering drugs. We anticipate that this study could provide an important avenue for the synthesis of HMSNs and further contribute to advancing practical applications of HMSNs in drug delivery systems.

Selective and sensitive turn-on detection of adenosine triphosphate and thrombin based on bifunctional fluorescent oligonucleotide probe

A bifunctional fluorescent oligonucleotide probe for small molecules and protein detection has been developed based on turn on fluorescence response via the target induced structure-switching of molecular beacon. The two loops of this molecular beacon are designed in such a manner that they consist of thrombin (Tmb) aptamer sequence and adenosine triphosphate (ATP) aptamer sequence, respectively, which are utilized to sense thrombin and ATP. The oligonucleotide forms double stem-loops in the absence of targets, yielding no fluorescence emission because of the FRET from the excited fluorophore to the proximal quencher. Upon addition of the target, the ATP or Tmb, its specific interaction with loop sequence of the hairpin structure induce the separation of reporter fluorophore and the fluorescence quencher of the molecular beacon, resulting in an increase of fluorescence response. Hence, the separate analysis of ATP and Tmb could be realized through only one designed molecular beacon. The detection limits were estimated to be 25 nM for ATP and 12 nM for Tmb, respectively. The results of this study should substantially broaden the perspective for future development of oligonucleotide probe for analysis of other analytes.

Simultaneous Visualization of Multiple mRNAs and Matrix Metalloproteinases in Living Cells Using a Fluorescence Nanoprobe

Simultaneous monitoring of multiple tumour markers is of great significance for improving the accuracy of early cancer detection. In this study, a fluorescence nanoprobe has been prepared that can simultaneously monitor and visualize multiple mRNAs and matrix metalloproteinases (MMPs) in living cells. Confocal fluorescence imaging results indicate that the nanoprobe could effectively distinguish between cancer cells and normal cells even if one tumour maker of normal cells was overexpressed. Furthermore, it can detect changes in the expression levels of mRNAs and MMPs in living cells. The current approach could provide new tools for early cancer detection and monitoring the changes in expression levels of biomarkers during tumour progression.

Visualizing the Conversion Process of Alcohol-Induced Fatty Liver to Steatohepatitis in Vivo with a Fluorescent Nanoprobe

Excess alcohol consumption and the associated development of alcoholic liver disease (ALD) are major public health challenges worldwide. Since patients with the severe stages of ALD no longer benefit from clinical therapies, early warning of ALD holds significant promise for increasing the cure rate of ALD. Herein, we develop a bicolor fluorescent nanoprobe for dynamically monitoring the conversion process of alcohol-induced fatty liver to steatohepatitis in vivo through simultaneous imaging of microRNA 155 and osteopontin mRNA, which are related to fatty liver and steatohepatitis, respectively. The fluorescence imaging results indicate that the nanoprobe can effectively differentiate alcohol-induced fatty liver and steatohepatitis. Moreover, the nanoprobe can monitor the transmutation process of alcohol-induced fatty liver to steatohepatitis and assess the remission effects of N-acetyl cysteine for alcohol-induced liver injury. We anticipate the developed nanoprobe and imaging method can provide new ways for early warning, treatments, and prognosis of ALD.

Dual-Ratiometric Fluorescent Nanoprobe for Visualizing the Dynamic Process of pH and Superoxide Anion Changes in Autophagy and Apoptosis

Autophagy and apoptosis are closely associated with various pathological and physiological processes in cell cycles. Investigating the dynamic changes of intracellular active molecules in autophagy and apoptosis is of great significance for clarifying their inter-relationship and regulating mechanism in many diseases. In this study, we develop a dual-ratiometric fluorescent nanoprobe for quantitatively differentiating the dynamic process of superoxide anion (O2•-) and pH changes in autophagy and apoptosis in HeLa cells. A rhodamine B-loaded mesoporous silica core was used as the reference, and fluorescence probes for pH and O2•- measurement were doped in the outer layer shell of SiO2. Then, chitosan and triphenylphosphonium were modified on the surface of SiO2. The experimental results showed that the nanoprobe is able to simultaneously and precisely visualize the changes of mitochondrial O2•- and pH in HeLa cells. The kinetics data revealed that the changes of pH and O2•- during autophagy and apoptosis in HeLa cells were significantly different. The pH value was decreased at the early stage of apoptosis and autophagy, whereas the O2•- level was enhanced at the early stage of apoptosis and almost unchanged at the initial stage of autophagy. At the late stage of apoptosis and autophagy, the concentration of O2•- was increased, whereas the pH was decreased at the late stage of autophagy and almost unchanged at the late stage of apoptosis. We hope that the present results provide useful information for studying the effects of O2•- and pH in autophagy and apoptosis in various pathological conditions and diseases.

A Highly Sensitive Strategy for Fluorescence Imaging of MicroRNA in Living Cells and in Vivo Based on Graphene Oxide-Enhanced Signal Molecules Quenching of Molecular Beacon

In situ imaging of microRNA (miRNA) in living cells and in vivo is beneficial for promoting the studies on miRNA-related physiological and pathological processes. However, the current strategies usually have a low signal-to-background ratio, which greatly affects the sensitivity and imaging performance. To solve this problem, we developed a highly sensitive strategy for fluorescence imaging of miRNA in living cells and in vivo based on graphene oxide (GO)-enhanced signal molecule quenching of a molecular beacon (MB). 2Cy5-MB was designed by coupling two Cy5 molecules onto the opposite ends of MB. The fluorescence intensities of two Cy5 molecules were reduced because of the self-quenching effect. After adsorbing on the GO surface, the fluorescence quenching of the molecules was enhanced by fluorescence resonance energy transfer. This double-quenching effect significantly reduced the fluorescence background. In the presence of one miRNA molecule, the fluorescence signals of two Cy5 molecules were simultaneously recovered. Therefore, a significantly enhanced signal-to-background ratio was obtained, which greatly improved the detection sensitivity. In the presence of miRNA, the fluorescence intensity of 2Cy5-MB-GO recovered about 156 times and the detection limit was 30 pM. Compared with 1Cy5-MB-GO, the elevated fluorescence intensity was enhanced 8 times and the detection limit was reduced by an order of magnitude. Furthermore, fluorescence imaging experiments demonstrated that 2Cy5-MB-GO could visually detect microRNA-21 in various cancer cells and tumor tissues. This simple and effective strategy provides a new sensing platform for highly sensitive detection and simultaneous imaging analysis of multiple low-level biomarkers in living cells and in vivo.