Haibin Xiao

A fluorescence nanosensor for glycoproteins with activity based on the molecularly imprinted spatial structure of the target and boronate affinity.

Glycoproteins are closely associated with the occurrence of diverse diseases, and they have been used as biomarkers and therapeutic targets in clinical diagnostics. Currently, mass spectrometry has proven to be a powerful tool for glycoprotein analysis, but it is almost impossible to directly identify glycoproteins without the preparation and pretreatment of samples. Furthermore, biological samples, especially proteins, are damaged by this process. Herein, we describe a novel fluorescence nanosensor based on a molecularly imprinted spatial structure and boronate affinity that is well-suited for monitoring glycoproteins selectively. Results showed that the recognition performance of the nanosensor for glycoproteins was regulated by controlling the pH value and temperature. Moreover, the nanosensor was successfully applied to the detection of HRP in biological fluids. This study provides a facile and efficient fluorescence tool for glycoprotein detection in clinical diagnostics.

Mitochondria-Targeted Reaction-Based Two-Photon Fluorescent Probe for Imaging of Superoxide Anion in Live Cells and in Vivo

A newly synthesized reaction-based two-photon (TP) fluorescence imaging probe, 9-butyltriphenylphosphoniumacylamino-2,7-dibenzothiazolineflurene (MF-DBZH), composed of a superoxide anion (O2(•-)) responsive group and a mitochondria-targeted site, has been shown to have high selectivity toward mitochondrial O2(•-) fluxes. The fluorescence intensity of MF-DBZH responds proportionally to changes in O2(•-) concentrations. Moreover, MF-DBZH was proved to be insensitive toward pH changes and has high photostability. Favorable features of this probe also include convenient cell loading, easy staining of both cells and small animals, and excellent biocompibility. Most importantly, MF-DBZH gives reliable TP fluorescent signal to changes of O2(•-) levels in vivo.

A New Polymer Nanoprobe Based on Chemiluminescence Resonance Energy Transfer for Ultrasensitive Imaging of Intrinsic Superoxide Anion in Mice.

Despite significant developments in optical imaging of superoxide anion (O2(•-)) as the preliminary reactive oxygen species, novel visualizing strategies that offer ultrahigh sensitivity are still imperative. This is mainly because intrinsic concentrations of O2(•-) are extremely low in living systems. Herein, we present the rational design and construction of a new polymer nanoprobe PCLA-O2(•-) for detecting O2(•-) based on chemiluminescence (CL) resonance energy transfer without an external excitation source. Structurally, PCLA-O2(•-) contains two moieties linked covalently, namely imidazopyrazinone that is capable of CL triggered by O2(•-) as the energy donor and conjugated polymers with light-amplifying property as the energy acceptor. Experiment results demonstrate that PCLA-O2(•-) exhibits ultrahigh sensitivity at the picomole level, dramatically prolonged luminescence time, specificity, and excellent biocompatibility. Without exogenous stimulation, this probe for the first time in situ visualizes O2(•-) level differences between normal and tumor tissues of mice. These exceptional features ensure that PCLA-O2(•-) as a self-luminescing probe is an alternative in vivo imaging approach for ultralow level O2(•-).

Simultaneous Quantitation of Na+ and K+ in Single Normal and Cancer Cells Using a New Near-Infrared Fluorescent Probe

Considering the important functions of cellular Na(+) and K(+) together with their cooperative efforts on various biological processes, it is significant to simultaneously detect Na(+) and K(+) at a single-cell level. Here, we present a novel method to discriminate and quantify simultaneously Na(+) and K(+) in single cells using a new near-infrared fluorescent probe associated with microchip electrophoresis. The fluorescent probe selectively responds to both Na(+) and K(+). The microchip electrophoresis allows accurate single-cell manipulation and effective distinction of Na(+) and K(+). Based on the method, the concentration of Na(+) and K(+) in single normal and cancer cells was compared, and the variation of Na(+) and K(+) in single cancer cells during the early stage of apoptotic volume decrease was monitored, which would help us to better understand the critical roles of Na(+) and K(+) in malignant cells and apoptosis. This method has paved a new way for the research of the synergistic function of Na(+) and K(+) in the regulation of various biological processes at a single-cell level.

A new endoplasmic reticulum-targeted two-photon fluorescent probe for imaging of superoxide anion in diabetic mice

Excessive or unfolded proteins accumulation in endoplasmic reticulum (ER) will cause ER stress, which has evolved to involve in various metabolic diseases. In particular, ER stress plays an important role in the pathogenesis of diabetes. Both ER stress and course of diabetes accompany oxidative stress and production of reactive oxygen species (ROS), among which superoxide anion (O2•-) is the first produced ROS and has been recognized as cell signaling mediator involved in the physiological and pathological process of diabetes. Hence, the development of effective monitoring methods of O2•- in live cells and in vivo is of great importance for ascertaining the onset and progress of related diseases. Herein, a new endoplasmic reticulum-targeted two-photon fluorescent probe termed ER-BZT is designed and synthesized for imaging of O2•-. The probe ER-BZT shows high sensitivity, selectivity, stability, and low cytotoxicity. Based on these superior properties, the rise of O2•- levels in endoplasmic reticulum induced with different stimuli is visualized by one- and two-photon fluorescence imaging. Most importantly, by utilizing ER-BZT, the two-photon fluorescence imaging results demonstrate that the endogenous O2•- concentration in abdominal or hepatic tissue of diabetic mice is higher than that in normal mice. Meanwhile, after treated with metformin, a broad-spectrum antidiabetic drug, the diabetic mice exhibit depressed O2•- level. The proposed two-photon probe, ER-BZT might serve as perfect tool to image the O2•- fluctuations and study the relevance between O2•- and various diseases in live cells and in vivo.

Simultaneous fluorescence visualization of mitochondrial hydrogen peroxide and zinc ions in live cells and in vivo

We report simultaneous fluorescence imaging of hydrogen peroxide and zinc ions within mitochondria using two fluorescent probes termed M-H2O2 and M-Zn.

Quantitative Fluorescence Ratio Imaging of Intralysosomal Chloride Ions with Single Excitation/Dual Maximum Emission

Fluorescence ratio imaging is currently being used to quantitatively detect biologically active molecules in biosystems; however, two excitations of most existing fluorescent ratiometric probes account for cumbersome operating conditions for imaging. Thus, a fluorescent ratiometric probe, 6-methoxyquinolinium-dansyl (MQ-DS), for Cl(-) with single excitation/dual maximum emission has been developed. MQ-DS can preferably localize into lysosomes and display excellent photostability. Upon excitation at a single wavelength, it responds precisely and instantaneously to changes in Cl(-) concentrations, and it can be conveniently utilized to implement real-time fluorescence ratio imaging to quantitatively track alterations in Cl(-) levels inside cells treated under various pH conditions, and also in zebrafish with acute wounds. The successful application of the new probe in bioimaging may greatly facilitate a complete understanding of the physiological functions of Cl(-) .

Illuminating Superoxide Anion and pH Enhancements in Apoptosis of Breast Cancer Cells Induced by Mitochondrial Hyperfusion Using a New Two-Photon Fluorescence Probe

Mitochondrial morphology regulated by fusion and fission processes determines mitochondrial function and cell fate. Some studies showed hyperfused mitochondria could induce apoptosis in cancer cells, but the relevant molecular mechanisms remain elusive. Superoxide (O2•-) and pH play vital roles in mitochondrial dysfunction and apoptosis. Therefore, it is worthwhile to explore if there is an intimate relationship between mitochondrial hyperfusion and simultaneous changes in O2•- and pH levels, which will be helpful to uncover relevant detailed mechanism. For this purpose, we have developed a new reversible two-photon fluorescent probe (CFT) to simultaneously monitor O2•- and pH in 4T1 cells and mice using dual-color imaging. With the assistance of probe, we found that inhibition of Dynamin-related protein 1 (Drp1) could transduce a signal through mitochondrial complexes I and II to enhance the O2•- and pH levels and eventually induced mitohyperfusion and apoptosis in breast cancer cells. Together, these data indicate that CFT provides a robust tool for unveiling the roles of O2•- and pH in signals associated with mitochondrial dysfunction in cells and in vivo.

Simultaneous Fluorescence Visualization of Endoplasmic Reticulum Superoxide Anion and Polarity in Myocardial Cells and Tissue

Diabetic cardiomyopathy (DCM) is a critical complication of diabetes, the accurate pathogenesis of which remains elusive. It is widely accepted that endoplasmic reticulum (ER) stress and abnormal fluctuations of reactive oxygen species (ROS) are considered to be closely associated with progress of DCM. In addition, DCM-induced changes of myocardial tissue and ROS-derived oxidation of proteins will cause changes of the hydrophilic and hydrophobic domains and may further seriously alter the myocardial cell polarity. Thus, real-time detection of ROS and polarity in ER of live cells and in tissue will contribute to revealing the exact molecular mechanisms of DCM. In this article, we first present an ER-targetable fluorogenic probe termed ER-NAPC for sensitive and selective detection of superoxide anion (O2•-). ER-NAPC can precisely target ER and visualize the increase of O2•- level in a live H9c2 cardiomyocyte cell during ER stress. Meanwhile, by combining ER-NAPC with a polarity-sensitive probe, ER-P, we accomplish the simultaneous fluorescence visualization of O2•- and polarity in ER of live cells and diabetic myocardial tissue. The dual-color fluorescence imaging results indicate that the O2•- level and polarity will synergistically rise during ER stress in live cells and diabetic myocardial tissue. The proposed dual-color imaging strategy may offer a proven methodology for studying coordinated variation of different parameters during ER stress oriented disease.

Two-photon Imaging of Endoplasmic Reticulum Thiol Flux in the Brains of Mice with Depression phenotypes

Depression is a common mental illness with high morbidity and mortality. Mounting evidence suggests that an imbalance of the oxidant-antioxidant defence system is strongly correlated with depression and the dysfunction of the endoplasmic reticulum (ER) is strongly related to the oxidative stress. Therefore, as vital and abundant antioxidants in the ER, biothiols may contribute to the etiology of depression. However, ideal two-photon (TP) fluorescent probes for in vivo imaging of ER-associated thiols in the brains of mice with depression phenotypes are still lacking. Hence, we describe a fluorescent probe (ER-SH) to visualize thiols in living systems. ER-SH displays high sensitivity, excellent ER-targeting ability, outstanding TP properties and low cytotoxicity. Using this ER-SH probe, we succeeded in revealing an increase in the endogenous thiol levels under ER stress induced by DTT. Significantly, TP in vivo imaging showed for the first time that the thiol levels are reduced in brains of mice with depression phenotypes. Collectively, this work can assist in further understanding the molecular mechanism of depression and offers a crucial dimension for diagnosis and anti-depression treatments.