Recent Progress of Small-Molecule Ratiometric Fluorescent Probes for Peroxynitrite in Biological Systems

Peroxynitrite (ONOO⁻) as a major reactive oxygen species plays important roles in cellular signal transduction and homeostatic regulation. Precise detection of ONOO⁻ in biological systems is vital for exploring its physiological and pathological function. Among numerous detection methods, fluorescence imaging technology using fluorescent probes offers some advantages, including simple operation, high sensitivity and selectivity, as well as real-time and nondestructive detection. In particular, ratiometric fluorescent probes, in which the built-in calibration of the two emission bands prevents interference from the biological environment, have been extensively employed to monitor the fluctuation of bioactive species. In this review, we will discuss small-molecule ratiometric fluorescent probes for ONOO⁻ in live cells or in vivo, which involves chemical structures, response mechanisms, and biological applications. Moreover, the challenges and future prospects of ONOO⁻-responsive ratiometric fluorescent probe are also proposed.     

Design of Turn-On Near-Infrared Fluorescent Probes for Highly Sensitive and Selective Monitoring of Biopolymers

Simple, sensitive, and selective detection of specific biopolymers is critical in a broad range of biomedical and technological areas. We present a design of turn-on near-infrared (NIR) fluorescent probes with intrinsically high signal-to-background ratio. The fluorescent signal generation mechanism is based on the aggregation/de-aggregation of phthalocyanine chromophores controlled by selective binding of small-molecule “anchor” groups to a specific binding site of a target biopolymer. As a proof-of-concept, we demonstrate a design of a sensor for EGFR tyrosine kinase—an important target in cancer research. The universality of the fluorescent signal generation mechanism, as well as the dependence of the response selectivity on the choice of the small-molecule “anchor” group, make it possible to use this approach to design reliable turn-on NIR fluorescent sensors for detecting specific protein targets present in the low-nanomolar concentration range.     

Recent advances in fluorescence imaging of alkaline phosphatase

Phosphatase plays a vital important role in many biological functions due to the dephosphorylation serves varied roles in cellular regulation and signaling.Among the family of phosphatase,alkaline phosphatase(ALP) could act as crucial prognostic indicators for many diseases such as bone diseases and cancer.However,the detection of ALP is mainly limited to in vitro colorimetric method in clinic.Therefore,huge efforts have been paid on the fluorescence imaging that provides a reliable method to detect the real-time and in vivo changes of the level of ALP.In this review,we summarize recent advances in fluorescence imaging of phosphatase,mainly focused on ALP.The imaging probes of phosphatase are mainly classified according to their luminescence mechanisms.In the end,we assessed the challenges and future prospects of phosphatase probes.     

Quantum dots for single bio-molecule imaging.

The emerging nanomaterial, quantum dots or QDs, offers numerous potential applications in the biological area. As cell labeling probes, QDs become now an alternative of existing organic fluorescent dyes and fluorescent proteins. In this short review, we cover typical and successful applications of QDs as fluorescent probes in cell labeling and genomic diagnosis. As a future important application, biomolecular detection at a single molecule level utilizing QDs is also discussed.     

Lysosome-targeting red fluorescent probe for broad carboxylesterases detection in breast cancer cells

Available online The abnormal carboxylesterase (CES) expression is closely related to many diseases such as hyperlipidemia, atherosclerosis, obesity, liver cancer, type 2 diabetes mellitus and gastrointestinal stromal tumors. The detection of a single enzyme in practical samples is often constrained by the structural diversity of CESs. Thus, the development of broad-carboxylesterase responsive fluorescent probe, which can detect the presence of wide variety of CESs, may provide overall or category information from another point of view, supplementing the deficiency of single detection for CES subspecies. Organelle lysosome is involved in various cell processes, such as cell signaling, apoptosis, secretion, and energy metabolism. Up to date, lysosome-targeted fluorescent probes, especially those with red emission (over 550 nm, with relatively low biological harmfulness), for CES detection are still rare. A lysosomes-targeted red fluorescent probe CES-Lyso was designed to monitor intracellular a variety of carboxylesterases alteration with wonderful selectivity and sensitivity, which was further applied to distinguish different derived breast cancer cells and monitor carboxylesterase activity in the anticancer drug treatment.     

AMP/GMP Analogs as Affinity ESIPT Probes for Highly Selective Sensing of Alkaline Phosphatase Activity in Living Systems

Current probes for alkaline phosphatase (ALP) detection had been developed mainly by adding a phosphate group to a dye, which would lead to indistinct performance when implemented in a living system as several phosphatases exist together. In this study, the nucleotides adenosine monophosphate (AMP) and guanosine monophosphate (GMP) were introduced into 2′‐(2′‐hydroxyphenyl)‐benzothiazole‐based probes, and highly fluorescent turn‐on probes with good selectivity towards ALP over several phosphatases, as well as high affinity and low toxicity were obtained. In the presence of l ‐phenylalanine, an ALP inhibitor, a strong decrease in fluorescence recovery was observed. These probes allowed for real‐time imaging of endogenous ALP activity in living cells as well as in a zebrafish model.     

Single‐Molecule Analysis Determines Isozymes of Human Alkaline Phosphatase in Serum

Alkaline phosphatase (ALP) is an important biomarker, as high levels of ALP in blood can indicate liver disease or bone disorders. However, current clinical blood tests only measure the total concentration of ALP but are unable to distinguish enzyme isotypes. Here, we demonstrate a novel and rapid approach to profile various ALP isozymes in blood via a single‐molecule‐analysis platform. The microarray platform provides enzyme kinetics of hundreds of individual molecules at high throughput. Using these single molecule kinetics, we characterize the different activity profiles of ALP isotypes. By analyzing both healthy and disease samples, we found the single molecule activity distribution of ALP in serum reflects the health status of patients. This result demonstrates the potential utility of the method for improving the conventional ALP test, as well as for analyzing other enzymatic biomarkers, including enzyme isotypes.     

Single-Molecule Analysis Determines Isozymes of Human Alkaline Phosphatase in Serum

Alkaline phosphatase (ALP) is an important biomarker, as high levels of ALP in blood can indicate liver disease or bone disorders. However, current clinical blood tests only measure the total concentration of ALP but are unable to distinguish enzyme isotypes. Here, we demonstrate a novel and rapid approach to profile various ALP isozymes in blood via a single-molecule-analysis platform. The microarray platform provides enzyme kinetics of hundreds of individual molecules at high throughput. Using these single molecule kinetics, we characterize the different activity profiles of ALP isotypes. By analyzing both healthy and disease samples, we found the single molecule activity distribution of ALP in serum reflects the health status of patients. This result demonstrates the potential utility of the method for improving the conventional ALP test, as well as for analyzing other enzymatic biomarkers, including enzyme isotypes.     

Organelle targetable fluorescent probes for hydrogen peroxide

Hydrogen peroxide(H_2 O_2), as important products of oxygen metabolism, plays an important role in many biological processes, such as immune responses and cellular signal transduction. However, abnormal production of H_2 O_2 can damage cellular biomolecules, which was closely associated with many diseases.Thus, it is urgent to monitor the level change of H_2 O_2 in living cells, particularly at subcellular levels.Toward this end, a wide variety of H_2 O_2 fluorescent probes have been designed, developed and applied for imaging of H_2 O_2 in subcellular levels. In this review, we highlight the representative cases of H_2 O_2 fluorescent probes with mitochondria, nuclei and lysosomes-targetable ability. The review contains organelle target strategies, structures, fluorescence behavior and biological applications of these probes.     

Development of Small-Molecule Fluorescent Probes Targeting Enzymes

As biological catalysts, enzymes are vital in controlling numerous metabolic reactions. The regulation of enzymes in living cells and the amount present are indicators of the metabolic status of cell, whether in normal condition or disease. The small-molecule fluorescent probes are of interest because of their high sensitivity and selectivity, as well as their potential for automated detection. Fluorescent probes have been useful in targeting particular enzymes of interest such as proteases and caspases. However, it is difficult to develop an ideal fluorescent probe for versatile purposes. In the future, the design and synthesis of enzyme-targeting fluorescent probes will focus more on improving the selectivity, sensitivity, penetration ability and to couple the fluorescent probes with other available imaging molecules/technologies.     

Recent advances in mitochondria-and lysosomes-targeted small-molecule two-photon fluorescent probes

This review summarized the recent advances in small-molecule two-photon fl uorescent probes for monitoring a wide variety of biomolecules and changes inside micro-environment in mitochondria and lysosomes, or served as mitotracker and lysotracker with the assistance of two-photon microscopy.     

Organelle targetable fluorescent probes for hydrogen peroxide

The review summarized organelle target strategies, structures, fluorescence behavior and biological applications of H₂O₂ fluorescent probes with mitochondria, nuclei and lysosomes-targetable ability.     

In vivo magnetic resonance detection of cancer by using multifunctional magnetic nanocrystals

The unique properties of magnetic nanocrystals provide them with high potential as key probes and vectors in the next generation of biomedical applications. Although superparamagnetic iron oxide nanocrystals have been extensively studied as excellent magnetic resonance imaging (MRI) probes for various cell trafficking, gene expression, and cancer diagnosis, further development of in vivo MRI applications has been very limited. Here, we describe in vivo diagnosis of cancer, utilizing a well-defined magnetic nanocrystal probe system with multiple capabilities, such as small size, strong magnetism, high biocompatibility, and the possession of active functionality for desired receptors. Our magnetic nanocrystals are conjugated to a cancer-targeting antibody, Herceptin, and subsequent utilization of these conjugates as MRI probes has been successfully demonstrated for the monitoring of in vivo selective targeting events of human cancer cells implanted in live mice. Further conjugation of these nanocrystal probes with fluorescent dye-labeled antibodies enables both in vitro and ex vivo optical detection of cancer as well as in vivo MRI, which are potentially applicable for an advanced multimodal detection system. Our study finds that high performance in vivo MR diagnosis of cancer is achievable by utilizing improved and multifunctional material properties of iron oxide nanocrystal probes.     

Hemin as a General Static Dark Quencher for Constructing Heme Oxygenase-1 Fluorescent Probes

The development of small-molecule probes suitable for live-cell applications remains challenging yet highly desirable. We report the first fluorescent probe, RBH, for imaging the heme oxygenase-1 (HO-1) activity in live cells after discovering hemin as a universal dark quencher. Hemin works via a static quenching mechanism and shows high quenching efficiency (>97 %) with fluorophores across a broad spectrum (λ_ex=400–700 nm). The favorable properties of RBH (e.g. long excitation/emission wavelengths, fast response rate and high magnitude of signal increase) enable its use for determining HO-1 activity in complex biological samples. As HO-1 is involved in regulating antioxidant defence, iron homeostasis and gasotransmitter carbon monoxide production, we expect RBH to be a powerful tool for dissecting its functions. Also, the discovery of hemin as a general static dark quencher provides a straightforward strategy for constructing novel fluorescent probes for diverse biological species.     

Recent advances in the targeted fluorescent probes for the detection of metastatic bone cancer

Tumors of the breast, prostate, and lung are most likely to metastasize to the bone and typically indicates a poor cure and survival rate in cancer patients. Detection of metastatic bone cancer in early stage would save many lives and greatly improve patients' quality of life. Clinically, bone scintigraphy is often utilized to visualize bone metastases due to its relatively low cost and high sensitivity. Recently, a growth number of analytical researches aimed at developing targeted fluorescent probes to noninvasively image bone metastases with improved spatial resolution and specificity has been reported. In this review, we will summarize and discuss the recent published fluorescent probes on the accurate detection of metastatic bone cancer. First, the design principles of various targeted probes for imaging bone metastases will be presented, highlighting the signal moieties, targeting ligands, and physicochemical properties of the bone-specific probes. Next, the up-to-date bone-targeting fluorescent probes will be summarized and overviewed. Finally, future perspectives and challenges confronting the researchers in this field will be discussed.We believe this review will encourage novel ideas to develop smart targeted molecular probes for bone metastasis imaging,image-guided surgery, and therapeutic imaging materials.     

In Situ Localization of Enzyme Activity in Live Cells by a Molecular Probe Releasing a Precipitating Fluorochrome

Current enzyme‐responsive, fluorogenic probes fail to provide in situ information because the released fluorophores tend to diffuse away from the reaction sites. The problem of diffusive signal dilution can be addressed by designing a probe that upon enzyme conversion releases a fluorophore that precipitates. An excited‐state intramolecular proton transfer (ESIPT)‐based solid‐state fluorophore HTPQ was developed that is strictly insoluble in water and emits intense fluorescence in the solid state, with λ _ex/em=410/550 nm, thus making it far better suited to use with a commercial confocal microscope. HTPQ was further utilized in the design of an enzyme‐responsive, fluorogenic probe (HTPQA), targeting alkaline phosphatase (ALP) as a model enzyme. HTPQA makes possible diffusion‐resistant in situ detection of endogenous ALP in live cells. It was also employed in the visualizing of different levels of ALP in osteosarcoma cells and tissue, thus demonstrating its interest for the diagnosis of this type of cancer.     

Small-molecule fluorescent probes for the detection of carbon dioxide

This review summarizes the design principles, recognition mechanisms, properties and functions of various kinds of small-molecule fluorescent probes for the detection of carbon dioxide     

Development of an osteoblast-based 3D continuous-perfusion microfluidic system for drug screening

In this work, we demonstrated that biological cells could be cultured in a continuous-perfusion glass microchip system for drug screening. We used mouse Col1a1GFP MC-3T3 E1 osteoblastic cells, which have a marker gene system expressing green fluorescent protein (GFP) under the control of osteoblast-specific promoters. With our microchip-based cell culture system, we realized automated long-term monitoring of cells and sampling of the culture supernatant system for osteoblast differentiation assay using a small number of cells. The system successfully monitored cells for 10 days. Under the 3D microchannel condition, shear stress (0.07 dyne/cm² at a flow rate of 0.2 μL/min) was applied to the cells and it enhanced the GFP expression and differentiation of the osteoblasts. Analysis of alkaline phosphatase (ALP), which is an enzyme marker of osteoblasts, supported the results of GFP expression. In the case of differentiation medium containing bone morphogenetic protein 2, we found that ALP activity in the culture supernatant was enhanced 10 times in the microchannel compared with the static condition in 48-well dishes. A combined system of a microchip and a cell-based sensor might allow us to monitor osteogenic differentiation easily, precisely, and noninvasively. Our system can be applied in high-throughput drug screening assay for discovering osteogenic compounds.     

Recent Advances in Excimer-Based Fluorescence Probes for Biological Applications

The fluorescent probe is a powerful tool for biological sensing and optical imaging, which can directly display analytes at the molecular level. It provides not only direct visualization of biological structures and processes, but also the capability of drug delivery systems regarding the target therapy. Conventional fluorescent probes are mainly based on monomer emission which has two distinguishing shortcomings in practice: small Stokes shifts and short lifetimes. Compared with monomer-based emission, excimer-based fluorescent probes have large Stokes shifts and long lifetimes which benefit biological applications. Recent progress in excimer-based fluorescent sensors (organic small molecules only) for biological applications are highlighted in this review, including materials and mechanisms as well as their representative applications. The progress suggests that excimer-based fluorescent probes have advantages and potential for bioanalytical applications.