Fluorescent Molecular Probes Research Articles

Construction of mercury ion fluorescence system in water samples and art materials and fluorescence detection method for rhodamine B derivatives

Abstract At present, the research on fluorescent molecular probe has become a hot topic in the field of environmental science, chemical materials, medicine, and other fields. Therefore, the detection of industrial mercury-containing wastewater (Hg2+) is of great significance. In this article, the fluorescent probe is used to detect mercury ions, and when compared with the traditional detection method, the fluorescent probe has the advantage of operation such as the effect of simplicity is evident. The experiments first synthesized rhodamine B derivatives and then the synthesized rhodamine B derivative fluorescent molecular probes were constructed and used to detect the mercury ions in water sample and oil paints. It was demonstrated that rhodamine B-derived probes have been constructed by UV and fluorescence spectroscopy. The different metal ions and rhodamine B-derived fluorescent molecular probes were compounded, resulting in the appearance of fluorescence peak centered at 583 nm only after the addition of metallic mercury ions with almost no response from other ions. The mercury ion rhodamine B derivative is more responsive to metallic mercury ions.

Molecular fluorescent probes for imaging and evaluation of peroxynitrite fluctuations in living cells and in vivo under hypoxic stress

Hypoxia stress causes cell damage and apoptosis by producing excessive reactive oxygen species (ROS), which is a universal concern in biology and medicine. Peroxynitrite (ONOO-) plays a vital part in the oxidative injury of tissue proteins in the pathogenic mechanism of many disorders, which is a one of ROS. Thus, excessive production of ONOO- may be one of the crucial factors leading to hypoxia stress injury. However, due to the unstable chemical property and strong reactivity of ONOO-, most of the existing detection ways cannot image and detect its level in real time. Herein, in order to selectively imaging and detecting ONOO- in living cells and in vivo, we synthesized a fluorescent probe Cy-CF3 (our fluorescent probe for detection of ONOO-). Cy-CF3 was capable of monitoring ONOO- with excellent sensitivity and selectivity. Using Cy-CF3, the relationship between hypoxia stress and ONOO- was evaluated. The probe was also employed to sense the change of ONOO- in zebrafish under hypoxia conditions as well as determine ONOO- in living mice with acute liver ischemia. Taken together, the proposed probe maybe a powerful chemical tool for the study of ONOO- in biological mechanisms.

Polymethine Molecular Platform for Ratiometric Fluorescent Probes in the Second near-Infrared Window.

Visualizing biomolecules such as enzymes in the deep tissue of living organisms via molecular ratiometric fluorescent probes in the second near-infrared window (NIR-II) with a built-in self-calibration function can provide reliable information about relevant pathophysiological processes directly but so far is not feasible due to the lack of a fluorescence modulation strategy in the NIR-II window. Here we present a molecular platform Py-2 by integrating the rhodamine 6G scaffold and polymethine. The maximal emission wavelength of Py-2 was 1010 nm and blue-shifted to 945 nm when its secondary amine was acylated. Based on Py-2, two molecular ratiometric NIR-II fluorescent probes, nitroreductase-responsive Rap-N and ROS-responsive Rap-R, were constructed and successfully demonstrated in vitro and in vivo. Overall, this report presents a unique approach to developing ratiometric NIR-II molecular probes for in vivo biosensing.

L007-0069 kills Staphylococcus aureus in high resistant phenotypes.

Staphylococcus aureus, a common gram-positive pathogenic bacterium, is a main cause of hospital infection. The prevalence rate of methicillin-resistant S. aureus (MRSA) has made its treatment difficult in recent decades. Moreover, S. aureus in the highly tolerant format of biofilm or persister often renders infections refractory. Thus, developing new active compounds against resistant S. aureus is urgently needed. In this study, by a high-throughput screening assay, we identified a small molecule, L007-0069, that exhibited strong and effective bactericidal activity against S. aureus and its high resistance patterns, such as biofilms and persisters, with a low probability of inducing resistance. By molecular dynamics and fluorescent probe analysis, mechanistic studies revealed that the bactericidal activity of L007-0069 was mainly mediated by membrane disruption and metabolic disorder induction. Furthermore, L007-0069 showed effective anti-MRSA effects in vivo in both a wound infection model and a peritonitis-sepsis model, with no detectable toxicity observed at the therapeutic dosage. In conclusion, L007-0069 has the potential to become an alternative for the treatment of highly resistant S. aureus-related infections.

Correlating viscosity and molecular crowding with fluorescent nanobeads and molecular probes: in vitro and in vivo.

In eukaryotes, intracellular physico-chemical properties like macromolecular crowding and cytoplasmic viscoelasticity influence key processes such as metabolic activities, molecular diffusion and protein folding. However, mapping crowding and viscoelasticity in living cells remains challenging. One approach uses passive rheology in which diffusion of exogenous fluorescent particles internalized in cells is tracked and physico-chemical properties inferred from derived mean square displacement relations. Recently, the crGE2.3 Förster resonance energy transfer biosensor was developed to quantify crowding in cells, though it is unclear how this readout depends on viscoelasticity and the molecular weight of the crowder. Here, we present correlative, multi-dimensional data to explore diffusion and molecular crowding characteristics of molecular crowding agents using super-resolved fluorescence microscopy and ensemble time-resolved spectroscopy. We firstly characterize in vitro and then apply these insights to live cells of budding yeast Saccharomyces cerevisiae. It is to our knowledge the first time this has been attempted. We demonstrate that these are usable both in vitro and in the case of endogenously expressed sensors in live cells. Finally, we present a method to internalize fluorescent beads as in situ viscoelasticity markers in the cytoplasm of live yeast cells and discuss limitations of this approach including impairment of cellular function.

The Dual Use of the Pyranine (HPTS) Fluorescent Probe: A Ground-State pH Indicator and an Excited-State Proton Transfer Probe.

ConspectusMolecular fluorescent probes are an essential experimental tool in many fields, ranging from biology to chemistry and materials science, to study the localization and other environmental properties surrounding the fluorescent probe. Thousands of different molecular fluorescent probes can be grouped into different families according to their photophysical properties. This Account focuses on a unique class of fluorescent probes that distinguishes itself from all other probes. This class is termed photoacids, which are molecules exhibiting a change in their acid–base transition between the ground and excited states, resulting in a large change in their pKa values between these two states, which is thermodynamically described using the Förster cycle. While there are many different photoacids, we focus only on pyranine, which is the most used photoacid, with pKa values of ∼7.4 and ∼0.4 for its ground and excited states, respectively. Such a difference between the pKa values is the basis for the dual use of the pyranine fluorescent probe. Furthermore, the protonated and deprotonated states of pyranine absorb and emit at different wavelengths, making it easy to focus on a specific state. Pyranine has been used for decades as a fluorescent pH indicator for physiological pH values, which is based on its acid–base equilibrium in the ground state. While the unique excited-state proton transfer (ESPT) properties of photoacids have been explored for more than a half-century, it is only recently that photoacids and especially pyranine have been used as fluorescent probes for the local environment of the probe, especially the hydration layer surrounding it and related proton diffusion properties. Such use of photoacids is based on their capability for ESPT from the photoacid to a nearby proton acceptor, which is usually, but not necessarily, water. In this Account, we detail the photophysical properties of pyranine, distinguishing between the processes in the ground state and the ones in the excited state. We further review the different utilization of pyranine for probing different properties of the environment. Our main perspective is on the emerging use of the ESPT process for deciphering the hydration layer around the probe and other parameters related to proton diffusion taking place while the molecule is in the excited state, focusing primarily on bio-related materials. Special attention is given to how to perform the experiments and, most importantly, how to interpret their results. We also briefly discuss the breadth of possibilities in making pyranine derivatives and the use of pyranine for controlling dynamic reactions.

A highly photostable and versatile two-photon fluorescent probe for the detection of a wide range of intracellular nitric oxide concentrations in macrophages and endothelial cells.

Nitric oxide (NO) is involved in many biological processes affecting the cardiovascular, nervous and immune systems. Intracellular NO can be monitored using fluorescent probes in combination with fluorescence imaging techniques. Most of the currently available NO fluorescent molecular probes are excited via one-photon excitation using UV or Vis light, which results in poor penetration and high photodamage to living tissues. Here, we report a two-photon fluorescent molecular probe, DANPY-NO, able to detect NO in live cells. The probe consists of an o-phenylenediamine linked to a naphthalimide core; and operates via photoinduced electron transfer. DANPY-NO exhibits good sensitivity (LOD of 77.8nM) and high selectivity towards NO, and is stable over a broad range of pHs. The probe targeted acidic organelles within macrophages and endothelial cells, and demonstrated enhanced photostability over a commercially available NO probe. DANPY-NO was used to selectively detect endogenous NO in RAW264.7ϒ NO- macrophages, THP-1 human leukemic cells, primary mouse (bone marrow-derived) macrophages and endothelial cells. The probe was also able to detect exogenous NO in endothelial cells and distinguish between increasing concentrations of NO. The NO detection was evidenced using confocal laser scanning and two-photon microscopies, and flow cytometry. Further evidence was obtained by recording the changes in the intracellular fluorescence emission spectrum of the probe. Importantly, the probe displayed negligible toxicity to the analysed biological samples. The excellent sensitivity, selectivity, stability and versatility of DANPY-NO confirm its potential for in vitro and in vivo imaging of NO.

Rational design of molecular rotor-based fluorescent probes with bi-aromatic rings for efficient in vivo detection of amyloid-β plaques in Alzheimer's disease

The presence of Aβ plaques in the brain is a hallmark of Alzheimer's disease. Here, we designed and synthesized a series of molecular rotors with various bi-aromatic rings and investigated their applications as near-infrared (NIR) probes for Aβ plaques. We found that the interaction with Aβ aggregates hindered the rotational freedom of the molecular rotors, which brought about a noticeable enhancement in fluorescence intensity. Among them, probe 4b (Kd = 8.5 nM) with a phenyl-pyridine ring showed a 98-fold increase in fluorescence intensity upon binding with Aβ aggregates. In addition, 4b could identify Aβ plaques in brain sections of both a transgenic (Tg) mouse and AD patients. Furthermore, 4b could readily penetrate the mouse blood-brain barrier (brain2min = 10.11% ID/g) and washed out rapidly. Finally, the NIR imaging with Tg mice confirmed the practical application of 4b in detecting Aβ plaques in vivo. Altogether, our work widens the landscape of Aβ NIR probes and offers a new tool for Aβ detection.

PET- and ICT-Based Ratiometric Probe: An Unusual Phenomenon of Morpholine-Conjugated Fluorophore for Mitochondrial pH Mapping during Mitophagy

Mitochondrial functions are heavily influenced by acid-base homeostasis. Hence, elucidation of the mitochondrial pH is essential in living cells, and its alterations during pathologies is an interesting question to be addressed. Small molecular fluorescent probes are progressively applied to quantify the mitochondrial pH by fluorescence imaging. Herein, we designed a unique small molecular fluorescent probe, PM-Mor-OH, based on the lipophilic morpholine ligand-conjugated pyridinium derivative of "IndiFluors". The morpholine-conjugated fluorescent probe usually localized the lysosome. However, herein, we observed unusual phenomena of morpholine-tagged PM-Mor-OH that localized mitochondria explicitly. The morpholine ligand also plays a pivotal role in tuning optical properties via photoinduced electron transfer (PET) during internal pH alteration (ΔpHi). In the mitophagy process, lysosomes engulf damaged mitochondria, leading to ΔpHi, which can be monitored using our probe. It exhibited "ratiometric" emission at single wavelength excitation (ex. 488) and is suitable for monitoring and quantifying the ΔpHi using confocal microscope high-resolution image analysis during mitophagy. The bathochromic emission shifts due to intramolecular charge transfer (ICT) in basic pH were well explained by the time-dependent density functional theory (TD-DFT/PCM). Similarly, the change in the emission ratio (green/red) with pH variations was also validated by the PET process. In addition, PM-Mor-OH can quantify the pH change during oxidative stress induced by rapamycin, mutant A53T α-synuclein-mediated protein misfolding stress in mitochondria, and during starvation. Rapamycin-induced mitophagy was further elucidated by the translocation of mCherry Parkin to damaged mitochondria, which well correlates with our probe. Thus, PM-Mito-OH is a valuable probe for visualizing mitophagy and can act as a suitable tool for the diagnosis of mitochondrial diseases.

Quantification of Engagement of Microtubules by Small Molecules in Living Cells by Flow Cytometry.

Drugs such as paclitaxel (Taxol) that bind microtubules are widely used for the treatment of cancer. Measurements of the affinity and selectivity of these compounds for their targets are largely based on studies of purified proteins, and only a few quantitative methods for the analysis of interactions of small molecules with microtubules in living cells have been reported. We describe here a novel method for rapidly quantifying the affinities of compounds that bind polymerized tubulin in living HeLa cells. This method uses the fluorescent molecular probe Pacific Blue-GABA-Taxol in conjunction with verapamil to block cellular efflux. Under physiologically relevant conditions of 37 °C, this combination allowed quantification of equilibrium saturation binding of this probe to cellular microtubules (Kd = 1.7 μM) using flow cytometry. Competitive binding of the microtubule stabilizers paclitaxel (cellular Ki = 22 nM), docetaxel (cellular Ki = 16 nM), cabazitaxel (cellular Ki = 6 nM), and ixabepilone (cellular Ki = 10 nM) revealed intracellular affinities for microtubules that closely matched previously reported biochemical affinities. By including a cooperativity factor (α) for curve fitting of allosteric modulators, this probe also allowed quantification of binding (Kb) of the microtubule destabilizers colchicine (Kb = 80 nM, α = 0.08), vinblastine (Kb = 7 nM, α = 0.18), and maytansine (Kb = 3 nM, α = 0.21). Screening of this assay against 1008 NCI diversity compounds identified NSC 93427 as a novel microtubule destabilizer (Kb = 485 nM, α = 0.02), illustrating the potential of this approach for drug discovery.

Detection of S2- in Water by a Glucose Enhanced Water-Soluble Fluorescent Bioprobe.

That sulfide anions (S2−) in aquatic environments are produced by microorganisms through degrading sulfur-containing proteins and other organics are harmful to human health. Thus, it is of significance to develop a convenient method for the detection of S2− in water. Small molecular fluorescent probes are very popular for their advantages of visualization, real-time, high sensitivity, and convenience. However, low solubility in water limits the application of existing S2− probes. In this work, we found that our previously developed water-soluble glycosylated fluorescent bioprobe Cu[GluC] can achieve detection of S2− in water. Cu[GluC] can restore fluorescence within 20 s when it encounters S2− and shows good sensitivity towards S2− with a detection limit of 49.6 nM. Besides, Cu[GluC] derived fluorescent test strips were obtained by immersion and realized conveniently visual S2− detection in water by coupling with a UV lamp and a smartphone app. This work provides a fluorescent bioprobe with good water solubility as well as its derived fluorescent test strip for sensitive and simple detection of S2− in water, which shows good prospects in on-site water quality monitoring.

Recent Advances in Molecular Fluorescent Probes for CYP450 Sensing and Imaging

Cytochrome P450 (CYP450) is a major drug-metabolizing enzyme system mainly distributed in liver microsomes and involved in the metabolism of many endogenous substances (such as fatty acids and arachidonic acids), and exogenous compounds (such as drugs, toxicants, carcinogens, and procarcinogens). Due to the similarity in structures and catalytic functions between CYP450 isoforms, the lack of effective selective detection tools greatly limits the understanding and the research of their respective physiological roles in living organisms. Until now, several small-molecular fluorescent probes have been employed for selective detection and monitoring of CYP450s (Cytochrome P450 enzymes) in vitro or in vivo owing to the tailored properties, biodegradability, and high temporal and spatial resolution imaging in situ. In this review, we summarize the recent advances in fluorescent probes for CYP450s (including CYP1, CYP2, and CYP3 families), and we discuss and focus on their identification mechanisms, general probe design strategies, and bioimaging applications. We also highlight the potential challenges and prospects of designing new generations of fluorescent probes in CYP450 studies, which will further enhance the diversity, practicality, and clinical feasibility of research into CYP450.

Advances in orderly assembly of fluorescent small molecular probes and related bioimaging applications

<p indent="0mm">Fluorescence imaging technology based on small molecule probes has the advantages of high sensitivity, simple operation, fast response speed, small sample damage and high spatial and temporal resolution, and is a powerful tool for obtaining biochemical information at the cellular and living level. Among them, the orderly assembled fluorescent small molecule probes have better imaging performance than traditional small-molecule fluorescent probes (such as strong anti-diffusion ability, high imaging contrast, and good photostability due to their unique assembly methods and recognition modes). This article summarizes the applications of several common organic dye aggregates and corresponding small molecule fluorescent probes in the field of bioimaging. Subsequently, the design and optimization strategies of organic dye assemblies represented by HPQ and its derivatives with hydrogen-bond-driven ordered assembly and their applications in the biomedical field are highlighted. Finally, the problems existing in the design and application of ordered assembled fluorescent small molecule probes are discussed, which provides ideas for the design of new ordered assembled fluorescent small molecule probes.

Lactosylation leads to a water-soluble fluorescent probe for detection of S2− in water

Detection of sulfide anion (S2−), which is a common environmental pollutant in water and a threat to human health through drinking or food, holds great significance. However, most of existing S2− probes are insensitive in aqueous solution due to poor water-solubility. Here, a water-soluble small molecular fluorescent probe L-CC was first developed via introducing lactose to coumarin derivative (L-C) and then coordinating with copper ions (Cu2+). Water solubility endowed by lactose moiety makes L-CC ultrasensitive toward S2− in water. The fluorescence of L-CC was recovered quickly within 10 s in aqueous solution by displacement of Cu2+ after adding S2− with an extremely low detection limit for S2− measured to be 5.20 nM, which make L-CC suitable for testing S2− spiked in some real water samples. What’s more, fluorescent test strips produced by filter papers embedded with L-CC were capable of visual and quantitative analysis of S2− in water with obvious fluorescent color changes. Thus, this work provides a fluorescent probe with good water solubility endowed by lactosylation as well as its derived fluorescent test strip for simple and sensitive detection of S2− in water, which shows good potential in on-site water quality monitoring.

Violet Phosphorus Quantum Dots as Distinguishable Environmental Biosensors

AbstractA molecular probe is demonstrated to be an effective semi‐invasive technique to measure the local environments of biomaterials. Environmental probes based on fluorescence intensity and lifetime are well studied as temperature sensors. However, usually both fluorescence intensity and lifetime of reported molecular probes depend on various local environments, such as temperature, pH, and ionic strength. It is a big challenge to distinguish the specific environmental effects on the fluorescence. Herein, the violet phosphorus quantum dots (VPQDs) are demonstrated to be effective fluorescence molecular probes to distinguish temperature or pH environment variation. The fluorescence intensity and bandwidth of VPQDs are demonstrated to be linearly correlated to a wide range of temperatures (283–363 K), while the fluorescence lifetime of VPQDs is demonstrated to be temperature‐independent. However, the fluorescence lifetime is demonstrated to be linearly correlated to pH value. Additionally, good water dispersibility, superior photo‐ and thermal stability, and excellent reversibility are also obtained by VPQDs. More importantly, the high sensitivity of fluorescence intensity to temperature instead of pH environments and the high sensitivity of fluorescence lifetime to pH environments instead of temperature allow VPQDs to be distinguishable biosensors to temperature and pH environments.

Synthesis of a dual-emissive pyrene-based fluorescent probe for imaging intracellular viscosity

A pyrene-based fluorescent molecular probe (Py2-5EG) was synthesized for optical detection of viscosity variation. Py2-5EG was synthesized by the reaction of two hydroxyl groups at the two ends of pentaethylene glycol with pyrene via a two-step reaction. Unlike typical pyrene compounds, the monomer fluorescence of Py2-5EG was not observed even in a dilute solution. Instead, the monomer fluorescence gradually increased with the increase in solvent viscosity. Blue fluorescent emission was observed from the monomer in a more viscous medium, and greenish blue emission from excimer was dominant in a less viscous medium in which dynamic excimer formation was involved. The observed fluorescence change of Py2-5EG was ratiometric and depended on the solution viscosity. A microscopic change in cellular viscosity was clearly observed inside and outside the mitochondria of living cells via different fluorescence colors of blue and green. The viscosity inside and outside mitochondria was determined using the fluorescence colors. Py2-5EG featured noticeable fluorescence emission shift in response to viscosity, and this phenomenon can be applied as a fluorescent probe, estimating the viscosity of various solutions as well as inside living cells.

Molecular to Supramolecular Self-Assembled Luminogens for Tracking the Intracellular Organelle Dynamics.

Deciphering the dynamics of intracellular organelles has gained immense attention due to their subtle control over diverse, complex biological processes such as cellular metabolism, energy homeostasis, and autophagy. In this context, molecular materials, including small-organic fluorescent probes and their supramolecular self-assembled nano-/microarchitectures, have been employed to explore the diverse intracellular biological events. However, only a handful of fluorescent probes and self-assembled emissive structures have been successfully used to track different organelle's movements, circumventing the issues related to water solubility and long-term photostability. Thus, the water-soluble molecular fluorescent probes and the water-dispersible supramolecular self-assemblies have emerged as promising candidates to explore the trafficking of the organelles under diverse physiological conditions. In this review, we have delineated the recent progress of fluorescent probes and their supramolecular self-assemblies for the elucidation of the dynamics of diverse cellular organelles with a special emphasis on lysosomes, lipid droplets, and mitochondria. Recent advancement in fluorescence lifetime and super-resolution microscopy imaging has also been discussed to investigate the dynamics of organelles. In addition, the fabrication of the next-generation molecular to supramolecular self-assembled luminogens for probing the variation of microenvironments during the trafficking process has been outlined.

High-fidelity imaging of lysosomal enzyme through in situ ordered assembly of small molecular fluorescent probes

As an organelle in cells, lysosomes play an important role in the degradation of biological macromolecules and pathogens. To elucidate the function of lysosomes in normal or disease states, recently, various fluorescent probes have been reported for imaging lysosomal analytes. However, because of the particularity of the lysosomal environment, most of the reported lysosomal fluorescent probes suffered from a series of practical issues such as easy diffusion, low detection signal-to-background ratio and false signal. To address these issues, based on an optimized in situ ordered assembly solid-state fluorophore HDPQ, we herein put forward a new strategy for the design of lysosomal enzymes probes. As a proof concept, we synthesized a fluorescent probe HDPQ-GLU for lysosomal enzyme β-glucuronidase (GLU). Experiment results displayed that compared with general lysosomal probe, the novel lysosomal probe not only exhibited excellent anti-pH interference ability and high signal-to-noise ratio in aqueous solution, but also has excellent long-term in situ imaging ability in the living system. Using this probe, we have realized high-fidelity and long-term in situ tracking GLU in lysosomes of living cells and evaluated the dynamic changes of GLU during the growth period of zebrafish. We anticipate that the new strategy based on the novel in situ ordered assembly solid-state fluorophore HDPQ may be a potential platform for developing fluorescent probes for high-fidelity imaging of lysosomal enzymes.

A potent estrogen receptor and microtubule specific purine-benzothiazole-based fluorescent molecular probe induces apoptotic death of breast cancer cells

Breast cancer is the most common malignancy in women and is a heterogeneous disease at molecular level. Early detection and specificity are the key prerequisite for the treatment of this deadly cancer. To address these issues attention on the breast cancer specific receptor protein(s) is the most realistic option. Herein estrogen (E) and progesterone (Pg) receptors(R) were considered to design fluorescent molecular probes with possible therapeutic option. We adopted QSAR technique to design a library of benzothiazole-purine hybrid molecules. Molecular docking offers us three screened molecules as most potential. Among these molecules one abbreviated as “CPIB” showed blue fluorescence and detected ER positive cancer cells at 1 nM concentration. At elevated concentration, CPIB induces apoptotic deaths of same cancer cells through targeting intracellular microtubules without affecting normal cells or ER negative cells. CPIB is one of its kind with two-in-one potential of “Detection and Destroy” ability targeting ER positive breast cancer cells.

Multi-dentate chelation induces fluorescence enhancement of pyrene moiety for highly selective detection of Fe(III).

Fluorescence enhancement has great advantages and various promising applications for a fluorescent molecular probe, which shows high sensitivity and high selectivity. In this report, a novel pyrene-based fluorescent probe with multidentate ligand (PPD) was synthesized for highly selective detection of Fe(III), which exhibited great fluorescence enhancement response upon the addition of Fe(III) in aqueous solution of pH 3.5 ~ 7.5, with a detection limit of 115nM. The probe also has good water solubility and photostability. Further fluorescence titration confirmed 1:1 stoichiometric ratio for the probe PPD-Fe(III), which can be applied for quantification of Fe(III). The probe was validated for ferric detection in real water samples by spike and recovery test.