Small Fluorescent Probes Research Articles

Small molecular fluorescent probes : application progress of specific bacteria detection and antibacterial phototherapy.

Bacterial infection is one of the leading causes of death worldwide and is easy to cause large-scale diseases. It is an urgent need to develop effective methods for the specific detection and treatment of bacterial infections. Recently, small molecular fluorescent probes, bridging the capability of imaging detection and sterilization, have attracted increasing attention. Fluorescence imaging assays have the benefit of being simple, specific, and fast, which is very advantageous in both in vitro and in vivo bacterial detection. Molecularly fluorophores for theranostics provide advantages of non-invasion, high specificity, and fewer side effects. In this review, we summarize the recent advances and design strategies of small molecular fluorescent probes for both targeted detection and treatment of bacteria. We hope that this review will provide guidance for the development of more effective fluorescent dyes in the future as well as encourage preclinical and clinical studies of phototherapy-mediated antimicrobial therapy.

A coumarin-based small molecular fluorescent probe for detection of the freshness of meat and shrimp

A coumarin-based small molecular fluorescent probe for detection of the freshness of meat and shrimp

Recent Advances in Small Molecular Fluorescence Probes for Fatty Liver Diseases

Fatty liver diseases are a spectrum of liver disorders consisting of the benign fatty liver, which could eventually lead to cirrhosis or even hepatocellular cancer (HCC) without timely treatment. Therefore, early diagnosis is crucial for fatty liver diseases. Liver biopsy is regarded as the gold standard in the diagnosis of fatty liver diseases. However, it is not recommended for routine use due to its invasiveness and complicated operation. Thus, it is urgent to diagnose fatty liver diseases with non-invasive and precise methods. In this regard, fluorescence imaging technology has attracted intensive attention and become a robust non-invasive method for fatty liver visualization, and a series of fluorescent probes are being intensively designed to track the biomarkers in fatty liver. In this brief review, the small molecular fluorescent probes employed in fatty liver are summarized, mainly focusing on the last four years. Moreover, current opportunities and challenges in the development of fluorescent probes for fatty liver will be highlighted.

Two-photon fluorescence imaging and specifically biosensing of norepinephrine on a 100-ms timescale

Norepinephrine (NE) is a key neurotransmitter in the central nervous system of organisms; however, specifically tracking the transient NE dynamics with high spatiotemporal resolution in living systems remains a great challenge. Herein, we develop a small molecular fluorescent probe that can precisely anchor on neuronal cytomembranes and specifically respond to NE on a 100-ms timescale. A unique dual acceleration mechanism of molecular-folding and water-bridging is disclosed, which boosts the reaction kinetics by ˃105 and ˃103 times, respectively. Benefiting from its excellent spatiotemporal resolution, the probe is applied to monitor NE dynamics at the single-neuron level, thereby, successfully snapshotting the fast fluctuation of NE levels at neuronal cytomembranes within 2 s. Moreover, two-photon fluorescence imaging of acute brain tissue slices reveals a close correlation between downregulated NE levels and Alzheimer’s disease pathology as well as antioxidant therapy.

A guide to small fluorescent probes for single-molecule biophysics

The explosive growth of single-molecule techniques is transforming our understanding of biology, helping to develop new physics inspired by emergent biological processes, and leading to emerging areas of nanotechnology. Key biological and chemical processes can now be probed with new levels of detail, one molecule at a time, from the nanoscopic dynamics of nature's molecular machines to an ever-expanding range of exciting applications across multiple length and time scales. Their common feature is an ability to render the underlying distribution of molecular properties that ensemble averaging masks and to reveal new insights into complex systems containing spatial and temporal heterogeneity. Small fluorescent probes are among the most adaptable and versatile for single-molecule sensing applications because they provide high signal-to-noise ratios combined with excellent specificity of labeling when chemically attached to target biomolecules or embedded within a host material. In this review, we examine recent advances in probe designs, their utility, and applications and provide a practical guide to their use, focusing on the single-molecule detection of nucleic acids, proteins, carbohydrates, and membrane dynamics. We also present key challenges that must be overcome to perform successful single-molecule experiments, including probe conjugation strategies, identify tradeoffs and limitations for each probe design, showcase emerging applications, and discuss exciting future directions for the community.

Recent progress in the development of small-molecule fluorescent probes for detection and imaging of selenocysteine and application in thyroid disease diagnosis.

Selenocysteine (SeCys) is the 21st genetically encoded amino acid present in proteins and is involved in various biological functions. Inappropriate levels of SeCys can be considered as a sign of various diseases. Therefore, small molecular fluorescent probes for the detection and imaging of SeCys in vivo in biological systems are considered to be of significant interest for understanding the physiological role of SeCys. Thus, this article mainly provides a critical evaluation of recent advances made in SeCys detection along with the biomedical applications based on small molecular fluorescent probes published in the literature during the past half a dozen years. Therefore, the article primarily deals with the rational design of fluorescent probes, wherein these were selective towards SeCys over other biologically abundant molecules, in particular the thiol-based ones. The detection has been monitored by different spectral techniques, such as fluorescence and absorption spectroscopy and in some cases even visual color changes. Further, the detection mechanism and the utility of fluorescent probes for in vitro and in vivo cell imaging applications are addressed. For clarity, the main features have been conveniently divided into four categories based on the chemical reactions of the probe, viz., in terms of the cleavage of the responsive group by the SeCys nucleophile: (i) 2,4-dinitrobene sulphonamide group, (ii) 2,4-dinitrobenesulfonate ester group, (iii) 2,4-dinitrobenzeneoxy group and (iv) miscellaneous types. Overall this article deals with the analysis of more than two dozen fluorescent probes demonstrated for selective detection of SeCys along with their applications towards disease diagnosis.

Improved synthesis of DA364, an NIR fluorescence RGD probe targeting αvβ3 integrin.

Optical imaging (OI) is gaining increasing attention in medicine as a non-invasive diagnostic imaging technology and as a useful tool for image-guided surgery. OI exploits the light emitted in the near-infrared region by fluorescent molecules able to penetrate living tissues. Cyanines are an important class of fluorescent molecules and by their conjugation to peptides it is possible to achieve optical imaging of tumours by selective targeting. We report here the improvements obtained in the synthesis of DA364, a small fluorescent probe (1.5 kDa) prepared by conjugation of pentamethine cyanine Cy5.5 to an RGD peptidomimetic, which can target tumour cells overexpressing integrin αvβ3 receptors.

Two-photon excited red-green "discoloration" bioprobes for monitoring lipid droplets and lipid droplet-lysosomal autophagy.

Lipid droplets (LDs) and their autophagy by lysosomes are closely related to a variety of physiological and pathological conditions. Therefore, identifying and tracking LDs and the dynamic process of autophagy can provide useful information for the diagnostics and treatment of related diseases. However, few organic small molecule-based fluorescent probes can specifically recognize LDs and dynamically track their autophagy process. Herein, we synthesized a "discoloration" fluorescent bioprobe DPABP-BI with distinguishable features including red fluorescence emission (630 nm), large Stokes shift (145 nm), two-photon excitation and outstanding photostability and biocompatibility. In particular, LDs could be specifically identified via the red fluorescence emission of DPABP-BI (colocalization constant of 0.98), while autophagolysosomes could be visualized via the green fluorescence emission of its acid-hydrolyzed product (colocalization constant of 0.90) to track the autophagy dynamic process. In addition, DPABP-BI enabled the specific recognition of fatty substances in zebrafish larvae. In this study, a two-photon excited red light small molecule probe was constructed to identify LDs and track their autophagy dynamic process by changing the fluorescence emission wavelength.

New insight into the application of fluorescence platforms in tumor diagnosis: From chemical basis to clinical application.

Early and rapid diagnosis of tumors is essential for clinical treatment or management. In contrast to conventional means, bioimaging has the potential to accurately locate and diagnose tumors at an early stage. Fluorescent probe has been developed as an ideal tool to visualize tumor sites and to detect biological molecules which provides a requirement for noninvasive,real-time, precise, and specific visualization of structures and complex biochemical processes in vivo. Rencently, the development of synthetic organic chemistry and new materials have facilitated the development of near-infrared small molecular sensing platforms and nanoimaging platforms. This provides a competitive tool for various fields of bioimaging such as biological structure and function imaging, disease diagnosis, in situ at the in vivo level, and real-time dynamic imaging. This review systematically focused on the recent progress of small molecular near-infrared fluorescent probes and nano-fluorescent probes as new biomedical imaging tools in the past 3-5years, and it covers the application of tumor biomarker sensing, tumor microenvironment imaging, and tumor vascular imaging, intraoperative guidance and as an integrated platform for diagnosis, aiming to provide guidance for researchers to design and develop future biomedical diagnostic tools.

Recent progress on small molecular temperature-sensitive fluorescent probes.

Temperature is an important biophysical parameter that is closely related with the metabolic activity in living cells. Therefore, the detection of intracellular temperature changes is crucial for exploring temperature-related biological processes. Fluorescence probe is an ideal tool for observing temperature changes in cells, which has many advantages, such as high sensitivity, good selectivity, and noninvasive, and thus aroused the great interest of researchers. In this paper, we summarize the recent progress of organic small molecule temperature-sensitive fluorescence probes in recent years was reviewed. Particularly, we describe the common response mode to the temperature and the practical applications of the probe in living cells and even animal models. Moreover, an outlook regarding temperature detection in clinical applications is discussed. The temperature-sensitive fluorescent probe is a "black box" to many researchers. This review aims to open a window on the prospect of the noninvasive in vivo detection of temperature which is helpful to deeper understand this rich research area.

Photophysical and Fluorescence Nitroaromatic Sensing Properties of Methylated Derivative of a Pamoic Acid Ester.

Rapid and selective detection of nitroaromatic explosives is very important for public safety, life, and environmental health. Current instrumental techniques suffer from high cost and poor site used. In order to investigate fluorescence sensing of nitroaromatics, we prepare a new small fluorescence probe derived from pamoic acid. This study covers the synthesis of Pamoic acid based [diisopropyl 4,4'-methylenebis(3-methoxy-2-naphthoate)] (2) material and characterization of its structure. The methylation of Pamoic acid ester, which we have successfully synthesized in our previous studies, was carried out in this study. Determination of the photophysical and fluorescent nitroaromatic detection properties of the compound forms the basis of the study. Structural characterization of the synthesized compound [diisopropyl 4,4'-methylenebis(3-methoxy-2-naphthoate)] (2) was characterized using spectroscopic methods. In addition, Molecular structure of the synthesized compound was determined by single crystal X-ray diffraction studies. In the final step, compounds [diisopropyl 4,4'-methylenebis(3-hydroxy-2-naphthoate)] (1) and [diisopropyl 4,4'-methylenebis(3-methoxy-2-naphthoate)] (2) were tested as fluorescent probes for the detection of some nitroaromatic explosives. It is seen that Nitrobenzene provides the best quenching effect on the compound [diisopropyl 4,4'-methylenebis(3-hydroxy-2-naphthoate)] (1) containing the -OH group, with lowest the limit of detection (LOD) value. It was observed that Picric acid provided the best quenching effect with lowest the limit of detection (LOD) value in the compound [diisopropyl 4,4'-methylenebis(3-methoxy-2-naphthoate)] (2) obtained by methylation of the -OH group in the compound [diisopropyl 4,4'-methylenebis(3-hydroxy-2-naphthoate)] (1).

Cell-imaging studies of highly substituted oxazole derivatives as organelle targeting fluorophores (OTFPs)

The field of biomedical science has progressed enormously in the past decade. With the advent of newer diagnostic tools for imaging and detection, identification of root cause of a disease is now remarkably accurate and specific. Small organic fluorophores in these connections are in great demand currently for cellular organelle sensing and detecting, due to their non-invasiveness, excellent accuracy and bio-sensitivity. Small molecule fluorescence probes offer most potent area for biological sensing with diagnostic imaging ability. These organelle targetable fluorescent probes are produced through synthetic manipulations to get the desired, decent fluorescence properties. When a suitable organelle specific functional group is installed within these highly fluorescent scaffolds, then these molecules turn out to be as lysotracker, mitotracker and cytoplasm-stainer in mammalian cells with high efficiencies (high Pearson co-efficient factors). The present work demonstrated an environmentally benign (green) one-pot, sp3 C–H functionalization of highly substituted oxazole derivatives with excellent photophysical properties. These molecules were further modified by installing organelle specific targetable groups (sensors/detectors) which selectively localize in specific intra-cellular organelles.

Benzothiazole Derivatives Targeting G‐Quadruplex DNA: Synthesis, DNA Interaction and Living Cell Imaging

AbstractThe small molecule fluorescence probes for G‐quadruplexes (G4) play a vital role in the detection of G4. In this work, two novel D−π−A benzothiazole derivatives were synthesized, and their interactions with different secondary structure DNA, cytotoxicity, and intracellular distribution were investigated in detail. Both compounds have a high affinity and about 100‐fold turn‐on fluorescence response to the promoter G4 in buffer solution, moreover, the compound also stabilizes effectively the promoter G4 structure. Besides, both compounds have no influence on the conformation of the promoter G4, but they induce the folding of bcl‐2 from the coiled chain into the mixed‐type G4 in salt‐free buffer solution. The binding stoichiometries of the compound to bcl‐2, c‐kit2, and c‐myc G4 are near 2 : 1, 1 : 1, and 3 : 1, respectively, and the interactions are an enthalpy‐driven process. Furthermore, both compounds inhibit the proliferation of HepG2 cells, with IC50 values of 3.23 and 38.9 μM. The compound mainly distributes in the cytoplasm of living cells and might interact with DNA. The current results provide the valuable information for design of fluorescence probes targeting G4 in the living cell.

Human-T-Cell-Selective Fluorescent Probe.

The identification of T and B lymphocytes has relied on using antibodies against different biomarkers as the gold standard. Emerging small molecule-based fluorescent probes have the potential to replace antibodies. Herein, we report the first human-T-cell-selective fluorescent probe, Mito thermo yellow (MTY), achieving the live T cells’ distinction from B cells, which was previously impossible without the help of antibodies. The unexpected cell selectivity of MTY is attributed to the higher mitochondria mass and membrane potential of T cells over B cells. This study enriches the toolbox for live cell distinction from complex cell communities.

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.

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.

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.

Ratiometric detection and imaging of endogenous alkaline phosphatase activity by fluorescein-coumarin-based fluorescence probe

Alkaline phosphatase (ALP) is a type of enzyme that widely exists in various tissues of the human body; it plays an important role in regulating many cell functions. The development of a sensitive and accurate tool to detect the changes of ALP activity in organisms can contribute to research in the fields of biochemistry, cytology, clinical medicine, etc. In this paper, a small organic molecule-based ratiometric fluorescent probe (FCP) was designed based on the hydroxyl electron-donating group in fluorescein-coumarin protected by the phosphate group. ALP can trigger the fluorescence change through the enzyme-catalyzed cleavage of phosphoryl ester groups, and the ratio of ALP can be measured at wavelengths of 465 nm and 530 nm. The probe had high selectivity and sensitivity to ALP, and the detection limit measured under the optimal conditions in an aqueous medium reached 0.006 mU/mL. The ALP activity of human serum samples was determined using the probe and found to be in good agreement with that measured using commercial ALP kits. Finally, the probe was also successfully applied to image ALP in living hepatocytes with good selectivity and sensitivity.

Intracellular Physical Properties with Small Organic Fluorescent Probes: Recent Advances and Future Perspectives.

The intracellular physical parameters i. e., polarity, viscosity, fluidity, tension, potential, and temperature of a live cell are the hallmark of cellular health and have garnered immense interest over the past decade. In this context, small molecule organic fluorophores exhibit prominent useful properties including easy functionalizability, environmental sensitivity, biocompatibility, and fast yet efficient cellular uptakability which has made them a popular tool to understand intra-cellular micro-environmental properties. Throughout this discussion, we have outlined the basic design strategies of small molecules for specific organelle targeting and quantification of physical properties. The values of these parameters are indicative of cellular homeostasis and subtle alteration may be considered as the onset of disease. We believe this comprehensive review will facilitate the development of potential future probes for superior insight into the physical parameters that are yet to be quantified.

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.