Fluorescence Signal Change Research Articles

BODIPY Based OFF-ON Fluorescent Probe for Endogenous Carbon Monoxide Imaging in Living Cells.

Carbon monoxide (CO) is one of the signaling molecules that are ubiquitous in humans, which involves in the regulation of human physiology and pathology. In this work, the probe PEC was designed and synthesized based on BODIPY fluorophore that can selectively detect CO through reducing the nitro group to amino group, resulting in a "turn-on" fluorescence response with a simultaneous increase in the concentration of CO. The response is selective over a variety of relevant reactive free radicals, ions, and amino acid species. PEC has the advantages of good stability, good water solubility, and obvious changes in fluorescence signals. In addition, PEC can be used to detect and track endogenous CO in living cells.

Naphthalimide-based probe as an in situ indicator of photochemical reaction for self-reporting imidazole ring formation

Fluorescent probes were promising ultrasensitive candidates in the sensing area. Herein, a fluorescent indicator bearing an active photochemical reactive site based on naphthalimide was designed and synthesized. Under UV irradiation, the photocyclization reaction to form imidazole occurred through the intramolecular addition reaction between α-amine and imine. This photochemical reaction was dynamically monitored in real time by significant fluorescence and UV spectrum changes. The reaction mechanism was further verified by NMR, MS, IR and free radical quenching reaction. The photocyclization reactions of analogous substrates based on this mechanism were also monitored by self-reporting spectrum changes.

Cell-Based Assay Approaches for Glycosaminoglycan Synthase High-Throughput Screening: Development and Applications.

Glycosaminoglycan synthases have immense potential in applications involving synthesis of oligosaccharides, using enzymatic approaches and construction of cell factories that produce polysaccharides as critical metabolic components. However, the use of high-throughput activity assays to screen for the evolution of these enzymes can be challenging because there are no significant changes in fluorescence or absorbance associated with glycosidic bond formation. Here, using incorporation of azido-labeled N-acetylhexosamine analogs into bacterial capsule polysaccharides via bacterial metabolism and bioorthogonal chemistry, fluorophores were specifically introduced onto cell surfaces. Furthermore, correlations between detectable fluorescence signals and the polysaccharide-synthesizing capacity of individual bacteria were established. Among 10 candidate genes, 6 members of the chondroitin synthase family were quickly identified in a recombinant Bacillus subtilis host strain. Additionally, directed evolution of heparosan synthase was successfully performed using fluorescence-activated cell sorting of recombinant Escherichia coli O10:K5(L):H4, yielding several mutants with increased activity. Cell-based approaches that selectively detect the presence or absence of synthases within an individual colony of bacterial cells, as well as their level of activity, have broad potential in the exploration and engineering of glycosaminoglycan synthases. These approaches also support the creation of novel strategies for high-throughput screening of enzyme activity based on cell systems.

Cell-Membrane-Localizing Fluorescence Probes for Aminopeptidase N.

Aminopeptidase N (APN), a transmembrane ectoenzyme, plays multifunctional roles in cell survival and migration, angiogenesis, blood pressure regulation, and viral uptake. Abnormally high levels of the enzyme can be found in some tumors and injured liver and kidney. Therefore, noninvasive detection methods for APN are in demand for diagnosing and studying the associated diseases, leading to two dozen activatable small-molecule probes reported up to date. All of the known probes, however, analyze the enzyme activity by monitoring fluorescent molecules inside cells, despite the enzymatic reaction taking place on the outer cell membrane. In this case, different cell permeability and enzyme kinetics can cause false signal data. To address this critical issue, we have developed two cell-membrane-localizing APN probes whose enzymatic products also localize the outer cell membrane. The probes selectively respond to APN with ratiometric fluorescence signal changes. A selected probe, which has two-photon imaging capability, allowed us to determine the relative APN levels in various organ tissues for the first time: 4.3 (intestine), 2.1 (kidney), 2.7 (liver), 3.2 (lung), and 1.0 (stomach). Also, a higher APN level was observed from a HepG2-xenograft mouse tissue in comparison with the normal tissue. Furthermore, we observed a significant APN level increase in the mouse liver of a drug (acetaminophen)-induced liver injury model. The probe thus offers a reliable means for studying APN-associated biology including drug-induced hepatotoxicity simply by ratiometric imaging.

A novel exonuclease III‐based turn‐on fluorescent aptasensor for aflatoxin B1 detection

AbstractBackgroundAflatoxin B1 (AFB1) is strongly carcinogenic and has been classified as a class I carcinogenic compound by the International Agency for Research on Cancer. AFB1 is widely contaminated in wheat, peanuts, corn and other crops and their products, posing a great threat to human health.ObjectiveTo establish a rapid, safe and accurate fluorescent aptasensor for sensitive detection of AFB1 in food.MethodsIn this study, single‐stranded DNA (S1) labeled with black hole quencher BHQ1 and carboxyfluorescein FAM at both ends was used as a signal probe. Nucleic acid exonuclease III (EXO‐III) was introduced as a signal amplification strategy. The basic principle is to exploit the fluorescence resonance energy transfer (FRET) effect generated between FAM and BHQ1. The fluorescent signal change of FAM in the presence or absence of the target is used for the quantitative detection of AFB1. And on this basis, the factors affecting the sensitivity of the sensor are explored in depth.ResultsUnder optimal experimental conditions, the sensor showed a good linear relationship between LgCAFB1 and relative fluorescence intensity in the range of 0.05‐500 ng/mL. The detection limit was as low as 18.2 pg/mL. In particular, the best performance of the aptasensor was achieved when the ratio of cDNA to Apt was 1.2 and the amount of EXO‐III was 25 U.ConclusionsThe combination of MBs, dual‐labeled S1 probes and Exo‐III was used to develop an accurate and sensitive fluorescent aptasensor for AFB1 detection. The developed aptasensor has satisfied recovery and selectivity in AFB1 detection of corn flour and wine samples, indicating that the designed aptasensor has the potential to be used in detecting AFB1 of food samples.

Biosensors Based on Inorganic Composite Fluorescent Hydrogels.

Fluorescent hydrogels are promising candidate materials for portable biosensors to be used in point-of-care diagnosis because (1) they have a greater capacity for binding organic molecules than immunochromatographic test systems, determined by the immobilization of affinity labels within the three-dimensional hydrogel structure; (2) fluorescent detection is more sensitive than the colorimetric detection of gold nanoparticles or stained latex microparticles; (3) the properties of the gel matrix can be finely tuned for better compatibility and detection of different analytes; and (4) hydrogel biosensors can be made to be reusable and suitable for studying dynamic processes in real time. Water-soluble fluorescent nanocrystals are widely used for in vitro and in vivo biological imaging due to their unique optical properties, and hydrogels based on these allow the preservation of these properties in bulk composite macrostructures. Here we review the techniques for obtaining analyte-sensitive fluorescent hydrogels based on nanocrystals, the main methods used for detecting the fluorescent signal changes, and the approaches to the formation of inorganic fluorescent hydrogels via sol-gel phase transition using surface ligands of the nanocrystals.

Fluorescence-Based Portable Assays for Detection of Biological and Chemical Analytes.

Fluorescence-based detection techniques are part of an ever-expanding field and are widely used in biomedical and environmental research as a biosensing tool. These techniques have high sensitivity, selectivity, and a short response time, making them a valuable tool for developing bio-chemical assays. The endpoint of these assays is defined by changes in fluorescence signal, in terms of its intensity, lifetime, and/or shift in spectrum, which is monitored using readout devices such as microscopes, fluorometers, and cytometers. However, these devices are often bulky, expensive, and require supervision to operate, which makes them inaccessible in resource-limited settings. To address these issues, significant effort has been directed towards integrating fluorescence-based assays into miniature platforms based on papers, hydrogels, and microfluidic devices, and to couple these assays with portable readout devices like smartphones and wearable optical sensors, thereby enabling point-of-care detection of bio-chemical analytes. This review highlights some of the recently developed portable fluorescence-based assays by discussing the design of fluorescent sensor molecules, their sensing strategy, and the fabrication of point-of-care devices.

A dual-site fluorescent probe for discriminately detecting low and high concentration of hypochlorite in living cells

Hypochlorite (ClO−) is an important bioactive molecule of living system which plays essential roles in many physiological and pathological processes. There is no doubt that the biological roles of ClO− depend highly on the concentration of ClO−. Unfortunately, the relationship between the concentration of ClO− and the biological process is unclear. Toward this purpose, in this work we addressed a core challenge for developing a powerful fluorescence tool for monitoring a wide concentration change (0–14 eq.) of ClO− via two distinct detection manners. The probe displayed fluorescence variation (red to green) upon addition of ClO− (0–4 eq.) and the color of test medium changed from red to colorless witnessed by the naked eyes. Surprisingly, in the presence of higher concentration of ClO− (4–14 eq.), the probe displayed another fluorescent signal change from green to blue. After demonstrating the excellent sensing properties of the probe with ClO− in vitro, it was successfully used to imaging different concentration of ClO− in living cells. We expected that the probe could act as an exciting chemistry tool for imaging of ClO− concentration-dependent oxidative stress event in biological system.

Rapid and accurate sensing platform based on lanthanide metal-organic framework and aptamer for Vibrio parahaemolyticus diagnosis

In this study, a rapid visual analysis strategy for pathogens was constructed based on lanthanide metal–organic frameworks (LnMOFs) and FAM-aptamer. The dual fluorescence signal changes were realized by the competitive binding effect of the target and LnMOFs on the FAM-aptamer. The nitrocellulose membrane (NC membrane) is used as the signal output platform, and the G/R value in Red, Green, Blue (RGB) of negative and positive signals is collected to output the detection results. Using Vibrio parahaemolyticus in seafood as model target, the detection performance of dual fluorescence sensing strategy was verified. Under the optimal conditions, the G/R difference between the positive and negative signals and the concentration of Vibrio parahaemolyticus was linear in the range of 102-105 colony forming units (CFU)/mL, and the detection limit was 102 CFU/mL. The method is simple to operate and the detection time can be controlled within 20 min. It can be developed as a portable detection kit, providing a new technical means for the safety and quality control of food supply chain.

A Handy Chemical Sensor Based on Benzaldehyde and Imidazo[1,2-a]pyridine Mixture for Naked-eye Colorimetric and Fluorescent Detection of F.

Upon the Schiff base condensation reaction of imidazo[1,2-a]pyridine-2-carbohydrazide and 2,5-dihydroxybenzaldehyde, a bimodal colorimetric and fluorescent chemosensor 1o for assaying fluoride (F-) in DMSO was synthesized. The characterization of 1o structure was obtained by 1H NMR, 13C NMR and MS.The structure of 1o was characterized by 1H NMR, 13C NMR and MS. Under the presence of various anions, 1o could be applied for naked-eye and fluorescent detection of F- (naked eye: colorless to yellow; fluorescence: dark to green) and displayed promising performance, such as high selectivity and sensitivity, as well as a low detection limit. Upon calculation, the detection limit of chemosensor 1o for F- was 193.5nM, which is well below the allowed maximum value of F- (1.5mg/L) by WHO. As the intermolecular proton transfer mechanism induced "turn-on" fluorescent signal and naked-eye color change of F- to 1o through deprotonation effect, which was confirmed by Job's plot curve, mass spectrometry and 1H NMR titration. Alternatively, the chemosensor 1o can be effectively manufactured into test strips to detect fluoride in solid state, which is user-friendly with no additional equipment required.

H2S-activated fluorescent probe enables dual-channel fluorescence tracking of drug release in tumor cells

Nowadays, the selective release of therapeutic drugs into tumor cells has become an important way of tumor treatment due to the high side effects of chemotherapy drugs. As one of the gas mediators, hydrogen sulfide (H2S) is closely related to cancer. Due to the high content of H2S in tumor cells, it can be used as a signaling molecule that triggers the release of drugs to achieve the selective release of therapeutic drugs. In addition, dual-channel fluorescence imaging technology can be better applied to monitor the drug delivery process and distinguish the state before and after drug release, so as to better track the effect of drug therapy. Based on this, we used NBD amines (NBD-NHR) as the recognition group of H2S and connected the tyrosine kinase inhibitor crizotinib to construct an activated dual-channel fluorescent probe CZ-NBD. After the probe enters the tumor cells, it consumes H2S and releases crizotinib, which is highly toxic to the tumor cells. Importantly, the probe displays significant fluorescence changes in different cells, enabling not only the screening of tumor cells, but also tracking and monitoring drug release and tumor cell activity. Therefore, the construction of probe CZ-NBD provides a new strategy for drug release monitoring in tumor cells.

A naphthalimide functionalized fluoran with AIE effect for ratiometric sensing Hg2+ and cell imaging application

The pollution caused by mercury ions (Hg2+) poses a potential threat to public health. Therefore, monitoring Hg2+ concentration in the environment is necessary and significant. In this work, a naphthalimide functionalized fluoran dye NAF has been prepared, which shows a new red-shift in emission at 550 nm with the maximum intensity in a mixture of water-CH3CN (v/v = 7/3) due to aggregating induced emission (AIE) effect. Meanwhile, NAF can be employed as a Hg2+ ions sensor, which displays a selective and sensitive response to Hg2+ ions by the reduced fluorescence of naphthalimide fluorophore and increased fluorescence of fluoran group, respectively, showing ratiometric fluorescence signal changes with more than 65-fold emission intensity ratio increase and naked eyes visible color change. In addition, the response time is fast (within 1 min) and the sensing can be conducted in a wide pH range (4.0–9.0). Moreover, the detection limit has been evaluated to be 5.5 nM. The sensing mechanism may be attributed to the formation of a π-extended conjugated system due to the Hg2+ ions-induced conversion of spironolactone to the ring-opened form, partially accompanied by the fluorescence resonance energy transfer (FRET) process. Significantly, NAF exhibits suitable cytotoxicity to living HeLa cells, which allows it to be utilized for ratiometric imaging of Hg2+ ions assisted by confocal fluorescence imaging.

Visualizing Efficacy of Antineoplastic Drug via Ratiometric Near-Infrared Fluorescence Imaging of Tumor-Associated Macrophage-Specific Nitric Oxide.

Elevated nitric oxide (NO) within tumor-associated macrophages (TAMs) suggests a reduction of TAM-mediated tumoral immune tolerance. This cellular event could be a reliable indicator for efficacy evaluation of antineoplastic drugs. However, a suitable method for TAM-specific NO measurement is still lacking. In this work, a simple and fast efficacy evaluation method for antineoplastic drugs is established based on a ratiometric TAM-specific NO near-infrared (NIR) fluorescence probe TAM-Cy-NO. Molecular fluorescence probe Cy-NO for NO response was encapsulated in the TAM-targeting peptide (M2pep)-functionalized liposome to construct TAM-Cy-NO. After TAM enters through M2pep, Cy-NO reacts with NO specifically, resulting in a dose-dependent ratiometric fluorescence signal (I 610/I 815) change manner. Utilizing this strategy, we observed that PLX-3397, metformin, and ibrutinib triggered NO generation within TAM greater than that with sorafenib. Notably, metformin and ibrutinib promoted TNF-α and reduced PD-L1 expressions, which suggest reductions of TAM-mediated immunosuppression. As expected, these drugs delayed tumor progression in mice. This method provides a promising efficacy evaluation strategy for rapid screening of antineoplastic drugs.

Shining light on plant health: Detecting salt stress with a near-infrared fluorescent probe

Salt stress remains a significant challenge for crop growth and food security. The development of effective analytical tools for salt stress-related studies is of great importance. Recently, Yang and Yin et al. have developed a novel biosensor that effectively traces salt stress in plants, based on salt-induced molecular J-aggregation and the corresponding changes in fluorescence signals.

Fluorescence Sensing of Monosaccharides by Bis-boronic Acids Derived from Quinolinium Dicarboxamides: Structural and Spectroscopic Studies.

Three new diboronic acid-substituted bisquinolinium salts were synthesized, structurally described by single-crystal X-ray diffraction, and studied in-depth as fluorescent receptors for six monosaccharides and two open-chain polyols in water at physiological pH. The dicationic pyridine-2,6-dicarboxamide-based receptors contain two N-quinolinium rings as the fluorescent units covalently linked to three different isomers of phenylboronic acid (ortho, 2; meta, 3; and para, 4) as chelating binding sites for polyols. Additions of glucose/fructose in the micromolar concentration range to receptors 2 and 3 induce significant fluorescence changes, but in the presence of arabinose, galactose, mannose, and xylose, only modest optical changes are observed. This optical change is attributed to a static photoinduced electron transfer mechanism. The meta-diboronic receptor 3 exhibited a high affinity/selectivity toward glucose (K = 3800 M-1) over other monosaccharides including common interfering species such as fructose and mannitol. Based on multiple spectroscopic tools, electrospray ionization high-resolution mass spectrometry, crystal structures, and density functional theory calculations, the binding mode between 3 and glucose is proposed as a 1:1 complex with the glucofuranose form involving a cooperative chelating diboronate binding. These results demonstrate the usefulness of a new set of cationic fluorescent diboronic acid receptors with a strong ability for optical recognition of glucose in the sub-millimolar concentration range.

Light activated simultaneous release and recognition of biological signaling molecule carbon monoxide (CO).

The therapeutic action of carbon monoxide (CO) is very well known and has been studied on various types of tissues and animals. However, real-time spatial and temporal tracking and release of CO is still a challenging task. This paper reported an amphiphilic CO sensing probe NP and phospholipid 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) based nanoscale vesicular sensing system Ves-NP consisting of NP. The liposomal sensing system (Ves-NP) showed good selectivity and sensitivity for CO without any interference from other relevant biological analytes. Detection of CO is monitored by fluorescence OFF-ON signal. Ves-NP displayed LOD of 5.94µM for CO detection with a response time of 5min. Further, in a novel attempt, Ves-NP is co-embedded with the amphiphilic CO-releasing molecule 1-Mn(CO)3 to make an analyte replacement probe Ves-NP-CO. Having a both CO releasing and sensing moiety at the surface of the same liposomal system Ves-NP-CO play a dual role. Ves-NP-CO is used for the simultaneous release and recognition of CO that can be controlled by light. Thus, in this novel approach, for the first time we have attached both the release and recognition units of CO in the vesicular surface, both release and recognition simultaneously monitored by the change in fluorescent OFF-ON signal.

Precise peripheral design enables propeller-like squaraine dye with highly sensitive and wide-range piezochromism.

Piezochromic fluorescent (PCF) materials that feature high sensitivity and wide-range switching are attractive in intelligent optoelectronic applications but their fabrication remains a significant challenge. Here we present a propeller-like squaraine dye SQ-NMe2 decorated with four peripheral dimethylamines acting as electron donors and spatial obstacles. This precise peripheral design is expected to loosen the molecular packing pattern and facilitate more substantial intramolecular charge transfer (ICT) switching caused by conformational planarization under mechanical stimuli. As such, the pristine SQ-NMe2 microcrystal exhibits significant fluorescence changes from yellow (λem = 554 nm) to orange (λem = 590 nm) upon slight mechanical grinding and further to deep red (λem = 648 nm) upon heavy mechanical grinding. Single-crystal X-ray diffraction structural analysis of two SQ-NMe2 polymorphs provides direct evidence to illustrate the design concept of such a piezochromic molecule. The piezochromic behavior of SQ-NMe2 microcrystals is sensitive, high-contrast, and easily reversible, enabling cryptographic applications.

A mitochondria-targetable fluorescent probe for sulfur dioxide detection and visualisation in living cells

Sulfur dioxide (SO2) is a one of reactive sulfur species (RSS) that plays significant roles in many physiological processes. While abnormal levels of SO2 in mitochondria have been related to various diseases. Hence, developing suitable fluorescent probe for monitoring SO2 is significant in living organisms. In this research, we designed and synthesized a mitochondrial-target probe Mito-NPH featuring the graft of a strong electron-withdrawing 4-pyridiniumylacrylonitrile unit to an electron-donating naphthalenic unit that intramolecular charge transfer (ICT) process happened. The probe Mito-NPH underwent a nucleophilic addition of HSO3−/SO32−to give fluorescent emission signal change from red to blue and exhibited specific response toward HSO3−/SO32−over other analytes. Moreover, Mito-NPH showed ultrafast response rate (within 10 s) for HSO3−. Importantly, cell imaging results demonstrated that the probe can sense endogenous SO2 in mitochondria.

Activity-Based Imaging of Macrophage Migration Inhibitory Factor with a Two-Photon Fluorescent Probe.

Macrophage migration inhibitory factor (MIF), as a cytokine, plays an important role in the pathogenesis of cancer and some other diseases, and it is also one of the potential drug targets for disease treatment. However, due to the lack of simple and effective MIF imaging detection tools, the fluctuation and distribution of MIF in living cells or at lesion sites remain difficult to track precisely and in real time. Here, we report activity-based fluorescent probes, named MIFP1-MIFP3, which are used for real-time imaging and tracking of intracellular MIF, thus establishing a relationship between the fluctuation of MIF and the change of fluorescence signal during the cancer disease process. With the excellent optical properties of two-photon probe imaging, we can easily distinguish multiple cancer cells from normal cells with the representative probe, MIFP3. Moreover, MIFP3 has also been successfully used to directly identify the pathological tissues of patients with clinical liver cancer. These potential MIF probes could provide powerful tools for further study of the physiological function of MIF and will be helpful to promote the accurate diagnosis and therapeutic evaluation of MIF-associated malignancies.

Redox-Controlled Energy Transfer Quenching of Fluorophore-Labeled DNA SAMs Enables In Situ Study of These Complex Electrochemical Interfaces.

Interfaces modified by a molecular monolayer can be challenging to study, particularly in situ, requiring novel approaches. Coupling electrochemical and optical approaches can be useful when signals are correlated. Here we detail a methodology that uses redox electrochemistry to control surface-based fluorescence intensity for detecting DNA hybridization and studying the uniformity of the surface response. A mixed composition single-strand DNA SAM was prepared using potential-assisted thiol exchange with two alkylthiol-modified ssDNAs that were either labeled with a fluorophore (AlexaFluor488) or a methylene blue (MB) redox tag. A significant change in fluorescence was observed when reducing MB to colorless leuco-MB. In situ fluorescence microscopy on a single-crystal gold bead electrode showed that fluorescence intensity depended on (1) the potential controlling the oxidation state of MB, (2) the surface density of DNA, (3) the MB:AlexFluor488 ratio in the DNA SAM, and (4) the local environment around the DNA SAM. MB efficiently quenched AlexaFluor488 fluorescence. Reduction of MB showed a significant increase in fluorescence resulting from a decrease in quenching or energy transfer efficiency. Hybridization of DNA SAMs with its unlabeled complement showed a large increase in fluorescence due to MB reduction for surfaces with sufficient DNA coverage. Comparing electrochemical-fluorescence measurements to electrochemical (SWV) measurements showed an improvement in detection of a small fraction of hybridized DNA SAM for surfaces with optimal DNA SAM composition and coverage. Additionally, this coupled electrochemical redox-fluorescence microscopy method can measure the spatial heterogeneity of electron-transfer kinetics and the influence of the local interfacial environment.