Fluorescence Of Fluorophore Research Articles

Spontaneous Raman spectroscopy for intracranial tumors diagnostics ex vivo

Neurosurgery of intracranial tumors, especially of glial origin, is a non-trivial task due to their infiltrative growth. In recent years, optical methods of intraoperative navigation have been actively used in neurosurgery. However, one of the most widely used approaches based on the selective accumulation of fluorescent contrast medium (5-ALA-induced protoporphyrin IX) by the tumor cannot be applied to a significant number of tumors due to its low accumulation. On the contrary, Raman spectroscopy, which allows analyzing the molecular composition of tissues while preserving all the advantages of the method of fluorescence spectroscopy, does not require the use of an exogenous dye and may become a method of choice when composing a system for intraoperative navigation or optical biopsy. This work presents the first results of using the principal component method to classify Raman spectra of human glioblastoma with intermediate processing of spectra to minimize possible errors from the fluorescence of both endogenous fluorophores and photosensitizers used in fluorescence navigation. As a result, differences were found in the principal component space, corresponding to tissue samples with microcystic components, extensive areas of necrosis, and foci of fresh hemorrhages. It is shown that this approach can serve as the basis for constructing a system for automatic intraoperative tissue classification based on the analysis of Raman spectra.

Recent Progress of Hybrid Optical Probes for Neural Membrane Potential Imaging.

The neural membrane potential of nerve cells is the basis of neural activity production, which controls advanced brain activities such as memory, emotion, and learning. In the past decades, optical voltage indicator has emerged as a promising tool to decode neural activities with high-fidelity and excellent spatiotemporal resolution. In particular, the hybrid optical probes can combine the advantageous photophysical properties of different components such as voltage-sensitive molecules, highly fluorescent fluorophores, membrane-targeting tags, and optogenetic materials, thus showing numerous advantages in improving the photoluminescence intensity, voltage sensitivity, photostability, and cell specificity of probes. In this review, the current state-of-the-art hybrid probes are highlighted, that are designed by using fluorescent proteins, organic dyes, and fluorescent nanoprobes as the fluorophores, respectively. Then, the design strategies, voltage-sensing mechanisms and the in vitro and in vivo neural activity imaging applications of the hybrid probes are summarized. Finally, based on the current achievements of voltage imaging studies, the challenges and prospects for design and application of hybrid optical probes in the future are presented.

A Facile and Sensitive DNA Sensing of Harmful Algal Blooms Based on Graphene Oxide Nanosheets.

Gene detection has important applications in biology, biomedical engineering, clinical, environmental, and marine fields. Rapid, sensitive, and selective recognition of specific genes is essential in practical applications. In this study, we describe a facile and sensitive DNA sensing platform for specific and quantitative detection of Heterosigma akashiwo, which is one of the causative agents of red tides. Fast and sensitive detection is achieved by using chemically synthesized graphene oxide (GO) nanosheets. Probe DNA is designed according to the specific DNA fragments of harmful algae and labeled with fluorescent molecules FAM (fluorescein-based dye). GO nanosheet solution is made, in which the strong interaction between FAM-labeled probe and GO nanosheets keeps them in close proximity, facilitating the fluorescence quenching of the fluorophore by GO nanosheets. In the presence of a complementary target DNA, the FAM-labeled DNA probe and the target DNA hybridize and desorb from the surface of GO nanosheets, resulting in restoration of fluorophore fluorescence. The concentration of target DNA fragments is analyzed by the fluorescence intensity at ~ 520nm with emission wavelength of 480nm. The sensitive detecting platform achieved stable measurement of 1pM specific genes from Heterosigma akashiwo. Our GO nanosheet-based DNA-sensing platform performs fast and sensitive detection of trace amount of DNA, and enables quantitative recognition of harmful algae, which has promising applications in real-time monitoring in the marine environment of red tide generative dynamics, allowing effective control, particularly in relation to marine aquaculture.

A track-regenerated DNA walker: Construction and its derived sensing application

Herein, a track-regenerated DNA walker is constructed and applied for sensitive detection. Blocker-silenced walking strands and linear DNAs serving as roadbed strands are co-modified onto gold nanoparticles. Fluorophore/quencher dually-labeled hairpin DNAs serving as rail strands are free in solution. Roadbed strands hybridize with rail strands to form the track. Activated by the stimulus specifically binding to blocker, the walking strand will be liberated and hybridize with the rail strand to convert its 3′-end from protruding to recessed state. The rail strand then gets digested by exonuclease III, accompanying by fluorophore release and fluorescence enhancement. The vacant roadbed strand anew binds free rail strands, resulting in the regeneration of track. The walking strand continues to interact with the next rail strand and constantly steps forward on the regenerative track, accumulating large amounts of signal. This feature allows the DNA walker to be tailored into a sensor. For Ebola virus detection, it obtains a detection limit down to 3.5 fM and detects the target in biological fluids with good recovery. The DNA walker offers a new mode of sustainable operation and will provide a sensitively and reliably analytical tool in diagnosis.

Easily Accessible Schiff Base ESIPT Molecules with Tunable Solid State Fluorescence: Mechanofluorochromism and Highly Selective Co2+ Fluorescence Sensing

AbstractSchiff base molecules of 1–4 ((E)‐N′‐benzylidene‐2‐hydroxybenzohydrazide derivatives) showed structure controlled solid state fluorescence tuning, reversible stimuli induced fluorescence switching and highly selective Co2+ sensing. The strong intramolecular H‐bonding between imine and amine facilitates excited state intramolecular proton transfer (ESIPT) and large red shifted solid state fluorescence. Positional isomers and substitutional change tuned the solid state fluorescence (460 to 605 nm). The substituents and positions control stimuli‐induced fluorescence switching. ESIPT fluorophores showed strong fluorescence in DMF (Φf=0.057 to 0.35 compared to quinine sulphate). ESIPT fluorophores (2–4) exhibited highly selective fluorescence sensing of Co2+ ions without significant interference from other metal ions. Concentration dependent studies of fluorophores show the detection limit (LOD) of 54 nM that is much lower than the Environmental Protection Agency (EPA) guideline value (1.7 μM) in drinking water. Solid state fluorescence of ESIPT fluorophores have been made use to fabricate solid fluorescence assay (PVA polymer composite thin films) and demonstrated highly selective fluorescence sensing of Co2+ upon dipping into aqueous metal ions solution. Thus, the present studies demonstrate the versatility of Schiff base molecules for developing ESIPT fluorophores for stimuli responsive materials and highly selective fluorescence sensing both in solution and solid state.

Tetrazine as a general phototrigger to turn on fluorophores.

Light-activated fluorescence affords a powerful tool for monitoring subcellular structures and dynamics with enhanced temporal and spatial control of the fluorescence signal. Here, we demonstrate a general and straightforward strategy for using a tetrazine phototrigger to design photoactivatable fluorophores that emit across the visible spectrum. Tetrazine is known to efficiently quench the fluorescence of various fluorophores via a mechanism referred to as through-bond energy transfer. Upon light irradiation, restricted tetrazine moieties undergo a photolysis reaction that generates two nitriles and molecular nitrogen, thus restoring the fluorescence of fluorophores. Significantly, we find that this strategy can be successfully translated and generalized to a wide range of fluorophore scaffolds. Based on these results, we have used this mechanism to design photoactivatable fluorophores targeting cellular organelles and proteins. Compared to widely used phototriggers (e.g., o-nitrobenzyl and nitrophenethyl groups), this study affords a new photoactivation mechanism, in which the quencher is photodecomposed to restore the fluorescence upon light irradiation. Because of the exclusive use of tetrazine as a photoquencher in the design of fluorogenic probes, we anticipate that our current study will significantly facilitate the development of novel photoactivatable fluorophores.

Апробация тонкоигольного оптического зонда для регистрации изменений флуоресценции коферментов клеточного дыхания

Currently, an optical biopsy is a rapidly developing and promising area of technologies for real-time diagnosing the state of tissues in various fields of medicine, especially in oncology. One of the methods – fluorescence spectroscopy - allows one to detect changes in tissue metabolism caused by pathological processes by evaluating the level of fluorescence of endogenous fluorophores involved in various biochemical reactions in the body's cells. However, in order to correctly interpret the data obtained, it is important to consider how accurately the configuration of the equipment used makes it possible to detect differences between normal and pathologically altered tissues with metabolic disorders. The paper describes the optical biopsy device including fluorescence spectroscopy channel and the fine needle optical probe for usage during fine-needle puncture-aspiration biopsy of liver tumors. To test the developed device, experimental measurements of internal organs fluorescence in a laboratory rat were performed in vivo. The surface of the tissues was affected with a mitochondrial uncoupler to induce changes in cellular respiration. The results of the pilot experiment showed the ability of the developed channel to register changes in fluorescence caused by oxidative phosphorylation changes of mitochondria.

Mixed metal zero-mode guides (ZMWs) for tunable fluorescence enhancement.

Zero-mode waveguides (ZMWs) are capable of modifying fluorescence emission through interactions with surface plasmon modes leading to either plasmon-enhanced fluorescence or quenching. Enhancement requires spectral overlap of the plasmon modes with the absorption or emission of the fluorophore. Thus, enhancement is limited to fluorophores in resonance with metals (e.g. Al, Au, Ag) used for ZMWs. The ability to tune interactions to match a wider range of fluorophores across the visible spectra would significantly extend the utility of ZMWs. We fabricated ZMWs composed of aluminum and gold individually and also in mixtures of three different ratios, (Al : Au; 75 : 25, 50 : 50, 25 : 75). We characterized the effect of mixed-metal ZMWs on single-molecule emission for a range fluorophores across the visible spectrum. Mixed metal ZMWs exhibited a shift in the spectral range where they exhibited the maximum fluorescence enhancement allowing us to match the emission of fluorophores that were nonresonant with single metal ZMWs. We also compared the effect of mixed-metal ZMWs on the photophysical properties of fluorescent molecules due to metal–molecule interactions. We quantified changes in fluorescence lifetimes and photostability that were dependent on the ratio of Au and Al. Tuning the enhancement properties of ZMWs by changing the ratio of Au and Al allowed us to match the fluorescence of fluorophores that emit in different regions of the visible spectrum.

Designing enhanced and ratiometric probes for detecting OCl- based on substituents influencing the fluorescence of HBT and their application in strips and bioimaging.

Nitro groups with a strong electron-withdrawing effect can powerfully influence the fluorescence of fluorophores. In this work, through adjusting the nitro group at the HBT fluorophore to construct a phenylhydrazone structure, two probes (HBTN and HBTH) were synthesized to detect OCl-. Consequently, HBTN with the nitro group quenched the fluorescence of HBT and HBTH without the nitro group causing a redshift of the fluorescence, which resulted in enhanced and ratiometric fluorescence signal changes during the detection process. Among them, HBTN shows good ability to selectively detect OCl- in the concentration range of 0-14 μM with the detection limit of 2.06 nM. Based on HBTN, a portable test strip for detecting OCl- was made for the convenient quantification of the OCl- concentration with a spectrophotometer, and exogenous and endogenous OCl- was successfully imaged in cells.

Controlled-synthesis of alumina-graphene oxide nanocomposite coupled with DNA/ sulfide fluorophore for eco-friendly “Turn off/on” H2S nanobiosensor

Hydrogen sulfide (H2S) is a toxic environmental pollutant and crucial gas-signaling agent of human physiology. There is an urgent need for developing a sensitive and selective detection approach for H2S. Herein, a novel turn off/on response approach using γ-Al2O3 nanorods anchored on the surface graphene oxide (GO) nanosheets and coupled with DNA/sulfide fluorophore (SF) for H2S detection at room temperature was developed. The fluorescent fluorophore, DNA/SF, remains quenched on the super quencher γ-Al2O3–GO hybrid surface. In the existence of H2S, DNA/SF fluorophore was detached from γ-Al2O3–GO hybrid surface because of the strong physical adsorption interaction between H2S molecules and γ-Al2O3–GO surface. The recovered fluorescence intensity gave direct insight about H2S concentration in the medium. The developed γ-Al2O3–GO/DNA/SF nanobiosensor shows high sensitivity with 75 to 2.5 μM linear detection range of H2S, and a high binding constant of 9.8 × 103 M−1. The nanobiosensor is very selective toward H2S in the presence of various interfering anions and thiols and used for H2S detection in real water samples. γ-Al2O3–GO/DNA/SF nanobiosensor provides a cheap, simple, and highly selective H2S detection method at room temperature.

DNA-Capped Silver Nanoflakes as Fluorescent Nanosensor for Highly Sensitive Imaging of Endogenous H2S in Cell Division Cycles.

Biochemical sensing is essential toward gaining a full understanding of various physiological and pathological events. The in vivo level of hydrogen sulfide (H2S), the third endogenous gaseous transmitter, is closely related to its biological functions at different phases of the cell division cycle. Here we report a facile strategy for H2S sensing in live cells at different phases of cell division by developing a fluorescent nanosensor with double-strand DNA (dsDNA)-stabilized silver nanoflakes (AgNF@dsDNA). The sensing principle is based on selective etching of AgNF@dsDNA by H2S, followed by conversion to Ag2S. AgNFs initially act as quenchers through surface energy transfer, and then its etching leads to fluorescence recovery of modified fluorophore and efficient fluorescence resonance energy transfer (FRET) between two fluorophores. The changes of FRET signal as the readout successfully enable semiquantitative imaging of endogenous H2S alterations in live cells at G1, S, and G2, followed by the cycle of mitosis and cytokinesis. The optimized nanosensor has an excellent linear response in the concentration range of 1-10 μM Na2S. It can also differentiate G0 from G1 and other cell cycle steps through fluorescence imaging of changes in the level of endogenous H2S in cytoplasm during cell division cycle. Thus, the present study paves the way toward utilizing new Ag nanomaterials for biological imaging and sensing in live cells during different phases of the cell division cycle.

Simultaneous detection of lead (II) and mercury (II) ions using nucleic acid aptamer molecular beacons

ABSTRACT We have developed a fluorescence quantitative analysis method for the simultaneous detection of Hg2+ and Pb2+ based on nucleic acid aptamer molecular beacon (MB) probes. In this analytical method, two MB probes for Hg2+ (PHg) and Pb2+ (PPb) were designed. The carboxyl fluorescein (FAM) and tetramethyl-6-carboxyrhodamine (TAMRA) were selected as fluorophores of PHg and PPb, Black Hole Quencher 1 (BHQ-1) and Black Hole Quencher 2 (BHQ-2) were selected as organic quenchers, and several continuous nucleotides with guanine (G base) were connected to organic quenchers. The aptamers are put in as a part of stem and loop. In general, the fluorescence of fluorophores was dually quenched by BHQ and G bases, so the fluorescence signals were weak. In the presence of Hg2+ and Pb2+, PHg and PPb bonded with them, and the stem-loop structure of MB was destroyed and the fluorescence recovered. Under the optimal conditions, the fluorescence intensity of FAM had a good linear relationship with the concentration of Hg2+ in the range from 0.7 nmol/L to 84 nmol/L, and that of TAMRA and Pb2+ in the range from 0.2 nmol/L to 24 nmol/L. The detection limit of Hg2+ is 0.36 nmol/L and that of Pb2+ is 0.16 nmol/L (3σ, n = 11). The relative standard deviations (RSD) for determination of Hg2+ and Pb2+ were both lower than 5%, the average recoveries of this method in real samples were 96.55–102.78%, which indicated that the method had a high accuracy.

Au nanoparticles based ultra-fast “Turn-On” fluorescent sensor for detection of biothiols and its application in living cell imaging

Au or other metal nanostructures have the ability to strongly quench the fluorescence of fluorophores. This feature has made AuNP-conjugates attractive for the construction of platforms for various bioanalytes to overcome the limitations of small molecule fluorophores (poor solubility, long reaction time). In this paper, an ultrafast “Turn-On” fluorescent sensor for biothiols was constructed. The sensor is based on the fluorescent resonance energy transfer (FRET) effect between the fluorophore (PN) and AuNPs, which effectively quenches the fluorescence of the fluorophore. In the presence of thiols, PN is displaced and released from AuNP surfaces, and thus, the fluorescence is rapidly restored. The sensor features appreciable water solubility and ultrafast response time (a few seconds for Cys). In addition, it exhibits high selectivity and a detection limit as low as 12 nmol/L for Hcy. Moreover, the sensor presents good biocompatibility and has been successfully applied for imaging biothiols in living cells.

Controllably prepared molecularly imprinted core-shell plasmonic nanostructure for plasmon-enhanced fluorescence assay

Plasmon-enhanced fluorescence (PEF) is an emerging technology for sensitive detection. It relies on the plasmonic effect of a noble metal nanostructure to dramatically enhance the fluorescence of target fluorophores around the metal surface. Because there is a compromise between plasmonic enhancement and fluorescence quenching, it is critical to control the distance between the fluorophore and the metal surface to an appropriate range. This makes the fabrication of plasmonic nanostructures for PEF assays a challenging task. Herein, we report a controllably prepared core-shell plasmonic nanostructure coated with molecularly imprinted polymer (MIP) for sensitive and specific PEF assay. Riboflavin (RF) was used as a test compound in this study. RF-imprinted Ag@SiO2 nanoparticles were prepared in a controllable manner, providing an optimal distance between the metal surface and RF molecules. The obtained hybrid nanostructure allowed for sensitive detection and specific recognition towards the target. Based on the plasmonic hybrid nanostructure, a sensitive and specific PEF assay of RF was developed and successfully applied to the determination of RF in human urine. Thus, the study paved the way for controllable preparation of molecularly imprinted plasmonic nanostructures for sensitive and specific PEF assays.

Transdermal fluorescence detection of a dual fluorophore system for noninvasive point-of-care gastrointestinal permeability measurement.

The intestinal mucosal barrier prevents macromolecules and pathogens from entering the circulatory stream. Tight junctions in this barrier are compromised in inflammatory bowel diseases, environmental enteropathy, and enteric dysfunction. Dual sugar absorption tests are a standard method for measuring gastrointestinal integrity, however, these are not clinically amenable. Herein, we report on a dual fluorophore system and fluorescence detection instrumentation for which gastrointestinal permeability is determined in a rat small bowel disease model from the longitudinal measured transdermal fluorescence of each fluorophore. This fluorophore technology enables a specimen-free, noninvasive, point-of-care gastrointestinal permeability measurement which should be translatable to human clinical studies.

S1/S0 Potential Energy Surfaces Experience Different Types of Restricted Rotation: Restricted Z/ E Photoisomerization and E/ Z Thermoisomerization by an Out-of-Plane Benzyl Group or In-Plane m-Pyridinium Group?

Any method that can enhance the fluorescence of fluorophores is highly desirable. Fluorescence enhancement accomplished by restricted Z/ E photoisomerization through intramolecular steric hindrance or relatively high bond order of a C═C double bond in a S1 excited state has rarely been studied. In this article, we used green fluorescent protein (GFP) chromophore analogues as a model to get new physical insights into the restricted Z/ E photoisomerization and E/ Z thermoisomerization phenomena. We found that the S1 and S0 potential energy surfaces (PESs) of the GFP chromophore analogues experience two dramatically different types of restricted rotation, and 2b can be a representative example. In its S1 PES, it is not the intramolecular steric hindrance between the out-of-plane benzyl group and the in-plane m-pyridinium group but the relatively high bond order of the I-bond in the S1 excited state of 2b that makes it have a higher barrier for the Z/ E photoisomerization, a smaller Z/ E photoisomerization quantum yield, and a higher fluorescence quantum yield. In its S0 PES, it is not the reduced bond order of the I-bond in the S0 ground state of 2b but the intramolecular steric hindrance between the out-of-plane benzyl group and the in-plane m-pyridinium group that makes it have an extra higher barrier for E/ Z thermoisomerization and a much smaller E/ Z thermoisomerization rate constant.

Photodynamic diagnostics of early gastric cancer: Complexity measures of gastric microcirculation and new model of metastatic adenocarcinoma of rat stomach

The detection of early gastric cancer that often develops asymptomatically is crucial for improving patient survival. The photodynamic diagnosis (PDD) of gastric cancer using 5-aminolevulinic acid/protoporphyrin IX (5-ALA/PpIX) has been reported in several studies. However, the selectivity of PDD of gastric tumor is poor with often false-positive results that require the development of new methods to improve PDD for early gastric cancer. Therefore, a measure of the complexity of gastric microcirculation (multi-scale entropy, MSE) and the detrended fluctuation analysis (DFA) were applied as additional tools to detect early gastric cancer in rats.In this experimental study, we used our original model of metastatic adenocarcinoma in the stomach of a rat. To induce a gastric tumor, we used a long-term combination (for 9 months, which is 1/2 of the life of rats) of two natural factors, such as chronic stress (overpopulation being typical for modern cities) and the daily presence of nitrites in food and drinks, which are common ingredients added to processed meat and fish to help preserve food. Our results clearly show that both methods, namely, PDD using 5-ALA/PpIX and complexity/correlation analysis, can detect early gastric cancer, which was confirmed by histological analysis. Pre-cancerous areas in the stomach were detected as an intermediate fluorescent signal or MSE level between normal and malignant lesions of the stomach. However, in some cases, PDD with 5-ALA/PpIX produced a false-positive fluorescence of exogenous fluorophores due to its accumulation in benign and inflammatory areas of the mucosa. This fact indicates that the PDD itself is not sufficient for the correct diagnosis of gastric cancer, and the use of additional characteristics, e.g., complexity measures or scaling exponents, can significantly improve the diagnostic accuracy of PDD of gastric cancer that should be confirmed in further clinical studies and applications.

Fluorescein or Green Fluorescent Protein: Is It Possible to Create a Sensor for Dehydration?

Dehydration occurs when more fluid leaves the body than enters it. Dehydration can cause symptoms ranging from headaches and dizziness, to more severe symptoms like fever and unconsciousness. Currently there is no early dehydration detection system using temperature and pH as indicators. A sensor could alert the wearer and others of low hydration levels, which would normally be difficult to catch prior to more serious complications resulting from dehydration. The temperature and pH of skin are known to increase and decrease, respectively, with dehydration. These variables are also known to affect the fluorescence of certain fluorophores, which could provide a visible marker of dehydration. In this study, a protein fluorophore, green fluorescent protein (GFP), and a chemical fluorophore, fluorescein, were tested for a change in fluorescence in response to increased temperature or decreased pH. Neither fluorophore was affected by the changes in temperature. However, both lost their fluorescence when the pH of their environment was decreased. We also tested whether the fluorescence returned to its standard brightness when the temperature and pH were normalized. Reversing the pH change did not restore GFP fluorescence, but that of fluorescein was re-established. This finding suggests that fluorescein could be used as a reusable sensor for a dehydration-related pH change.

Orientation of nitro-group governs the fluorescence lifetime of nitrobenzoxadiazole (NBD)-labeled lipids in lipid bilayers.

Nitrobenzoxadiazole (NBD) labeled lipids are popular fluorescent probes of membrane structure and dynamics, and have been widely used in both model systems and living cells. Irrespective of attachment to the lipid head group or hydrocarbon chains, the NBD fluorophore generally adopts a transverse bilayer location near the host lipid carbonyl/glycerol moieties. Still, considerable variability is observed in the measured fluorescence lifetimes, indicating that overall fluorophore location is not the determinant of NBD fluorescence properties. Combining fluorescence experiments and molecular dynamics simulations, we show that for two almost identical NBD probes, significant differences in fluorophore orientation and fluorescence lifetime are observed. Integrating these findings with literature data, we demonstrate a correlation between NBD orientation and fluorescence lifetime. The latter is longer when the NBD nitro group is predominantly oriented towards the bilayer interior, compared to probes for which it points to the water medium.

Effect of Fixation and Mounting on Fluorescence Lifetime of Cellular Autofluorescence

Fluorescence lifetime measurements are often performed on live as well as fixed cells and tissues. Fixation and mounting processes are routinely used in cellular research or clinical diagnosis. In this paper, the effects of fixation and mounting on the fluorescence lifetime of cellular autofluorescence were studied by fluorescence lifetime imaging microscopy over time. Two endogenous fluorescent fluorophores, reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), showed different results between live cells and fixed cells. The average lifetime of NADH in live HeLa cells was about 1.02 ns, while maintained about 1.57 ns during the fixation periods of 14 days. The average lifetimes of FAD in live and fixed HeLa cells within 11 days were similar around 1.75 ns but increased to 2.10 ns after 12 days. The free and bound states of the two kinds of fluorophores were further analyzed. It was found that the bound-FAD had two different groups, which was related to the cell division cycle. The effect of mounting medium on fluorescence lifetimes was also studied, indicating glycerol has a negative impact on the fluorescence lifetime compared with neutral balsam.