Fluorescein-based Fluorescence Research Articles

A fluorescein-based fluorescent probe for real-time monitoring hypochlorite

Hypochlorite is an important reactive oxygen species in environment and pathophysiological processes. Abnormal level of ClO− can be destructive and lead to many diseases. Herein, to real-time and sensitively monitoring ClO−, a fluorescein-based fluorescence turn-on probe for ClO− in almost pure PBS buffer solution was successfully developed. After sensing hypochlorite, the spirolactam ring of the probe was opened and fluorescence at 525 nm greatly enhanced (∼1800 fold). The probe has a low detection limit (9.6 nM), short response time (∼1 min) and wide pH work range (5.0–9.0). Furthermore, the probe has been successfully applied to detect ClO− in disinfectant and actual water samples. Based on its fast response rate and almost pure buffer detection condition, the probe can be used to real time monitor ClO− in actual water environment.

Sodium fluorescein in brain tumor surgery: Assessing relative fluorescence intensity at tumor margins

Purpose:The use of intraoperative 5-aminolevulinic acid fluorescence has been shown to increase the extent of resection in high-grade glioma surgery. Sodium fluorescein is an alternate fluorescence agent with advantages of low cost, low adverse effect profile, and ability to visualize anatomical detail under the fluorescence filter. Sodium fluorescein-based fluorescence is not specific to tumor cells, and the significance of residual fluorescence at tumor margins has been questioned. In this article, the authors sought to correlate fluorescence intensity at tumor margins with the presence of residual contrast-enhancing tumor on magnetic resonance imaging (MRI).Methods:Eleven patients with a total of 12 lesions were enrolled in the study. Sodium fluorescein was administered at a dose of 5 mg/kg on induction of anesthesia. Relative intensity of fluorescence was extrapolated from intraoperative photographs through isolation of the green channel from the red/green/blue image, then graphically representing of pixel intensity through application of a thermal map. The correlation between areas of avid fluorescence at tumor cavity margins and the presence of residual contrast-enhancing tumor on postoperative MRI was evaluated.Results:All tumors demonstrated fluorescence. The presence of avid fluorescence at tumor cavity margins had a sensitivity of 66.7% and specificity of 75% for the presence of residual contrast-enhancing tumor on postoperative MRI. There were no adverse effects of fluorescein administration.Conclusion:Quantification of relative fluorescence intensity allows easy identification of areas that are high risk for residual contrast-enhancing tumor. Graphical representation of green pixel intensity requires validation through histopathological analysis but has the potential for real-time clinical application.

Bifunctional Molecular Probes for Activity-Based Visualization of Quinone-Dependent Amine Oxidases.

The design, synthesis, and evaluation of two bifunctional molecular probes that can be used to visualize quinone-dependent amine oxidase enzymes in an activity-dependent manner are described. These probes use alkylhydrazines to irreversibly bind the target enzymes, which can then be visualized with either Western blotting or in-gel fluorescence. The results show that the Western blotting readout, which utilizes commercially available anti-nitrophenyl antibodies to detect a simple dinitrophenyl antigen, provides a stronger readout than the fluorescein-based fluorescence readout. This visualization strategy can be used to measure the potency of enzyme inhibitors by selectively visualizing the active enzyme that remains after treatment with an inhibitor. Looking forward, this probe molecule and visualization strategy will enable activity-based protein-profiling experiments, such as determining inhibitor selectivity values within full proteome mixtures, for this family of amine oxidase enzymes.

The Effects of Heteroatoms Si and S on Tuning the Optical Properties of Rhodamine- and Fluorescein-Based Fluorescence Probes: A Theoretical Analysis.

The effects of the incorporated heteroatoms Si and S on tuning the optical properties of rhodamine- and fluorescein-based fluorescence probes is investigated using DFT and time-dependent DFT with four different functionals. As previously proposed, the large redshift (90 nm) produced by a Si atom in both the absorption and emission spectra can be attributed to the σ*-π* conjugation between the σ* orbital of the Si atom and the π* orbital of the adjacent carbon atoms. However, the presence of a Si atom does not alter the fluorescence quenching mechanism of the nonfluorescent forms of the investigated compounds. For the first time, these theoretical results indicate that the n orbital of the S atom plays an important role in determining the optical properties of the nonfluorescent form of rhodamine-based fluorescence probes. It alters the fluorescence quenching mechanism by lowering the energy of the dark nπ* state, which is due to breakage of the C10-S52 bond upon photoexcitation.

A fluorescein-based fluorescence probe for the fast detection of thiol

A new turn-on fluorescent probe for the selective detection of thiol over other amino acids was synthesized. Probe possesses the widely-used thiol-selective 2,4-dinitrobenzenesulfonyl (DNBS) group which can react with thiol and release the fluorescein which has strong fluorescence. Fluorescein, a well known xanthene fluorescent dye, has two states at different environment. Fluorescein is in the state of spirocyclization when connected with 2,4-dinitrobenzenesulfonyl (DNBS) group which has no fluorescence. However, it is in the state of open form when it reacts with thiol which has a strong fluorescence. The transition of the two states can be used to selectively detect thiol and the color can change from colorless to yellow which can be differentiated by naked eyes. Upon the titration of thiol, the absorption band at 454nm rises gradually and the fluorescence emerges at 521nm and the detection limit can be as low as 0.16μM. All of such good properties prove it to be a good sensor for the selective detection of thiol and it shows a potential use in bioimaging applications.

A highly sensitive and selective fluorescein-based fluorescence probe for Au3+ and its application in living cell imaging

A new fluorescein-based fluorescent probe for the detection of Au3+ has been developed. This probe was derived from 4′,5′-fluoresceindicarboxaldehyde and exhibited highly selective and sensitive response toward gold(III) ions in aqueous solution. There is a good linear relationship between the fluorescence intensity at 523nm and the concentrations of Au3+ ranging from 0μM to 60μM, which makes it effective for the detection of Au3+. The limit of detection (LOD) was evaluated to be 0.07μM based on S/N=3. The fluorescence enhancement was attributed to the irreversible CN bond hydrolysis that occurred to this probe in the presence of Au3+, generating the fluorescent 4′,5′-fluoresceindicarboxaldehyde. Furthermore, the imaging experiments indicated that this probe is cell-permeable and can be used to detect Au3+ within living cell.

Bispyrene-fluorescein hybrid based FRET cassette: a convenient platform toward ratiometric time-resolved probe for bioanalytical applications.

Pyrene excimer possesses a large Stokes shift and long fluorescence lifetime and has been widely applied in developing time-resolved biosensing systems to solve the autofluorescence interference problems in biological samples. However, only a few of pyrene excimer-based small molecular probes have been reported so far. Ratiometric probes, on the other hand, can eliminate interferences from environmental factors such as instrumental efficiency and environmental conditions by a built-in correction of the dual emission bands but are ineffective for endogenous autofluorescence in biosystems. In this work, by combining the advantages of time-resolved fluorescence technique with ratiometric probe, we reported a bispyrene-fluorescein hybrid FRET cassette (PF) as a novel ratiometric time-resolved sensing platform for bioanalytical applications, with pH chosen as a biorelated target. The probe PF showed a fast, highly selective, and reversible ratiometric fluorescence response to pH in a wide range from 3.0 to 10.0 in buffered solution. By employing time-resolved fluorescence technique, the pH-induced fluorescence signal of probe PF can be well-discriminated from biological autofluorescence background, which enables us to detect pH in a range of 4.0-8.0 in cell media within a few seconds. It has also been preliminarily applied for ratiometric quantitative monitoring of pH changes in living cells with satisfying results. Since many fluorescein-based fluorescence probes have been developed, our strategy might find wide applications in design ratiometric time-resolved probes for detection of various biorelated targets.

Evaluation of Fluorescent Compound Interference in 4 Fluorescence Polarization Assays: 2 Kinases, 1 Protease, and 1 Phosphatase

With the increasing use of fluorescence-based assays in high-throughput screening (HTS), the possibility of interference by fluorescent compounds needs to be considered. To investigate compound interference, a well-defined sample set of biologically active compounds, LOPAC, was evaluated using 4 fluorescein-based fluorescence polarization (FP) assays. Two kinase assays, a protease assay, and a phosphatase assay were studied. Fluorescent compound interference and light scattering were observed in both mixture- and single-compound testing under certain circumstances. In the kinase assays, which used low levels (1-3 nM) of fluorophore, an increase in total fluorescence, an abnormal decrease in mP readings, and negative inhibition values were attributed to compound fluorescence. Light scattering was observed by an increase in total fluorescence and minimal reduction in mP, leading to false positives. The protease and phosphatase assays, which used a higher concentration of fluorophore (20-1200 nM) than the kinase assays, showed minimal interference from fluorescent compounds, demonstrating that an increase in the concentration of the fluorophore minimized potential fluorescent compound interference. The data also suggests that mixtures containing fluorescent compounds can result in either false negatives that can mask a potential "hit" or false positives, depending on the assay format. Cy dyes (e.g., Cy3B and Cy5 ) excite and emit further into the red region than fluorescein and, when used in place of fluorescein in kinase 1, eliminate fluorescence interference and light scattering by LOPAC compounds. This work demonstrates that fluorescent compound and light scattering interferences can be overcome by increasing the fluorophore concentration in an assay or by using longer wavelength dyes.

Rational design of fluorescein-based fluorescence probes. Mechanism-based design of a maximum fluorescence probe for singlet oxygen.

Fluorescein is one of the best available fluorophores for biological applications, but the factors that control its fluorescence properties are not fully established. Thus, we initiated a study aimed at providing a strategy for rational design of functional fluorescence probes bearing fluorescein structure. We have synthesized various kinds of fluorescein derivatives and examined the relationship between their fluorescence properties and the highest occupied molecular orbital (HOMO) levels of their benzoic acid moieties obtained by semiempirical PM3 calculations. It was concluded that the fluorescence properties of fluorescein derivatives are controlled by a photoinduced electron transfer (PET) process from the benzoic acid moiety to the xanthene ring and that the threshold of fluorescence OFF/ON switching lies around -8.9 eV for the HOMO level of the benzoic acid moiety. This information provides the basis for a practical strategy for rational design of functional fluorescence probes to detect certain biomolecules. We used this approach to design and synthesize 9-[2-(3-carboxy-9,10-dimethyl)anthryl]-6-hydroxy-3H-xanthen-3-one (DMAX) as a singlet oxygen probe and confirmed that it is the most sensitive probe currently known for (1)O(2). This novel fluorescence probe has a 9,10-dimethylanthracene moiety as an extremely fast chemical trap of (1)O(2). As was expected from PM3 calculations, DMAX scarcely fluoresces, while DMAX endoperoxide (DMAX-EP) is strongly fluorescent. Further, DMAX reacts with (1)O(2) more rapidly, and its sensitivity is 53-fold higher than that of 9-[2-(3-carboxy-9,10-diphenyl)anthryl]-6-hydroxy-3H-xanthen-3-ones (DPAXs), which are a series of fluorescence probes for singlet oxygen that we recently developed. DMAX should be useful as a fluorescence probe for detecting (1)O(2) in a variety of biological systems.

A novel multifunctionally labelled DNA probe bearing an intercalator and a fluorophore

A 15-mer DNA labelled with a novel multifunctional fluorescent agent bearing acridine and fluorescein moieties is synthesized and the intensity of fluorescein-based fluorescence detected by the excitation of acridine is shown to be strongly affected by the DNA's secondary structure.

Multiple Releasable Fluorescein Labels for Immunoassay: The Principle Illustrated by an Immunoassay for Antibodies to the Human Immunodeficiency Virus

Attempts to increase the sensitivity of fluorescein-based fluorescence immunoassays by using multiple labelling have generally been unsuccessful because of concentration quenching. We have labelled antibodies to human immunoglobulin G with multiple fluorescein fluorophores attached by means of a disulphide linkage: this linkage can be rapidly and easily broken by treatment with dithiothreitol, allowing fluorescein to be released from the antibody and measured in free solution. Application of this technique to a fluorescence labelled immunosorbent assay for antibodies to the human immunodeficiency virus gave an approximately 20-fold increase in signal compared with an equivalent assay using fluorescein isothiocyanate.