Fluorescein Probe Research Articles

Moxifloxacin HCl -loaded Cellulose Acetate Butylate In Situ Forming Gel for Periodontitis Treatment.

Periodontitis presents significant treatment challenges due to its complexity and potential complications. In response, an in situ forming gel (ISG) loaded with moxifloxacin HCl (Mx) and cellulose acetate butyrate (CAB) was developed for targeted periodontitis therapy. Mx-loaded 10-45% CAB-based ISGs were developed, and their physicochemical properties such as rheology, viscosity, contact angle, gel morphology and gel formation, interface interaction were investigated. Moreover, the formulation performance studies including drug release and kinetics, in vitro degradation, and antimicrobial activities were also evaluated. The Mx-loaded ISGs containing 25-45% CAB demonstrated rapid matrix formation in both macroscopic and microscopic examinations and presented plastic deformation matrix. Tracking with sodium fluorescein and Nile red fluorescence probes indicated delayed solvent movement owing to CAB matrix formation. Adequate CAB content sustained Mx release for one week, following Peppas-Sahlin model and indicating a predominantly Fickian diffusion mechanism. Higher CAB content likely contributed to a denser matrix structure, leading to a slower in vitro degradation rate. Synchrotron radiation X-ray tomographic and SEM imaging provided insights into the CAB matrix structure and porous network formation. These ISG formulations effectively inhibited Staphylococcus aureus, Escherichia coli, Candida albicans, and Porphyromonas gingivalis. The Mx-loaded 40% CAB-based ISG shows promise as a dosage form for treating periodontitis. Further clinical trials are necessary to ensure the safety of this new ISG formulation, despite existing safety data for other medicinal uses of CAB. HIGHLIGHTS: Moxifloxacin HCl-loaded 10-45% cellulose acetate butyrate (CAB)-based in situ forming gels (ISG) were developed. They were evaluated for physicochemical properties, drug release, in vitro degradation, and antimicrobial activities. ISGs with 25-45% CAB showed swift matrix formation and plastic deformation Adequate CAB content sustained Mx release with Fickian diffusion mechanism They promise for periodontitis treatment because of effective inhibition of related pathogens.

Fluorescent cyclopropyl ester probes are efficiently cleaved by endogenous carboxylesterase in mouse blood: implications for preclinical fluorescence imaging

ABSTRACT Prior studies have shown that fluorescent molecular probes based on cyclopropyl esters are good substrates for butyrylcholine esterase (BChE), which is a biomarker of several human diseases. We tested two fluorescent cyclopropyl ester derivatives as BChE-activated fluorogenic probes. One was a known fluorescein probe, and the other was a newly designed near-infrared probe based on a heptamethine cyanine dye. As expected, both probes were good substrates for BChE, but they were also good substrates for carboxylesterase (CE). The probes were efficiently cleaved in mouse blood and serum which contains high levels of CE, but they were not cleaved in human serum which contains negligible CE. There are two major implications of these results. One is a cautionary note that esterase levels in different organisms can vary substantially. Researchers developing fluorescent cyclopropyl ester probes for BChE imaging should anticipate high levels of background signal in preclinical mouse models due to ester cleavage by the abundant CE in mouse blood. However, there is very little cleavage of fluorescent cyclopropyl ester probes in human blood, which contains low levels of CE. Therefore, fluorescent cyclopropyl ester probes should be viable in humans as imaging agents that identify disease sites with overexpressed levels of CE or BChE.

Neuroprotective Effect of Sterculia setigera Leaves Hydroethanolic Extract.

Plants are a valuable source of information for pharmacological research and new drug discovery. The present study aimed to evaluate the neuroprotective potential of the leaves of the medicinal plant Sterculia setigera. In vitro, the effect of Sterculia setigera leaves dry hydroethanolic extract (SSE) was tested on cultured cerebellar granule neurons (CGN) survival when exposed to hydrogen peroxide (H2O2) or 6-hydroxydopamine (6-OHDA), using the viability probe fluorescein diacetate (FDA), a lactate dehydrogenase (LDH) activity assay, an immunocytochemical staining against Gap 43, and the quantification of the expression of genes involved in apoptosis, necrosis, or oxidative stress. In vivo, the effect of intraperitoneal (ip) injection of SSE was assessed on the developing brain of 8-day-old Wistar rats exposed to ethanol neurotoxicity by measuring caspase-3 activity on cerebellum homogenates, the expression of some genes in tissue extracts, the thickness of cerebellar cortical layers and motor coordination. In vitro, SSE protected CGN against H2O2 and 6-OHDA-induced cell death at a dose of 10µg/mL, inhibited the expression of genes Casp3 and Bad, and upregulated the expression of Cat and Gpx7. In vivo, SSE significantly blocked the deleterious effect of ethanol by reducing the activity of caspase-3, inhibiting the expression of Bax and Tp53, preventing the reduction of the thickness of the internal granule cell layer of the cerebellar cortex, and restoring motor functions. Sterculia setigera exerts neuroactive functions as claimed by traditional medicine and should be a good candidate for the development of a neuroprotective treatment against neurodegenerative diseases.

RBOHF activates stomatal immunity by modulating both reactive oxygen species and apoplastic pH dynamics in Arabidopsis.

Stomatal defences are important for plants to prevent pathogen entry and further colonisation of leaves. Apoplastic reactive oxygen species (ROS) generated by NADPH oxidases and apoplastic peroxidases play an important role in activating stomatal closure upon perception of bacteria. However, downstream events, particularly the factors influencing cytosolic hydrogen peroxide (H2 O2 ) signatures in guard cells are poorly understood. We used the H2 O2 sensor roGFP2-Orp1 and a ROS-specific fluorescein probe to study intracellular oxidative events during stomatal immune response using Arabidopsis mutants involved in the apoplastic ROS burst. Surprisingly, the NADPH oxidase mutant rbohF showed over-oxidation of roGFP2-Orp1 by a pathogen-associated molecular pattern (PAMP) in guard cells. However, stomatal closure was not tightly correlated with high roGFP2-Orp1 oxidation. In contrast, RBOHF was necessary for PAMP-mediated ROS production measured by a fluorescein-based probe in guard cells. Unlike previous reports, the rbohF mutant, but not rbohD, was impaired in PAMP-triggered stomatal closure resulting in defects in stomatal defences against bacteria. Interestingly, RBOHF also participated in PAMP-induced apoplastic alkalinisation. The rbohF mutants were also partly impaired in H2 O2 -mediated stomatal closure at 100 μm while higher H2 O2 concentration up to 1 mm did not promote stomatal closure in wild-type plants. Our results provide novel insights on the interplay between apoplastic and cytosolic ROS dynamics and highlight the importance of RBOHF in plant immunity.

Fundamental Differences in Inactivation Mechanisms of Escherichia coli O157:H7 Between Chlorine Dioxide and Sodium Hypochlorite.

Chlorine dioxide (ClO2) and sodium hypochlorite (NaClO) are two chlorinated oxidizing agents that are implemented in water treatment and postharvest processing of fresh produce. While the antibacterial mechanisms of NaClO have been investigated, there are comparatively few studies that have looked at how ClO2 kills bacteria. Therefore, the objective of this study was to compare the inactivation pathways of ClO2 and NaClO against Escherichia coli O157:H7. Treatments consisted of 2.5, 5, and 10 ppm ClO2 or 50, 100, and 200 ppm NaClO for 5, 10, and 15 min. Maximum log reductions of E. coli O157:H7 were 5.5 and 5.1 after treatment with ClO2 or NaClO, respectively. Bacterial inactivation was measured using log reductions, intracellular reactive oxygen species (ROS) using with 2′,7′–dichlorofluorescin diacetate (DCFDA) or aminophenyl fluorescein (APF) probes, relative values of NAD+, NADH, NADP+, and NADPH cofactors. Additionally, the expression of three key genes involved in ROS stress was measured via RT-PCR. Levels of intracellular ROS measured by DCFDA after ClO2 treatment were significantly higher than those found after treatment in NaClO. Additionally, NaClO treatment resulted in upregulation of ROS-defense genes, while expression of the same genes was typically at base levels or downregulated after ClO2 treatment. As the concentrations of both treatments increased, the NADP+:NADPH ratio shifted to the cofactor being predominantly present as NADP+. These data indicate that ClO2 and NaClO damage E. coli O157:H7 via measurably different mechanisms and that ClO2 does not appear to cause substantial oxidative stress to E. coli O157:H7 directly.

Selective blue-filtering spectacle lens protected primary porcine RPE cells against light emitting diode-induced cell damage.

This study aimed to investigate whether use of a selective-blue-filtering (S-BF) lens can protect cultured primary porcine RPE cells against photo-irradiation. Transmittance of S-BF and UV-filtering (UVF) lenses was characterised spectrophotometrically. RPE cells were exposed to 1700 lux of white (peak λ at 443 and 533 nm; 0.44 mW/cm2) or blue (peak λ at 448 and 523 nm; 0.85 mW/cm2) LED light for 16 h to evaluate the influence of light source on the culture. The effect of the S-BF and UVF ophthalmic lenses on RPE cell cultures under blue light irradiation was then investigated. Cell viability was compared using trypan blue and MTT assays. Intracellular ROS production was detected by a fluorescein probe CM-H2DCFDA. Expression levels of catalase and Prdx3 were analysed by western blot. Trypan blue staining showed blue light caused more cell death than no light (p = 0.001) or white light (p = 0.005). MTT assay supported the hypothesis that exposure to blue light damaged RPE cells more severely than no light (p = 0.002) or white light (p = 0.014). Under blue light, use of the S-BF lens, which blocked 17% more blue light than the UVF lens, resulted in higher cellular viability (S-BF: 93.4±1.4% vs UVF: 90.6±1.4%; p = 0.022; MTT: 1.2-fold; p = 0.029). Blue and white light both significantly increased ROS production. The S-BF lens protected cells, resulting in lower levels of ROS and higher expression of catalase and Prdx3. To conclude, blue LED light exposure resulted in significant cytotoxicity to RPE cells. Partial blockage of blue light by an S-BF lens led to protective effects against retinal phototoxicity, which were mediated by reduction of ROS and increased levels of antioxidant enzymes.

Amphiphilic N-oxyethylimidazolium calixarenes: Synthesis, micellar solubilization and molecular recognition of Adenine-containing nucleotides

For the first time amphiphilic calix[4]arenes bearing oxyethyl-imidazolium polar headgroups on one side and alkyl fragments on another side of the macrocyclic platform were synthesized. Critical aggregation concentration (CAC) value was estimated using two methods: (a) pyrene fluorescent probe; (b) fluorescein probe based on both its fluorescent and absorbance properties in micellar solution. All plots were adequately described by a sigmoidal Boltzmann type function. A comparative analysis of the CACs were detected by using two types of dyes was held. The system calixarene - fluorescein was found to different change the absorbance and colorimetric response toward various adenosines phosphates.

Dual mode of cyanide detection by Fluorescein-Based “Turn-ON” Bi-Signaling fluorescence and colorimetric sensing: Agricultural product and cellular studies

A “Turn-ON” fluorescence chemosensor was developed as a new reaction-based fluorescein probe for detecting the cyanide ion (CN−). The sensor’s spectroscopic properties and binding abilities with several anions were investigated in phosphate-buffered saline and acetonitrile solutions via fluorometric titration. The sensor exhibited high CN− selectivity via fluorescence enhancement against other competing anions, including F−, Cl−, I−, Br−, SO42−, NO3−, HSO4−, ClO4−, CO32−, HPO42−, and CH3COO−. Moreover, the sensor exhibited fluorescence and chromogenic changes from pale pink to orange upon sensing CN−, which was easily visible to the naked eye. The sensor was used to detect CN− in agricultural samples (i.e., cassava root and bamboo shoot) and locate CN− in the HepG2 cellular system.

Natural antioxidants attenuate mycolactone toxicity to RAW 264.7 macrophages.

Mycobacterium ulcerans produces a macrolide exotoxin, mycolactone which suppresses immune cells activity, is toxic to most cells and the key virulence factor in the pathogenesis of Buruli ulcer disease. Mycolactone is reported to mediate the production of reactive oxygen species in keratinocytes; cells that play critical role in wound healing. Increased levels of reactive oxygen species have been shown to disrupt the well-ordered process of wound repair; hence, the function of wound-healing cells such as macrophages, keratinocytes, and fibroblast could be impaired in the presence of the reactive oxygen species mediator, mycolactone. To ensure regeneration of tissues in chronic ulcers, with proper and timely healing of the wounds, natural antioxidants that can combat the effects of induced reactive oxygen species in wound-healing cells ought to be investigated. Reactive oxygen species activity was determined in mycolactone-treated RAW 264.7 macrophages and the scavenging ability of the antioxidants (ascorbic acid, gallic acid, and green tea kombucha) against mycolactone-induced reactive oxygen species (superoxide anions) was assessed using fluorescein probe (DCF-DA) and nitroblue tetrazolium dye. Cytotoxicity of the antioxidants, mycolactone, and the protective effect of the antioxidants on the cells upon treatment with mycolactone were determined using the Alamar blue assay. The expression levels of endogenous antioxidant enzyme genes (superoxide dismutase, catalase, and glutathione peroxidase) in response to mycolactone-mediated reactive oxygen species were determined using RT-qPCR. Mycolactone induced the production of reactive oxygen species in RAW 264.7 macrophages, and the resulting superoxide anions were scavenged by some of the antioxidants. The selected endogenous antioxidant enzyme genes in the macrophages were upregulated in the presence of the antioxidants and mycolactone. The exogenously supplied ascorbic acid and green tea kombucha offered moderate protection to the macrophages against the toxicity of mycolactone. We conclude that the results provide insights into alternate and adjunct therapeutic approaches in Buruli ulcer treatment, which could significantly attenuate the toxicity of the pathogenic factor; mycolactone.

Mercury speciation at trace level using fluorescein hydrazide as fluorescent probe

The fluorescent probe fluorescein hydrazide was designed for mercury sensing by fluorescence assay. The probe was color less and non-fluorescent in the absence of Hg2+ ions and highly fluorescent and green colored in the presence of Hg2+ ions. The probe is highly specific to Hg2+ ions under pH 12 buffer conditions. No other ions interfered during fuolrimetric analysis of the Hg2+ ions. The fluorescence assay was done at excitation wavelength of 502 nm and emission wavelength at 520 nm. The emission wavelength measured at 520 nm is proportional to the concentration of Hg2+ ions in the assay solution. The developed fluorescence assay protocol based on fluorescein hydrazide as fluorescent probe was used for mercury speciation in lead acid battery samples successfully.

Peroxide-responsive boronate ester-coupled turn-on fluorogenic probes: Direct linkers supersede self-immolative linkers for sensing peroxides

Turn-on fluorogenic probes are commonly utilized for an efficient estimation of reactive oxygen species such as H2O2. In the present study, three different sets of turn-on fluorogenic probes for sensing H2O2 were rationally designed by coupling boronate ester group with three important fluorescent dyes either by linking directly or via self-immolative linkers. Interestingly, our results reveal that directly-linked boronate ester probes are superior and sensitive than the self-immolative linker-containing probes in detecting traces of exogenous and endogenous levels of H2O2. The difference in probe efficiency was also dependent on the nature of fluorophore unit being used. Considering these aspects, our detailed studies reveal that the directly-linked boronate ester-based fluorescein probe 19 is the best probe for efficiently sensing H2O2 both in aqueous as well as cellular medium with very high level of sensitivity. The present observation would be useful while designing the fluorogenic sensors for ROS as well as during their utilization in ROS-associated pathologies.

A Novel Turn on Fluorescence Sensor for Determination Enoxaparin, a Low Molecular Weight Heparin.

A sensor system was designed for the detection of Enoxaparin (Enox), a low molecular weight heparin (LMWH) that was run over the fluorescence quenching mechanism of fluorescein (FL) dye. At nanomolar concentrations, FL probe was subjected to fluorescence quenching by Fe(III). Fluorescence quenching mechanism of FL by Fe(III) was examined using various analytical techniques such as UV-vis absorption, fluorescence, and Fourier transform Infrared spectroscopy techniques, as well as with scanning electron microscope. The results indicated that photoinduced electron transfer process occurred between FL and Fe and that FL was quenched both statically and dynamically. Thermodynamic parameters showed that the interactions between them were predominantly hydrophobic interactions. Enox caused FL to recover its lost fluorescence properties and an increase was observed in the intensity of the fluorescence. Enox was detected successfully with the turn on fluorescence sensor. The developed Enox biosensor exhibited linearity in the range of 0-1.1μg/ml. For Enox detection, the limit of detection was measured as 255ng/mL. Enox biosensor was presented as a practical, simple, and applicable sensor system with high sensitivity and good selectivity. Enox is a medication usually monitored indirectly over anticoagulation. This study was presented as an alternative method for monitoring Enox directly. HIGHLIGHTS: Fluorescence quenching of Fluorescein dye by Fe(III) was studied in detail. The presence of enoxaparin enhanced the fluorescence properties of the fluorescein dye. A sensitive, simple and effective sensor system for determination of Enoxaparin, a low molecular weight heparin was shaped in the aqueous media. It was presented as a new method for Enoxaparin to be followed directly.

Novel fluorescein- and pyridine-conjugated schiff base probes for the recyclable real-time determination of Ce3+ and F−

The effect of aminopyridines substituted at different positions on the fluorescence properties deserves to be studied. Since 2-aminopyridyl-based probes have been reported, the effects of 3-aminopyridine and 4-aminopyridine on the performance of fluorescein probes were discussed in here. Two Schiff base fluorescein probes FN-1, FN-2 were designed and synthesized. Among them, since the ligand shows a highly selective and sensitive response to metal charge transfer (LMCT), the fluorescence of FN-1 can be quenched by Ce3+ ions in PBS buffer. At the same time, a specific precipitation reaction between Ce3+ and F− releases the free probe to restore the fluorescence of FN-1. Therefore, FN-1 can be used for the recyclable ‘ON-OFF-ON’ detection of Ce3+and F−. The detection limits for Ce3+and F− are 4.48 μM and 11.58 μM in concentration range of 0–50 μM and 0–150 μM. However, due to the para position of N and amino groups on 4-aminopyridine, the spatial structure of FN-2 cannot be complexed with ions, resulting in poor selectivity. Polyvinylidene fluoride (PVDF) membrane containing FN-1 were prepared for the real-time qualitative detection of Ce3+and F− in real water samples. FN-1 exhibits high water solubility and biocompatibility and has been successfully applied to biological imaging in vascular smooth muscle cells (VSMCs).

Mapping out the Degree of Freedom of Hosted Enzymes in Confined Spatial Environments

Summary The integration of enzymes with solid materials is crucial for promoting their industrialization. Understanding the enzyme behavior upon association within a confined space, though of fundamental importance for biocomposite development, remains a persistent, unresolved challenge. Here, we present a comprehensive elucidation of the spatial environment’s impact on hosted enzymes’ degree of freedom and, consequently, their accompanying reactivity. Site-directed spin labeling in combination with electron paramagnetic resonance spectroscopy allows the direct detection of host-guest interactions at atomic resolution, while the tailorable synthesis of covalent organic frameworks (COFs) enables an evaluation of factors affecting such interactions. Specifically, lysozyme is found to be more constrained and less active along with increasing hydrophilicity of the COFs. These results support the establishment of a connection between the hydrophilicity of the spatial environment and the resulting biocomposites’ reactivity, enabling the prediction of the performance of unknown biocomposites. This study provides a unique insight into the mechanistic pathways underpinning biocatalysis.

Synthesis and Characterization of FITC Labelled Ruthenium Dendrimer as a Prospective Anticancer Drug.

Metallodendrimers—dendrimers with included metals—are widely investigated as biocompatible equivalents to metal nanoparticles. Applications can be expected in the fields of catalysis, as chemical sensors in molecular recognition and as anticancer drugs. Metallodendrimers can also mimic certain biomolecules, for example, haemoprotein in the case of using a dendrimer with a porphyrin core. In previous papers, we showed the promising anticancer effects of carbosilane ruthenium dendrimers. The present paper is devoted to studying biocompatibility and the cytotoxic effect on normal and cancer cells of carbosilane ruthenium dendrimers labelled with fluorescent probe fluorescein isothiocyanate (FITC). The addition of fluorescent probe allowed tracking the metallodendrimer in both normal and cancer cells. It was found that carbosilane ruthenium dendrimer labelled with FITC in concentration up to 10 µmol/L was more cytotoxic for cancer cells than for normal cells. Thus, FITC labelled carbosilane ruthenium dendrimer is a good candidate for diagnostic imaging and studying anticancer effects of metallodendrimers in cancer therapy.

Abstract 217: Development of a rapid and efficient methodology to quantitate thiol detection in antibody-drug conjugates (ADCs)

Abstract Antibody-Drug Conjugates (ADCs) are comprised of three distinct components, an antibody, a payload (usually a cytotoxic agent) and a linker (a synthetic chemical chain joining the antibody and payload). Payloads can be conjugated to antibodies in either a stochastic or site-specific manner, with stochastic conjugations usually accomplished through either amine groups of lysine residues or through thiol groups in disulfide bonds located in the hinge region of antibodies. In the latter case, an electrophilic Michael acceptor grouping (e.g., maleimide) of a linker-payload construct reacts with a nucleophilic thiol group of the antibody, forming the ADC. In order for this to occur, disulfide bonds need to be reduced to free thiols. However, knowing the concentration of reactive thiols at different stages during the reduction process can be challenging, and so a more accurate assessment of thiol concentration may allow greater control of the desired average Drug-Antibody Ratio (DAR). Currently, different spectrophotometric (i.e. Ellman’s reagent) and fluorometric assays are used to monitor thiol concentration, but these methods can be expensive, time consuming and inaccurate. To address these issues, a rapid and efficient fluorescence-based method has been developed to allow the reduction of thiols to be monitored. The method is based on a fluorescein derivative produced via a four-step synthesis starting from commercially available fluorescein sodium salt. In its unreacted form, this novel probe exists in a lactone form. However, once it reacts with a free thiol group, it converts stoichiometrically via opening of the lactone ring to a fluorescent form, thus allowing quantitative determination of the concentration of thiol groups present in solution. The three antibodies used to trial this procedure (i.e., Trastuzumab, Cetuximab and Rituximab) were reduced using TCEP over a 120 minute period. Aliquots of solution were collected at various time intervals, and incubated with the fluorescein probe for 5 minutes, monitoring fluorescence with a simple fluorimeter. Using this approach, it was possible to accurately assess thiol concentration at each time interval, and relate the overall reduction profile of each antibody to the concentration of reducing agent used. Payloads were then conjugated to the three antibodies at specific timepoints (selected through monitoring thiol reduction) in an effort to generate species with a defined DAR. Overall, the resulting ADCs were found to have average DAR profiles consistent with the measured number of thiols in the reduced antibodies. When compared with other assays, the developed method gave results with improved reproducibility. Furthermore, the fluorescein probe was less complex and faster to use than other fluorometric kits, and was easy to synthesize, making it a useful new tool in the generation of ADCs. Citation Format: Ilona Pysz, Paolo Andriollo, Paul J. Jackson, K M. Rahman, David E. Thurston. Development of a rapid and efficient methodology to quantitate thiol detection in antibody-drug conjugates (ADCs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 217.

Determination of the critical micelle concentration by absorbance and fluorescence techniques using fluorescein probe

We reported a unique and sensitive probe (fluorescein) for determination the critical micelle concentration(CMC) of ionic surfactants based on both fluorescence and absorbance properties of probe in micellar solution. We found that the spectral properties (fluorescence and absorbance) of fluorescein are affected by different types of surfactants. For this purpose spectrophotometric technique using fluorescein as probe to determine the critical micelle concentration of cationic, anionic and zwitterionic surfactants. The critical micelle concentrations of surfactants were determined from the sigmoidal shaped plots or inflection points. The obtained CMC values were in good agreement with the values obtained with conductivity measurements or with literature values.

Optical Recognition of Ammonia and Amine Vapor Using "Turn-on" Fluorescent Chitosan Nanoparticles Imprinted on Cellulose Strips.

A practical fluorescent test dipstick for an efficient recognition of ammonia and amines vapors was developed. The prepared testing strip was based on a composite of molecularly imprinted chitosan nanoparticles, supported on cellulose paper assay, with artificial fluorescent receptor sites for ammonia/amines recognition in aqueous and gaseous phases. A modified chitosan nanoparticles containing fluorescein molecules, were successfully prepared and employed on cellulose paper strip creating fluorescent cellulose (FL-Cell) to act as "turn-on" fluorescent sensor for sensing and determining ammonia and organic amine vapor. We employed chitosan nanoparticles that had fluorescein incorporated as the fluorescent probe molecule, with a readout limit achieved for aqueous ammonia as low as 280ppm at room temperature and atmospheric pressure. The sensor responded linearly relying on the aqueous ammonia concentration in the range of 0.13-280ppm. The chromogenic fluorescent cellulose platform response depended on the acid-base characteristic effects of the fluorescein probe. The protonated form of fluorescein molecules immobilized within the chitosan nanoparticles were in a nanoenvironment demonstrating only weak fluorescence. When binding to ammonia/amine vapor, the fluorescein active sites were deprotonated and exhibited higher "turned-on" fluorescence as a result of exposure to those alkaline species. The simple fabrication and abovementioned characteristics of such fluorescent chitosan nanoparticles are such that they should be applicable for monitoring of ammonia/amines in either aqueous or vapor states. We studied the distribution of the fluorescent chitosan onto paper sheets fabricated from bleached bagasse pulp and coated with two different thicknesses of a fluorescent nanochitosan and blank nanochitosan solutions. A thin fluorescent nanochitosan layer was created on the surface of cellulose strips using an applicator. Its distribution was assessed by scanning electron microscopic (SEM) and transmission electron microscopic (TEM) analysis as well as Fourier-transform infrared spectroscopic (FT-IR) measurements. The mechanical properties were also tested. The exploitation of this "turn-on" fluorescence sensor invented platform should be amenable to different situations where determination of ammonia/amine vapor or aqueous solution is required.

A simple approach for rapid and cost-effective quantification of extracellular vesicles using a fluorescence polarization technique

Extracellular vesicles (EVs) are membrane-bound phospholipid vesicles actively secreted by all cells. As they carry specific markers expressed by their parental cells, EVs are utilized to identify specific cells via liquid biopsy. To facilitate EV-based clinical diagnosis, a fast and reliable method to count EVs is critical. We developed a method for rapid and cost-effective quantification of EVs which relies on the fluorescence polarization (FP) detection of lipophilic fluorescein probe, 5-dodecanoylamino fluorescein (C12-FAM). The alkyl tail of C12-FAM is specifically incorporated into the EVs, producing high FP values due to a slow diffusional motion. We quantified EVs derived from two cell lines, HT29 and TCMK1 using the new strategy, with good sensitivity that was at par with the commercial method. The new method involves minimal complexity and hands-on time. In addition, FP signaling is inherently ratiometric and is robust against environmental noise.

Synthesis and spectral analysis of fluorescent probes for Ce4+ and OCl- ions based on fluorescein Schiff base with amino or hydrazine structure: Application in actual water samples and biological imaging.

Two Schiff base fluorescein probes (FDA, FDH) based on fluorescein-aldehyde and nitroaniline derivatives were synthesized. The effects of amino and hydrazine substituents in fluorescein backbones were examined via fluorescence and absorbance spectra. In the presence of Ce4+, the fluorescence of FDA was quenched due to the ligand to metal charge transfer (LMCT). Hypochloric acid can react with the CN bond, and blocking the photo induced electron transfer (PET) of FDH leads to enhancement of the fluorescence. FDA showed detection limits for Ce4+ and OCl- as low as 63 nM in concentration range of 0-4 μM. FDH showed detection limits for OCl- as low as 0.8 μM in concentration rang 0-100 μM. Polyvinylidene fluoride (PVDF) membrane containing the probes was prepared for the real-time qualitative detection of Ce4+ and OCl- in real water samples. The probes were successfully applied to biological imaging in vascular smooth muscle cells (VSMCs) and are expected to find applications in biosensing.