Fluorescein Isothiocyanate Isomer Research Articles

High biocompatible FITC-conjugated silica nanoparticles for cell labeling in both in vitro and in vivo models

Fluorescence nanosilica-based cell tracker has been explored and applied in cell biological research. However, the aggregation of these nanoparticles at physiological pH is still the main limitation. In this research, we introduced a novel fluorescence nano-based cell tracker suitable for application in live cells. The silica-coated fluorescein isothiocyanate isomer (FITC-SiO2) nanoparticles (NPs) were modified with carboxymethylsilanetriol disodium salt (FITC-SiO2-COOH), integrating the dianion form of FITC molecules. This nanosystem exhibited superior dispersion in aqueous solutions and effectively mitigated dye leakage. These labeled NPs displayed notable biocompatibility and minimal cytotoxicity in both in vitro and in vivo conditions. Significantly, the NPs did not have negative implications on cell migration or angiogenesis. They successfully penetrated primary fibroblasts, human umbilical vein endothelial cells and HeLa cells in both 2D and 3D cultures, with the fluorescence signal enduring for over 72 h. Furthermore, the NP signals were consistently observed in the developing gastrointestinal tract of live medaka fish larvae for extended periods during phases of subdued digestive activity, without manifesting any apparent acute toxicity. These results underscore the promising utility of FITC-SiO2-COOH NPs as advanced live cell trackers in biological research.

Enhanced detection of acrylamide using a versatile solid-state upconversion sensor through spectral and visual analysis

Acrylamide (AM) generally forms in high-temperature processes and has been classified as a potential carcinogen. In this study, we put forward a maneuverable solid-state luminescence sensor using polydimethylsiloxane (PDMS) as the matrix coupled with upconversion nanoparticles as the indicator. The core-shell upconversion nanoparticles emitting cyan light were uniformly encapsulated in PDMS. Then it was further modified with complementary DNA of AM aptamer. The nanocrystalline fluorescein isothiocyanate isomer (FITC), coupled with AM aptamer, was attached to the surface of PDMS. FITC effectively quenched the upconversion luminescence through fluorescence resonance energy transfer (FRET). The introduction of AM resulted in preferentially bound to aptamer caused the separation of the quencher and the donor, and led to luminescence recovery. The developed sensor was applied for both spectral and visual monitoring, demonstrating a detection limit (LOD) of 1.00 nM and 1.07 nM, respectively. Importantly, in the actual foodstuffs detection, there is no obvious difference between the results of this study and the standard method, which indicates the developed method has good accuracy. Therefore, this solid-state sensor has the potential for on-site detection using a smartphone device and an Android application.

Gd-MOF composites luminescent arrays for highly sensitive detection of epileptic drug and biomarkers

Nowadays, more than 50 million people suffer from epilepsy in worldwide. Epilepsy can be diagnosed more efficiently by detecting epileptic biomarkers such as human serum albumin (HSA) and relative nucleic acid. Pregabalin is an effective curing drug for epilepsy, but the dosage of it needs to be strictly controlled. In this work, to realize highly efficiently detect and discriminate epileptic curing drugs and biomarkers (pregabalin, epilepsy-DNA-1, and HSA), a novel Gd-MOF has been synthesized by H2L ligand (H2L = 5-(4-(imidzol-1-yl)phenyl)isophthalic acid), namely [Gd2L3(DMF)(H2O)2]n, which can be further functionalized with 3-Bromo-L-phenylalanine (L-3-Br-PHE-OH), thioflavin T (ThT) and fluorescein isothiocyanate isomer I labeled anti-HSA (anti-HSA/FITC), respectively. Through the post-synthetic modification (PSM) method, three Gd-MOF composite materials with good luminescence performance can be successfully prepared, namely Gd-MOF@L-3-Br-PHE-OH (Gd-MOF-1), Gd-MOF@ThT (Gd-MOF-2), Gd-MOF@anti-HSA/FITC (Gd-MOF-3). These three composite materials can be used for the detection of pregabalin, epilepsy-DNA-1, and HSA with high selectivity and sensitivity, respectively. The limit of detection (LOD) is 45.41 nM for pregabalin, 5.56 nM for epilepsy-DNA-1, and 5.11 nM for HSA. Development of detection application of Gd-MOF composite materials for drugs and biomarkers was also performed in the real biological samples. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) results demonstrated that Gd-MOF based luminescent array can simultaneously identify and detect epileptic drugs and biomarkers in the human serum environment. Finally, detection mechanisms for this epileptic drug and biomarkers were also investigated and discussed in detail.

On-line preconcentration of fluorescein derivatives of essential amino acids using sweeping in a nonionic micellar system

A rapid and effective method of nonionic micellar electrokinetic chromatography with laser-induced fluorescence detection and sweeping as an online preconcentration method was developed for the simultaneous determination of free essential amino acids in supplement formulations. The analytes were derivatized using fluorescein isothiocyanate isomer I dissolved in dimethyl sulfoxide. Optimal separation conditions were achieved using an uncoated fused silica capillary (40.0 cm effective length, 50.2 cm total length, 50 µm internal diameter), 20 mM Na2B4O7, (pH 9.3), 50 mM Brij-35, and 15% (v v-1) methanol as running buffer. Complete separation of all analytes was achieved within 15 min. The optimized method exhibited good linearity (r2 > 0.997), acceptable precision (1.3 – 12.4%), and accuracy (88 – 112%) in concentration range 20 – 1000 ng mL−1. The limit of detection was 6 ng mL−1. The method successfully determined the quantity of free essential amino acids in a dietary supplement containing spirulina.

Carbon dot and FITC conjugated dual-emission nanoprobe and its electrospun film for the sensing of ammonia

Nanomaterials are at the forefront of next-generation sensing technologies because of their unique and tunable properties. For monitoring trace ammonia (NH3) in human metabolism, we synthesized a dual-emission nanoprobe with an average diameter of 4 nm by conjugating the NH3-sensitivity fluorescein derivative, the fluorescein isothiocyanate isomer (FITC, green emitter), onto the surface of carbon dots (CDs, blue emitter). A significant Förster resonance energy transfer (FRET) effect was observed, where the CDs acted as energy donors and FITC moieties acted as energy acceptors. The fluorescent color changed obviously from blue to green once ammonia was introduced into the test chamber. Subsequently, the dual-emission nanoprobe was immobilized in polyvinyl pyrrolidone nanofibers with an average diameter of 400 nm via electrospinning, thus obtaining a ratiometric NH3 sensor. Results showed that the fabricated sensor with a high surface area exhibited high sensitivity (theoretical detection limit was 0.53 ppm), short response time (∼4.3 s), excellent selectivity, and good reversibility in tracing 0–300 ppm NH3. Further prototyped application studies reveal that the sensor is expected to serve as an indicator for monitoring breath/blood ammonia concentration. Thus, the newly developed fluorescent sensor was able to be a feasible solution for the application of nanomaterials in health and medicine.

Acrylates-based hydrophilic co-polymeric nanobeads as nanocarriers for imaging agents

Acrylates-based co-polymeric nanoparticles (PNPs) are widely used in nanomedicine applications due to their tunable hydrophilic surface, physical and chemical versatility. Particularly attracting is their use as nanocarriers for imaging agents. Herein, methyl methacrylate (MMA) and N,N-dimethylacrylamide (DMAA) monomers were used to synthesize hydrophilic p(MMA-co-DMAA) nanoparticles in the 200–600 nm size range via surfactant-free radical emulsion polymerization technique. Different MMA/DMAA molar ratios, temperatures, and reaction times were investigated to evaluate their role in determining the average particle size, polydispersity, and optimizing surface properties of PNPs. Nanoparticles formation, stability (in water and culture medium for cell growth), swelling behavior, structural features and molecular weights were assessed by spectroscopic, non-spectroscopic, and chromatographic techniques. Morphological profiles confirming spherical-shaped NPs were obtained at solid state via microscopies (FESEM, AFM). To use such colloids as potential imaging agents, PNPs were loaded with Y3+(aq) ions by the addition of aqueous solutions of YCl3 at different concentrations, and results compared with p(MMA-co-AA)-DTPA NPs (AA = acrylic acid) functionalized with DTPA chelating agent. Yttrium ions loading percentage was ca. 90% for both p(MMA-co-DMAA) and p(MMA-co-AA)-DTPA, with negligible release (<15%) over a month. Parallelly, optical imaging nanoprobes were obtained by physical encapsulation of fluorescein isothiocyanate isomer I (FITC) dye during the synthesis process, and the spontaneous FITC incorporation was evaluated by spectroscopic studies and fluorescence microscopy. Cytotoxicity studies on pristine and yttrium-loaded nanoparticles were done in vitro on human glioblastoma T98G cell line within 24 h of treatment. Transmission electron microscopy (TEM) studies on cancer cells treated with NPs confirmed an active uptake of PNPs through multiple endocytic pathways to reach the perinuclear region of the cell. Overall, this work elucidated the role of synthetic parameters for a rational design of hydrophilic PNPs as nanocarriers for imaging agents with potential applications in theranostics.

Smart Protein-Based Fluorescent Nanoparticles Prepared by a Continuous Nanoprecipitation Method for Pesticides' Precise Delivery and Tracing.

It is highly desirable to develop smart and green pesticide nanoformulations for improving pesticide targeting and reducing their inherent toxicity. Herein, we demonstrate a continuous nanoprecipitation method to construct a novel type of enzyme-responsive fluorescent nanopesticides (denoted as ABM@BSA-FITC/GA NPs) based on abamectin, fluorescein isothiocyanate isomer (FITC)-modified protein, and food-grade gum arabic. The as-prepared ABM@BSA-FITC/GA NPs exhibit good water dispersibility, excellent storage stability, and enhanced wettability compared to commercial formulations. The controlled release of pesticides can be achieved through protein degradation caused by trypsin. Most importantly, the deposition, distribution, and transport of the ABM@BSA-FITC/GA NPs are precisely tracked on target plants (cabbage and cucumber) by fluorescence. Furthermore, the ABM@BSA-FITC/GA NPs show the high control efficacy against Plutella xylostella L., which is comparable with commercial emulsifiable concentrate formulation. In consideration of its eco-friendly composition and absence of organic solvent, this pesticide nanoformulation has promising potential in sustainable plant protection.

Trace Explosive Detection Based on Photonic Crystal Amplified Fluorescence.

With increasing demand for public security and environmental protection, it is highly desirable to develop strategies to identify trace explosives (e. g., 2,4,6-trinitrotoluene (TNT)). Herein, we report novel photonic crystal (PC)-based sensor chips for trace TNT detection by using amplification effect of PCs on fluorescence (FL) signals. The sensor chips are constructed by integrating silica nanoparticles (NPs) modified with (3-aminopropyl)triethoxysilane (APTES) and fluorescein isothiocyanate isomer (FITC) and PC substrates. The amino groups on FITC-APTES-silica NPs can specifically bind with TNT molecules to form Meisenheimer complexes and strongly quench the FL signal of neighboring fluorophores FITC through Förster resonance energy transfer. PCs with matched PBG can amplify the FL signal of FITC-APTES-silica NPs about 24.4-fold and significantly improve sensitivity and resolution of trace TNT detection with the limit of detection of 0.23 nM. The PC-based sensor chips are stable, sensitive, and reliable TNT sensing platforms, showing great potential in homeland safety and environmental protection.

Determination of Nicotinic Acid in Food and Pharmaceuticals by a Simple and Rapid Fluorescence Polarization Immunoassay (FPIA)

A simple and rapid fluorescence polarization immunoassay (FPIA) based on polyclonal antibodies is reported for the first time for the determination of nicotinic acid. First, six nicotinic acid analogues were coupled with fluorescein isothiocyanate isomer I (FITC) to obtain tracers. The influences of tracer structure on the performance of FPIA as well as the structure-activity relationships were explored. Subsequently, the tracer concentration and antibody dilution ratio on the performance of FPIA were investigated. For the optimal conditions, the detection range of FPIA was from 1.1 to 145.6 μg mL−1 and its IC20 value as the limit of detection (LOD) was 1.1 μg mL−1. The cross-reactivity of antibodies with common vitamins was evaluated. The intra-assay recovery was from 92.0% to 101.2% in food and tablets, with inter-assay recoveries between 92.0% and 103.8%. The developed FPIA provided good sensitivity and reproducibility and therefore provided efficient and economic screening for nicotinic acid in food and tablets.

PolyJet-Based 3D Printing against Micromolds to Produce Channel Structures for Microchip Electrophoresis.

In this work, we demonstrate the ability to use micromolds along with a stacked three-dimensional (3D) printing process on a commercially available PolyJet printer to fabricate microchip electrophoresis devices that have a T-intersection, with channel cross sections as small as 48 × 12 μm2 being possible. The fabrication process involves embedding removable materials or molds during the printing process, with various molds being possible (wires, brass molds, PDMS molds, or sacrificial materials). When the molds are delaminated/removed, recessed features complementary to the molds are left in the 3D prints. A thermal lab press is used to bond the microchannel layer that also contains printed reservoirs against another solid 3D-printed part to completely seal the microchannels. The devices exhibited cathodic electroosmotic flow (EOF), and mixtures of fluorescein isothiocyanate isomer I (FITC)-labeled amino acids were successfully separated on these 3D-printed devices using both gated and pinched electrokinetic injections. While this application is focused on microchip electrophoresis, the ability to 3D-print against molds that can subsequently be removed is a general methodology to decrease the channel size for other applications as well as to possibly integrate 3D printing with other production processes.

Optimized synthesis of layered double hydroxide lactate nanosheets and their biological effects on Arabidopsis seedlings

BackgroundLayered double hydroxide lactate nanosheets (LDH-lactate-NS) are powerful carriers for delivering macro-molecules into intact plant cells. In the past few years, some studies have been carried out on DNA/RNA transformation and plant disease resistance, but little attention has been paid to these factors during LDH-lactate-NS synthesis and delamination, nor has their relationship to the DNA adsorption capacity or transformation efficiency of plant cells been considered.ResultsSince the temperature during delamination alters particle sizes and zeta potentials of LDH-lactate-NS products, we compared the LDH-lactate-NS stability, DNA adsorption rate and delivery efficiency of fluorescein isothiocyanate isomer I (FITC) of them, found that the LDH-lactate-NS obtained at 25 °C has the best characters for delivering biomolecules into plant cell. To understand the potential side effects and cytotoxicity of LDH-lactate-NS to plants, we compared the root growth rate between the Arabidopsis thaliana seedlings grown in the culture medium with 1–300 μg/mL LDH-lactate-NS and equivalent raw material, Mg(lactate)2 and Al (lactate)3. Phenotypic analysis showed LDH in a range of 1–300 μg/mL can enhance the root elongation, whereas the same concentration of raw materials dramatically inhibited root elongation, suggesting the nanocrystallization has a dramatical de-toxic effect to Mg(lactate)2 and Al (lactate)3. Since enhancing of root elongation by LDH is an unexpected phenomenon, we further designed experiments to investigate influence of LDH to Arabidopsis seedlings. We further used the gravitropic bending test, qRT-PCR analysis of auxin transport proteins, non-invasive micro-test technology and liquid chromatography-mass spectrometry to investigate the auxin transport and distribution in Arabidopsis root. Results indicated that LDH-lactate-NS affect root growth by increasing the polar auxin transport.ConclusionsOptimal synthesized LDH-lactate-NS can delivery biomolecules into intact plant cells with high efficiency and low cytotoxity. The working solution of LDH-lactate-NS can promote root elongation via increase the polar auxin transport in Arabidopsis roots.

Precisely Encoded Barcodes through the Structure-Fluorescence Combinational Strategy: A Flexible, Robust, and Versatile Multiplexed Biodetection Platform with Ultrahigh Encoding Capacities.

With the rapid development of suspension array technology, microbeads-based barcodes as the core element with sufficient encoding capacity are urgently required for high-throughput multiplexed detection. Here, a novel structure-fluorescence combinational encoding strategy is proposed for the first time to establish a barcode library with ultrahigh encoding capacities. Based on the never revealed transformability of the structural parameters (e.g., porosity and matrix component) of mesoporous microbeads into scattering signals in flow cytometry, the enlargement of codes number has been successfully realized in combination with two other fluorescent elements of fluorescein isothiocyanate isomer I (FITC) and quantum dots (QDs). The barcodes are constructed with precise architectures including FITC encapsulated within mesopores and magnetic nanoparticles as well as QDs immobilized on the outer surface to achieve the ultrahigh encoding level of 300 accompanied with superparamagnetism. To the best of knowledge, it is the highest record of single excitation laser-based encoding capacity up to now. Moreover, a ten-plexed tumor markers bioassay based on the tailored-designed barcodes has been evaluated to confirm their feasibility and effectiveness, and the results indicate that the barcodes platform is a promising and robust tool for practical multiplexed biodetection.

Life-Inspired Endogenous Dynamic Behavior of Lipid Droplet-like Microcompartments in Artificial Adipocyte-like Structures

Chemical systems that can replicate cellular behaviors are gaining increasing attention and are being used to study various biological processes. Here, a protein- or amylose-based assembly at an oi...

Anomalously Shaped Functional Particles Prepared by Thiol-Isocyanate Off-Stoichiometric Click Dispersion Polymerization.

Anomalously shaped microparticles have attractive advantages in applications. They are usually prepared by chain-growth polymerizations in heterogeneous systems. Recently, thiol-X step-growth polymerizations have been used to produce functional particles with a regular shape but rarely anomalous shapes. Herein, we report the preparation of anomalously shaped particles by thiol-isocyanate dispersion polymerization (Dis.P) in ethanol using polyvinylpyrrolidone (PVP) as a stabilizer and catalyst. Papillae-shaped, raspberry-like, and multibulged particles are prepared by tuning monomer combinations, contents, and feed ratios. Particle morphology evolutions during polymerization are observed by scanning electron microscopy (SEM). Distinct from previous works, particles with residual -SH groups are obtained even with equal moles of monomers added initially. The residue of -SH groups is revealed by Fourier transform infrared spectroscopy (FT-IR) analyses and confirmed by detection with a fluorescent probe containing disulfide linkage. Moreover, fluorescent particle probes are formed by the reaction of excess -NCO groups on particles with fluorescein isothiocyanate isomer I (FITC) and dithioacetal-functionalized perylenediimide (DTPDI). The probes are sensitive in detection of glutathione (GSH) and Hg2+ in water. Hg2+ as low as 1-0.1 ppb is detected using a raspberry-like particle probe with DTPDI.

Water-soluble superparamagnetic dysprosium-doped iron oxide flowerlike nanoclusters for high-resolution MR imaging

Magnetic nanomaterials have great application prospects in water treatment, disease diagnosis and therapy, surface adsorption and other fields. Herein, the water-soluble, biocompatible and superparamagnetic dysprosium-doped iron oxide flowerlike nanoclusters (DyIO NCs) were synthesized by thermal decomposition of iron oleate and dysprosium oleate in high-boiling solvent of phenyl ether with α,ω-dicarboxyl poly(ethylene glycol) as a capping agent. The size of the nanoclusters and the amount of Dy ions in the clusters can be controlled by simply adjusting the reaction parameters. The DyIO NCs exhibited excellent colloid stability under physiological conditions. The saturation magnetization and transverse relaxivity (r2) of DyIO NCs were significantly improved by the doping of Dy ions. The r2 value of 21 nm Dy0.209Fe2.791O4 NCs was 485.6 mM−1s−1, which is 9 times that of Dy-doped iron oxide nanoparticles (55.6 mM−1s−1), 4 times that of commercial Feridex (123.6 mM−1s−1) and 2 times that of iron oxide nanoclusters (253.9 mM−1s−1). In vivo imaging showed that DyIO NCs exhibited long blood circulation time and excellent enhancement for T2-weighted magnetic resonance imaging. In addition, the DyIO NCs can be functionalized with tumor targeting ligand cyclic Arg-Gly-Asp peptide and fluorescein isothiocyanate isomer I. This study provides an effective strategy for the development of new sensitive nanoprobes and drug delivery systems, and other applications requiring strong magnetic responses.

Cell membrane-coated gold nanoparticles for apoptosis imaging in living cells based on fluorescent determination

A nanoprobe was developed to achieve apoptosis detection by cell membrane-functionalized gold nanoparticles (AuNP-pep@Mem). The fluorescence of the fluorescein isothiocyanate isomer I (FITC)-labeled caspase-3 substrates was quenched by the attachment to AuNPs. The fluorescence signal was recovered via the cleavage of caspase-3 under apoptotic conditions. It exhibited a low detection limit of 1.3pg·mL-1 with a linear range from 3.2 to 100pg·mL-1 for caspase-3 detection with excitation wavelength of 490nm. After wrapped by thecell membrane, the nanoprobe was effectively delivered into cells with high cell permeability. AuNP-pep@Mem nanoprobe provided signal enhancement of 1.8 times in living cells compared to non-membrane-coated nanoparticles (AuNP-pep). In combination with its excellent stability, low LOD and good specificity, the AuNP-pep@Mem probe can be an ideal probe for fluorescence imaging of apoptosis. Graphical abstractSchematic representation of fluorescent determination for apoptosis in living cells based on cell membrane-coated gold nanoparticls.

Preparation and evaluation of fluorescent poly(p-phenyleneethylene) covalently coated microspheres with reactive sites for bioconjugation

Fluorescent microspheres with reactive sites for interacting with biomolecules are greatly demanded in flow cytometry based suspension array. Aiming to develop a new method for preparing fluorescent microspheres, two poly(p-phenyleneethylene) (PPE) conjugated polymers (CPs) with pedant carboxylic groups were synthesized via Sonogashira coupling and followed with hydrolysis of ester groups; then the conjugated polymers were immobilized onto monodispersed amino-modified porous poly(glycidylmethacrylate) (APGMA) microspheres via coupling reaction between carboxylic and amino groups to give APGMA-CP fluorescent microspheres. The fluorescent microspheres were found to have good photo- and thermal stability as well as negligible influence from rigorous washing. The emission was uniform all across the inner and surface of the spheres. To evaluate the effectiveness of bioconjugation on the fluorescent microspheres, fluorescein isothiocyanate isomer I (FITC) labeled bovine serum albumin (BSA) (BSA-FITC) was chosen as the representative biomolecule to react with the fluorescent microspheres to give APGMA-CP-BSA-FITC. In the flow cytometry study, fluorescence compensation between the V500 and FITC detectors (receiving signals from fluorophores excited by 405 nm and 488 nm, respectively), to remove the interference between the emission of FITC and CPs, was realized using singly-stained microspheres. Finally, APGMA-CP-BSA-FITC microspheres were found to be double positive for CP and FITC with very high percentage (>95%), suggesting the bioconjugation is very effective. This study provides a facile method for simultaneous introduction of fluorescence and reactive sites onto the microspheres, which is very promising to be used as general strategy for fabricating fluorescence microspheres for application in high-throughput technology.

Super Stable Fluorescein Isothiocyanate Isomer I Monolayer for Total Internal Reflection Fluorescence Microscopy.

Total internal reflection fluorescence microscopy (TIRFM) is an important method in surface science and for the analysis of surface-bound macromolecules. Here, we developed and explored the use of a novel fluorescein isothiocyanate isomer I (FITC)-adsorbed monolayer for alignment and validation of TIRFM measurements and configurations. Aqueous solutions of FITC exist as several different protolytic forms (dianionic, anionic, neutral, and cationic) with each form having different emission characteristics. However, the emission behavior of FITC adsorbed on hydrophilic, hydrophobic, and unmodified glass surfaces at different pH was unknown. TIRFM imaging and spectroscopy were used to study FITC and FITC-labeled bovine serum albumin (BSA-FITC) monolayers generated on three different glass surfaces. Monolayer emission intensity, spectra, and the photobleaching profiles were all dependent on pH and the surface properties of the glass. Very strangely, however, at pH 5.0 on hydrophobic surfaces, the FITC monolayers produced were both bright and apparently unbleachable over ∼20 min of imaging (60 s total exposure). During monolayer formation at pH 5.0, we saw clear evidence for concentration-based quenching, indicating high surface coverage. When the monolayer had been rinsed with buffer to remove unbound FITC, we observed an increase in emission intensity during illumination indicative of some form of photoactivated species being present. Eventually, the fluorescence emission stabilized and remained constant for extended periods of time with no evidence of photobleaching. We hypothesize that during the adsorption process (a hydrophobic-hydrophobic interaction) there was conversion to the fluorescent quinoid form of FITC. In contrast, at pH 7.4 and 9.6 on hydrophobic surfaces, FITC monolayers had well-defined, fast photobleaching kinetics (decay to ∼50% intensity in 5-10 s). The equivalent BSA-FITC monolayers were slightly brighter, with similar photobleaching kinetics. While the precise mechanism for this unusual behavior is still unknown, all these low-cost monolayers were easily prepared, were reproducible, and can serve as convenient test samples for TIRFM alignment, calibration, and validation prior to undertaking measurements with more sensitive biogenic or biological specimens.

Visualization and quantification of the cellular and extracellular components of Salmonella Agona biofilms at different stages of development.

Salmonella is a major food-borne pathogen able to persist in food processing environments because of its ability to form biofilms. A Salmonella enterica serotype Agona isolate from poultry (S24) was grown at 37°C in biofilms for up to 144 hours (H144) in attachment to polystyrene surfaces. Biofilm structures were examined at different stages in their development (H3, H24, H48, H72, H96 and H144) using confocal laser scanning microscopy (CLSM) in conjunction with fluorescent dyes for live cells (SYTO 9), dead cells (propidium iodide), proteins (fluorescein isothiocyanate isomer I), lipids (DiD’oil), α-polysaccharides (concanavalin A, tetramethylrhodamine conjugate), and β-polysaccharides (calcofluor white M2R). Strain S24 developed a robust biofilm at H72 (biovolume of 166,852.5 ± 13,681.8 μm3 in the observation field of 16,078.2 μm2). The largest biovolume of live cells was also detected at H72 (128,110.3 ± 4,969.1 μm3), decreasing thereafter, which was probably owing to the detachment of cells prior to a new phase of colonization. The percentage of dead cells with regard to total cells in the biofilms increased throughout the incubation, ranging from 2.3 ± 1.1% (H24) to 44.2 ± 11.0% (H144). Proteins showed the greatest biovolume among the extracellular components within the biofilms, with values ranging from 1,295.1 ± 1,294.9 μm3 (H3) to 19,186.2 ± 8,536.0 μm3 (H96). Maximum biovolume values of 15,171.9 ± 660.7 μm3 (H48), 7,055.3 ± 4,415.2 μm3 (H144), and 2,548.6 ± 1,597.5 μm3 (H72) were observed for β-polysaccharides, α-polysaccharides and lipids, respectively. A strong (P < 0.01) positive correlation was found between the total biovolume of biofilm and the biovolume of live cells, proteins and β-polysaccharides, which may serve as useful markers of biofilm formation. The present work provides new insights into the formation of S. Agona biofilms. Our findings may contribute to the designing of reliable strategies for preventing and removing these bacterial communities.

Use of fluorescein isothiocyanate isomer I to study the mechanism of intestinal absorption of fucoidan sulfate in vivo and in vitro.

A new method to label fucoidan sulfate was established with tyramine and fluorescein isothiocyanate isomer I (FITC). Fluorescence spectrophotometry and high performance liquid chromatography verified the successful labelling of fucoidan by FITC. The results of the single-pass intestinal perfusion indicated that the jejunum and ileum are the main absorption sites, and there was carrier saturation. In addition, fucoidan sulfate at 1mg/ml had no inhibitory effect on Caco-2 cell proliferation. Studies on the transmembrane transport mechanism showed that fucoidan can be absorbed because the apparent permeability coefficient of the drugs (Papp ) A→B was 3.78+0.03 ×10-6 and that of B→A was 1.42+0.19 ×10-6 . The peak absorption of fucoidan occurred at 120min after administration; moreover, the higher the concentration used, the worse the absorption was, suggesting the saturation of transport carriers. The absorption was temperature dependent: the absorption at 37°C was much better than that at 4°C. Further, the absorption of fucoidan sulfate might rely on clathrin endocytosis as chlorpromazine (10μg/ml) significantly inhibited it.