Fluorescence Intensity Of Fluorescein Isothiocyanate Research Articles

Development of peptide-based biosensors for detecting cross-linking and deamidation activities of transglutaminases.

Transglutaminases (TGs) are a protein family that catalyzes isopeptide bond formation between glutamine and lysine residues of various proteins. There are eight TG isozymes in humans, and each is involved in diverse biological phenomena due to their characteristic distribution. Abnormal activity of TG1 and TG2, which are major TG isozymes, is believed to cause various diseases, such as ichthyosis and celiac disease. To elucidate TGs' mechanisms of action and develop new therapeutic strategies, it is essential to develop bioprobes that can specifically examine the activity of each TG isozyme, which has not been sufficiently studied. We previously have identified several substrate peptide sequences containing Gln residues for each isozyme and developed a method to detect isozyme-specific activities by incorporating a labeled substrate peptide into lysine residues of proteins. We prepared the fluorescein isothiocyanate (FITC)-labeled Gln substrate peptide (FITC-K5 and FITC-T26) and Rhodamine B-labeled Lys substrate peptide (RhoB-Kpep). Each TG reaction specifically cross-linked these probe pairs, and the proximity of FITC and Rhodamine B significantly decreased the fluorescence intensity of FITC depending on the concentration and reaction time of each TG. In this study, we developed a peptide-based biosensor that quickly and easily measures TG isozyme-specific activity. This probe is expected to be helpful in elucidating TG's physiological and pathological functions and in developing compounds that modulate TG activity.

Local pH mapping in the cell adhesion nano-interfaces on a pH-responsive fluorescence-dye-immobilized substrate.

Cell-substrate adhesion nano-interfaces can, in principle, exhibit a spatial distribution of local pH values under the influence of the weakly acidic microenvironment of glycocalyx grafted on lipid bilayer cell membrane which is compressed and closely attached to culture substrate in the vicinity of integrin-adhesion complexes. However, a simple local pH distribution imaging methodology has not been developed. In this study, to visualize the local pH distribution at the cell adhesion interface, we prepared glass substrates chemically modified with a pH-responsive fluorescent dye fluorescein isothiocyanate (FITC), observed the distribution of FITC fluorescence intensity at the adhesion interface of fibroblast (NIH/3T3) and cancer cells (HeLa), and compared the FITC images with the observed distribution of focal adhesions. FITC images were converted to pH mapping based on the pH-fluorescence calibration data of surface-immobilized FITC pre-measured in different pH media, which showed significantly larger regions with lowered pH level (6.8-7.0) from outside the cell (pH 7.4) were observed at the thick inner periphery of HeLa cells while 3T3 cells exhibited smaller lowered pH regions at the thin periphery. The lowered pH regions overlapped with many focal adhesions, and image analysis showed that larger focal adhesions tend to possess more lowered pH sites inside, reflecting enhanced glycocalyx compression due to accumulated integrin-adhesion ligand binding. This tendency was stronger for HeLa than for 3T3 cells. The role of glycocalyx compression and the pH reduction at the cell adhesive interface is discussed.

CCL25 Inhibition Alleviates Sepsis-Induced Acute Lung Injury and Inflammation.

PurposeAcute lung injury (ALI) is a common clinical syndrome with high mortality. The chemokine ligand 25 (CCL25) is involved in inflammation, leukocyte trafficking and immunoregulation. However, the role and mechanism of CCL25 in ALI are not fully understood yet. The aim of this study was to explore the relationship between acute lung injury and CCL25.Patients and MethodsIn this study, we first examined chemokine expression in sepsis patients and found that serum CCL25 expression levels were relatively high in sepsis patients compared to healthy individuals. Based on this, we designed in vitro and in vivo experiments to verify the validity of the theory. In vitro, we used lipopolysaccharide-stimulated human pulmonary microvascular endothelial cells (HPMECs). In vivo, we established male C57BL/6 mice cecal ligation puncture (CLP) model of sepsis.ResultsIn vitro, we used lipopolysaccharide-stimulated human pulmonary microvascular endothelial cells (HPMECs) and found significantly higher expression of CCL25 by enzyme-linked immunosorbent assay. Inhibition of CCL25 resulted in a significant decrease in the expression of inflammatory cytokines in HPMECs. In addition, we found that CCL25 promoted increased endothelial permeability by reducing the expression of tight junction proteins and was associated with activation of the P38 MAPK pathway by measuring the transepithelial electrical resistance and fluorescence intensity of fluorescein isothiocyanate. Results from luciferase assays and chromatin immunoprecipitation assays showed that inhibition of NF-κB activity in HPMECs decreased CCL25 expression, but addition of recombinant CCL25 increased cell permeability and inflammatory cytokine expression. In vivo, we established male C57BL/6 mice cecal ligation puncture (CLP) model of sepsis. We found that inhibition of CCL25 significantly reduced inflammatory cytokine expression in a CLP-induced sepsis model, thereby alleviating lung tissue damage in mice.ConclusionOur study suggests that CCL25 contributed to the development of ALI by modulating the functions of microvascular endothelial cells.

DNAzyme-functionalized porous carbon nanospheres serve as a fluorescent nanoprobe for imaging detection of microRNA-21 and zinc ion in living cells.

The present study shows that a dual-signal nanoprobe consisting of DNAzyme-functionalized porous carbon nanospheres (PCNs) responds to microRNA-21 and zinc ion (Zn2+). The fluorescent probe undergoes anincrease in the fluorescence intensity of fluorescein isothiocyanate (FITC) (with excitation/emission wavelengths at 488/517nm) and the fluorescence intensity of cyanine-5 (Cy5) (with excitation/emission wavelengths at 633/670nm) in the presence of microRNA-21 and Zn2+. The recognition between microRNA-21 and its complementary strand in the PCNs induces the separation of Zn2+-specific DNAzyme from PCNs, thus resulting in the increase of green fluorescence, and the exogenous Zn2+ triggers the rupture of cleavage strand of DNAzyme and recovery of red fluorescence. This nanoprobe allows us to acquire in vitro the determination of microRNA-21 in the range of 2-300nM with a detection limit of 0.57nM and the determination of Zn2+ in the range 2-100nM with a detection limit of 0.43nM, and in situ simultaneous imaging in MCF-7 breast cancer cells. Therefore, this strategy permits to obtain the expression levels of different biomarkers in living cells, providing a useful tool for diagnosis of cancers and understanding their biological process. Graphical abstract Schematic representation of the DNAzyme-functionalized porous carbon nanospheres for the imaging analysis of microRNA-21 and Zn2+ in living cells.

Fluorescent nanosensor designing via hybrid of carbon dots and post-imprinted polymers for the detection of ovalbumin

We reported a facile strategy to assemble a ratiometric nanosensor for the ovalbumin (OVA) fluorescence determination and meanwhile it can be utilized for selective visual identification by naked eyes with fluorescent test papers under 365 nm UV lamp. The nanosensor was prepared through simply mixing blue color carbon dots (CDs) and green color core-shell imprinted polymers. Blue CDs were used directly as the internal reference without participating in the imprinting process and modified molecularly imprinted polymers (MIPs) were synthesized by post-imprinting, using fluorescein isothiocyanate (FITC) as fluorescence enhanced signal. Upon the addition of different concentrations of OVA, the fluorescence intensity of FITC was enhanced, while the fluorescence intensity of CDs was almost unchanged, leading to a detection limit as low as 15.4 nM. Accordingly, the fluorescence color was gradually changed from blue to dark olive green to green with naked eyes observation. Moreover, the ratiometric nanosensor was successfully applied to detect OVA in the human urine samples with satisfactory recoveries attaining of 92.0–104.0% with relative standard deviation (RSD) of 3.3–3.9% and 93.3–101.0% with RSDs of 2.7–3.8% for the spiked chicken egg white samples. This strategy reported here opens a novel pathway for biomacromolecule detection in real applications and can realize the visual observation on fluorescent test papers.

Ultrasensitive fluorescence detection of Fe3+ ions using fluorescein isothiocyanate functionalized Ag/SiO2/SiO2 core–shell nanocomposites

Fluorescein isothiocyanate (FITC) functionalized Ag/SiO2/SiO2 core–shell (CS) nanoparticles (NPs) and nanorods (NRs) are synthesized by chemical method to perform the fluorescence detection of Fe3+ in aqueous medium. The Fe3+ ion sensing is performed on the basis of metal-enhanced fluorescence and subsequent fluorescence quenching of fluorophore (fluorescein isothiocyanate) in aqueous medium. The intensity of fluorescence of FITC is found to get enhanced with increasing the silica shell thickness. The fluorescence intensity of FITC of spherical Ag/SiO2/SiO2 (FITC) CS NPs (AgSiF50) is amplified up to 2.25-folds whereas, in case of Ag/SiO2/SiO2 (FITC) CS NRs (AgSiFNR), the fluorescence intensity of FITC is amplified up to 3.63-folds compared to that of pure FITC. Owing to the amplified fluorescence of FITC, AgSiF50 and AgSiFNR samples are evaluated for detection of Fe3+ in an aqueous medium and the proposed nanoprobes exhibit good sensitivity towards Fe3+ detection because of strong binding affinity of Fe3+ with isothiocyanate group of FITC. The limit of detections for AgSiF50 and AgSiFNR are found to be 19.4 nM and 0.8 nM, respectively. The BET surface areas of AgSiF50 and AgSiFNR are calculated to be 157.64 m2 g−1 and 193.42 m2 g−1, respectively. The higher sensitivity of AgSiFNR compared to AgSiF50 for Fe3+ ion sensing is accredited to the higher adsorption capacity and higher fluorescence of FITC. This Ag/SiO2/SiO2 (FITC) CS NRs probe is a very simple and efficient sensor for Fe3+ ion detection and thus, using this sample, a very simple Arduino based electronic device is fabricated which can give real-time application of the idea.

A Tf-modified tripterine-loaded coix seed oil microemulsion enhances anti-cervical cancer treatment.

PurposeA transferrin-modified microemulsion carrying coix seed oil and tripterine (Tf-CT-MEs) was developed for improved tumor-specific accumulation and penetration to enhance cervical cancer treatment.Materials and methodsTripterine-loaded coix seed oil microemulsion (CT-MEs) was prepared through one-step emulsion method. The morphology, size, and zeta potential of CT-MEs and Tf-CT-MEs were examined by transmission electron microscopy and dynamic light scattering. The cellular uptake and mechanisms of HeLa cells were investigated by flow cytometry. Intratumor penetration was investigated using a HeLa three-dimensional (3D) tumor spheroid as the model. The cytotoxicity of the CT-MEs and Tf-CT-MEs against HeLa cells were evaluated by the MTT assay. Additionally, the apoptotic rate of CT-MEs and Tf-CT-MEs inducing apoptosis in HeLa cells was evaluated.ResultsIn the physicochemical characterization, coix seed oil and CT-MEs exhibited a small size (32.47±0.15 nm) and nearly neutral surface charge (−0.36±0.11 mV). After modification with transferrin, the particle size of Tf-CT-MEs slightly increased to 40.02±0.21 nm, but the zeta potential decreased remarkably to -13.63±1.31 mV. The IC50 of Tf-CT-MEs against HeLa cells was 0.7260 µM, which was 2.58-fold lower than that of CT-MEs. In cellular studies, the intracellular fluorescence intensity of fluorescein isothiocyanate (FITC)-labeled Tf-CT-MEs (FITC/Tf-CT-MEs) was 2.28-fold higher than that of FITC-labeled CT-MEs (FITC/CT-MEs). The fluorescence signal of Tf-CT-MEs was observed at 350 µm below the surface of the 3D tumor spheroid. The apoptotic rate of cells treated with Tf-CT-MEs was 1.73- and 2.77-fold higher than that of cells treated with CT-MEs and tripterine, respectively, which was associated with mitochondrial-targeted delivery of tripterine. Moreover, Tf-CT-MEs was capable of significantly downregulating the cellular level of antiapoptotic proteins and arrested cell proliferation in the G2/M phase.ConclusionTaken together, Tf-CT-MEs holds promising potential to be an efficient drug delivery system for combinational therapy of cervical cancer.

The Influence of Virus Infection on the Extracellular pH of the Host Cell Detected on Cell Membrane.

Influenza virus infection can result in changes in the cellular ion levels at 2–3 h post-infection. More H+ is produced by glycolysis, and the viral M2 proton channel also plays a role in the capture and release of H+ during both viral entry and egress. Then the cells might regulate the intracellular pH by increasing the export of H+ from the intracellular compartment. Increased H+ export could lead indirectly to increased extracellular acidity. To detect changes in extracellular pH of both virus-infected and uninfected cells, pH sensors were synthesized using polystyrene beads (ϕ1 μm) containing Rhodamine B and Fluorescein isothiocyanate (FITC). The fluorescence intensity of FITC can respond to both pH and temperature. So Rhodamine B was also introduced in the sensor for temperature compensation. Then the pH can be measured after temperature compensation. The sensor was adhered to cell membrane for extracellular pH measurement. The results showed that the multiplication of influenza virus in host cell decreased extracellular pH of the host cell by 0.5–0.6 in 4 h after the virus bound to the cell membrane, compared to that in uninfected cells. Immunostaining revealed the presence of viral PB1 protein in the nucleus of virus-bound cells that exhibited extracellular pH changes, but no PB1 protein are detected in virus-unbound cells where the extracellular pH remained constant.

Graphene-Based Fluorescence-Quenching-Related Fermi Level Elevation and Electron-Concentration Surge.

Intermolecular p-orbital overlaps in unsaturated π-conjugated systems, such as graphene and fluorescent molecules with aromatic structure, serve as the electron-exchanged path. Using Raman-mapping measurements, we observe that the fluorescence intensity of fluorescein isothiocyanate (FITC) is quenched by graphene, whereas it persists in graphene-absent substrates (SiO2). After identifying a mechanism related to photon-induced electron transfer (PET) that contributes to this fluorescence quenching phenomenon, we validate this mechanism by conducting analyses on Dirac point shifts of FITC-coated graphene. From these shifts, Fermi level elevation and the electron-concentration surge in graphene upon visible-light impingements are acquired. Finally, according to this mechanism, graphene-based biosensors are fabricated to show the sensing capability of measuring fluorescently labeled-biomolecule concentrations.

Bitargeted microemulsions based on coix seed ingredients for enhanced hepatic tumor delivery and synergistic therapy

A hepatic tumor bitargeted microemulsions drug delivery system using coix seed oil and coix seed polysaccharide (CP) acting as anticancer components, as well as functional excipients, was developed for enhanced tumor-specific accumulation by CP-mediated enhancement on passive tumor targeting and modification of galactose stearate (tumor-targeted ligand). In the physicochemical characteristics studies, galactose stearate-modified coix seed multicomponent microemulsions containing 30% CP (w%) (Gal-C-MEs) had a well-defined spherical shape with a small size (47.63±1.41nm), a narrow polydispersity index (PDI, 0.101±0.002), and a nearly neutral surface charge (−4.37±1.76mV). The half-maximal inhibitory concentration (IC50) of Gal-C-MEs against HepG2 cells was 70.2μg/mL, which decreased by 1.8-fold in comparison with that of coix seed multicomponent microemulsions (C-MEs). The fluorescence intensity of fluorescein isothiocyanate (FITC)-loaded Gal-C-MEs (FITC-Gal-C-MEs) internalized by HepG2 cells was 1.8-fold higher than that of FITC-loaded C-MEs (FIT C-C-MEs), but the cellular uptake of the latter became reduce by 1.6-fold when the weight ratio of CP decreased up to 10%. In the cell apoptosis studies, C-MEs (containing 30% CP) did not show a significant difference with Gal-C-MEs, but exhibited 3.3-fold and 1.5-fold increase relative to C-MEs containing 10% CP and 20% CP, respectively. In the in vivo tumor targeting studies, Cy5-loaded Gal-C-MEs (Cy5-Gal-C-MEs), notably distributed in the tumor sites and still found even at 48h post-administration, displayed the strongest capability of tumor tissue accumulation and retention among all the test groups. Most importantly, Gal-C-MEs had stronger inhibition of tumor growth, prolonged survival time and more effectively tumor cell apoptosis induction in comparison with C-MEs containing different amounts of CP, which further confirmed that a certain amount of CP and tumor-targeted ligand were of great importance to potent anticancer efficacy. The aforementioned results suggested that Gal-C-MEs presented promising potential as a highly effective and safe anticancer drug delivery system for enhanced liver cancer delivery.

Residual cancer lymphocytes in patients with chronic lymphocytic leukemia after therapy show increased expression of surface antigen CD52 detected using quantitative fluorescence cytometry.

Rituximab and alemtuzumab, mAbs used in recent years to treat CLL, are directed against antigens CD20 and CD52. CD20 is not highly expressed by CLL tumor cells, and rituximab does not have significant effectiveness in CLL unless combined with chemotherapy. Alemtuzumab targets CD52, which is much more highly expressed, and is currently the most effective agent used alone for CLL. Variability in expression of both antigens among these patients might be related to different individual therapeutic responses to mAb therapy. A total 95 patients diagnosed with CLL and/or SLL were divided into 4 groups: (1) untreated; (2) in complete or partial remission; (3) disease in progression; and (4) diagnosed with SLL. Flow cytometry of peripheral blood cells included gating of the CD5(+)CD19(+) tumor population, within which mean fluorescence intensity of fluorescein isothiocyanate (FITC) conjugated with anti-CD20 or anti-CD52 antibody was measured. The resulting expression of the 2antigens was deduced from the calibration curve using Quantum FITC particles. Expression of CD20 showed no significant differences among the 4 groups of patients. However, significantly greater expression of surface antigen CD52 was recorded in patient group 2 in complete or partial remission (P< .001). The residual population of CLL cells after therapy is characterized by increased surface detection of CD52. Although the exact cause of this phenomenon is unknown, our results provide a basis to consider the potential for CLL consolidation therapy using alemtuzumab.

Honey-Induced Protein Stabilization as Studied by Fluorescein Isothiocyanate Fluorescence

Protein stabilizing potential of honey was studied on a model protein, bovine serum albumin (BSA), using extrinsic fluorescence of fluorescein isothiocyanate (FITC) as the probe. BSA was labelled with FITC using chemical coupling, and urea and thermal denaturation studies were performed on FITC-labelled BSA (FITC-BSA) both in the absence and presence of 10% and 20% (w/v) honey using FITC fluorescence at 522 nm upon excitation at 495 nm. There was an increase in the FITC fluorescence intensity upon increasing urea concentration or temperature, suggesting protein denaturation. The results from urea and thermal denaturation studies showed increased stability of protein in the presence of honey as reflected from the shift in the transition curve along with the start point and the midpoint of the transition towards higher urea concentration/temperature. Furthermore, the increase in ΔG D H2O and ΔG D 25°C in presence of honey also suggested protein stabilization.

Highly sensitive turn-on fluorescent detection of captopril based on energy transfer between fluorescein isothiocyanate and gold nanoparticles

A novel approach for highly sensitive detection of captopril was developed based on the fluorescence resonance energy transfer (FRET) between gold nanoparticles (Au NPs) and fluorescein isothiocyanate (FITC), in which FITC acts as the donor and Au NPs as the acceptor. The fluorescence intensity of fluorescein isothiocyanate (FITC) was strictly quenched as a result of noncovalently adsorbed on Au NPs. Upon the addition of captopril, the fluorescence intensity of FITC “turn-on” due to the competition between captopril and FITC towards the surface of Au NPs. Under the optimum conditions, the fluorescence intensity of the released FITC displays a linear relationship in the range of 20μgL−1 to 500μgL−1 of captopril. Lower limit of detection for captopril, at the signal-to-noise ratio of 3 (3σ), was 2.6μgL−1. The developed methodology was successfully applied for the determination of captopril in human plasma.

Comparison of Photostability and Photobleaching Properties of FITC- and Dylight488- Conjugated Herceptin

ABSTRACT Nowadays, fluorescent dyes have become significant tools in immunostaining experiments. Different fluorescent dyes have various physicochemical traits. Some of these traits, for instance photobleaching and photostability, are applicable calibers in such experiments as immunocytochemistry (ICC), immunohistochemistry (IHC), and flow cytometry (FC). In this study, photobleaching, photostability, and mean fluorescence intensity (MFI) of FITC (fluorescein isothiocyanate)- and Dylight488- conjugated Herceptin, the humanized anti Her2 monoclonal antibody drug, were examined. After conjugation of FITC and Dylight488 to Herceptin, their degree of labeling (DOL) was calculated by a spectrophotometer. Photobleaching, photostability, and MFI of conjugates were subsequently investigated by ICC and FC on BT-474 cells. Dylight488 labeled Herceptin revealed higher fluorescent lifetime and photostability than FITC conjugated Herceptin.

A study on increased permeability and morphological changes in actin cytoskeleton and tight junction of vascular endothelial cells induced by tumor necrosis factor-alpha

To study the effect of human tumor necrosis factor-alpha (TNF-alpha) on permeability of human vascular endothelial cell (EA.hy926) monolayer and its mechanism. 5, 10, 20 microg/L TNF-alpha was respectively added to the cultured endothelial cell monolayer for 24 hours, or 10 microg/L TNF-alpha for 6, 12, 24 hours. Human vascular endothelial cell (EA.hy926) monolayer permeability was measured by detecting fluorescence intensity of fluorescein isothiocyanate (FITC) labeled dextran. Immunofluorescence and laser confocal microscopy were used to assess vascular endothelial actin cytoskeleton (F-actin) and tight junction protein (ZO-1) distribution. Western blotting was used to assess ZO-1 expression. Compared with control group, TNF-alpha significantly increased endothelial permeability and induced F-actin redistribution and stress fiber formation with ZO-1 derangement. Gaps increased obviously between endothelial cells. Furthermore, Western blotting showed that TNF-alpha reduced ZO-1 expression in a dose- and time-dependent manner. TNF-alpha increased endothelial cell permeability by damaging integrity of endothelial barrier function.

Fluoroimmunoassay for Antigen Based on Fluorescence Quenching Signal of Gold Nanoparticles

A unique, sensitive, and highly specific fluoroimmunoassay system for antigen detection using gold and magnetic nanoparticles has been developed. The assay is based on the fluorescence quenching of fluorescein isothiocyanate caused by gold nanoparticles coated with monoclonal antibody. To demonstrate its analytical capabilities, the magnetic nanoparticles were coated with anti-alpha-fetoprotein polyclonal antibodies, which specifically bound with alpha-fetoprotein. Gold nanoparticles coated with anti-alpha-fetoprotein monoclonal antibodies could sandwich the alpha-fetoprotein captured by the magnetic nanoparticle probes. The sandwich-type immunocomplex was formed on the surface of magnetic nanoparticles and could be separated by a magnetic field. The supernatant liquid, which contained the unbound gold nanoparticle probes, was used to quench the fluorescence, and the fluorescence intensity of fluorescein isothiocyanate at 516 nm was proportional to the alpha-fetoprotein concentration. The result showed that the limit of detection of alpha-fetoprotein was 0.17 nM. This new system can be extended to detect target molecules with matched antibodies and has broad potential applications in immunoassay and disease diagnosis.

A long lifetime chemical sensor: study on fluorescence property of fluorescein isothiocyanate and preparation of pH chemical sensor

The fluorescence property of fluorescein isothiocyanate (FITC) in acid–alkaline medium was studied by spectrofluorimetry. The characteristic of FITC response to hydrogen ion has been examined in acid–alkaline solution. A novel pH chemical sensor was prepared based on the relationship between the relative fluorescence intensity of FITC and pH. The measurement of relative fluorescence intensity was carried out at 362 nm with excitation at 250 nm. The excellent linear relationship was obtained between relative fluorescence intensity and pH in the range of pH 1–5. The linear regression equation of the calibration graph is F=66.871+6.605 pH ( F is relative fluorescence intensity), with a correlation coefficient of linear regression of 0.9995. Effects of temperature, concentration of FITC on the response to hydrogen ion had been examined. It was important that this chemical sensor was long lifetime, and the property of response to hydrogen ion was stable for at least 70 days. This pH sensor can be used for measuring pH value in water solution. The accuracy is 0.01 pH unit. The results obtained by the pH sensor agreed with those by the pH meter. Obviously, this pH sensor is potential for determining pH change real time in biological system.

Reagentless and regenerable immunosensor for monitoring of immunoglobulin G based on non-separation immunoassay

Based on the enhancement of fluorescein isothiocyanate (FITC) fluorescence caused by reactions between proteins, we developed a reagentless, regenerable and rapid immunosensing system to determine immunoglobulin G (IgG). Fluorescence intensity of the immobilized FITC depends on IgG concentration, ranging from 10 to 50 μg/ml, specifically, even with co-existing proteins. The response time is 30 min during steady-state measurement and is less than a minute during transient measurement. When the FITC-labeled protein A binds to IgG, the surrounding atmosphere of FITC becomes hydrophobic. Since the fluorescence intensity of fluorescent substances generally increases at a hydrophobic environment, FITC fluorescence intensity increases with the concentration of protein A bonding to IgG. This system is regenerable because the fluorescence enhancement repeatedly occurs every time the immobilized fluorescent reagent is immersed in sample solutions.

A total internal reflection fluorescence biosensor for aluminum (III)

Total internal reflection fluorescence (TIRF) is widely used for investigating interfacial interactions. It has proven to be a well-suited technique for biosensing applications due to its surface sensitivity and minimal sample consumption. In this paper, the protein phosvitin has been investigated as a molecular sensor for the detection of aluminum (III). Phosvitin was labeled with fluorescein isothiocyanate (FITC) and immobilized on a quartz surface via EDC/NHS chemistry. Phosvitin–FITC and aluminum complexation, both in free solution and surface-bound form, was studied. A TIRF biosensor selective for Al 3+ was constructed using surface-immobilized phosvitin–FITC. An enhancement of phosvitin–FITC fluorescence intensity was observed upon addition of Al 3+. The response was linear over the range 0.1–10 μM of aluminum concentration.

Mycelial pellet intrastructure and visualization of mycelia and intracellular lipid in a culture of Mortierella alpina.

The intrastructure of mycelial pellets of Mortierella alpina, which accumulate fatty acids in mycelia, was visualized following labeling with fluorescein isothiocyanate (FITC) and Nile red using fluorescence microscopy. The pellet was an ellipse shape, but its intrastructure was shaped as a doughnut with a cave inside. Using three-dimensional image analysis, it was shown that the lipid was produced on the edge of the pellet, which corresponded to the area where the mycelial density was high. The cavity ratio of the pellet section was determined on the basis of the FITC fluorescence intensity, and in the early culture stage remained at 0.2 in a 10-kl fermentor culture, but finally increased to 0.35. Mycelial pellet volume paralleled the cavity ratio. Application of the technique used here allows analysis of the intrastructure of fungal pellets and new types of fungal biological study.