Nerve growth factor from Indian Russell's viper venom (RVV-NGFa) shows high affinity binding to TrkA receptor expressed in breast cancer cells: Application of fluorescence labeled RVV-NGFa in the clinical diagnosis of breast cancer.

Nerve growth factor (NGF) is a minor and neglected component of snake venom. Present study describes the purification and characterization of a NGF isoform (RVV-NGFa) from Indian Russell's viper venom (RVV). RVV-NGFa showed a protonated molecular ion [MH+] at m/z 17388.725 Da. The RVV-NGFa induced neuritogenesis in PC-12 cells but did not show cytotoxicity in mammalian cells, hemolytic activity, platelet modulation, and interference in blood coagulation system which are the characteristic pharmacological properties of RVV. By ELISA and immunofluorescence microplate reader assay, the RVV-NGFa showed appreciable binding to TrkA receptor expressed in breast cancer MDA-MB-231 and MCF-7 cells; nevertheless, pre-incubation of cells with anti-TrkA (and not TrkB or TrkC) or anti-p75NTR antibody significantly decreased (p < 0.05) this binding. The RVV-NGFa demonstrated insignificant binding with non-cancerous cells (HEK-293, L6) lacking TrkA receptor. The binding of RVV-NGFa to TrkA receptor of breast cancer cells resulted in internalization of ligand (RVV-NGFa)-receptor (TrkA) complex to cell cytoplasm in a time-dependent manner. The spectrofluorometric study demonstrated an interaction between RVV-NGFa and cytosolic domain of the purified TrkA receptor. The fluorescence (FITC)-tagged RVV-NGFa depicted a strong fluorescence signal that was observed under a fluorescence microscope and determined by fluorescence microplate reader assay post binding to breast cancer cells; but no fluorescence signal was detected after incubating FITC-RVV-NGFa with non-cancerous L6 and HEK-293 cells. The clinical application of FITC/fluorescence nanoparticle tagged RVV-NGFa for the ex vivo and in vivo diagnosis of breast cancer is highly promising.

Low-concentration trypsin detection from a metal-enhanced fluorescence (MEF) platform: Towards the development of ultra-sensitive and rapid detection of proteolytic enzymes.

Proteolytic enzymes, which serve to degrade proteins to their amino acid building blocks, provide a distinct challenge for both diagnostics and biological research fields. Due to their ubiquitous presence in a wide variety of organisms and their involvement in disease, proteases have been identified as biomarkers for various conditions. Additionally, low-levels of proteases may interfere with biological investigation, as contamination with these enzymes can physically alter the protein of interest to researchers, resulting in protein concentration loss or subtler polypeptide clipping that leads to a loss of functionality. Low levels of proteolytic degradation also reduce the shelf-life of commercially important proteins. Many detection platforms have been developed to achieve low-concentration or low-activity detection of proteases, yet many suffer from limitations in analysis time, label stability, and ultimately sensitivity. Herein we demonstrate the potential utility of fluorescein derivatives as fluorescent labels in a new, turn-off enzymatic assay based on the principles of metal-enhanced fluorescence (MEF). For fluorescein sodium salt alone on nano-slivered 96-well plates, or Quanta Plates™, we report up to 11,000x enhancement for fluorophores within the effective coupling or enhancement volume region, defined as ~100 nm from the silver surface. We also report a 9% coefficient of variation, and detection on the picomolar concentration scale. Further, we demonstrate the use of fluorescein isothiocyanate-labeled YebF protein as a coating layer for a MEF-based, Quanta Plate™ enzymatic activity assay using trypsin as the model enzyme. From this MEF assay we achieve a detection limit of ~1.89 ng of enzyme (2.8 mBAEE activity units) which corresponds to a minimum fluorescence signal decrease of 10%. The relative success of this MEF assay sets the foundation for further development and the tuning of MEF platforms for proteolytic enzyme sensing not just for trypsin, but other proteases as well. In addition, we discuss the future development of ultra-fast detection of proteases via microwave-accelerated MEF (MAMEF) detection technologies.

An efficient method for FITC labelling of proteins using tandem affinity purification.

Fluorescence-based assays are extremely diverse, sensitive and robust experimental methods for investigating the conformational changes, enzyme kinetics, dynamics and molecular interactions. A prerequisite for most of these experimental approaches is to label the protein of interest with one or more extrinsic fluorophores with desired photophysical properties. Fluorescein isothiocyanate (FITC), due to its high quantum efficiency and conjugate stability, is most widely used fluorescence labelling reagent for such experimental approaches. However, the bottlenecks in this labelling reaction is requirement of high protein concentration, maintenance of protein stability during the labelling process as well as high background fluorescence due to ineffective removal of unreacted FITC, prior to fluorescence studies. Therefore, to overcome these inadequacies or limitations, we have modified the existing protocol by introducing tandem affinity purification tags at the N- and C-terminus of target protein. Using this modified method, we have efficiently labelled target protein with significant decrease in precipitation, degradation and background fluorescence of unreacted FITC. This facile and rapid technique may also be used as a basis for labelling procedures with other fluorophores and hence has a broad application in spectroscopic studies.

Direct fluorescent labeling for efficient biological assessment of inhalable particles.

Labeling of aerosol particles with a radioactive, magnetic, or optical tracer has been employed to confirm particle localization in cell compartments, which has provided useful evidence for correlating toxic effects of inhaled particles. However, labeling requires several physicochemical steps to identify functionalities of the inner or outer surfaces of particles, and moreover, these steps can cause changes in size, surface charge, and bioactivity of the particles, resulting in misinterpretations regarding their toxic effects. This study addresses this challenging issue with a goal of introducing an efficient strategy for constantly supplying labeled aerosol particles in a single-pass configuration without any pre- or post-physicochemical batch treatments of aerosol particles. Carbon black (CB, simulating combustion-generated soot) or calcium carbonate (CC, simulating brake-wear fragments) particles were constantly produced via spark ablation or bubble bursting, respectively. These minute particles were incorporated with fluorescein isothiocyanate-poly(ethylene glycol) 2-aminoethyl ether acetic acid solution at the orifice of a collison atomizer to fabricate hybrid droplets. The droplets successively entered a diffusion dryer containing 254-nm UV irradiation; therefore, the droplets were dynamically stiffened by UV to form fluorescent probes on particles during solvent extraction in the dryer. Particle size distributions, morphologies, and surface charges before and after labeling were measured to confirm fluorescence labeling without significant changes in the properties. In vitro assays, including confocal imaging, were conducted to confirm the feasibility of the labeling approach without inducing significant differences in bioactivity compared with untreated CB or CC particles.

Sensitive and inexpensive digital DNA analysis by microfluidic enrichment of rolling circle amplified single-molecules.

Single molecule quantification assays provide the ultimate sensitivity and precision for molecular analysis. However, most digital analysis techniques, i.e. droplet PCR, require sophisticated and expensive instrumentation for molecule compartmentalization, amplification and analysis. Rolling circle amplification (RCA) provides a simpler means for digital analysis. Nevertheless, the sensitivity of RCA assays has until now been limited by inefficient detection methods. We have developed a simple microfluidic strategy for enrichment of RCA products into a single field of view of a low magnification fluorescent sensor, enabling ultra-sensitive digital quantification of nucleic acids over a dynamic range from 1.2 aM to 190 fM. We prove the broad applicability of our analysis platform by demonstrating 5-plex detection of as little as ∼1 pg (∼300 genome copies) of pathogenic DNA with simultaneous antibiotic resistance marker detection, and the analysis of rare oncogene mutations. Our method is simpler, more cost-effective and faster than other digital analysis techniques and provides the means to implement digital analysis in any laboratory equipped with a standard fluorescent microscope.

Lysozyme as a recognition element for monitoring of bacterial population.

Bacterial infections remain a significant challenge in biomedicine and environment safety. Increasing worldwide demand for point-of-care techniques and increasing concern on their safe development and use, require a simple and sensitive bioanalysis for pathogen detection. However, this goal is not yet achieved. A design for fluorescein isothiocyanate-labeled lysozyme (FITC-LYZ), which provides quantitative binding information for gram-positive bacteria, Micrococcus luteus, and detects pathogen concentration, is presented. The functional lysozyme is used not only as the pathogenic detection platform, but also as a tracking reagent for microbial population in antibacterial tests. A nonlinear relationship between the system response and the logarithm of the bacterial concentration was observed in the range of 1.2×10(2)-1.2×10(5) cfu mL(-1). The system has a potential for further applications and provides a facile and simple method for detection of pathogenic bacteria. Meanwhile, the fluorescein isothiocyanate -labeled lysozyme is also employed as the tracking agent for antibacterial dynamic assay, which show a similar dynamic curve compared with UV-vis test.

An assessment of the ability of the obstruction-scaling model to estimate solute diffusion coefficients in hydrogels.

The ability to estimate the diffusion coefficient of a solute within hydrogels has important application in the design and analysis of hydrogels used in drug delivery, tissue engineering, and regenerative medicine. A number of mathematical models have been derived for this purpose; however, they often rely on fitted parameters and so have limited predictive capability. Herein we assess the ability of the obstruction-scaling model to provide reasonable estimates of solute diffusion coefficients within hydrogels, as well as the assumption that a hydrogel can be represented as an entangled polymer solution of an equivalent concentration. Fluorescein isothiocyanate dextran solutes were loaded into sodium alginate solutions as well as hydrogels of different polymer volume fractions formed from photoinitiated cross-linking of methacrylate sodium alginate. The tracer diffusion coefficients of these solutes were measured using fluorescence recovery after photobleaching (FRAP). The measured diffusion coefficients were then compared to the values predicted by the obstruction-scaling model. The model predictions were within ±15% of the measured values, suggesting that the model can provide useful estimates of solute diffusion coefficients within hydrogels and solutions. Moreover, solutes diffusing in both sodium alginate solutions and hydrogels were demonstrated to experience the same degree of solute mobility restriction given the same effective polymer concentration, supporting the assumption that a hydrogel can be represented as an entangled polymer solution of equivalent concentration.

Three-dimensional human arterial wall models for in vitro permeability assessment of drug and nanocarriers.

Monolayers of endothelial cells (1L-ECs) have been generally used as in vitro vascular wall models to study the vascular mechanisms and transport of substances. However, these two-dimensional (2D-) system cannot represent the properties of native vascular walls which have a 3D-structure and are composed of not only ECs, but also smooth muscle cells (SMCs) and other surrounding tissues. Here in, 5-layered (5L) 3D-arterial wall models (5L-AWMs) composed of EC monolayer and 4-layered SMCs were constructed by hierarchical cell manipulation. We applied the 5L-AWMs to evaluate their barrier function and permeability to nano-materials in order to analyze drug, or drug nanocarrier permeability to the blood vessel in vitro. Barrier property of the 3D-AWMs was confirmed by Zonula occludens (ZO-1) staining and their transendothelial electrical resistance (TEER), which was comparable to 1L-ECs, while the SMCs showed close to zero. The effect of substance size to permeability across the 5L-AWMs was clearly observed from dextrans with various molecular weights, which agreed well with the known phenomena of the in vivo blood vessels. Importantly, transport of nano-materials could be observed across the depth of 5L-AWMs, suggesting the advantage of 3D-AWMs over general 2D-systems. By using this system, we evaluate the transport of 35 nm phenylalanine-modified poly(γ-Glutamic Acid) nanoparticles (γ-PGA-Phe NPs) as a candidate of biodegradable drug carrier. Interestingly, despite of having comparable size to dextran-2000 k (28 nm), the γ-PGA-Phe NPs distinctly showed approximately 20 times faster transport across the 5L-AWMs, suggesting the effect of intrinsic properties of the substance on the transport. This in vitro evaluation system using the 3D-AWMs is therefore useful for the design and development of nano-drug carriers for treatment of vascular diseases, such as atherosclerosis.

Single-step turn-on homogeneous fluorescent immunosensor for rapid, sensitive, and selective detection of proteins.

Quick & easy: A simple and positive-readout fluorescent immunosensor was developed based on a quencher composed of a 3 nm gold nanoparticle (GNP) covalently linked to a Fab fragment, which proved to be stable and specific. The method allows for direct and sensitive detection of target antigens in homogeneous solutions in one step without any separation steps. It has been successfully applied to rapidly quantify the amount of secretory immunoglobulin A (sIgA) in human saliva samples.

In vitro assessment of alkylglyceryl-functionalized chitosan nanoparticles as permeating vectors for the blood-brain barrier.

A series of O-substituted alkylglyceryl chitosans with systematically varied alkyl chain length and degree of grafting has been employed for the formulation of aqueous nanoparticulate systems, which were in turn investigated for their effects on a modeled blood-brain-barrier system of mouse-brain endothelial cells. Barrier function measurements employing electric cell-substrate impedance sensing and analyses of tight junction-specific protein profiles have indicated that the alkylglyceryl-modified chitosan nanoparticles impact upon the integrity of the model blood-brain barrier, whereas confocal microscopy experiments have demonstrated the efficient cellular uptake and the perinuclear localization of these nanoparticles. The application of nanoparticles to the model blood-brain barrier effected an increase in its permeability, as demonstrated by following the transport of the tracer molecule fluorescein isothiocyanate.

A low-cost intracellular delivery system based on microbubble and high gravity field.

In this paper, we developed a low-cost intracellular delivery system based on microbubble and high gravity field. We successfully delivered FITC-Dextran (40kD) into hard-to-deliver THP-1 cells. The results showed that our method achieved high delivery efficiency up to 80%. It was found that the delivery efficiency and cell viability were closely related to the centrifuge speed. We speculated that the burst of microbubbles causes transient pore opening thus increasing the chance of biomolecules entering cells. This fast, low-cost and easy-to-operate protocol is very promising for delivering therapeutic genes and drugs into any cells which do not actively take up extracellular materials. This method is most effective for in-vitro delivery, but after delivery, treated cells might be injected back to human for in-vivo imaging.

First assessment of diffusion coefficients in model cheese by fluorescence recovery after photobleaching (FRAP).

Mass transfer of solutes like salt, moisture and metabolites, is very important for the final quality of cheese, through the control of the brining and ripening processes. Numerous studies have reported salt and water transfer properties in cheese, but few have dealt with other solutes. Moreover, most diffusion coefficients have been obtained by macroscopic and destructive methods. We developed the fluorescence recovery after photobleaching (FRAP) technique on a confocal microscope to measure in situ and at the microscopic scale diffusion properties inside cheese. A model matrix based on ultrafiltrated milk was used. FITC-dextran molecules were chosen as models of migrant solutes. Diffusion coefficients were estimated with a modelling approach which takes into account diffusion during the bleach phase. The FITC-dextrans (4 and 20 kDa) were able to migrate in the proteinic network, but their mobility was 2.2-3 times lower than in water, depending on their size.

Rapid identification of fluorochrome modification sites in proteins by LC ESI-Q-TOF mass spectrometry.

Conjugation of either a fluorescent dye or a drug molecule to the ε-amino groups of lysine residues of proteins has many applications in biology and medicine. However, this type of conjugation produces a heterogeneous population of protein conjugates. Because conjugation of fluorochrome or drug molecule to a protein may have deleterious effects on protein function, the identification of conjugation sites is necessary. Unfortunately, the identification process can be time-consuming and laborious; therefore, there is a need to develop a rapid and reliable way to determine the conjugation sites of the fluorescent label or drug molecule. In this study, the sites of conjugation of fluorescein-5'-isothiocyanate and rhodamine-B-isothiocyanate to free amino groups on the insert-domain (I-domain) protein derived from the α-subunit of lymphocyte function-associated antigen-1 (LFA-1) were determined by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF MS) along with peptide mapping using trypsin digestion. A reporter fragment of the fluorochrome moiety that is generated in the collision cell of the Q-TOF without explicit MS/MS precursor selection was used to identify the conjugation site. Selected ion plots of the reporter ion readily mark modified peptides in chromatograms of the complex digest. Interrogation of theses spectra reveals a neutral loss/precursor pair that identifies the modified peptide. The results show that one to seven fluorescein molecules or one to four rhodamine molecules were attached to the lysine residue(s) of the I-domain protein. No modifications were found in the metal ion-dependent adhesion site (MIDAS), which is an important binding region of the I-domain.

Quantitative assessment of intestinal injury using a novel in vivo, near-infrared imaging technique.

Intestinal injury owing to inflammation, severe trauma, and burn is a leading cause of morbidity and mortality. Currently, animal models employed to study the intestinal response to injury and inflammation depend on outdated methods of analysis. Given that these classic intestinal assays are lethal to the experimental animal, there is no ability to study the gut response to injury in the same animal over time. We postulated that by developing an in vivo assay to image intestinal injury using fluorescent dye, it could complement other expensive, time-consuming, and semiquantitative classic means of detecting intestinal injury. We describe a novel in vivo, noninvasive method to image intestinal injury using a charge-coupled device (CCD) camera that allows for serial visual and quantitative analysis of intestinal injury. Our results correlate with traditional, time-consuming, semiquantitative assays of intestinal injury, now allowing the noninvasive, nonlethal assessment of injury over time.

Detection enhancement in nano-channels using micro-machined silicon groove.

The present paper reports on an experimental study of the possibility to use a micro-machined detection groove to enhance the detection sensitivity in flat-rectangular nano-channels for ultra-rapid liquid chromatography separations. Transversally running detection grooves with three different axial widths (respectively, 2, 4 and 6 microm) and one depth (4.75 microm) were tested in glass and silicon channels for the whole range of detectable fluorescein isothiocyanate isomer I, FITC, concentrations. The groove with the most square-like cross-section (i.e., 4 microm wide and 4.75 microm deep) yielded the best combination of detection gain and minimal additional band broadening. In a 1cm long channel, the effective plate loss caused by the 4 microm wide groove would only be of the order of 20%, while the gain in S/N-ratio was of the order of a factor of 5. The detection groove concept yields larger gains in silicon channel substrates than in glass channel substrates, due to the larger stray light losses occurring in the latter.

Cytometric assessment of histone H2AX phosphorylation: a reporter of DNA damage.

DNA damage that leads to formation of DNA double-strand breaks (DSBs) induces phosphorylation of histone H2AX on Ser-139 at sites flanking the breakage. Immunocytochemical detection of phosphorylated H2AX (denoted as gammaH2AX) thus provides a marker of DSBs. The method presented in this chapter describes the detection of gammaH2AX for revealing the presence of DSBs, combined with differential staining of cellular DNA for revealing the cell cycle phase. The detection of gammaH2AX is based on indirect immunofluorescence using secondary antibody tagged with fluorescein isothiocyanate (FITC) while DNA is counterstained with propidium iodide (PI). Intensity of cellular green (FITC) and red (PI) fluorescence is measured by flow cytometry and bivariate analysis of the data is used to correlate the presence of DSBs with the cell cycle phase.

A simplified flow cytometry method of CD4 and CD8 cell counting based on thermoresistant reagents: implications for large scale monitoring of HIV-infected patients in resource-limited settings.

OBJECTIVE: To validate a simplified flow cytometry assay for CD4 and CD8 T cell counting based on monoclonal antibodies which are made resistant to high temperatures (simplified thermoresistant assay (STRA)). METHOD: The STRA employs FITC-conjugated anti-CD4 and anti-CD8 monoclonal antibodies, predispensed into test tubes and chemically treated to be resistant to high temperatures. Five correlation studies were performed in three different laboratories on a total of 560 blood samples from HIV-1 infected patients. Each study correlated the STRA with either double or single platform assays currently available. Accelerated stability tests on the FITC-conjugated monoclonal antibodies were performed to assess the resistance of the STRA to high temperatures. RESULTS: Comparison of STRA with both single platform and double platform assays gave correlation coefficients ranging 0.957-0.987 for CD4+ T cells and 0.946-0.968 for CD8+ T cells. In all correlation studies there was a perfect data overlapping in the low-pathological interval of CD4+ T cells (0-400 cells/ml). The FITC-conjugated CD4 and CD8 monoclonal antibodies maintained intact binding activity and fluorescence brightness after storage for 4 weeks at 45 degrees C and can be stored for up to 8 years in regular conditions (+4 degrees C). CONCLUSIONS: The STRA correlates well with both single-platform and double-platform flow-cytometry assays currently used to assess CD4+ T cells. The test procedure is simple, rapid, and easy to perform. The reagents can be stored under unfavorable environmental conditions for long period of time. These features should facilitate access to flow cytometry testing in resource-poor settings.

Flow cytometric assessment of effects of fluvastatin on low-density lipoprotein receptor activity in stimulated T-lymphocytes from patients with heterozygous familial hypercholesterolemia.

To test the effects of fluvastatin on low-density lipoprotein (LDL) receptor activity in patients with heterozygous familial hypercholesterolemia, the authors measured LDL receptor activity in stimulated T-lymphocytes prepared from 34 patients before and after treatment with 40 mg fluvastatin daily for 12 weeks. Maximally induced pretreatment LDL receptor activities did not correlate with pretreatment plasma cholesterol levels or with changes in plasma cholesterol levels during treatment, and there were no significant changes in LDL receptor activity during treatment. Barring methodological problems, two explanations are possible. Insofar that LDL receptor activity in lymphocytes reflects LDL receptor activity in the liver, the results suggest that the primary response to treatment with fluvastatin in heterozygous familial hypercholesterolemia (FH) patients is not enhanced LDL receptor activity. Alternatively, fluvastatin increases LDL receptor activity in hepatocytes but has little effect on receptor-dependent lipoprotein catabolism in extrahepatic tissues in vivo.

Double fluorescent flow cytometric assessment of bacterial internalization and binding by epithelial cells.

This study describes a new flow cytometric method for assessment of phagocytosis of specific bacteria (bacillus Calmette-Guérin (BCG) and Escherichia coli) by bladder epithelial cells. The internalization assay consisted of labeling bacteria chemically with fluorescein isothiocyanate (FITC). Subsequent to incubation of fluoresceinated bacteria with internalizing cells, adherent nonphagocytosed bacteria were marked by two-step labeling using specific antibodies and phycoerythrin (PE)-conjugated antibodies. Double fluorescent FACS analysis differentiated between bacterial phagocytosis and adherence. The validity of the method was shown by inhibition of BCG phagocytosis at 4 degrees C by cytochalasin B, by removal of excess free bacteria, and by anti-BCG antibodies. BCG-phagocytizing and -nonphagocytizing cell lines were discriminated by applying this technique to a series of bladder carcinoma cell lines. There seemed to be a relationship between phagocytic capacity and grade of differentiation in these cell lines, which may have implications for topical BCG immunotherapy in superficial bladder cancer. In conclusion, a new, reliable, rapid, and relatively simple double fluorescent method is described for quantification of specific bacterial internalization by large numbers of (bladder) epithelial cells. This method should be generally applicable to the study of in vitro interaction between bacteria and different types of host cells.

Ataxonomic assessment of phytoplankton integrity by means of flow cytometry.

Flow cytometry, a method well established in medicine and biotechnology, can also make an important contribution to (applied) limnological as well as ecotoxicological studies on phytoplankton. Flow cytometry can, for instance, contribute to the ataxonomic structural and functional assessment of phytoplankton. This approach may serve as a supplement to the well-established taxonomic evaluation by means of various microscope techniques. We present some examples for such ataxonomic phytoplankton evaluation. These examples include phytoplankton of eutrophicated and acidified water bodies as well as slowly flowing rivers. Phytoplankters may be differentiated by their pigment contents into carotinoid-rich ones (such as Chrysophyceae, Bacillariophyceae, and Dinophyceae) and carotinoid-poor ones (such as Euglenophyceae and Chlorophyceae). As a useful biomass parameter of phytoplankton algae we tested successfully protein staining by fluorescein isothiocyanate. We discuss the advantage of this approach as compared with results obtained by Coulter counter or by biomass calculations from microscope analyses. Up to now, evaluation of the biological quality of pelagic water bodies is still laborious and time consuming because of the microscopical examination of planktic communities usually practiced. As a possible improvement we present a structural ataxonomic approach for assessing the integrity of individual phytoplankters (on the basis of physiological parameters) as well as of the phytoplankton communities that is based on annual means of biomass spectra. Flow cytometry can provide considerable relief.