Simple Fluorescence Technique Research Articles

Sensing the melting transition of semicrystalline polymers via a novel fluorescence technique

The melting point (Tm) is an important parameter that dictates the physical properties and thereby applications of semicrystalline thermoplastics. Conventional melting transition characterization techniques, e.g., differential scanning calorimetry, ellipsometry, optical microscopy, and X-ray based methods, probe Tm by monitoring temperature (T)-dependent thermal properties, morphologies, and microstructures. Although these techniques have been well developed, they tend to have their own limitations, e.g., X-ray based techniques require X-ray beams and most previous techniques can only measure spatially averaged properties throughout a film cross section or an entire bulk sample. Here, we have developed a new, simple, and versatile fluorescence technique for probing the melting transitions of semicrystalline thermoplastics. With this approach, fluorescent probes are incorporated into a semicrystalline polymer, either by physical doping or covalent labeling, and their T-dependent fluorescence intensity data exhibit a stepwise decrease nearby Tm because of the reduced restriction of intramolecular motion when crystals start to melt. Interestingly, the first derivative of the obtained T-dependent fluorescence intensity data can reveal more details of the underlying melting transition, e.g., the onset and endset of the melting transition as well as the peak melting temperature. The melting point values determined by fluorescence agree well with those characterized by conventional differential scanning calorimetry, confirming the validity of our fluorescence technique for probing melting transitions. In addition, this fluorescence technique can be applied with various types of fluorescent probes (e.g., fluorophores exhibiting either aggregation-induced emission or aggregation-caused quenching effects) and generalized to many semicrystalline thermoplastics (e.g., poly(l-lactic acid), poly(caprolactone), and poly(ethylene oxide)), while maintaining excellent sensitivity to melting transitions. Overall, our fluorescence technique represents an easy and contact-free melting point characterization approach that may allow for novel location-specific Tm investigations within heterogeneous polymeric systems (e.g., multilayer films, blends, and composites). Such a novel fluorescence technique can expand the toolbox for Tm determination and potentially lead to unprecedented insights into the design principles for semicrystalline thermoplastics.

Correlating Quantitative Measurements of Radical Production by Photocatalytic TiO2 with Daphnia magna Toxicity.

Increased use of titanium dioxide (TiO2 ) nanoparticles (NPs) in domestic and industrial applications has increased the risk for adverse environmental outcomes based on an elevated likelihood of organism exposure. Anatase TiO2 NP exposure to ultraviolet A (UV-A) radiation in aquatic environments generates radical oxygen species (ROS), which may ultimately be responsible for increased organism toxicity. We have identified and measured the 2 most relevant ROS species, hydroxyl and superoxide radicals, and described that ROS can be modeled using the highly reactive hydroxyl radical to provide an upper bound for toxicity. The TiO2 NPs were co-exposed to increasing natural organic matter (NOM) amounts (measured as concentration of dissolved organic carbon [DOC]) and simulated-sunlight UV-A intensities. Radical production rate was determined using fluorescence spectroscopy and was positively correlated with increases in TiO2 concentration and UV-A intensity, and negatively correlated with increased DOC concentration. Daphnia magna toxicity was also found to decrease with NOM addition, which is attributed to the decreased radical production rate with increased DOC concentrations. We demonstrate that the rate of ROS production from simulated-sunlight-irradiated TiO2 NPs can be quantified using relatively simple fluorescent techniques. We show that toxicity to TiO2 NP varies greatly with conditions, and that concentration alone is a poor predictor of toxicity. Describing toxicity/hydroxyl radical measurement may be a more accurate way to describe overall risk. We provide a framework for a simple model to describe toxicity/hydroxyl radical. These conclusions demonstrate the importance of considering exposure conditions as a means of risk management during TiO2 NP toxicity testing, waste management, and regulatory decisions. Environ Toxicol Chem 2021;40:1322-1334. © 2021 SETAC.

Fluorescent visualization of oil displacement in a microfluidic device for enhanced oil recovery applications.

A microfluidic device was developed to mimic the reservoir pore-scale and track the oil/water phases during air flooding. The chip was generated by combining soft-lithography and NOA81 replication. A unique feature of this approach is the inclusion of fluorescent dyes into the oil/water phases, allowing for real-time visualization of oil recovery without altering the phases' surface properties. As a proof of concept, the air was injected into the water/oil-flooded device for enhanced oil recovery applications.

Femtomolar Detection of Dengue Virus DNA with Serotype Identification Ability.

Dengue surveillance trusts only on reverse transcription-polymerase chain reaction (RT-PCR) type methodologies for confirmation of dengue virus serotypes; however, its real time application is restricted due to the expensive, complicated, and time-consuming process. In search of a new sensing system, here, we have reported a two-way-detection method for Dengue virus (DENV) serotype identification along with DNA quantification by using a new class of nanocomposite of gold nanoparticles (AuNP) and nitrogen, sulfur codoped graphene quantum dots (N,S-GQDs). The N,S-GQDs@AuNP has been used for serotype detection via a simple fluorescence technique using four dye-combined probe DNAs which is further validated by confocal microscopy. The quantification of the DNA has been measured by the differential pulse voltammetric (DPV) technique using methyelene blue as a redox indicator. Results obtained in this study, clearly demonstrate that the N,S-GQDs@AuNP can efficiently detect the four serotypes of DENV individually in the concentration range of 10-14 to 10-6 M with the LOD of 9.4 fM. In addition, to confirm its applicability in long chained complex DNA system, the sensor was also applied to the clinically isolated DENV DNA and showed satisfactory performances for serotype identification as well as quantification. We hope this simple and reliable method can pave an avenue for the development of sensitive and robust sensing probes in biomedical applications.

Non-invasive Neurite Mechanics in Differentiated PC12 Cells.

Thermal Fluctuations Spectroscopy (TFS) in combination with novel optical-based instrumentation was used to study mechanical properties of cell-cultured neurites with a spatial resolution limited only by the light diffraction. The analysis of thermal fluctuations together with a physical model of cellular elasticity allow us to determine relevant mechanical properties of neurite as axial tension σ, flexural rigidity , plasma membrane tension γ, membrane bending rigidity , and cytoskeleton to membrane-coupling ρbk, whose values are consistent with previously reported values measured using invasive approaches. The value obtained for the membrane-coupling parameter was used to estimate the average number of coupling elements between the plasma membrane and the cytoskeleton that fell in the range of 30 elements per area of the laser spot used to record the fluctuations. Furthermore, to expand the TFS analysis, we investigate the correlation between F-actin linear density and the mechanical features of PC12 neurites. Using a hybrid instrument that combines TFS and a simple fluorescent technique, our results show that the fluctuations are related with the F-actin concentration. These measurements have an advantage of not requiring the application of an external force, allowing as to directly establish a correlation between changes in the mechanical parameters and cytoskeleton-protein concentrations. The sensibility of our method was also tested by the application of TFS technique to PC12 neurite under Paraformaldehyde and Latrunculin-A effect. These results show a dramatic modification in the fluctuations that are consistent with the reported effect of these drugs, confirming the high sensitivity of this technique. Finally, the thermal fluctuation approach was applied to DRG axons to show that its utility is not limited to studies of PC12 neurites, but it is suitable to measure the general characteristic of various neuron-like cells.

Discerning the effects of photoinhibition and photoprotection on the rate of oxygen evolution in Arabidopsis leaves

Higher plants possess a set of interconnected processes to regulate light harvesting. Non-photochemical quenching of chlorophyll a fluorescence (NPQ) is the fastest process activated to protect the photosystem (PS) II from the absorption of excess light energy. However, damage of PSII reaction centers (RCIIs) is often inevitable, a phenomenon known as photoinhibition. Both NPQ and photoinhibition undermine PSII quantum yield (ΦPSII). Recently, we devised a fluorescence-based methodology that uses the coefficient of photochemical quenching measured in the dark following illumination (qPd) to assess the intactness of RCIIs. This procedure enables to express ΦPSII as a function (ƒ) of NPQ and qPd, ΦPSII=ƒ(NPQ,qPd), thus allowing to efficiently discern between the effects of protective NPQ and photoinhibition upon the efficiency of electron transport. In this study, we addressed the relationship between qPd and ΦPSII measured by photosynthetic oxygen evolution in intact leaves of Arabidopsis. We found a linear correlation between qPd and ΦPSII of oxygen evolution (as well as Fv/Fm). This relates to the fact that qPd reflects the onset of photoinhibition. These results further demonstrate the validity of the qPd parameter and underlying theory in quantitatively assessing PSII efficiency solely by using this effective and simple fluorescence technique.

Phosphoprotein Detection on Protein Electroblot Using a Phosphate-Specific Fluorophore.

The reversible phosphorylation of phosphoproteins is a vital regulatory process for many cellular pathways. A reliable and simple fluorescent detection technique for phosphoproteins has been developed using a small-molecule organic fluorophore, Pro-Q Diamond dye. This was originally developed for use in gel staining, but a new formulation has allowed for its use in protein blotting. The dye binds noncovalently and selectively to the phosphate moiety, so proteins lacking phosphate groups and other macromolecules, such as DNA or RNA, are not detected. It uses a standard electrophoresis and electroblotting technique, which can blot the sample onto nitrocellulose membranes or polyvinylidene fluoride (PVDF). The electroblotting is followed by staining with the dye and destaining. The blot can then be read by multiple types of imaging devices such as a laser-based gel scanner. This process is compatible with matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) and Edman sequencing. It can also be followed by standard chemiluminescent, colorimetric, and fluorogenic detection techniques used in immunoblotting.

Optical percolation and oxygen diffusion in poly(divinylbenzene) doped poly(methyl methacrylate) latex flms

AbstractA simple fluorescence technique is proposed for the measurement of the diffusion coefficient of oxygen into poly(methyl methacrylate) (PMMA) latex‐poly(divinylbenzene) (PDVB) composite films. Percolation model was used by using photon transmission (PT) technique to interpret the distribution of PDVB particles in PMMA lattice. Optical results were interpreted according to site percolation theory. The optical percolation threshold value and critical exponent were calculated as, Rc = 0.03 and, β = 0.34, respectively. PT measurements were performed for eight different PDVB content (0, 1.5, 3, 5, 10, 20, 40, and 60) wt%. Pyrene (P) functionalized PDVB cross‐linked spherical microspheres with diameters of 2.5 μm were synthesized by using precipitation polymerization technique followed by click coupling reaction. The diameter of the PMMA particles prepared by emulsion polymerization was in the range of 0.5–0.7 μm. PMMA/PDVB composite films were then prepared by physically blending of PMMA latex with PDVB microspheres at various compositions. The steady‐state fluorescence method was used to monitor oxygen diffusion into these (0, 5, 10, 20, and 40 wt%) latex films. Diffusion coefficients, D, of oxygen were determined by the fluorescence quenching method by assuming Fickian transport and were found to be increased from 1.8 × 10−11 to 36.6 × 10−11 cm2 s−1 with increasing PDVB content. This increase in D values was explained with formation of microvoids in the film by using PT technique. POLYM. COMPOS., 2013. © 2012 Society of Plastics Engineers

A simple fluorescent labeling technique to study virus adsorption in Newcastle disease virus infected cells

The present study demonstrates that the fluorescent general membrane dyes PKH67 and PKH26 are suitable to label Newcastle disease virus, an enveloped virus belonging to the family of paramyxoviridae. Adsorption of the labeled virus particles was tracked, visualized and quantitated using confocal laser scanning microscopy. The specificity of PKH-labeling was determined by colocalization analysis of the PKH signal with NDV-specific immunolabeling, and by using mock-infected controls and infection with detergent-pretreated labeled virus particles. The infectivity of the NDV particles was not affected by the labeling procedure as indicated by the results of a cytotoxicity ATP assay, an apoptosis assay and detection of virus-specific RNA and protein by qPCR and Western blotting, respectively, in cells infected with PKH-labeled and unlabeled virus particles. This technique can be used as an inexpensive, sensitive and rapid alternative method in the analysis of adsorption and internalization of enveloped viruses by the infected cells.

Oxygen Diffusion into Polymer‐Clay Composite Films as a Function of Clay Content and Temperature

AbstractSummary: A simple fluorescence technique is proposed for the measurement of the diffusion coefficient of oxygen into polystyrene‐clay composite films as a function of clay content and temperature. The composite films were prepared from a mixture of surfactant‐free pyrene‐labeled polystyrene latexes and modified Na‐montmorillonite clay of various compositions at room temperature. Diffusion measurements were performed with films at room temperature for seven different clay contents (0, 5, 10, 20, 30, 50 and 60 wt.%). The diffusion coefficients of oxygen increased from 7.4 × 10−10 to 26.9 × 10−10 cm2s−1 with increasing clay content. On the other hand, diffusion measurements were performed over a temperature range of 25–70 °C for 0, 5 and 20 wt.% clay content films. The calculated diffusion activation energies decreased from 2.44 to 0.44 kcal/mol with increasing clay content. No clay content and temperature effects were observed on quenching rate constant and mutual diffusion coefficient values. The results showed that the diffusion coefficients are strongly dependent on both the temperature and clay content in the film.

Oxygen diffusion into polystyrene–bentonite films

A simple fluorescence technique is proposed for the measurement of the diffusion coefficient of oxygen into polystyrene–clay composite films. The composite films were prepared from the mixture of surfactant-free pyrene (P)-labeled polystyrene latexes (PS) and modified bentonite (MNaLB) at various compositions at room temperature. These films were annealed at 200 °C above the glass transition ( T g) temperature of polystyrene for 10 min. Oxygen diffusion into the films was monitored with steady state fluorescence (SSF) measurements. Measurements were performed at room temperature for different film compositions (0, 5, 10, 20, 30, 50 and 60 mass% modified bentonite) films to evaluate the effect of MNaLB content on oxygen diffusion. The diffusion coefficient, D of oxygen was determined by the fluorescence quenching method by assuming Fickian transport and increased from 7.4 × 10 − 10 to 26.9 × 10 − 10 cm 2 s − 1 with increasing MNaLB content. This increase in D value was explained by formation of microvoids in the film. These voids are large enough to contribute to the penetration of oxygen molecules through the films. The montmorillonite content did not affect the quenching rate constant, k q and mutual diffusion coefficient, D m values.

Fluorescent sensor responsive to local viscosity and its application to the imaging of liquid-ordered domain in lipid membranes

A novel fluorescent sensor responsive to local viscosity in the lipid bilayer membranes was designed and synthesized. The sensor changes its fluorescence intensity reflecting the local viscosity of its surrounding medium. The fluorescence measurement showed that the sensor is capable of discriminating between different phase states of lipid bilayer. In addition, the sensor visualized liquid-ordered microdomains on giant vesicles in terms of the microviscosity with a simple fluorescence technique.

Analysis of SCEs in Vicia faba chromosomes by a simple fluorescent plus Giemsa technique

Cortés, F. and Andersson, H.C. 1987. Analysis of SCEs in Viciafaba chromosomes by a simple fluorescent plus Giemsa technique. —Hereditas 207: 7–13. Lund, Sweden. ISSN 0018–0661. Received October 3, 1986 A modified fluorescent plus Giemsa (FPG) technique for differential staining of sister chromatids and for studying sister chromatid exchanges (SCE) in root tip cells of Viciafaba is described. This new technique is quick and simple and has the advantage of a high reproducibility. The best quality of the sister chromatid differentiation (SCD) is obtained when seedlings are grown for two rounds of chromosome replication in the presence of 5-bromodeoxyuridine (BrdUrd). The technique stains the bifilarly substituted chromatid light and the unifilarly substituted one dark. However. SCD is also obtained after one round of replication in BrdUrd followed by another in the presence of thymidine. There was no difference in the baseline level of SCE in the two types of experimental protocol. The influence of various concentrations of BrdUrd on the yield of SCEs was investigated, as well as the frequency of SCEs in root tip cells grown in the absence and presence of 5-fluorodeoxyuridine. The results of these experiments are discussed.

Covalent surface chemistry gradients for presenting bioactive peptides

The activation of surfaces by covalent attachment of bioactive moieties is an important strategy for improving the performance of biomedical materials. Such techniques have also been used as tools to study cellular responses to particular chemistries of interest. The creation of gradients of covalently bound chemistries is a logical extension of this technique. Gradient surfaces may permit the rapid screening of a large range of concentrations in a single experiment. In addition, the biological response to the gradient itself may provide new information on receptor requirements and cell signaling. The current work describes a rapid and flexible technique for the covalent addition of bioactive peptide gradients to a surface or gel and a simple fluorescence technique for assaying the gradient. In this technique, bioactive peptides with a terminal cysteine are bound via a heterobifunctional coupling agent to primary amine-containing surfaces and gels. A gradient in the coupling agent is created on the surfaces or gels by varying the residence time of the coupling agent across the surface or gel, thereby controlling the extent of reaction. We demonstrate this technique using poly( l-lysine)-coated glass surfaces and fibrin gels. Once the surface or gel has been activated by the addition of the coupling agent gradient, the bioactive peptide is added. Quantitation of the gradient is achieved by measuring the reaction kinetics of the coupling agent with the surface or gel of interest. This can be done either by fluorescently labeling the coupling agent (in the case of surfaces) or by spectrophotometrically detecting the release of pyridine-2-thione, which is produced when the thiol-reactive portion of the coupling agent reacts. By these methods, we can obtain reasonably precise estimates for the peptide gradients without using expensive spectroscopic or radiolabeling techniques. Validation with changes in fibroblast cell migration behavior across a bioactive peptide gradient illustrates preservation of peptide function as well as the usefulness of this technique.

Fluorescence screening of antioxidant capacity in pumpkin seed oils and other natural oils

AbstractPlant oils provide a rich source of dietary polyunsaturated fatty acids (PUFAs) and mostly lipophilic antioxidants. PUFAs are both in their free form and as components of glycerolipids preferred targets of free radical‐induced oxidation, leading to the formation of highly atherogenic compounds. Thus, stabilization of polyunsaturated lipids by radical scavengers in the oils is important in order to avoid pathophysiological side effects of these essential components of our diet. To determine lipid oxidizability and its inhibition by endogenous antioxidants, we developed a simple fluorescence technique. It is based on solubilisation of the oils in aqueous buffer, labeling of the resulting emulsions with a suitable reporter fluorophore, which reflects lipid oxidation, and continuous monitoring of the decomposition process. Using this method, we found that oxidizability of the oils depended only to a limited extent on the content of lipophilic antioxidants. In addition, a smaller fraction of polar (phenolic) compounds showed comparable protective effects, especially in pumpkin seed oil, which is a non‐refined product therefore containing antioxidative components that are removed from most other edible oils during processing. Therefore, the contribution of these “minor” compounds has to be taken into account when potential biological effects of plant oils are evaluated.

Oxygen Diffusion into Latex Films Annealed at Various Temperatures: A Fluorescence Study

A simple fluorescence technique is proposed for the measurement of the diffusion coefficient of oxygen into latex films. These latex films were prepared by annealing pyrene (P) labeled polymethyl methacrylate (PMMA) particles above the glass transition temperature. Transparency of these films were measured by monitoring the transmitted photon intensity, Itr, which increased as the annealing temperature was increased. Steady state fluorescence was used to monitor oxygen diffusion into latex films. Diffusion coefficients, D, of oxygen were determined using fluorescence quenching method and are found to be around 5.6 × 10−11 cm2s−1.

Phospholipid subclass specific alterations in the passive ion permeability of membrane bilayers: separation of enthalpic and entropic contributions to transbilayer ion flux.

Alterations in phospholipid class, subclass, and individual molecular species contribute to the diversity of biologic membranes, but their effects on membrane passive ion permeability have not been systematically studied. Herein, we developed a simple and efficient fluorescence technique based upon the loss of valinomycin-inducible membrane potential to characterize the passive flux of ions across phospholipid bilayers. Detailed kinetic characterization of ion flux across membrane bilayers composed of discrete chemical entities demonstrated that the class, subclass, and individual molecular species of each phospholipid have substantive effects on membrane passive ion permeability properties. Increasing the degree of unsaturation in either the sn-1 or sn-2 aliphatic chains in phosphatidylcholine markedly enhanced transmembrane ion flux, with over 10-fold differences in the first-order rate constant manifested in molecular species containing four double bonds in comparison to those possessing three double bonds (e.g., kapp = 0.0014 min-1 for 1-octadec-9'-enoyl-2-octadec-9', 12'-dienoyl-sn-glycero-3-phosphocholine (18:1-18:2 phosphatidylcholine) while kapp = 0.021 min-1 for 1,2-dioctadec-9', 12'-dienoyl-sn-glycero-3-phosphocholine (18:2-18:2 phosphatidylcholine)). Moreover, although the apparent first-order rate constants for transmembrane ion flux in vesicles composed of phosphatidylcholine or plasmanylcholine containing palmitate at the sn-1 position and arachidonate at the sn-2 position were similar (kapp = 0.04 min-1 at 22 degreesC for both), the kapp for corresponding vesicles composed of plasmenylcholine was 20-fold less (kapp = 0.002 min-1 at 22 degreesC). Examination of the temperature dependence of passive membrane ion permeability demonstrated that altered ion flux across membranes composed of choline glycerophospholipids was primarily due to entropic effects without substantial changes in the activation energy for ion translocation. For example, Ea = 19.7 +/- 0.5 and 20.7 +/- 0.6 kcal.mol-1 for 1-hexadecanoyl-2-eicosa-5',8',11', 14'-tetraenoyl-sn-glycero-3-phosphocholine (16:0-20:4 phosphatidylcholine) and 1-O-(Z)-hexadec-1'-enyl-2-eicosa-5',8',11', 14'-tetraenoyl-sn-glycero-3-phosphocholine (16:0-20:4 plasmenylcholine), respectively, while their difference in the entropies of activation (DeltaS) was 4.3 +/- 0.5 cal.mol-1.K-1. Collectively, these results identify substantial differences in the membrane passive ion permeability properties of phospholipid classes, subclasses, and molecular species present in biologic membranes of eukaryotic cells and identify entropic alterations as an important contributor to these differences.

Orthotopic transplant mouse models with green fluorescent protein-expressing cancer cells to visualize metastasis and angiogenesis.

There have been major efforts in metastasis research in recent years, especially on the role of angiogenesis in the metastatic process. Much of the information in this area has been obtained from model systems that are not representative of clinical cancer. The technique of surgical orthotopic implantation (SOI) has allowed the development of clinically relevant metastatic models of human cancer in immunodeficient rodents such as the nude and SCID mouse. In order to allow direct visualization of the metastatic process, we took advantage of the green fluorescent protein (GFP) of the jellyfish, Aequorea victoria. A series of cancer cell lines have been stably transfected with vectors containing humanized GFP cDNA. To utilize GFP expression for metastasis studies, fragments of subcutaneously growing tumor, which were comprised of GFP-expressing cells, were implanted by SOI in nude mice. Subsequent metastases were visualized in systemic organs by GFP fluorescence in the lung, liver, bones, brain and other organs down to the single-cell level. With this fluorescent tool, we detected and visualized for the first time tumor cells at the microscopic level in fresh viable tissue in their normal host organs even in the live animal. Angiogenesis is readily visualized in the transplanted GFP-expressing tumors in real time in situ in the live animal using simple laparotomy and fluorescent techniques. The results with the GFP-transfected tumor cells, combined with the use of SOI, demonstrate a fundamental advance to visualize and study cancer metastasis and the role of angiogenesis and other factors in the metastatic process.

Fluorescent staining for proteins on polyacrylamide gels with 5-dimethylamino-1-naphthalenesulfonyl chloride (dansyl chloride)

A simple and sensitive post electrophoresis fluorescent staining technique for proteins on polyacrylamide gels using 5-dimethylamino-1-naphthalene sulfonyl chloride (dansyl chloride) has been developed. Dansyl chloride staining increases the sensitivity, 0.125 μg protein per band can be visualised by this technique. The staining method appears to be applicable to all types of proteins including proteoglycans.

Intestinal permeability measurements - a new application for water soluble contrast media?

The detection and quantification of altered intestinal barrier function (intestinal permeability) have been addressed frequently during the last year, but the tests available for determination of intestinal permeability are both flawed and have limitations. The new water-soluble roentgen contrast media (CM) of low- and iso-osmolar type have been proposed as intestinal permeability probes. In this article this possible new application for water-soluble CM is discussed in terms of their inherent properties and the experimental and clinical results obtained so far. The 2 iso-osmolar dimers, iodixanol (Visipaque) and iotrolan (Isovist), seem to be as good as the well-documented radio-labeled permeability probe (51)Cr-EDTA in detecting injury of the intestinal mucosal barrier. The CM offer the advantage of allowing direct control of bowel wall exposure with the aid of fluoroscopy. Current permeability probes lack this quality. Iodine-containing substances may be analyzed by the rapid, simple and reliable X-ray fluorescence technique, which is suitable for application in routine clinical practice. We conclude that the new water-soluble roentgen CM have properties which may be of use in the diagnosis and quantification of altered intestinal barrier function.