Intrinsic Fluorescence Methods Research Articles

유산균 측정을 위한 형광측정법의 안전성 평가

The intrinsic fluorescence method is effective for microbial identification and characterization, but there is a need for such a method for monitoring fermentation-related bacteria in fermented food. This methodology is based on monitoring live bacteria that contain a variety of intracellular biomolecules harboring specific emitting photons following excitation by ultraviolet (UV) radiation that characterize their intrinsic fluorescence; however, UV photons can damage cells. Therefore, a safety assessment of the developed method is required before it is used in the food industry to distinguish and enumerate food-associated bacteria. In this study, six lactic acid bacteria (LAB) strains were exposed to UV for four weeks (total irradiation time: 3,009 min) and their safety, including hemolytic activity, undesirable biochemical characteristics, and antibiotic resistance, was evaluated. Moreover, LAB growth inhibition by a UV treatment was also investigated. The results indicated that the six UV-treated LAB strains did not pose any health risk, as they were not hemolytic, exhibited no undesirable biochemical activity, and no antibiotic resistance. Nevertheless, prolonged UV radiation (3,009 min) caused LAB growth inhibition, and that the effect was different depending on the LAB strain and LAB culture medium. Among the six LAB strains, the growth of two strains cultivated in MRS at -1.5℃ was inhibited by long-term UV radiation. On the other hand, the same treatment with UV radiation did not affect the growth of any of the six strains cultivated in kimchi filtrate. Therefore, the intrinsic fluorescence method can be used safely in the food (kimchi) industry for the identification of LAB strains and monitoring their growth during fermentation.

An intrinsic fluorescence method for the determination of protein concentration in vaccines containing aluminum salt adjuvants

Determination of protein concentration in vaccines containing aluminum salt adjuvant typically necessitates desorption of the protein prior to analysis. Here we describe a method based on the intrinsic fluorescence of tyrosine and tryptophan that requires no desorption of proteins. Adjuvanted formulations of three model Bordetella pertussis antigens were excited at 280 nm and their emission spectra collected from 290 to 400 nm. Emission spectra of protein antigens in the presence of aluminum salt adjuvants were able to be detected, the effects of adjuvants on the spectra were analyzed, and linear regressions were calculated. The fluorescence method proved to be very sensitive with a limit of quantification between 0.4 and 4.4 µg/mL and limit of linearity between 100 and 200 µg/mL, across the formulations tested. The fluorescence method was found to be influenced by adjuvant presence, type of adjuvant, adjuvant concentration, buffer and pH conditions. The method also demonstrated ability to monitor the percent adsorption of antigens to the adjuvants. Furthermore, intrinsic fluorescence showed good correlation with micro-Kjeldahl elemental assay in quantifying protein concentration. Being a non-invasive, quick and sensitive method, intrinsic fluorescence has the potential to be utilized as a high throughput tool for vaccine development and conceivably implemented in-line, using in-line fluorimeters, to monitor antigen concentration during formulation processing.

Effects of ruthenium complexes on prion amyloid peptide PrP115-135 aggregation and their interaction mechanism

Protein conformational change and amyloid fibrils deposition are associated with severe diseases. Those proteins include amyloid- β protein (A β , Alzheimer’s disease), human islet amyloid polypeptide (hIAPP, type 2 diabetes mellitus, T2DM), and prion protein (PrP, transmissible spongiform encephalopathies). Scientists have focused on studying these amyloid proteins to develop potential drugs against the diseases. Prion disease is a type of chronic progressive fatal neurodegenerative disease, whose pathogenic mechanism is the conformational conversion of normal prion protein PrPC to a pathogenic type PrPSc. The transition produces amyloid deposition and consequent neurotoxicity. PrP115-135 (115-AAAAGAVVGGLGGYMLGSAMS-135) is an N-terminal fragment of PrP with self-aggregation and cytotoxicity. Metal complexes act as traditional antitumor agents, and non-platinum-based metallodrugs are developed to improve clinical effectiveness in terms of their general toxicity and spectral activity. Besides, they interact with various proteins, enzymes and bioactive peptides. Ruthenium complexes can effectively bind to proteins, such as apotransferrin, apolactoferrin, and serum albumin, resulting in the altered protein structure and binding ability toward other molecules. They are also considered as potential inhibitors of amyloid proteins because of their low cytotoxicity and disaggregation ability to amyloid fibrils. This work studied the interactions of PrP115-135 with two ruthenium complexes NAMI-A and KP418 in order to compare the action mechanisms between different prion neuropeptides and ruthenium complexes. The intrinsic fluorescence method and cyclic voltammetry (CV) were used to study the binding mode and binding affinity of the two complexes to PrP15-135. In addition, we used Thioflavin T fluorescence assay to display the effects of ruthenium complexes on amyloid peptide aggregation. Furthermore, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to reveal the effects of ruthenium complexes on amyloid peptide-induced cytotoxicity. The change in peptide fluorescence upon metal complex addition could be assumed to reflect the amounts of peptide–ruthenium complex produced and the conformational change of the peptide. The dissociation constant K d values were (6.2±1.4) and (6.4±1.1) μ mol/L for KP418 and NAMI-A respectively, indicating a strong binding affinity between the peptide and ruthenium complexes. CV method showed that, the reduction potential of the complexes changed with the addition of the peptide, reducing the peak current as well. The change of reduction potential indicated that the interaction between them was attributed to electrostatic interaction, and the decrease of the reduction peak current implied that there were other binding forces, such as hydrophobic interaction existed. ThT assay showed that, after incubating ruthenium complexes with aggregated PrP115-135, the fluorescence intensity decreased dramatically. It suggested that the two complexes effectively inhibited the aggregation of peptide. The aggregation kinetics indicated that the complex could significantly prolong the nucleation period and aggregation time of amyloid peptide. Ruthenium complexes could bind to PrP115-135 and inhibit their aggregation, forming oligomers or monomers, thereby reducing the cytotoxicity of PrP115-135 and improving the survival rate of cells. The inhibitory effect of the two complexes on the aggregation of PrP115-135, and their rescue of peptide induced cytotoxicity make it possible for them to be inhibitors of peptide aggregation and potential drugs against amyloidosis. Exploring metal complexes with low toxicity, better disaggregation ability and good water solubility that can penetrate the blood-brain barrier is a principal research direction in this field. So our next step will keep on configuration design of the complexes so as to find potential metallodrugs against prion disease.

β-Lactoglobulin–naringenin complexes: Nano-vehicles for the delivery of a hydrophobic nutraceutical

Naringin, a glycosylated polyphenol of the flavanones class and its aglycone, naringenin, have been shown to possess many health benefits, therefore they show promise for supplementation of staple foods and beverages, as a mean of preventive medicine. Major obstacles to the successful implementation of these flavanones as nutraceuticals are their low water-solubility and the related low oral bioavailability. Naringin is more soluble but is also very bitter. To allow the enrichment of clear beverages with these important health promoters, we have used native and preheated β-Lactoglobulin (β-Lg) based nanovehicles. Using UV spectrophotometry and intrinsic fluorescence methods, we have found that naringenin forms complexes with preheated and non-preheated β-Lg, with Ka in the order of 104 M−1. No binding was found between naringin and β-Lg, probably due to the lower hydrophobicity of naringin and the steric interference of its sugar. Naringenin: β-Lg binding stoichiometry was 2:1, suggesting naringenin was probably bound at the two main hydrophobic binding sites of the protein, the calyx and the cleft near the alpha-helix. The complex formation with both native and preheated protein solubilized naringenin up to 3 times its solubility limit and prevented crystal formation in the aqueous medium. Such crystallization is responsible for both reduced bioavailability and hampered visual attractiveness of the product. Dynamic light scattering revealed the existence of nanoparticles of ∼10 nm, marginally larger than those of the pure protein (∼7 nm), therefore the solution was clear, hence suitable for clear beverages. Reconstitution after freeze drying was good, but only after quench freezing.

Direct quantitative analysis of aromatic amino acids in human plasma by four-way calibration using intrinsic fluorescence: Exploration of third-order advantages

A novel intrinsic fluorescence method for the direct determination of l-phenylalanine, l-tyrosine, and l-tryptophan in human plasma is presented. By using fluorescence excitation-emission-pH-sample data array in combination with four-way calibration method based on the quadrilinear component model, the proposed approach successfully achieved quantitative analysis of the aromatic amino acids in human plasma, even in the presence of an unknown, uncalibrated serious interferent. It needs little preparation, uses the "mathematical separation" instead of "analytical separation", what makes it fast and environmentally friendly. Satisfactory results have been achieved for calibration set, validation set, and prediction set. The ranges for phenylalanine, tyrosine, and tryptophan are 2.0 × 10(3)-20.0 × 10(3), 50.0-500.0, and 20.0-200.0 ng mL(-1) respectively. Average spike recoveries (mean ± standard deviation) are 93.3 ± 7.7%, 104.3 ± 6.6%, and 99.5 ± 9.0% respectively. The real concentrations in human plasma are 10.2 ± 0.3, 6.6 ± 0.1, and 5.3 ± 0.1 μg mL(-1) respectively, which are consistent with the results obtained by LC-MS/MS method and reference values. In addition, we explored the third-order advantages through the real four-way array; it has shown that higher resolving power is one of the main advantages of higher-order tensor calibration method. These results demonstrated that the proposed method is sensitive, accurate, and efficient for direct quantitative analysis of aromatic amino acids in human plasma.

Formulation Design and High-Throughput Excipient Selection Based on Structural Integrity and Conformational Stability of Dilute and Highly Concentrated IgG1 Monoclonal Antibody Solutions

A systematic approach is presented to characterize and stabilize the higher order structural integrity of an immunoglobulin G (IgG1) monoclonal antibody (mAb) formulated at both low concentrations and as a highly concentrated solution. The conformational and colloidal stabilities of a recombinant humanized IgG1κ mAb at both 1 and 100 mg/mL were investigated as a function of solution temperature (10°C-87.5°C) and pH (3-8). Protein secondary structure was characterized using circular dichroism, whereas intrinsic (tryptophan) and extrinsic (8-anilino-1-naphthalenesulfonic acid) fluorescence spectroscopy measurements were used to evaluate the tertiary structure of the protein. Light scattering analysis was employed to monitor mAb aggregation behavior as a function of temperature and solution pH. These biophysical data sets were analyzed and summarized using a previously described empirical phase diagrams (EPDs) approach. The different phases observed in the EPD were correlated with the individual physical states of the IgG1 in solution (aggregated, native, unfolded, etc.). The temperature-dependent conformational stability profile of the mAb, at both 1 and 100 mg/mL, generally followed the order pH 6 ≥ pH 7 ≥ pH 8 > pH 5 > pH 4 ≥ pH 3. Analysis of the EPD apparent phase boundaries identified solution conditions of pH 4.5 near 60°C for the development of an excipient screening assay. A supplemented generally regarded as safe excipient library was screened using an aggregation assay (optical density at 350 nm) at low mAb concentrations (4 mg/mL) and potential stabilizers were identified. The ability of these excipients to prevent conformational alterations in high concentration mAb solutions (100 mg/mL) was determined by monitoring tertiary structure changes using an intrinsic fluorescence method. The results suggest that substantial increases in the onset temperature of thermal transitions (>5°C) are obtained in the presence of (a) 20% dextrose, (b) 20% sorbitol, and (c) 5% dextrose + 10% sorbitol. Similar stabilization effects were obtained at an intermediate (50 mg/mL) as well as low mAb concentrations (1 mg/mL).

Entanglement and phase separation in hyperbranched polymer/linear polymer/solvent ternary blends

Hyperbranched polyethylene (HBPE)/linear polystyrene (PS)/chloroform (CF) solution was selected as a model system to investigate the effect of branching structure on entanglement and phase separation behavior in semi-dilute ternary polymer solutions. All the HBPE materials in this work were found to have similar chain architectures and the critical molecular weight was estimated to be 81.2 kDa. The results obtained by elastic light scattering and intrinsic fluorescence methods suggested that all ternary solutions exhibited UCST transition behavior upon cooling. Also, it was found that the increase in the molecular weight of PS led to increase in the phase separation rate, consistent with de Gennes prediction. However, the increase of molecular weight of HBPE did not monotonously reduce the compatibility of polymer components and the phase separation rate in ternary blends is as follows: medium molecular weight HBPE (HBPE-M) > high molecular weight HBPE (HBPE-H) > low molecular weight HBPE (HBPE-L). This abnormal behavior can be explained by the fact that, (i) for HBPE-L, no entanglements between HBPE chains occurred and the branching effect can be ignored, and (ii) for HBPE-M and HBPE-H, entanglement of HBPE chains can be formed, and the dilution of branches on entanglement of backbones should be taken into consideration, that is, the shorter the branches of HBPE, the higher the possibility of interpenetration of HBPE backbones between neighboring molecules and, consequently, the faster aggregation of HBPE during phase separation. Furthermore, a simple model based on decomposition reaction was proposed to quantitatively describe the phase separation kinetics and the apparent activation energies of phase separation were calculated to be −150.3 and −52.3 kJ/mol for HBPE-M/PS/CF and HBPE-H/PS/CF systems, respectively.

Temperature‐induced Molecular Chain Motions of Styrenic Triblock Copolymers Studied by Intrinsic Fluorescence Spectra

AbstractThe temperature‐induced molecular chain motions of styrenic triblock copolymers (SBC), i.e. polystyrene‐block‐polybutadiene‐block‐polystyrene (SBS) and polystyrene‐block‐poly(ethylene‐co‐1‐butene)‐block‐polystyrene (SEBS), were studied by intrinsic fluorescence method. For SBS, the glass transition temperatures (Tgs) of B block and S block obtained by intrinsic fluorescence method were in good agreement with differential scanning calorimetry measurements (DSC). In the case of SEBS, an isoemission point was observed at about 310 nm at elevated temperatures, suggesting the slight conversion between the monomer and excimer emission. On this basis, the molecular chain motion of SEBS was monitored by both fluorescence intensity and excimer/monomer fluorescence ratio. Besides the Tgs of S block and EB blocks, a melting point (Tm) of weak crystalline in EB block was unambiguously determined by intrinsic fluorescence. Furthermore, it was found that the melting process directly led to the slight loosening of PS segments in interface and consequently the reduction of the amount of excimer. A reasonable mechanism was proposed to describe the molecular chain movements and phase transitions of SEBS upon heating. Moreover, the influence of temperature on the apparent activation energy of non‐radiative process (EaT) around Tg of S block was much stronger than that around Tg of B or EB blocks.

Inhibitor of inflammation, peptide fragment (65–76) of monocyte chemotactic protein-1 (MCP-1), inhibits binding of MCP-1 to heparin

Monocyte chemotactic protein-1 (MCP-1, CCL2) is one of the most important chemokines involved in inflammation. MCP-1 stimulates migration of monocytes and certain lymphocyte populations to the affected area, in particular to the sites of atherosclerotic plaque formation. Development of drugs inhibiting MCP-1 is now a topical task. We earlier designed and synthesized a dodecapeptide from C-terminal domain of MCP-1 (65–76, peptide X) that possessed an anti-inflammatory activity. The mechanism of action of chemokines (in particular, of MCP-1) in vivo is based on activation of CCR2 receptor on target cells and binding to glycosaminoglycans (GAGs) on the cell surface of and extracellular matrix. Peptide X did not affect the MCP-1-CCR2 interaction. Thus, we hypothesized that peptide X could impair MCP-1 binding to GAGs. Here we studied the effect of peptide X on the MCP-1 binding to heparin using the label-free biosensing device Picoscope®, enzyme-linked immunoassay (ELISA), and the intrinsic fluorescence method. According to the data obtained, peptide X interfered with the MCP-1-heparin binding, which may be due to the competition of peptide X with MCP-1 for heparin binding sites. Probably, this effect determines the anti-inflammatory activity of peptide X in vivo.

Intrinsic fluorescence studies of compatibility in thermoplastic phenol formaldehyde resin / poly(ε-caprolactone) blends

Intrinsic fluorescence method was applied to study the miscibility and interactions of thermoplastic phenol formaldehyde resin (TPF) / poly(!-caprolactone) (PCL) blends. The characteristic intrinsic fluorescence emission of TPF at 313 nm showed the very good sensitivity to monitor the macromolecular chain motion in the TPF/PCL blends. The glass transition (Tg), crystallization (Tc), and melting transition point (Tm) of TPF/PCL blends were measured by the temperature dependence of intrinsic fluorescence intensities upon heating or cooling process. Interestingly, when TPF/PCL 5/5, besides a Tg for the amorphous phase of blend, another transition at temperature a little higher than Tg of PCL can be observed by intrinsic fluorescence method. This microheterogeneity can be explained by the so-called 'rigid amorphous phase' (RAP) due to the good flexibility and the strong self-association of PCL chains in amorphous phase. Besides, the analysis of the dependence of Tg on the content of PCL suggests that this microheterogeneity can attenuate the interactions between TPF and PCL chains and result in a lowering of Tgs of blends. In view of the simplicity and sensitivity of measurement as well as affordability of instrument, intrinsic fluorescence proved to be an effective means for characterization of microstructural variation in polymer blends.

The inhibition of Saccharomyces cerevisiae cells by acetic acid quantified by electrochemistry and fluorescence

In this paper, the effect of acetic acid on S. cerevisiae metabolism was studied for the first time with the combinational utilization of the mediated electrochemical method and intrinsic fluorescence method, and the results from both methods were compared with each other. It was confirmed in vivo that not only the glycolysis enzymes but also the NADH dehydrogenase were sensitive to acetic acid. The established technique might also be applicable in studying the toxicity of other environmental pollutants, such as phenol and its derivatives.

Enzymatic hydrolysis of microcrystalline cellulose in reverse micelles

The activities of cellulases from Trichoderma reesei entrapped in three types of reverse micelles have been investigated using microcrystalline cellulose as the substrate. The reverse micellar systems are formed by nonionic surfactant Triton X-100, anionic surfactant Aerosol OT (AOT), and cationic surfactant cetyltrimethyl ammonium bromide (CTAB) in organic solvent mediae, respectively. The influences of the molar ratio of water to surfactant ω 0, one of characteristic parameters of reverse micelles, and other environmental conditions including pH and temperature, on the enzymatic activity have been studied in these reverse micellar systems. The results obtained indicate that these three reverse micelles are more effective than aqueous systems for microcrystalline cellulose hydrolysis, and cellulases show “superactivity” in these reverse micelles compared with that in aqueous systems under the same pH and temperature conditions. The enzymatic activity decreases with the increase of ω 0 in both AOT and Triton X-100 reverse micellar systems, but reaches a maximum at ω 0 of 16.7 for CTAB reverse micelles. Temperature and pH also influence the cellulose hydrolysis process. The structural changes of cellulases in AOT reverse micelles have been measured by intrinsic fluorescence method and a possible explanation for the activity changes of cellulases has been proposed.

Stability of equine lysozyme: I. Thermal unfolding behaviour

The thermal denaturation of Ca 2+- and apo-forms of equine lysozyme was followed by using far and near UV circular dichroism and intrinsic fluorescence methods. The difference found between the temperature dependence of the ellipticity at 222 nm and 287 nm, which show two stages in the thermal transition, and those at 228 nm and 294 nm, which indicate only one stage over a wide range of temperatures reflects that different subdivisions of the protein molecule are characterized by a different stability, cooperativity and pathway of denaturation. The first transition, reflected in the increase of the cilipticity at 222 nm and 287 nm, coincides with the transition detected by fluorescence and occurs at 30–50°C for the apo-form and at 50–60°C for the Ca 2+-form of lysozyme. It seems to correlate with the transfer of some tryptophan residues to a more hydrophobic environment and with a local rearrangement of the tertiary and secondary structures. The unfolding transition detected by the decrease of the ellipticity at all wavelengths occurs nearly in the same temperature region for the apo- and Ca 2+-forms, i.e. 50–80°C and 55–80°C, respectively. The presence of a Ca 2+-binding loop in equine lysozyme may be partly responsible for the drastic destabilization of its structure as a whole both in the presence but especially in the absence of Ca 2+ in comparison with hen and human lysozymes.

Comparative study of the cytotoxins of the venom of the central asian cobraNaja naja oxiana by the intrinsic-fluorescence method

The intrinsic fluorescence of cytotoxin VC1 and VC5 from the venom of the Central Asian cobra due to the tyrosine residues and differing with respect to quantum yield for these two polypeptides because of the nonidentity of the microenvironments of the tyrosyl residues has been measured and characterized. The dependence of the quantum yield on the pH and the temperature also indicate this nonidentity.

Some neutron experiments on flexible polymers

Hyperbranched polyethylene (HBPE)/linear polystyrene (PS)/chloroform (CF) solution was selected as a model system to investigate the effect of branching structure on entanglement and phase separation behavior in semi-dilute ternary polymer solutions. All the HBPE materials in this work were found to have similar chain architectures and the critical molecular weight was estimated to be 81.2 kDa. The results obtained by elastic light scattering and intrinsic fluorescence methods suggested that all ternary solutions exhibited UCST transition behavior upon cooling. Also, it was found that the increase in the molecular weight of PS led to increase in the phase separation rate, consistent with de Gennes prediction. However, the increase of molecular weight of HBPE did not monotonously reduce the compatibility of polymer components and the phase separation rate in ternary blends is as follows: medium molecular weight HBPE (HBPE-M) > high molecular weight HBPE (HBPE-H) > low molecular weight HBPE (HBPE-L). This abnormal behavior can be explained by the fact that, (i) for HBPE-L, no entanglements between HBPE chains occurred and the branching effect can be ignored, and (ii) for HBPE-M and HBPE-H, entanglement of HBPE chains can be formed, and the dilution of branches on entanglement of backbones should be taken into consideration, that is, the shorter the branches of HBPE, the higher the possibility of interpenetration of HBPE backbones between neighboring molecules and, consequently, the faster aggregation of HBPE during phase separation. Furthermore, a simple model based on decomposition reaction was proposed to quantitatively describe the phase separation kinetics and the apparent activation energies of phase separation were calculated to be −150.3 and −52.3 kJ/mol for HBPE-M/PS/CF and HBPE-H/PS/CF systems, respectively.