Applied Fluorescence Spectroscopy Research Articles

Applying fluorescence spectroscopy coupled with Gaussian band fitting to reveal dynamic variation process of humus fractions from riparian soils along an urbanized river

Humus, an important fraction of soil organic matter, play an environmental role on nutrients, organic and inorganic pollutants in riparian zones of urbanized rivers. In this study, dynamic variation process of humus fractions from riparian soils was revealed along Puhe River. Composite soil samples of four depths were collected from four land-uses, i.e., eco-conservation area (ECA), industrial area (INA), urban/town area (UTA), rural/agricultural area (RAA). Based on synchronous fluorescence spectra coupled with Gaussian band fitting, fulvic/humic acid predominantly contained tyrosine-like (TYLF), tryptophan-like (TRLF), microbial-like (MLF), fulvic-like (FLF) and humic-like (HLF) substances within each soil profile. TRLF, MLF and FLF (89.43–90.30 %) are the representative components in fulvic-acid, while MLF and HLF (52.81–59.97 %) in humic-acid. Phenolic, carboxylic and humified materials were present in both humus. According to 2-dimensitonal correlation spectroscopy and canonical correlation analysis, fulvic/humic acid within the ECA soil profile could be mainly derived from the degradations of terrestrial plant metabolites and residuals. Within the INA, fulvic-acid could be associated with treated/untreated wastewater, which entered the river and flew into the riparian during high flow period; whereas humic-acid could be relative to the terrestrials. Fulvic-acid had the same source as humic-acid in the UTA, which might be concerned with scattered domestic sewage and livestock wastewater, rather than the fluvial water. Furthermore, the source of fulvic/humic acid in the RAA was the crop metabolites and residuals, apart from the livestock wastewater. Noticeably, the variations of humus fractions in the ECA and RAA roughly occurred in 0–60 cm, while approximately in 20–80 cm in the INA and UTA. This proved that humus fractions in the former were referred to the plant/crop residuals, whereas humus fractions in the latter were those the terrestrials and fluvial water. This study could provide a key support for the construction and restoration of the urbanized riparian zone.

Synchronous Front-Face Fluorescence Spectra: A Review of Milk Fluorophores.

Milk is subjected to different industrial processes, provoking significant physicochemical modifications that impact milk's functional properties. As a rapid and in-line method, front-face fluorescence can be used to characterize milk instead of conventional analytical tests. However, when applying fluorescence spectroscopy for any application, it is not always necessary to determine which compound is responsible for each fluorescent response. In complex matrixes such as milk where several variables are interdependent, the unique identification of compounds can be challenging. Thus, few efforts have been made on the chemical characterization of milk' fluorescent spectrum and the current information is dispersed. This review aims to organize research findings by dividing the milk spectra into areas and concatenating each area with at least one fluorophore. Designations are discussed by providing specific information on the fluorescent properties of each compound. In addition, a summary table of all fluorophores and references cited in this work by area is provided. This review provides a solid foundation for further research and could serve as a central reference.

Applying fluorescence spectroscopy and DNA pyrosequencing with 2D-COS and co-occurrence network to deconstruct dynamical DOM degradation of air-land-water sources in an urban river

In an urban river, comprehending the interplay between dissolved organic matter (DOM) and atmospheric, terrestrial, and aquatic sources is crucial. This encompassed investigating temporal variations in DOM and its association with the bacterioplankton community to gain profound insights into the biogeochemical dynamics and biodegradability of DOM. DOM was extracted from PM2.5, soil, sediment, bait, and terrestrial/aquatic plant residuals collected along the Wenyuhe River in Beijing, China - a region predominantly supplied with reclaimed water. Subsequently, mixed microbial communities from the river were introduced into DOM samples originating from each source and incubated for 10 days. Principal component analysis (PCA) applied to reassembled excitation-emission matrix (EEM) data revealed two distinct clusters: cluster 1 comprising soil, sediment, and PM2.5 samples; and cluster 2 consisting of bait as well as terrestrial/aquatic plant residuals. According to parallel factor analysis, C1 (microbial humic-like) and C2-C3 (fulvic-like) dominated the DOM from soil, sediment, and PM2.5. These components were continuously degraded during incubation, except for PM2.5. DOM from bait and terrestrial/aquatic plants contained representative components of C6 (phenolic-like) and C7 (tryptophan-like), which underwent extensive decomposition. Interestingly, DOM in PM2.5 contained aliphatic compounds and polycyclic aromatic hydrocarbons (PAHs) but exhibited weak degradation with the complete disappearance of C6 and C7. Rhodococcus was a unique species capable of degrading PAHs, which might be particularly important considering the specificity of PM2.5 pollution. Based on two-dimensional correlation spectroscopy (2D-COS), variations in DOM components such as C6, and C7 were significantly larger compared to those of C1, C2, C3, and C5 (terrestrial humic-like) from bait samples, sediments, and residual terrestrial plants. MW-2D-COS analysis revealed that DOM from bait samples and terrestrial/aquatic plants experienced substantial degradation by the second day while DOM from soil or sediment decomposed mainly on the fourth day. Notably, the decomposition of DOM fractions in PM2.5 occurred throughout the entire four-day period. Co-occurrence network analysis classified sources of DOM into two clusters similar to PCA results: cluster 1 showed significant microbial degradation of fulvic-like compounds while cluster 2 demonstrated deep microbial decomposition of tyrosine-like and phenolic compounds. Therefore, the artificial loading of DOM into rivers not only expands the chemical diversity within DOM but also perturbs bacterioplankton diversities.

Modulation of Insulin Amyloid Fibrillization in Imidazolium-Based Ionic Liquids with Hofmeister Series Anions.

Amyloid fibrils have immense potential to become the basis of modern biomaterials. The formation of amyloid fibrils in vitro strongly depends on the solvent properties. Ionic liquids (ILs), alternative solvents with tunable properties, have been shown to modulate amyloid fibrillization. In this work, we studied the impact of five ILs with 1-ethyl-3-methylimidazolium cation [EMIM+] and anions of Hofmeisterseries hydrogen sulfate [HSO4-], acetate [AC-], chloride [Cl-], nitrate [NO3-], and tetrafluoroborate [BF4-] on the kinetics of insulin fibrillization and morphology, and the structure of insulin fibrils when applying fluorescence spectroscopy, AFM and ATR-FTIR spectroscopy. We found that the studied ILs were able to speed up the fibrillization process in an anion- and IL-concentration-dependent manner. At an IL concentration of 100 mM, the efficiency of the anions at promoting insulin amyloid fibrillization followed the reverse Hofmeister series, indicating the direct binding of ions with the protein surface. At a concentration of 25 mM, fibrils with different morphologies were formed, yet with similar secondary structure content. Moreover, no correlation with the Hofmeister ranking was detected for kinetics parameters. IL with the kosmotropic strongly hydrated [HSO4-] anion induced the formation of large amyloid fibril clusters, while the other kosmotropic anion [AC-] along with [Cl-] led to the formation of fibrils with similar needle-like morphologies to those formed in the IL-free solvent. The presence of the ILs with the chaotropic anions [NO3-] and [BF4-] resulted in longer laterally associated fibrils. The effect of the selected ILs was driven by a sensitive balance and interplay between specific protein-ion and ion-water interactions and non-specific long-range electrostatic shielding.

Flapping motion as a fluorescent probe for assembly process involving highly viscous liquid-like cluster intermediates during evaporative crystallization.

Fluorescence probes are widely used to assess the molecular environment based on their photo-physical properties. Specifically, flexible and aromatic photo-functional system (FLAP) is unique viscosity probe owing to the excited-state planarization of anthracene wings. We have previously applied fluorescence spectroscopy to monitor the evaporative crystallization of solvents. The fluorescence color and spectral changes, which depend on the aggregation form, enable direct fluorescence visualization during evaporative crystallization. The fluorescence visualization of the liquid-like cluster intermediate proposed in the two-step nucleation model for the nucleation process has been achieved. However, the physical properties of these clusters, especially the viscosity, molecular motion, and intermolecular interactions, are still unclear. In this study, FLAPs are used as probes for local-viscosity changes and space limitations of the liquid-like cluster state during evaporative crystallization by observing the fluorescence-spectral changes and using hyperspectral-camera (HSC) imaging. Green emission originates from the monomer in the solution owing to the free-flapping motion. The fluorescence color turns blue with increasing viscosity under crowding conditions. If the survival time of the liquid-like cluster state is sufficient, crystalline phase (R-phase) formation proceeds via a 2-fold π-stacked array of the V-shaped molecules. It is difficult to form the V-shaped stacked columnar structures in the liquid-like cluster state region, resulting in the deposition of head-to-tail dimer structures, such as the yellow-emissive phase (Y-phase). In the case of the FLAP, the stacking intermediate does not form during solvent evaporation in the liquid-like cluster; rather, it is deposited in an amorphous form that exhibits blue emission (B-phase). These findings suggest that it is important to the maintenance of the survival time of the liquid-like cluster states to organize and rearrange the stacking forms. We have achieved the fluorescence probing of viscosity changes at local molecular motion with solvent depletion during solvent evaporation for the first time.

Fluorescence signal of proteins in birch pollen distorted within its native matrix: Identification of the fluorescence suppressor quercetin-3-O-sophoroside

The properties of biogenic aerosol strongly depend on the particle’s proteinaceous compounds. Proteins from primary biological aerosol particles (PBAPs) can cause allergic reactions in the human respiratory system or act as ice and condensation nuclei in clouds. Consequently, these particles have high impact on human health and climate. The detection of biogenic aerosol is commonly performed with fluorescence-based techniques. However, many PBAPs (i.e., pollen of birch, mugwort, or ragweed) show weak or rather low fluorescence signals in the particular protein region (λex ~ 255–280 nm, λem ~ 280–350 nm). We hypothesize that the fluorescence signal of proteins present in birch pollen is being distorted within its native matrix. In this study, we conducted in vitro quenching experiments and employed UV/Vis spectroscopy, capillary zone electrophoresis (CZE), liquid chromatography (LC), electrospray ionization mass spectrometry (ESI–MS), and multistage MS (MS2 and MS3) to target major components in birch pollen washing water (BPWW) possibly quenching the fluorescence activity of proteins and thus explaining the lack of corresponding protein fluorescent signals. We identified quercetin-3-O-sophoroside (Q3OS, MW 626 g mol−1) to be the main UV/Vis absorbing component in BPWW. Our results point out that Q3OS suppresses the fluorescence of proteins in our samples predominantly due to inner filter effects. In general, when applying fluorescence spectroscopy to analyze and detect PBAPs in the laboratory or the atmosphere, it is important to critically scrutinize the obtained spectra.Graphical abstract

Evaluation of ovalbumin nanocarriers to promote the vehiculization and antifungal properties of cinnamaldehyde in aqueous media

This paper is aimed to evaluate the vehiculization of trans-cinnamaldehyde (CIN) through the formation of complexes with ovalbumin (OVA) and heat-induced OVA nanoparticles (OVAn) to promote its application in aqueous media. OVAn production variables (pH and protein concentration) and some physicochemical features (size, surface hydrophobicity, and molecular weight) were explored. Complex formation was examined in terms of stoichiometric and thermodynamic (ΔH°, ΔS°, and ΔG°) parameters by applying fluorescence spectroscopy. Results revealed the strong influence of the OVA conformational state (native or aggregated) on CIN vehiculization. However, all systems showed similar encapsulation efficiencies (~62%). Besides, nanosized complexes were colloidally stable at pH 3.0 and 7.0. Finally, their antifungal properties against the Aspergillus niger species (as a microbial model) were determined, being the OVA-CIN complex the most efficient. The preservation of the conformational structure of OVA as a nanocarrier was relevant to promote the CIN antifungal properties in aqueous media.

Abstract 1922: Tamoxifen detection by molecular fluorescence in breast cancer patients as a screening for metabolizer classification

Abstract Breast cancer patients with polymorphic changes in the CYP2D6 gene show considerable variations in tamoxifen (TMX) bioavailability due to the need for bioactivation of this drug. Therefore, patients with such allelic changes have a worse prognosis compared to controls. Biospectroscopy allows the analysis of biofluids, providing the complete characterization of all molecules present in the biological matrix. Among spectroscopic techniques, molecular fluorescence stands out for its high analytical sensitivity in the broad identification of information obtained by the excitation-emission fluorescence matrix (EEM). This allows the specific measurement of fluorescent species in the sample. Quantification of TMX by fluorescence spectroscopy provides a phenotypic assessment of the patient's pharmacological metabolizing status, leading to a personalized and effective pharmacological therapy. We applied fluorescence spectroscopy to blood samples from n=20 women, who had genotyping of the CYP2D6*4_g.1846G>A allele, which confers decreased protein activity. We were able to demonstrate that the fluorescence signal generated by the TMX structure in plasma solution could be detected using our methodology. The data collected exhibited signal interference due to the numerous molecular species present in this matrix. However, using multivariate statistical algorithms we could extract relevant information from the analyte, improving the signal / noise of the emission-excitation matrix. Finally, we were able to demonstrate that low cost method, which can be performed rapidly, may allow for a simple point of care stratification of patients by the CYP2D6 status, which in turn may allow for increased personalization of tamoxifen therapy for breast cancer patients. Citation Format: Lorenna Larissa Ferreira Costa, Daniel Lucas Dantas de Freitas, Kassio Michell Gomes de Lima, Tirzah Braz Petta Lajus. Tamoxifen detection by molecular fluorescence in breast cancer patients as a screening for metabolizer classification [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1922.

Biophysical insight into the interaction of levocabastine with human serum albumin: spectroscopy and molecular docking approach

Interaction of levocabastine with human serum albumin (HSA) is investigated by applying fluorescence spectroscopy, circular dichroism spectroscopy and molecular docking methods. Levocabastine is an important drug in treatment of allergy and currently a target drug for drug repurposing to treat other diseases like vernal keratoconjuctivitis. Fluorescence quenching data revealed that levocabastine bind weakly to protein with binding constant in the order of 103 M−1. Förster resonance energy transfer results indicated the binding distance of 2.28 nm for levocabastine. Synchronous fluorescence result suggest slight blue shift for tryptophan upon levocabastine binding, binding of levocabastine impelled rise in α-helical structure in protein, while there are minimal changes in tertiary structure in protein. Moreover, docking results indicate levocabastine binds to pocket near to the drug site-I in HSA via hydrogen bonding and hydrophobic interactions. Understanding the interaction of levocabastine with HSA is significant for the advancement of therapeutic and diagnostic strategies for optimal treatment results. Communicated by Ramaswamy H. Sarma

Fluorescence spectroscopic and dynamics simulation studies on isoorientin binding with human serum albumin.

Isoorientin (ISOO) a glycosylated flavonoid found in acai berry exhibits relevant activities such as antidiabetic and antidepressant. However, its physicochemical action on any molecular target is scarcely known. In this work, we tackle the problem about the binding of ISOO to human serum albumin (HSA) applying fluorescence spectroscopy bimodal analysis aided by computational simulations. A static quenching process was detected having hypsochromic shift with implication in the polarizability around the endogenous probe (Trp 214) during complex formation. The binding mechanism reveals that all sites are equivalents and independents with binding constant value of 9.1 × 104 M-1 and, a total of six sites accessed whereas three of them were identified experimentally. The thermodynamic evaluation indicates that the complex formation is spontaneous (ΔG<0). The dynamics and docking simulations corroborated the experimental data by adding details of each site and its respective microenvironment.

Enabling PAT in insect cell bioprocesses: In situ monitoring of recombinant adeno-associated virus production by fluorescence spectroscopy.

The process analytical technology (PAT) initiative shifted the bioprocess development mindset towards real-time monitoring and control tools to measure relevant process variables online, and acting accordingly when undesirable deviations occur. Online monitoring is especially important in lytic production systems in which released proteases and changes in cell physiology are likely to affect product quality attributes, as is the case of the insect cell-baculovirus expression vector system (IC-BEVS), a well-established system for production of viral vectors and vaccines. Here, we applied fluorescence spectroscopy as a real-time monitoring tool for recombinant adeno-associated virus (rAAV) production in the IC-BEVS. Fluorescence spectroscopy is simple, yet sensitive and informative. To overcome the strong fluorescence background of the culture medium and improve predictive ability, we combined artificial neural network models with a genetic algorithm-based approach to optimize spectra preprocessing. We obtained predictive models for rAAV titer, cell viability and cell concentration with normalized root mean squared errors of 7%, 4%, and 7%, respectively, for leave-one-batch-out cross-validation. Our approach shows fluorescence spectroscopy allows real-time determination of the best time of harvest to maintain rAAV infectivity, an important quality attribute, and detection of deviations from the golden batch profile. This methodology can be applied to other biopharmaceuticals produced in the IC-BEVS, supporting the use of fluorescence spectroscopy as a versatile PAT tool.

Empirical Modeling of Zn/ZnO Nanoparticles Decorated/Conjugated with Fotolon (Chlorine e6) Based Photodynamic Therapy towards Liver Cancer Treatment

The current study is based on Zn/ZnO nanoparticles photodynamic therapy (PDT) mediated effects on healthy liver cells and cancerous cells. The synthesis of Zn/ZnO nanoparticles was accomplished using chemical and hydrothermal methods. The characterization of the synthesized nanoparticles was carried out using manifold techniques (e.g., transmission electron microscopy (TEM), X-ray diffraction (XRD), and energy dispersive X-ray spectroscopy (EDS)). In order to study the biotoxicity of the grown nanoparticles, they were applied individually and in conjunction with the third generation photosensitiser Fotolon (Chlorine e6) in the in vivo model of the normal liver of the Wister rat, and in the in vitro cancerous liver (HepG2) model both in the dark and under a variety of laser exposures (630 nm, Ultraviolet (UV) light). The localization of ZnO nanoparticles was observed by applying fluorescence spectroscopy on a 1 cm2 selected area of normal liver, whereas the in vitro cytotoxicity and reactive oxygen species (ROS) detection were carried out by calculating the loss in the cell viability of the hepatocellular model by applying a neutral red assay (NRA). Furthermore, a statistical analysis is carried out and it is ensured that the p value is less than 0.05. Thus, the current study has highlighted the potential for applying Zn/ZnO nanoparticles in photodynamic therapy that would lead to wider medical applications to improve the efficiency of cancer treatment and its biological aspect study.

Nanosized Phase Segregation of Sphingomyelin and Dihydrosphigomyelin in Unsaturated Phosphatidylcholine Binary Membranes without Cholesterol.

In this study, we applied fluorescence spectroscopy, differential scanning calorimetry (DSC), and 2H NMR to elucidate the properties of nanoscopic segregated domains in stearoylsphingomyelin (SSM)/dioleoylphosphatidylcholine (DOPC) and dihydrostearoylsphingomyelin (dhSSM)/DOPC binary membranes. The results obtained from fluorescence measurements suggest the existence of gel-like domains with high fluidity in both SSM and dhSSM macroscopic gel phases. The DSC thermograms showed that DOPC destabilizes SM-rich gel-like domains to a much lesser extent compared to the same amount of cholesterol. It was also found that a stable lateral segregation occurs without cholesterol, indicating that SSM itself undergoes homophilic interactions to form small gel-like domains. 2H NMR experiments disclosed differences in the temperature-dependent ordering of SSM/DOPC and dhSSM/DOPC bilayers; the dhSSM membrane showed less miscibility with the DOPC fluid phase, higher thermal stability, and tighter packing. In addition, the NMR results suggest the formation of mid-sized gel-like aggregates consisting of dhSSM. These differences could be accounted for by homophilic interactions, as previously reported ( Yasuda Biophys. J. 2016 , 110 , 431 - 440 ). In the absence of cholesterol, the moderately strong sphingomyelin (SM)/SM affinity results in the formation of small gel-like domains, whereas a stronger dhSSM/dhSSM affinity leads to larger gel-like domains. Considering the similar physicochemical features of SSM and dhSSM, the present results suggest that the formation of nanosized domains of SM is better characterized by homophilic interactions than by SM-cholesterol interplay. These effects are considered important to the ordered domain formation of SMs in biological membranes.

Probing the binding of two 19-nortestosterone derivatives to human serum albumin: insights into the interactions of steroid hormone drugs with functional biomacromolecule.

Norethindrone acetate (NETA) is a fatty acid ester of norethindrone (NET) that can convert to its more active parent compound NET when orally administered. To study the interactions of NETA and NET with human serum albumin (HSA), we applied fluorescence spectroscopy, circular dichroism (CD), and molecular docking. The effects of metal ions on the HSA-NETA/NET system were also explored. Fluorescence data showed that the quenching mechanism of HSA by NETA and NET was consistent with a static model and that the binding constant of NETA was higher than that of NET. Thermodynamic parameters indicated that hydrogen bonds and van der Waals forces were the main forces maintaining the stability of the HSA-NETA/NET complex. Molecular modeling studies revealed that NETA and NET were bound within subdomain IIA of HSA, in accordance with the site probe results. Synchronous fluorescence spectroscopy, CD, and three-dimensional fluorescence spectroscopy further confirmed that the binding of NETA/NET to HSA changed the secondary structure of the protein. All other metal ions, except for Ca(2+) , decreased the K value of the HSA-NETA/NET system with enhancement of the maximum effectiveness of NETA/NET. Three commercially available steroid hormone drugs influenced the binding ability of NETA on HSA to different extents. This study provides novel insights into the interactions between HSA and NETA/NET, as well as a solid foundation for future research on drug pharmacokinetics and pharmacodynamics. Copyright © 2016 John Wiley & Sons, Ltd.

Pentavalent antimony uptake pathway through erythrocyte membranes: molecular and atomic fluorescence approaches

Previous studies by our group have shown that Sb(V) is able to enter red blood cells in a dynamic process and is reduced to Sb(III) by glutathione. The present study aims to investigate a possible entry pathway for Sb(V) through the erythrocyte membrane. Applying fluorescence spectroscopy studies with Laurdan and diphenylhexatriene (DPH) probes, it was found that there was no interaction between Sb(V) and membrane lipids. By comparing the Sb(V) entry percentages through lipid vesicles and sealed erythrocyte membranes, it was found that Sb(V) required protein channels to pass through the membrane. The competitive inhibition results using HCO3 (-) and Cl(-) showed that the Sb(V) uptake rate through the membrane fell approximately 50-70% until full inhibition was reached, which was possibly due to the inhibition of the anion exchanger 1 (AE1) channel. Finally, the fluorescence measurements with the 5-iodoacetamidofluorescein (5-IAF) probe showed that Sb(V) interacted with membrane protein SH groups during this process.

The Effect of pH on Globular State of Lipase-3646; an Appropriate Model for Molten Globule Investigations.

Secondary structure content of proteins in molten globule state is relatively constant while the quantity of tertiary structures clearly declines due to alterations in side-chain packing. In the present study, we analyze the MG state of lipase-3646 for the first time. We introduce lipase-3646 as an appropriate model for investigating the properties and behavior of a protein in MG state as well as folding pathway. Applying fluorescence spectroscopy we measured both intrinsic and extrinsic fluorescence of lipase-3646 in a pH range from 1.0 to 12.0. It was found that at pH 3.0 the protein acquires a MG state. Applying far-UV circular dichroism (CD), our analysis on the secondary structure of lipase-3646 revealed a slight change in the MG state intermediate (pH 3.0) compared to the native state (pH 8.5), which this amount of change is common for MG. Measurements in near-UV CD also showed a significant change in the enzyme conformation at pH 3.0 in comparison with the pH 8.5 wherein the protein acquires its native structure. Quenching the fluorescence by applying acrylamide, the amount 23 and 35M(-1) were measured at pHs 8.5 and 3.0 respectively for stern-volmer constant (KSV). An increase in the enzyme molecular volume in the MG state was confirmed by gel filtration chromatography.

Applying fluorescence spectroscopy and multivariable analysis to characterize structural composition of dissolved organic matter and its correlation with water quality in an urban river

Excitation–emission matrix fluorescence spectroscopy combined with multivariable analysis was employed to discriminate structural component of dissolved organic matter (DOM) in Xihe River, an urban river, located in western Shenyang City of China, and to reveal its correlation with water quality. Eight water samples were collected along the river across the centralized wastewater-discharge region and the dispersed wastewater-discharge region. Water quality had deeply deteriorated in the river. Based on DOM fluorescence spectroscopic properties, DOM was composed of tyrosine-like, tryptophan-like, soluble microbial by-product-like, fulvic-like and humic-like materials, among which protein-like material was dominant in the DOM fractions. The DOM in the centralized wastewater-discharge region had a lower level of aromaticity than that in the dispersed wastewater-discharge region, and its humification degree was lower than that in the latter too. Key factors for the trophic state contained NH4-N, DO, DOC, TP, Φ 2,n and Φ 4,n by multivariable analysis. DOM was mainly derived from treated and untreated industrial wastewater with weak biodegradability.

Fluorescent Properties of Cancerous Liver Tissue

Fluorescence spectroscopy is a technology used fruitfully for biomedical diagnostics as well as for therapeutic purposes. The fluorescent analysis method are applied in biomedical diagnosticsbases on fluorescence of endogenous or exogenous fluorophores. We initially applied fluorescence spectroscopy for the study of human liver cancer (hepatocellular carcinoma) with an exogenous fluorophores, Radachlorin\(^{\circledR }\) 0.35%. The main aim of this study is to determine the spectral variation between normal and malignant liver tissues in 2 cases: the samples expose and non-expose to Radachlorin. The excitation wavelength used for the fluorescence measurements is 405 nm.

Purification of porcine pancreatic lipase by aqueous two-phase systems of polyethylene glycol and potassium phosphate

An aqueous two-phase system (ATPS) was applied for the purification of porcine pancreatic lipase (PPL) from crude PPL using polyethylene glycol (PEG) and potassium phosphate. Phase diagrams for polyethylene glycol (PEG) and potassium phosphate dibasic were determined at room temperature to find an operating region to first form the ATPS. The PPL was preferentially partitioned into the PEG-rich phase in systems with molecular weights of 1000 and 1500 and concentrated in the phosphate-rich phase in systems with PEG of 4000. Moreover, instead of tie line length (TLL), we used a stability ratio without NaCl in the system, and we first applied fluorescence spectroscopy for the protein conformational analysis of the ATPS. The molecular weight of the purified lipase was determined to be approximately 52 kDa by SDS-PAGE. The enzyme was efficiently purified in PEG 1500/potassium phosphate (17/13, %) at a pH of 7.0 at 4 °C. This system obtained an enzyme partition coefficient of 12.7, an extraction efficiency of 94.7% and a purification factor of approximately 4. These results demonstrate that the aqueous two-phase system is a highly efficient method for PPL purification.

The application of fluorescence spectroscopy to organic matter characterisation in drinking water treatment

Key to effective disinfection byproduct (DBP) management is source water control and management, and more specifically, organic matter (OM) control and management. However, the content and character of OM in source waters is spatially and temporally variable, and the prediction of its composition is challenging. Water treatment companies require adequate analytical techniques for OM characterisation to maintain the operation of the water supply and treatment systems adjusted to constantly changing environmental conditions. There is a requirement, therefore, for an improved understanding of OM composition and character in source water, how that composition and character varies with flow conditions, and how this impacts on drinking water treatment. This paper demonstrates that fluorescence spectroscopy offers a potential alternative to other analytical methods of OM characterisation. The advantages of fluorescence include rapid, sensitive and selective characterisation of OM, no sample pre-treatment, small sample volume, and the potential for on-line monitoring incorporation. Fluorescence can provide useful information on OM reactivity and treatability together with an indication of the OM sources (allochthonous or autochthonous). The paper discusses a body of literature which has identified relationships between fluorescence spectra and OM physico-chemical properties (i.e. degree of hydrophobicity, microbial content), has applied fluorescence spectroscopy to characterise the changes in OM upon disinfection, and has related the fluorescence properties to DBP formation. Further work is required in the robust management of data arising from fluorescence spectroscopy analysis and, in particular, Excitation Emission Matrices. Consideration must be given as to how the data might best be employed to greatest effect on a routine basis at WTW.