Fluorimetry Method Research Articles

A high-throughput differential scanning fluorimetry method for rapid detection of thermal stability and iron saturation in lactoferrin

Thermal stability and iron saturation of lactoferrin (LF) are of great significance not only for the evaluation of the biological activities of LF but also for the optimization of the isolation and drying process parameters. Differential scanning calorimetry (DSC) is a well-established and efficient method for thermal stability and iron saturation detection in LF. However, multiple DSC measurements are typically performed sequentially, thus time-consuming and low throughput. Herein, we introduced the differential scanning fluorimetry (DSF) approach to overcome such limitations. The DSF can monitor LF thermal unfolding with a commonly available real-time PCR instrument and a fluorescent dye (SYPRO orange or Glomelt), and the measured melting temperature of LF is consistent with that determined by DSC. On the basis of that, a new quantification method was established for determination of iron saturation levels using the linear correlation of the degree of ion saturation of LF with DSF measurements. Such DSF method is simple, inexpensive, rapid (<15 min), and high throughput (>96 samples per experiment), and provides a valuable alternative tool for thermal stability detection of LF and other whey proteins.

The effect of fingerprint enhancement methods applied on adhesive surfaces on DNA recovery: a preliminary study

The presence of body fluids such as blood, saliva, semen or urine during fingerprint research on the evidence taken from the crime scene makes it necessary to protect biological materials to examine the evidence in multiple ways. Therefore, it is crucial that fingerprint development (FD) techniques do not disrupt the structure of biological materials during FD procedures. In this sense, it is essential to determine whether biological material or fingerprints should be the priority during the collection of evidentiary materials, to determine the systematic order and to determine whether the FD methods to be applied cause damage to the genetic material used in the identification of individuals and to evaluate them in terms of their evidentiary quality. This study investigated the effects of the application of trace detection methods on DNA profiling processes in evidence where fingerprints and biological samples are found at the same time. In this study, blood, saliva, semen and urine samples were taken from a male individual who signed an informed consent form at the laboratory stage. The samples were applied 50 µL on the adhesive tape surface. After application, the samples were treated with crystal violet (CV) and sticky side (SS) fingerprint development chemicals suitable for the surface type. The prepared samples were dried under room conditions. After 1 day and 45 days under normal room conditions, silica-based DNA extraction was performed. After extraction, DNA quantification was performed using the fluorimetry method. In the study, biological samples with known DNA content were used to focus on DNA quantification. Among the fresh samples prepared in the study, DNA recovery was higher in the SS-treated urine, blood and saliva samples and in the CV-treated semen sample group compared to the other groups. This shows that chemical treatment of some biological samples on adhesive tape increases the efficiency of DNA recovery. When the 45-day waiting samples were compared with the control group samples, DNA recovery decreased in CV-treated urine and blood samples, while DNA recovery increased in SS-treated urine and blood samples. In semen samples, both CV and SS treatment negatively affected DNA recovery. In saliva samples, DNA recovery increased ~2-fold in the CV-treated sample group, while SS treatment caused a ~75% decrease in DNA recovery. The results show that the non-porous adhesive tape does not adversely affect the amount of DNA in terms of STR profiling of latent FD chemicals used on the surfaces and that adhesive tape treated with fingerprint enhancement chemicals can actually be used for advanced forensic genetic analyses for DNA extraction on surfaces.

Biomonitoring of the surface waters of the Volyntsevo reservoir using the fluorimetry method

The article examines the state of the drinking Volyntsevo reservoir and its tributaries in conditions of significant changes in the hydraulic regime and pollution by waste mine waters. The state of algoflora of a water body is considered, the results of studying the species composition of phytoplankton, the content of basic photopigments and photosynthetic activity of microalgae are presented. The study presents the results of the application of the method of fluorimetric assessment of the state of phytoplankton cells as the main bioindicator of the state of the aquatic environment. The research was conducted in the vicinity of Yenakievo for the Volyntsevo reservoir and the Bulavin river. These water bodies are subject to significant man-made impacts: as a result of intensive water use of reservoir resources, there has been a significant decrease in water levels and, as a result, a deterioration in water quality. The conducted studies have shown a significant excess of the permissible standards for the content of magnesium ions, sulfates, total hardness and alkalinity in water samples. The negative impact of surface water pollution on the species composition of algoflora, as well as the photosynthetic activity of phytoplankton cells, has been shown. The obtained materials can serve as a basis for further monitoring of drinking reservoirs in the region. The current situation requires constant monitoring observations aimed at identifying and eliminating all sources of pollution of the studied water bodies.

Methodological approaches to the electromagnetic radiation effects on microorganisms

All living organisms are surrounded by natural and artificial origin electromagnetic radiation (EMR), which affects organisms. The artificial radiation sources number is increasing, but the EMR influence mechanisms on living organisms have not yet been fully investigated, despite the large number of studies. However, understanding the EMR action mechanisms can give impetus to the development of various biotechnologies. To solve the problem of understanding the EMR effects on organisms and develop new biotechnologies, it is necessary to apply new research methods. Therefore, the study of research methods is an urgent task. This research is a review of studies and research methods on the effects, of EMR on organisms. It brings together studies that consider different approaches to the study and existing hypotheses of the mechanisms of radiation effects. The aim of the paper is to review and evaluate methodological approaches to investigate the effects of EMR. The paper considers research on the EMR effects on microorganisms, eubacteria. Based on the results of the work, it is proposed to study the effect of radiation lasting several hours. As well as to apply spectrophotometry and fluorimetry methods.

Modification of bimetal Zn/ Mg MOF with nanoparticles Fe3O4 and Fe3O4@SiO2, investigation of the peroxidase-like activity of these compounds by calorimetry and fluorimetry methods

In this article; the bimetal metal-organic framework Zn/Mg (Zn/Mg MOF) is synthesized.Then Zn/Mg MOF bimetal was combined with Fe3O4 and Fe3O4@SiO2, and composites of Fe3O4@ SiO2/MOF/Dextrin, Fe3O4@SiO2/MOF, Fe3O4@MOF/Dextrin and Fe3O4@MOF made. The peroxidase-like activity of these compounds was investigated and compared by calorimetric Resazurin (Rz) and O-phenylenediamine (OPD); (Rz-H2O2, OPD-H2O2) and fluorimetric Rz and terephtalic acid (TA); (Rz-H2O2, TA-H2O2). The Fe3O4@ MOF/Dextrin composite has the highest peroxidase-like activity. The effect factors (amount of pH (6), the values of TA (1.37 mM), H2O2 (0.025 mM), reaction time (8.15 min), and amount of Composite (116.67 mg)) to increase the catalytic activity of Fe3O4@ MOF/Dextrin measured by chemometrics method. The most suitable linear range of the calibration curve by the TA-H2O2 -Composite fluorimetric method is 1–600 μg L−1, and the detection limit is 2.27 μg L−1. The relative standard deviation (RSD%) for measuring concentration atropine 1 μg L−1 (n = 6) is 1.18%. Finally, from this system for measuring atropine extracted by the Liquid-liquid extraction (LLE) method in two types of plants, D. Innoxia north and west and D. stramonium north and west of Iran (118.25 μg L−1, 79.80 μg L−1) and (18.477 μg L−1, 9.27 μg L−1) used, respectively.

Comparative analysis of soil pollution oil products of industrial and landscape-recreational zones of the city of Usolye-Sibirskoe using the fluorimetry method

The purpose of the work is to compare soil pollution with oil products on the territory of industrial and landscape-recreational zones of the city of Usolye-Sibirskoye using the fluorometry method. The content of oil products in the samples of the four upper layers of soils taken from the production area of the chemically contaminated city of Usolye-Sibirskoye and from its landscape and recreational zone was determined. Although the maximum permissible concentration of oil products in the soils of these zones has not been established, it is known that the mass fraction of oil products in uncontaminated soils should not exceed 0.1%. The obtained results of fluorimetric analysis of soils from two practically diametrically opposite areas of the city in terms of pollution made us think and conduct a deeper analysis, taking into account the wind rose of the city and adjacent territories and the sufficiency of the fluorimetry method performed using the Fluorat-0.2 luminescent photometric analyzer. Using the results of the analysis of soil samples from the territories of "Usolekhim-prom" and "Ski base" in Usolye-Sibirskoye, it was revealed that the mass fractions of oil products exceed the indicators for all four upper soil layers in the case of focusing on the principles of rationing, which today are not legally established. The results of the study showed that the highest content of oil products in both cases is in the layer of forest litter. Based on the fact that the activities of Usolekhimprom have been completely stopped since 2017, and pollution with oil products exceeding the approximate standard values was found only in the uppermost layer of the soil, it was suggested that the main cause of soil pollution with oil products in the territory of the city of Usolye - Sibirskoye is, basically, the current largest enterprise in Eastern Siberia for the production of petroleum products and petrochemistry - JSC Angarsk Petrochemical Company, and this is annually confirmed by statistics.

Microtiter Plate-Based Differential Scanning Fluorimetry: A High-Throughput Method for Efficient Formulation Development

Nano/microparticles are widely used as vaccine adjuvants to improve antigen stability and enhance immune response. Conformational stability of a given protein was normally assessed using differential scanning calorimetry (DSC) for the optimization of formulation and for ensuring antigen stability in vaccine products. Here, a higher throughput version, namely the microtiter plate-based differential scanning fluorimetry (DSF) method was developed and optimized for assessing the protein thermal stability in the particulate adjuvant-adsorbed form. Using recombinant human papillomavirus (HPV) vaccine antigens, along with several model proteins, enhanced sensitivity and correlation to the well-established differential scanning calorimetry were demonstrated. Higher throughput and much smaller sample consumption (1/10 ∼ 1/20 of the amount needed as compared to DSC) make the plate-based DSF a method of choice for formulation development, particularly during the early developmental phase of a project where the sample amount is usually quite limited.

Nutrient Detection Sensors in Seawater Based on ISI Web of Science Database

Marine ecosystem is increasingly deteriorating. In order to assess anthropogenic influence and instigate appropriate remedial actions, it is still of great significance to develop the technology of sensors applied for nutrient detection (e.g., nitrate, phosphate, and silicate) in seawater. This brief review shows an important direction for the development of nutrient detection sensors in seawater and also the limitations and challenges based on data from the ISI Web of Science database. Being different from previous review papers, in this short critical review paper (1) we unified the unit of limit of detection (LOD) for making the comparison within different researches possible; (2) only the literatures focusing on the technological development of sensors in seawater were used; and (3) not only the detection methods but also the detected analytes and publication years were discussed to supply more valuable information for the development of nutrient sensors applied in seawater. In total, 109 literatures were collected with regard to technological development. The quantity of literatures has increased most during 2011-2020. For analytes, literatures related to nitrate, phosphate, ammonium, and phosphate will continue to increase with more accurate data. For detection methods, spectrophotometry, colorimetry, fluorimetry, and electrochemistry are the most widely used sensors. LODs show thousands of orders. In general, there are lower LOD to nitrite and ammonium and fluorimetry method. Now, for analytes, nitrate 1.0983 &gt; silicate 0.5495 &gt; phosphate 0.4823 &gt; ammonium 0.1324 &gt; nitrite 0.0568 . For detection methods, microfluidics 1.7617 &gt; electrochemistry 1.2607 &gt; colorimetry 0.4462 &gt; spectrophotometry 0.2941 &gt; fluorimetry 0.0558 . This result indicated that the development level of detection methods is closer for nitrate, nitrite, phosphate, and silicate. For ammonium, spectrophotometry has significantly lower LOD than electrochemistry ( p &lt; 0.05 ), and fluorimetry also has significantly lower LOD than electrochemistry ( p &lt; 0.05 ). Our results imply that sensors with accurate LOD should be developed in the future. In addition, more detection methods should be considered by future sensors.

Correction pen as a hydrophobic/lipophobic barrier plotter integrated with paper-based chips and a mini UV-torch to implement all-in-one device for determination of carbazochrome

The design of a cheap, simple, and handy sensing system for rapid quantitation of pharmaceuticals becomes mandatory to ease drug development procedures, quality control, health care, etc. This work describes a simple, innovative, and easily manufactured paper-based device using a correction pen as a plotter for hydrophobic/lipophobic barriers and graphene quantum dots for recognition and quantification of the hemostatic drug carbazochrome, via fluorescence turn-off mechanism mediated by the inner filter effect. A smartphone-based all-in-one device fitted with an inexpensive 365 nm flashlight as a UV light source and a free image processing software was developed for rapid and reliable interpretation of the fluorescence change from the paper-based device upon introduction of the drug. The simple and convenient steps permit the analysis of many samples in a very short time. The smartphone-based all-in-one device featured excellent sensitivity for carbazochrome with a limit of detection equals to 12 ng/detection zone and good %recovery (100.0 ± 0.4). The reliability of the device was ascertained by favorable statistical comparison with the analogous optimized conventional fluorimetry method and a reference HPLC method. The device has been successfully applied for versatile quantitation of carbazochrome in tablets and on manufacturing equipment surfaces with excellent recoveries. The device offers many green aspects that definitely assist the implementation of the sustainability concept to analytical laboratories. The cost-efficiency, reliability, and ease of fabrication as well as the greenness and user friendship qualify the device for wide application in low-income communities.

Activation of Nanocomposite Liposomal Capsules in a Conductive Water Medium by Ultra-Short Electric Exposure

In a system that simulates aquatic biological media, the possibility of activating nanocomposite liposomal capsules (NLCs) containing spherical electrically conductive nanoparticles on the outer and inner surfaces of the liposomal membrane, using an external ultrashort electric action, has been shown. The decapsulation effect was registrated by fluorimetry methods. The key role of conducting nanoparticles in increasing the sensitivity of NLCs to external ultrashort electrical impact is shown. A theoretical model of nonthermal interaction of NLCs with ultrashort electric pulses is constructed, within the frame of which an expression is obtained for the critical value of the electric field strength, which determines the threshold for the appearance of the decapsulation effect in a conducting medium. The described mechanism of decapsulation explains the selective nature of ultrashort pulsed electrical impact on the NLCs.

Investigation of micellar and interfacial phenomenon of amitriptyline hydrochloride with cationic ester-bonded gemini surfactant mixture in different solvent media.

Herein, the interaction among the antidepressant drug amitriptyline hydrochloride (AMT) and a green gemini surfactant, ethane-1, 2-diyl bis(N,N-dimethyl-N-tetradecylammoniumacetoxy) dichloride (14-E2-14), via numerous techniques such as tensiometry, fluorimetry, FT-IR and UV-visible spectroscopy in three different media (aqueous 0.050 mol·kg-1 NaCl, 0.50 and 1.0 mol·kg-1 urea) were investigated. AMT is used to treat mental illness or mood problems, such as depression. The aggregation of biologically active ingredients can enhance the bioavailability of hydrophobic drugs. A significant interaction between AMT and 14-E2-14 was detected by tensiometric study as the critical micelle concentration (cmc) of AMT+14-E2-14 is reduced upon an increase of mole fraction (α1) of 14-E2-14. The decrease in cmc indicates the nonideality of studied mixtures of different compositions. Although, employed drug AMT is freely soluble in the aqueous and non-aqueous system but is not hydrophobic enough to act as its carrier. Instead, gemini surfactant formed spherical micelles in an aqueous system and their high solubilization capability, as well as their relatively lower cmc value, makes them highly stable in vivo. The cmc values of AMT+14-E-14 mixtures in all cases were further decreased and increased in NaCl and urea solutions respectively as compared with the aqueous system. Numerous micellar, interfacial, and thermodynamic parameters have been measured by applying various theoretical models. The obtained changes in the physicochemical assets of AMT upon adding of 14-E2-14 are likely to enhance the industrial and pharmaceutical applications of gemini surfactants. The negative interaction parameters (βm and βσ), indicate synergistic attraction is occurring in the mixed systems. The aggregation number (Nagg), Stern–Volmer constant (Ksv), etc. are attained through the fluorescence method, also supporting the attractive interaction behavior of AMT+14-E2-14 mixtures in all solvents. The Nagg was found to increase in the salt solution and decrease in the urea system compared with the aqueous solution. FT-IR and UV-visible analysis also depict the interaction between the constituent alike tensiometry and fluorimetry methods. The results suggested that gemini surfactants may serve as a capable drug delivery agent for antidepressants, improving their bioavailability.

Measuring the Microphase Separation Scale of Polyurethanes with a Vibration-Induced Emission-Based Ratiometric "Fluorescent Ruler".

Polyurethanes (PUs) are a very attractive type of segmented polymer with unique mechanical properties derived from thermodynamic incompatibility between flexible soft segments and hard segments. The performance of PUs is closely related to their microphase separation structures, in which the hard domains serve as physical cross-linking points in the soft matrix. Studying the microphase separation of PUs in a facile manner is thus of great significance but challenging due to the complexity of the internal structures of PUs. N,N'-disubstituted-dihydrophenazine (DPAC) derivatives, the typical molecules featured with vibration-induced emission (VIE) attribute, can emit fluorescence varying with surrounding environment. In this proof-of-concept work, a series of DPAC derivatives were employed as built-in ratiometric "fluorescent rulers" to measure the degree of microphase separation in PUs modulated by temperature variation. The fluorescence of selected DPAC-doped PU films is dependent on the temperature, providing a theoretical basis for this concept. The feasibility of these fluorescent rulers was further validated by the small-angle X-ray scattering (SAXS) analysis. The SAXS curves show significant change in q range of 0.02 to 0.15 Å-1 with the variation in temperature, showing the changes in the internal microstructure. The polydisperse hard sphere model analysis of the scattering data revealed that the volume fraction of hard spheres has a defined relationship with the fluorescence intensity ratio of orange-red light and blue light, thereby demonstrating a novel fluorimetry method for measuring and monitoring the microphase separation of polyurethanes.

Green conventional and first-order derivative fluorimetry methods for determination of trimebutine and its degradation product (eudesmic acid). Emphasis on the solvent and pH effects on their emission spectral properties.

In this report, the fluorescence properties of the antimuscarinic drug trimebutine maleate (TRB) were fully studied and characterized. TRB exhibited intrinsic fluorescence that is greatly dependent on the local environmental factors including the solvent nature and the pH. Yet, its fluorescence was not significantly influenced by the existence of some surface active agents and polymer. The outcomes of this investigation verified that TRB fluorescence emission is intense in ethanol: 1.0 M aqueous acetic acid (9:1, v/v) with emission maxima at 357 nm and excitation maxima at 270 nm. Whereas, going towards higher pH causes fluorescence quenching. These conditions permitted ultrasensitive fluorimetric determination of TRB over the concentration range of 2.00-1500.0 ng/mL with a lower detection limit of 0.40ng/mL Application for the determination of TRB in tablets, ampoule and suspension was successfully achieved with %recoveries ranged between 98.21-100.17%. Furthermore, a first order derivative fluorimetric method was validated for resolving and simultaneous determination of TRB and its degradation product and impurity, eudesmic acid (EUA) making use of the pH-mediated fluorescence spectral shift of EUA. An ethanolic solution containing acetate buffer (pH 5.3) was used for this goal with excitation at 255 nm and measurement of the first order derivative peak amplitudes at respective zero-crossing points of 375 and 351 nm over the corresponding concentration ranges of 20.00-500.00 and 10.00-300.00 ng/mL for TRB and EUA, respectively. The two methods were assessed regarding greenness and eco-friendship by the National Environmental Methods Index and analytical eco-scale score approaches which confirmed their excellent greenness and safety.

Separation and Preconcentration of Trace Uranium(VI) by Solid Phase Extraction with 2,3-Dihydroxynaphthalene and Cetyltrimethylammonium Bromide on Molten Naphthalene and Its LED Fluorimetric Determination in Water Samples.

A simple and rapid solid phase extraction (SPE) procedure has been developed for the extraction and determination of uranium in water samples. The method is based upon the adsorption of uranium(VI)-2,3-dihydroxynaphthalene complex on microcrystalline naphthalene at the pH range 10 - 12 in the presence of a counter cation cetyltrimethylammonium ion. The solid mass consisting of uranium-2,3-dihydroxynaphthalene-CTA complex and naphthalene is ignited in the furnace at a temperature of 700°C for 1 h and then digested in dil. HNO3 solution. Uranium is then determined by LED fluorimetry using fluorescence enhancing pyrophosphate buffer. The effects of different variables like pH of the solution, reagent concentrations, counter cations, stirring time, interfering ions etc. have been investigated thoroughly for the quantitative recovery of uranium. The accuracy of the developed method has been ascertained by standard addition method as well as conventional pellet fluorimetry method involving co-precipitation of uranium using aluminum phosphate as a carrier.

Ultrasound processed cuminaldehyde/2-hydroxypropyl-β-cyclodextrin inclusion complex: Preparation, characterization and antibacterial activity

Ultrasound is an energy saving, simple, high efficiency and eco-friend physical technology. In this study, the inclusion complex of cuminaldehyde (CUM), a major constituent of cumin essential oil, with 2-hydroxypropyl-β-cyclodextrin (HPβCD) was synthesised with the aid of ultrasound. The solid CUM/HPβCD-IC was characterized using Fourier transform infrared spectroscopy (FT-IR), powder X-ray diffraction (PXRD), thermal gravimetric analysis (TGA) and differential scanning calorimetry (DSC) techniques. The stability constants were evaluated by phase solubility, absorption and fluorimetry methods, and were found to be 168, 122 and 256 M−1, respectively. FT-IR and molecular modeling studies indicated that the phenyl ring with the aldehyde group of CUM was inserted into the hydrophobic HPβCD cavity. Further, the efficacy of CUM/HPβCD-IC for inactivation of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was investigated through plate colony counting method. According to the results, the inactivation was 100 ± 0.06% for both E. coli and S. aureus bacteria. The results demonstrated the inclusion of CUM within the harmless HPβCD cavity assisted by ultrasound technology, contributing the improvements of water solubility, thermal stability and antibacterial activities.

Оцінка стану перекисного окислення ліпідів методом хемілюмінесценції у самців кролів за гонадодистрофій

Reducing the reproductive capacity of males is one of the major problems in andrology. Poor quality of semen and its reproductive capacity, impaired structural and functional characteristics of sperm is a consequence of gonadodystrophy. The leading link in the pathogenesis of various types of gonadodystrophy is the violation of the prooxidant-antioxidant system. Early diagnosis of a complex of pathological processes that develop in gonadodystrophy is possible due to the registration of the intensity of the processes of free radical oxidation. It is known that a highly sensitive way of detecting subtle biochemical shifts is chemiluminescence, which is used to detect free radical oxidation, the development of generalized oxidative stress. The aim of the study was to estimate the state of lipid peroxidation in rabbits with gonadodystrophy by the chemiluminescent method. The main tasks of the study: to investigate the state of the lipid peroxidation system by determining the final product of peroxidation – malondialdehyde in the serum of rabbits with gonadodystrophy; to determine the intensity of rabbit serum chemiluminescence and to determine the use of this technique for the diagnosis of gonadodystrophy. Three groups were formed: control – clinically healthy animals kept on a standard diet; experimental group I – rabbits with alimentary type of gonadodystrophy (prolonged maintenance on a diet deficient in carotene (vitamin A), Zinc); experimental group II – rabbits with gonadodystrophy of toxic type (chronic nitrate-nitrite toxicosis, caused by feeding of sodium nitrate). Serum samples were taken in 15, 30, and 45 day of the study. The concentration of malondialdehyde was determined by a fluorimetry method based on the reaction between malondialdehyde and thiobarbituric acid, which occurs under conditions of high temperature and acidic environment, with the formation of a colored trimethin complex. Spectra of spontaneous chemiluminescence of serum samples were measured on a chemiluminometer "Lum-5773", manufactured by the Russian Federation, which was connected to an interface with a personal computer to record the instrument performance with PowerGraph software (version 3.3). The rate of light chemiluminescence was estimated in 5 minutes. The state of lipid peroxidation in rabbits according to the malondialdehyde content showed a significant increase in the indicator in the experimental groups I, II compared with the control. It was noted that in the animals of the experimental group I with gonadodystrophy of alimentary type there was increased number of malondialdehyde in the serum by 15 days (by 58,3 %, P ˃ 0.001), the figure reached the maximum value by 45 days of the experiment (increase by 76 %, P ˃ 0.001), however, there was a minimal difference between the 30 and 45 day samples. In serum samples of male rabbits with toxic gonadodystrophy, there was a marked increase in malondialdehyde concentration by the 15th day of the experiment (by 75 %, P ˃ 0.001), and by an increase in the number during the experiment (up to 96 % by 45 days, P ˃ 0.001). Data obtained from a chemiluminescent serum study had a similar upward trend as with malondialdehyde in both groups. This may indicate a lack of activity of the body antioxidant protection system and the need for antioxidant therapy, due to the complexity of the chemiluminescence indicator, which is a concomitant marker of free radical oxidation. Chemiluminescent analysis of serum samples of rabbit males showed high efficiency as a diagnostic tool for gonadodystrophy and showed similar results to the malondialdehyde determination. Obtained during chemiluminescent analysis, data indicate the need of therapeutic measures as early as the 15th day of the experiment, due to the significant accumulation of lipid peroxidation products and the lack of adequate response of the organism to the action of external factors.

Synthesis, characterization and application of a new fluorescence reagent for the determination of aluminum in food samples

A new sensitive fluorimetry method for the simple and rapid measurement of aluminum in some food samples, which used 3′,6′-bis(diethylamino)-2-{[(1E)-(4-ethoxyphenyl) methylene] amino}spiro[isoindole-1,9′-xanthen]-3(2H)-one (DEMAX) for Al(III) chelation is described herein. The fluorescence intensity of the aluminum-DEMAX complex remains unaltered for over 24h at room temperature and is a linear function of the concentration of aluminum in the 0–1000mg/L range with a detection limit (LOD, defined as 3s/b) of 3.02µgL−1 and quantification limit (LOQ, defined as 10Sb/m) of 30µgL−1. The relative standard deviation (RSD) was 0.1% for a 1µgL−1 aluminum level. The influences of the analytical parameters such as pH, waiting time of aluminum-DEMAX complex, amount of reagent, effect of temperature etc. were investigated. The developed method was successfully used for assaying aluminum in some food samples and dialysis solution, measured by the new home-made fluorimetric system. The results of analysis of the certified reference material (INCT-TL-1) are in good agreement with the certified value.

Extraction of Protein Thermodynamic Parameters from High‐Throughput Differential Scanning Fluorimetry Assays

Thermodynamic properties of protein unfolding have been extensively studied; however, the methods used have typically required significant preparation time and high protein concentrations. Here, we present a facile, simple, and high‐throughput differential scanning fluorimetry (DSF) method that enables a complete set of thermodynamic parameters of protein unfolding to be extracted. This method assumes a two‐state, reversible protein unfolding mechanism and provides the capacity to quickly analyze the biophysical mechanisms of changes in protein stability and to more thoroughly characterize the effect of additives, inhibitors, or pH. We show the utility of the DSF method by analyzing the thermal denaturation of lysozyme, carbonic anhydrase, chymotrypsin, horseradish peroxidase, and cellulase. Compared to similar biophysical analyses by circular dichroism, DSF provides greater than 24‐fold reduction in experimental time. This study opens to door to rapid characterization of protein stability on low concentration protein samples.Support or Funding InformationThe authors acknowledge financial support from the US National Science Foundation (Award No. MCB 1552113 to RCP) and institutional support from Miami University through the Robert H. and Nancy J. Blayney Professorship (to RCP).

Extraction of Thermodynamic Parameters of Protein Unfolding Using Parallelized Differential Scanning Fluorimetry.

Thermodynamic properties of protein unfolding have been extensively studied; however, the methods used have typically required significant preparation time and high protein concentrations. Here we present a facile, simple, and parallelized differential scanning fluorimetry (DSF) method that enables thermodynamic parameters of protein unfolding to be extracted. This method assumes a two-state, reversible protein unfolding mechanism and provides the capacity to quickly analyze the biophysical mechanisms of changes in protein stability and to more thoroughly characterize the effect of mutations, additives, inhibitors, or pH. We show the utility of the DSF method by analyzing the thermal denaturation of lysozyme, carbonic anhydrase, chymotrypsin, horseradish peroxidase, and cellulase enzymes. Compared with similar biophysical analyses by circular dichroism, DSF allows for determination of thermodynamic parameters of unfolding while providing greater than 24-fold reduction in experimental time. This study opens the door to rapid characterization of protein stability on low concentration protein samples.

C-phycocyanin from Spirulina maxima as a Green Fluorescent Probe for the Highly Selective Detection of Mercury(II) in Seafood

In this work, a highly selective and sensitive fluorimetric method has been developed for sensing mercury ions in seafood using a fluorescent probe of C-phycocyanin (C-PC) from Spirulina maxima. The interactions between C-PC and mercury ions were systematically characterized by UV-vis spectrophotometer, microscopy imaging, and fluorescence measurements. It was found that the C-PC fluorescence could be quenched by mercury ions triggering the C-PC aggregation phenomenon. Importantly, the fluorescence intensities of C-PC probes could be linearly changed depending on the concentrations of mercury ions, showing the detection limit of 0.0080 μM, and the relative standard deviation of this method was about 1.9%. Moreover, the developed fluorimetry method was successfully applied to detect mercury ions in the real seafood samples (oysters and Pampus argenteus samples), with the acceptable rate of recovery between 93.6 and 107.2%. Such a green, efficient, and cost-effective fluorimetric method of C-PC probe should promise the wide applications for tracing mercury ions levels in seafood.