Determination of biogenic amines by thermal lens microscopic detection of enzymatically released ammoniumMladen Franko
, Mojca Žorž
, 2016, objavljeni povzetek znanstvenega prispevka na konferenci
Opis: Biogenic amines (BAs) are organic amines present in meat, fish, dairy produce and wine due to the breakdown of amino acids, catalysed by microbial decarboxylases. BAs determination in food is important not only because of possible toxicological effects such as nausea, sweating and headache but also due to their possible role as indicators of food spoilage. Chromatographic methods are traditionally applied for determination of BAs in food , which usually require preliminary operations for sample pre-treatment that are laborious and difficult to automate. On the other hand, screening analytical systems provide simple, low cost and rapid analysis with the possibility of subjecting high number of samples to the screening system in a short time . In this work we present a novel method for screening determination of BAs using a microfluidic system with the detection by highly sensitive thermal lens microscope (μFIA-TLM).
Four biogenic amines (putrescine, cadaverine, histamine and tyramine) were subjected to enzymatic catalysis by transglutaminase, where ammonia was released as a product of acyl transfer reaction between the peptide bound glutamine (Gln) and the amino group of BAs. Ammonia was further transformed into indophenol blue by the Berthelot reaction. The coloured product was detected in batch (static) mode in a 100 μm sample cell or in μFIA (flowing) mode in a microchip with the same optical path length. The detection was performed on a TLM system applying a solid-state diode as an excitation source (660 nm). Organic solvents were tested for signal enhancement.
For evaluation of the sensitivity and determination of LOD values (S/N = 3 basis), the NH4Cl standard solution was applied in Berthelot reaction with further detection on TLM system. The LODs for NH4+ in batch mode and in μFIA were 24 μg/L and 109 μg/L, respectively. Both LOD values are lower than the LOD achieved with conventional spectrophotometry (180 μg/L). When mixtures of the indophenol standard solutions and EtOH in the ratio of 1:1 were prepared the LOD in batch mode was improved to 3 μg/L, achieving 60-times improvement compared to spectrophotometry.
Najdeno v: ključnih besedah
Povzetek najdenega: ...Biogenic amines, Thermal lens microscopy, microfluidics, translgutaminase...
Ključne besede: Biogenic amines, Thermal lens microscopy, microfluidics, translgutaminase
Objavljeno: 05.07.2016; Ogledov: 1610; Prenosov: 0
Polno besedilo (74,85 KB)
TLS Spectrometry & Detection in Liquid Chromatography, FIA and Microfluidic SystemsMladen Franko
, predavanje na tuji univerzi
Opis: This presentation provides theoretical backround for thermal lens spectrometry (TLS) and TLS microscopy (TLM) and describes the basic instrumentation for TLS and TLM measurements. Presented applications of TLS and TLM include detection in HPLC (carotenoids, bilirubin, biliverdin), FIA (alergens, Ag, Ag+, phytoplankton and cyanobacterial pigments, organophospate pesticides) and TLM detection in microfluidic systems (Cr(VI), microcystin, NGAL, study of diffusion proceses)
Najdeno v: ključnih besedah
Povzetek najdenega: ...Spekrometry TLS, TLM, HPLC, FIA, microfluidics, bilirubin, biliverdin, carotenoids, alergens, mikrocystin, pigments, organophos...
Ključne besede: Spekrometry TLS, TLM, HPLC, FIA, microfluidics, bilirubin, biliverdin, carotenoids, alergens, mikrocystin, pigments, organophosphate pesticides, NGAL, Ag, Cr(VI), diffusion
Objavljeno: 16.09.2016; Ogledov: 1464; Prenosov: 0
Polno besedilo (35,40 MB)
NOVEL METHODS FOR DETECTION AND REMOVAL OF POLLUTANTS FROM WATERSFranja Prosenc
, 2017, doktorska disertacija
Opis: Water security and quality are a global issue of concern, which have recently become alarming due to the growth of the human population, industrialisation and expanded agricultural activities. Biologically active compounds, such as pharmaceuticals and personal care products can have major adverse effects on aquatic organisms, and are therefore one of the biggest threats in water quality. Another major concern is the spread of waterborne pathogens, including multidrug resistant (MDR) bacteria, which can cause serious illnesses in humans. In order to maintain water abundance and quality, it is necessary that adequate wastewater treatment and analytical techniques allowing for sensitive and fast-response detection of water hazards are in place.
Conventional (waste)water treatment technologies often fail to adequately remove all of the water hazards detailed above. Moreover, conventional analytical techniques currently used in water quality control are, although highly selective and sensitive, time-extensive, with throughput of merely 2 to 3 samples per hour, excluding the time for sample preparation. With respect to these drawbacks, research was proposed to explore new approaches for degradation of recalcitrant compounds, inactivation of microorganisms, and fast screening methods, which are listed in the second chapter of this dissertation as research objectives.
In the third chapter, an extensive theoretical background on the hazards found in aquatic environment, namely pharmaceuticals and waterborne pathogens, is given. Pharmaceuticals enter the environment through several routes (disposal of unused medication via the toilet, pharmaceuticals passing through the human body unchanged/slightly transformed, animal excretions of pharmaceutically active compounds, insufficient wastewater treatment, etc.); therefore, traces of pharmaceuticals have repeatedly been reported in surface waters, groundwater, wastewater effluents, and even drinking water. Iodinated contrast agents (ICAs), as the compounds of interest in this project are further described. ICAs are eliminated from the human body practically unchanged; therefore a large proportion of them end up in municipal and hospital wastewater, where they can be present in concentrations of up to 2.4 g/L. Their ecotoxicity, degradation attempts, as well as detection monitoring in the environment are reviewed within the chapter. Additionally, waterborne pathogens, which account for 2.2 million deaths per year, are reviewed in this chapter, with emphasis on multidrug-resistant (MDR) bacteria. Although MDR infections are mostly prevalent in hospital environments, the presence of MDR bacteria in the environment is not a rarity. A high percentage of bacterial isolates in waters have been shown to be of an MDR phenotype. The theoretical background in analytical methods in water quality monitoring is also given in this chapter. Vanguard and rearguard techniques are explained, the first offering simple, cheap, and rapid sample screening, but sacrificing sensitivity and selectivity, whereas the second providing the highest quality information, excellent sensitivity and selectivity, but in expense of complicated and timely sample handling and high-cost instruments. By combining the two techniques the benefits of both can be exploited in a single system. The basic principles of thermal lens spectrometry (TLS) and its miniaturised version - the thermal lens microscopy (TLM) as fast screening methods providing high sensitivity are further explained, and their practical applications are reviewed. Furthermore, composite materials have recently been finding applications in water treatment technologies, as filter materials, adsorptives for pollutants, catalysts for degradation reactions, and disinfectants. The applications of three main types of composites: synthetic composites, biocomposites, and nanocomposites, are also reviewed within this chapter.
The core of this dissertation is presented in the fourth and the fifth chapter, which examine two separate approaches for water treatment, as well as analytical methods for fast screening purposes. The fourth chapter is investigating options for degradation of iodinated X-ray agents (ICAs), namely diatrizoate, through biodegradation with extracellular enzymes of white rot fungus Dichomitus squalens, and chemical oxidation with manganese(III) acetate. Enzymatic degradation with laccase (Lac) and manganese peroxidase (MnP) at low enzymatic activities was unsuccessful, whereas at approximately 3-times higher activities the enzymes were capable of 60 % degradation in 12 days. Chemical oxidation of diatrizoate with manganese(III) acetate resulted in 85 % degradation in 12 days. Moreover, the suitability of microfluidic flow injection analysis coupled with thermal lens microscopy (μFIA-TLM) as a fast screening method for diatrizoate degradation was examined. The degradation was monitored through the release of iodide from the diatrizoate molecule. μFIA-TLM proved to be a preferable method over UV-Vis spectrophotometry, due to its higher sensitivity, sample throughput, and simple sample handling. Limit of detection (LOD) for μFIA-TLM method was estimated to be 0.14 µM in a 100 µm channel, which is 9 times lower than LOD obtained in UV-Vis measurements. In addition to μFIA-TLM and UV-VIS, high-pressure liquid chromatography (HPLC) was used to monitor the remaining parent compound in the reaction mix.
In the fifth chapter, the second water treatment approach is described. This includes synthesis of biocomposite materials from cellulose (CEL) and keratin (KER), with metal (Ag0, AgCl, Au0) nanoparticles (NPs). Materials were characterised for presence, species, and size of NPs with X-ray diffraction (XRD) and with scanning electron microscopy (SEM). Nanoparticles were confirmed to be of expected species, with sizes as follows: 6.3 ± 0.5 nm for Au NPs, 12 ± 2 nm for Ag NPs, and 22 ± 1 nm for AgCl NPs. In order to evaluate antibacterial properties of the materials, contact tests with gram-negative (Escherichia coli and Pseudomonas aeruginosa) and gram-positive bacteria (Staphylococcus aureus, Methicillin-resistant S. aureus (MRSA), and Vancomycin-resistant Enterococcus faecalis (VRE)) were conducted. Direct-contact assay over 24 hours showed a dose and species-dependent antibacterial activity of [CEL:KER + Ag NPs] materials. The highest potency against the selected bacteria (up to 6-log of reduction) was observed for the material with 500 mg of Ag NPs. AgCl NPs appeared to be less potent than Ag NPs, whereas Au NPs exhibited antibacterial activity only against MRSA and VRE. In addition, antiviral properties of materials were investigated on selected bacteriophages (MS2, phiX174, and fr). However, biocomposite materials with 500 mg of Ag NPs and AgCl NPs, as well as, 240 mg of Au NPs did not exhibit any activity against selected bacteriophages. Biocompatibility with human fibroblasts was evaluated through a direct contact assay for 3 and 7 days of exposure. High concentrations of metal NPs turned out to be cytotoxic for human fibroblasts, whereas the amount of 69 mg of Ag NPs in [CEL:KER] was low enough not to affect the viability of the fibroblasts after 3 days of exposure. Composites with Ag NPs and AgCl NPs were also tested for leachability of NPs out of the materials. Both types of NPs were leaching out in two different forms, as silver ions, and as colloidal silver. Leaching of ionic silver from both materials stabilised after 3 days, whereas colloidal silver was still leaching out on the 7th day. The overall percentage of the total silver (ionic + colloidal) leached was only 0.04 % of silver incorporated in the material.
In summary, this dissertation investigates alternative approaches for water treatment technologies, which could potentially serve as unit improvements of existing technologies, or as on-point pre-treatment technologies to facilitate further conventional water treatment techniques. It also demonstrates the suitability of μFIA-TLM for fast screening measurements in aquatic samples, offering high sample throughput, simple handling of the samples and superior sensitivity over the UV-Vis spectrophotometry.
Najdeno v: ključnih besedah
Ključne besede: Antibacterial biocomposites, nanomaterials, water treatment technologies, multidrug-resistant bacteria, iodinated contrast agents, thermal lens microscopy, flow-injection analysis, microfluidics
Objavljeno: 26.04.2017; Ogledov: 1883; Prenosov: 44
Polno besedilo (27,08 MB)
Immobilization of microbial transglutaminase on carboxyl-functionalized magnetic iron oxide maghemite (γ-Fe2O3) nanoparticle clusters for application in microfluidic flow injection analysis of biogenic aminesMladen Franko
, Slavko Kralj
, Mojca Žorž
, 2017, objavljeni povzetek znanstvenega prispevka na konferenci
Najdeno v: ključnih besedah
Povzetek najdenega: ...immobilization, microbial transglutaminase, biogenic amines, microfluidics, thermal lens microscope...
Ključne besede: immobilization, microbial transglutaminase, biogenic amines, microfluidics, thermal lens microscope
Objavljeno: 11.07.2017; Ogledov: 920; Prenosov: 0
Polno besedilo (740,53 KB)
Simulation Of Gas Focused Liquid JetsRizwan Zahoor
, 2018, doktorska disertacija
Opis: The main aim of dissertation is to develop an experimentally verified computational fluid dynamic (CFD) model of micron-sized liquid jet, produced by an injection molded Gas Dynamic Virtual Nozzle (GDVN). In these nozzles, liquid jets are efficiently orientedly transporting mass and momentum. They are produced by intelligently projecting hydrodynamic focusing effect from a high-speed stream of a co-flowing lower density and lower viscosity gas on a stream of liquid from a feeding capillary. Liquid micro-jets are used for delivery of protein crystal samples in a hard X-ray beam in serial femtosecond crystallography experiments. The diffraction patterns of crystals are collected just before their destruction. The samples are hard to crystallize and very precious, so a thorough knowledge of the jet used in delivering them is required. The jet characteristics are analyzed as a function of operating parameters, geometry and material properties.
The physical model is described by mixture formulation and Navier-Stokes equations for transient, Newtonian, two-phase, compressible flow. Multiphase flow problem is solved with finite volume method (FVM), where fluid-fluid interface tracking is obtained with volume of fluid (VOF). The implementation of FVM-VOF CFD model is available in open source codes OpenFOAM and Gerris. They are validated by performing a series of standard interface advection and multiphase flow test cases. Both open source codes are compared for their abilities in solving GDVN flow problem. Due to the compressible nature of the focusing gas flow, OpenFOAM was chosen for GDVN simulations, since Gerris has no compressible flow option.
Constant effective material properties are used in the phases together with ideal gas density constitutive relation. A mixture model of the two-phase system is solved in axisymmetry. The discretization of the nozzle and chamber system uses approximately 300 000 finite volumes. Mesh independent results are obtained with the finite volumes of the size 0.25 µm in the vicinity of the jet and drops. The simulations are compared with experimental results according to the jet thickness and length for distilled water jet and helium focusing gas, discharging into low-pressure environment of 150 Pa. Reynolds numbers of the liquid and gas are in the range 413-3828 and 17-1222, respectively and Weber number in the range 3-353. A reasonably good agreement with experimental and scaling results is found for the range of nozzle operating parameters never tackled before.
Subsequently, a numerical study of effects of nozzle geometry on stability, shape and flow characteristics of micron-sized liquid jets is performed. The jet characteristics are described as a function of (i) capillary-to-orifice distance, (ii) nozzle outlet orifice diameter and (iii) liquid feeding capillary angle. The study is performed for two sets of liquid flow rates while keeping the gas flow rate unchanged. It is observed that for each value of capillary-to-orifice distance and nozzle outlet diameter, there exists a minimum liquid flow rate below which the jet stability cannot be achieved. It is found that the changes in the nozzle outlet diameter have the biggest influence on the jet diameter, length and velocity, while the liquid capillary angle has no observable effect on the jet characteristic. Change in capillary-to-orifice distance does not affect the flow field around micro jet, so the jet stability and shape is found to be affected by the way liquid-gas interacts near meniscus.
The same numerical model is used to additionally analyze the jet performance under the influence of Argon, Carbon dioxide and Nitrogen focusing gases. The study shows that the helium gas at the same mass flow rate provides twice the length of the jet compared to other gases. The jet focused with helium is also much thinner, faster and interestingly shows no considerable temperature drop at the nozzle outlet.
This work for the first time discuss the computational model of an injection molded micron-sized nozzle and produces valuable information for their design.
Najdeno v: ključnih besedah
Povzetek najdenega: ... Microfluidics, gas dynamic virtual nozzle, flow focusing, micro-jet,...
Ključne besede: Microfluidics, gas dynamic virtual nozzle, flow focusing, micro-jet, convective instability, absolute instability, compressible multiphase flows, dripping, spurting, jetting, jet thickness, jet length, computational fluid dynamics, finite volume method, volume of fluid method
Objavljeno: 27.03.2018; Ogledov: 1088; Prenosov: 27
Polno besedilo (11,47 MB)
The detection and study of biologically active compounds in environmental processes and samplesMojca Žorž Furlan
, doktorska disertacija
Opis: Environmental pollution in the 21th century still represents a global problem for human and animal health. Despite general awareness about released substances and their degradation products their fate and possibilities of removal are not well investigated. Even though the chemicals released are dispersed and diluted in water cycles, their poor biodegradability and/or strong accumulation can result in the intoxication of exposed organisms. Similarly, as a part of the environment, food can get contaminated by bioactive substances during different steps of preparation. Not only artificial compounds such as pesticides or pharmaceuticals, but also natural toxins enter the food chain and impact negatively on humans' and animals' health. In addition, the activity of some bacteria can influence the production of amines from amino acids after fermentation, to which the human body responds with several symptoms of intoxication.
Several analytical methods for the determination of trace levels of broad range contaminants have been developed. Due to the largely robust, selective and sensitive features of the conventional (rearguard) techniques, they represent the first choice for analysing multiple organic compounds in frequently very complex matrices. However, screening (vanguard) methods are paving the way in the chemical analytics as a solution that provides simplicity and rapid analytical responses with binary (yes/no) answers. They require little or no sample treatment as well as more economically-efficient instrumentation. The combination of vanguard-rearguard analytical strategies hence offers a compromise between classical analytical figures of merit and productivity-related characteristics.
In the first part of our research feasibility studies for the application of TLS and/or TLM in novel analytical methods for the determination of lipid-lowering drug atorvastatin and a mycotoxin ochratoxin A . The survey on atorvastatin performed spectrophotometrically has shown a decrease of ATV-sulpho-vanillin product at the wavelength of its maximum absorbance after dilution by organic solvent, which was investigated due to the possible increasing of the method sensitivity. As the predicted LODs that could be obtained by TLM (0.3 mg/L) could not reach the concentration of ATV usually present in the environment (ng/L-g/L) further experiments on this subject were therefore not justified. On the other hand, the ELISA assay for the determination of ochratoxin A was performed. In case of μFIA-TLM, the measurements were influenced by high background signal resulting in high LODs of TLM (470 pg/mL), which is known as a background limited technique. It was estimated that the LODs of standard ELISA assay could not be significantly improved, therefore no further research was conducted in this direction.
In the second part of the dissertation, a sensitive rearguard system by coupling HPLC and TLS for the determination of biogenic amines in wine samples was developed. Putrescine, cadaverine, histamine and tyramine were separated and detected on a HPLC-TLS system after derivatization by dabsyl chloride. The method was optimized in terms of chromatographic conditions and in terms of TLS parameters. Also, the sensitivity of the newly developed method was evaluated by comparing the TLS detection with DAD detection in terms of LOD values, where TLS showed 3.6-fold improvement compared to DAD. Afterwards, the standard addition calibration was performed and evaluated for its recoveries (86−117%) in the determination of the four BAs. The applicability of the novel method was tested by the analysis of real white and red wine samples and by comparing the results to the standard HPLC-FL method and concentrations of BAs in wine samples were in good accordance. In addition, the dabsylated BAs showed better stability compared to the OPA derivatives as they have not lost the peak intensity after 17h of storage.
In the third part, a vanguard system for detection of the overall biogenic amines concentration was developed by employing μFIA-TLM. Initially, NH4Cl standard solutions were applied in the indophenol reaction for batch mode, off-line μFIA-TLM and in an on-line indophenol formation for μFIA-TLM detection. By adding 50 % of EtOH to indophenol we obtained 9-fold improvement. In addition, indophenol showed good stability under TLM conditions. We optimized the microfluidic and TLM parameters in the off-line and on-line indophenol reaction. The addition of 5% ethanol to the reagent in the on-line reaction resulted in the 3-fold improvement of the signal-to-noise ratio. Further on, the overall reaction, including the enzymatic and the following indophenol reaction, was optimized by choosing the optimal buffer (pH=7, 0.5 M) and alkaline conditions (2M NaOH). The influence of interferences from amino compounds was also evaluated and discussed. The off-line and on-line μFIA-TLM were evaluated by their performance characteristics. The LOD for ammonia detection reached 2.3 μM and the applicability in ammonia detection in water samples was discussed. Similar LOD of 3.2 μM was obtained for the overall concentration of BAs and LOD of 3.8 μM for histamine, which is more than 4-folds lower value as the lowest suggested limits of intake for histamine in wine samples (2 mg/L; 18 μM). Finally, an immobilization procedure on magnetic nanoparticles was developed for the possible implementation of the selected enzyme in a miniaturized biosensor.
Najdeno v: ključnih besedah
Povzetek najdenega: ...thermal lens microscopy, high performance liquid chromatography, microfluidics, biogenic amines, microbial transglutaminase, indophenol (Berthelot) reaction...
Ključne besede: thermal lens spectrometry, thermal lens microscopy, high performance liquid chromatography, microfluidics, biogenic amines, microbial transglutaminase, indophenol (Berthelot) reaction
Objavljeno: 04.06.2018; Ogledov: 697; Prenosov: 105
Polno besedilo (2,90 MB)