dissertation
Mojca Žorž (Author), Mladen Franko (Mentor)

Abstract

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.

Keywords

thermal lens spectrometry;thermal lens microscopy;high performance liquid chromatography;microfluidics;biogenic amines;microbial transglutaminase;indophenol (Berthelot) reaction;

Data

Language: English
Year of publishing:
Typology: 2.08 - Doctoral Dissertation
Organization: UNG - University of Nova Gorica
Publisher: [M. Žorž Furlan]
UDC: 543
COBISS: 5157371 Link will open in a new window
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Other data

Secondary language: Slovenian
Secondary title: Detekcija in proučevanje biološko aktivnih snovi v procesih in vzorcih iz okolja
Secondary abstract: Kljub splošni ozaveščenosti ljudi glede okoljske problematike, onesnaženje v 21. stoletju še vedno ogroža zdravje ljudi in živali na globalni ravni. Splošne težave, povezane s kemičnimi snovmi v okolju, so slaba biološka razgradljivost ter njihovo kopičenje v okolju, kar lahko privede do zastrupitve organizmov, ki pridejo z določeno snovjo v neposreden stik. Škodljive snovi lahko pristanejo v prehranjevalni verigi. Te pa niso nujno umetne, kot so pesticidi in farmacevtske učinkovine, temveč tudi naravni toksini rastlinskega ali živalskega izvora. Tudi slabe higienske razmere so lahko še vedno zelo pogost razlog za bolezni, ki izvirajo iz hrane. Znane so zastrupitve z biogenimi amini, ki med postopki za pripravo živil s fermentacijo, pri aktivnem delovanju nekaterih vrst bakterij nastajajo kot stranski produkt iz aminokislin. Te snovi večinoma tvorijo Gram negativne bakterije, zato so biogeni amini tudi pokazatelji higienskih razmer med pridelovanjem in predelovanjem hrane. Za določitev več organskih spojin hkrati v zelo kompleksni matrici, so se za najbolj primerne izkazale konvencionalne (rearguard) metode. Te metode se odlikujejo tako po robustnosti, kot tudi po selektivnosti in občutljivosti. Podrugi strani se na tem področju uveljavljajo tudi t. i. presejalne metode (screening ali vanguard methods), ki predstavljajo preprostejšo analizno metodo s hitrimi da/ne odgovori na analizna vprašanja. Omenjene metode so cenejše in vključujejo minimalno predhodno obdelavo vzorca, kot je čiščenje ali pred-koncentriranja. Kombinacija vanguard-rearguard strategije tako ponuja vmesno pot med klasično analitiko s poudarkom na občutljivosti, točnosti in natančnosti ter produktivnostjo v smislu cenejše in hitrejše analize z manjšo, a še vedno zadovoljivo točnostjo in natančnostjo. V prvem delu disertacije so predstavljene raziskave o izvedljivosti uporabe novih metod na osnovi TLS in/ali TLM pri določitvi atorvastatina (ATV) ter ohratoksina A (OTA). Po redčenju ATV derivata z organskim topilom se absorpcijski vrh premakne h krajšim valovnim dolžinam, ki niso več v dosegu našega detekcijskega sistema. Napovedane meje detekcije (LOD) pri določitvi ATV z uporabljenim TLM sistemom dosežejo tako le 0,3 mg/L, zaradi česar je detekcija ng/L-g/L koncentracij, ki so navadno prisotne v okolju, neizvedljiva. Pri ELISA testu za določanje ohratoksina A z mikrofluidno injekcijsko analizo v kombinaciji z detekcijo TLM (μFIA-TLM) pa se je pokazal velik vpliv visokega signala in šuma ozadja, ki se je odrazil v visokih mejah detekcije, ter slabša ponovljivost meritev. Ocenili smo, da mej detekcije nove metode za OTA ne bomo mogli bistveno znižati, zato se je nadaljevanje raziskav izkazalo za neupravičeno. Konvencionalno metodo na osnovi tekočinske kromatografije visoke zmogljivosti in dvožarkovnrga spektrometera na osnovi toplotnih leč (HPLC-TLS) smo uporabili za detekcijo štirih najpogosteje prisotnih biogenih aminov (BA) v vinu, tj. putrescina, kadaverina, histamina in tiramina. Optimizacija metode je obsegala optimizacijo kromatografskega dela ter detekcijskega dela z optimizacijo TLS parametrov. Izvedena je bila tudi študija stabilnosti dabsil-BA derivatov pri izpostavljenosti laserski svetlobi. TLS detekcija je pokazala 3,6-krat nižje meje detekcije od DAD detekcije. Z metodo standardnega dodatka smo dosegli izkoristke v območju 86−117 %. Ustreznost metode smo preverili na vzorcih belega in rdečega vina. Rezultate smo primerjali s tistimi, pridobljenimi z uporabo fluorescenčne metode za detekcijo BA (OIV metoda). Metoda standardnega dodatka se je izkazala za uspešno pri odpravi učinka matrice vina in s HPLC-TLS izmerjene koncentracije so se dobro ujemale z rezultati OIV metode. Z uporabo dabsil klorida namesto fluorescenčnega orto-ftalaldehida (OPA) pri OIV metodi, pa se izognemo nestabilnosti fluorescenčnih derivatov. Razvili smo tudi novo presejalno metodo za določevanje celokupne koncentracije biogenih aminov. Nova metoda sestoji iz mikrofluidnega dela, kombiniranega z mikroskopom s termičnimi lečami (μFIA-TLM). Za detekcijo smo BA najprej s pomočjo mikrobne transglutaminaze (MTG) encimsko deaminirali in nato sproščeni amonijak z Berthelotovo reakcijo pretvorili v modro obarvani indofenol. V začetnih raziskavah smo za optimizacijo meritev indofenola v šaržnem načinu, v μFIA-TLM sistemu nepovezanem z indofenolno reakcijo (off-line) in v μFIA-TLM sistemu, v katerem je potekala tudi indofenolna reakcija (on-line), uporabili raztopino amonijevega klorida (NH4Cl). Po dodatku 50 % etanola v šaržnem sistemu smo izboljšali meje detekcije za kar 9-krat. Preverili smo tudi stabilnost indofenola pod TLM pogoji ter optimizirali mikrofluidne pogoje ter TLM pogoje v dveh pretočnih μFIA-TLM sistemih. Ugotovili smo, da 5% dodatek etanola v nosilno tekočino, ki je hkrati tudi reagent pri on-line indofenolni reakciji, izboljša razmerje signal-šum za faktor tri. Z določitvijo optimalnega pufra (pH=7, 0,5 M) in alkalnih pogojev (2M NaOH) smo optimizirali tudi skupno reakcijo. Ugotovili smo tudi vpliv že prisotnih aminov v vzorcu na celotno reakcijo, ki se lahko odraža v pozitivnih napakah meritev ter predstavili tudi možne rešitve za zmanjšanje njihovega vpliva na določanje biogenih aminov. Z novo on-line μFIA-TLM metodo smo dosegli mejo detekcije 2,3 μM, ki je obenem tudi meja detekcije za amonij. S tem smo μFIA-TLM tehniko potrdili tudi kot dovolj občutljivo za določevanje amonija v odpadnih vodah, kjer so koncentracije le-tega v območju mg/L. Podobne LOD vrednosti (3,2 μM) smo dosegli tudi za določitev celokupne koncentracije štirih BA. Ta vrednost predstavlja mejo detekcije za histamin 3,8 μM, kar je več kot štirikrat nižja vrednost od najnižje priporočene meje za ta toksični biogeni amin v vinu, ki znaša 2 mg/L ali 18 μM. Da bi izkoristili možnosti popolnoma avtomatiziranega sistema, smo encim imobilizirali na magnetne nanodelce. Uspešna imobilizacija encima tako odpira možnosti za vpeljavo encima v miniaturiziran biosenzor.
Secondary keywords: toplotne leče;spektrometrija;mikroskopija;tekočinska kromatografija visoke ločljivosti;mikrofluidika;biogeni amini;mikrobna transglutainaza;indofenolna reakcija;disertacije;
URN: URN:SI:UNG
Type (COBISS): Doctoral dissertation
Thesis comment: Univ. v Novi Gorici, Fak. za podiplomski študij
Pages: XXII, 126 str.
ID: 10939310