Označevanje polielektrolitov s fluorescentnimi barvili in spremljanje elektrostatskega oblaganja bakterijskih celic z mikroskopijo in kalorimetrijo

Grilc, Nina Katarina (avtor), Kristl, Julijana (mentor), Mirtič, Janja (komentor)

Žive bakterijske celice so danes nepogrešljive na mnogih področjih biotehnologije in biomedicine. Za te namene pogosto spreminjamo površinske lastnosti celic. Ena od možnosti za modifikacijo celične površine je oblaganje celic z metodo nanoplastenja, pri kateri se plasti polielektrolitov nalagajo na nabito površino oblaganca na podlagi elektrostatskih interakcij. V magistrski nalogi smo spremljali tvorbo polikationske plasti na negativno nabiti površini bakterijskih celic. To je prva naloga, ki preučuje nanoplastenje celic s fluorescenčno mikroskopijo z vzbujenim praznjenjem emisije (STED). Namen je bil optimizacija postopka označevanja polikationov s fluorescentnim barvilom in izbor barvila, ki na uporabljenem STED mikroskopu izkazuje boljšo ločljivost metode kot na konfokalnem mikroskopu. Sistematično smo nanoplastenje s polielektroliti preučili na dveh vrstah celic (E. coli in P. stutzeri) z meritvijo zeta potenciala (ZP) in STED mikroskopijo. Nanooblaganje smo spremljali še z izotermno titracijsko kalorimetrijo (ITC). Rezultati kažejo izboljšanje ločljivosti na STED v primerjavi s konfokalnim mikroskopom pri barvilu STAR RED, ne pa pri ATTO 594 in rodamin-B-izotiocianatu. S STAR RED smo označili polikatione (visoko- in nizkomolekularna polietilenimin - PEI in polialilamin klorid - PAH) in z njimi obložili celice E. coli in P. stutzeri. ZP celic se je spremenil iz negativnega pred nanooblaganjem v pozitivnega po nanooblaganju. STED slike kažejo nastanek nanoobloge na celični površini. Ugotovili smo, da polikationi prehajajo v notranjost P. stutzeri, ne pa v E. coli, nismo pa dokazali razlik v prehajanju polikationov glede na molekulsko maso. Pri ITC so krivulje odvisnosti toplotnega toka od količine dodanega polielektrolita pri E. coli dosegale plato, pri katerem predpostavimo zasedenost celotne celične površine s polikationom. Pri P. stutzeri je bilo prisotno sipanje podatkov okoli pričakovane (sigmoidne) oblike krivulje zaradi prehajanja polielektrolita v notranjost. Nanooblaganje preučevanih bakterijskih celic s PEI in PAH je bilo uspešno, saj se je površinski naboj spremenil iz negativnega v pozitivnega, tvorba nanoobloge pa je vidna tudi s STED. Permeabilnost celične stene za polikation je manj odvisna od njegove molekulske mase in bolj od vrste celic, kar smo dokazali z opazovanjem lokalizacije polikationov po nanooblaganju s STED mikroskopijo. Z ITC potrdimo doseganje nasičenja celične površine s polikationom.

• Jezik: Slovenski jezik
• Ključne besede: bakterije, nanooblaganje, polielektrolit, STED, kalorimetrija
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2020
• PID: 20.500.12556/RUL-113691
• Datum objave v RUL: 25.01.2020

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Labelling of polyelectrolytes with fluorescent dyes and monitoring of electrostatic coating of bacterial cells with microscopy and calorimetry

Izvleček

Live bacterial cells are used in many biotechnological and biomedical applications that often require modifications of the cellular surface. A possible method for cell surface modification is nanocoating via layer-by-layer method, which is based on electrostatically driven deposition of polyelectrolytes onto a charged cell surface. We observed the formation of the polycation layer on a negatively charged bacterial cell surface. This is the first master thesis studying nanocoating of cells with stimulated emission depletion microscopy (STED). The aim of this work was to optimize a protocol for fluorescent labelling of polycations with a fluorescent dye and determine the appropriate dye, manifesting an improvement in resolution on the used STED microscope when compared to confocal microscope. A systematic study of nanocoating of two bacterial species (E. coli and P. stutzeri) was done by measuring zeta potential (ZP) and observing the nanocoated cells with STED. We also followed the nanocoating process with isothermal titration calorimetry (ITC). STAR RED showed an improved resolution in STED when compared to confocal microscopy, whereas ATTO 594 and rhodamine-B-isothiocyanate did not. We labelled polycations (high and low molecular weight polyethyleneimines - PEI and polyallylamine hidrochlorides - PAH) with STAR RED and used them to coat E. coli and P. stutzeri. STED microscopy showed the formation of a polyelectrolyte layer on the cell surface. Polycations permeated into P. stutzeri and not into E. coli. We did not observe a difference in cell wall penetration between high and low molecular weight polymers. ZP changed from negative before to positive after nanocoating. ITC termograms, showing the dependance of heat rate on the amount of added polymer, reach a plateau in the case of E. coli, suggesting achievement of cell surface saturation with polycation. In case of P. stutzeri the curve is not the expected sigmoidal shape as polycations permeate into the cell. We conclude the nanocoating of studied bacterial cells with PEI and PAH was successful, as surface charge changed from negative to positive. Permeation of polycations through the cell wall depends less on molecular weight and more on species, which was proven by observing the localization of fluorescently labelled polycations after nanocoating with STED. ITC confirmed the saturation of the cellular surface with the polycation.

• Ključne besede: bacteria, nanocoating, polyelectrolytes, STED, calorimetry