Simultaneous optical and electrochemical biofilm analysis

Filipović, Filip (author), Mandić Mulec, Ines (mentor), Richter Dahlfors, Agneta (comentor)

Microorganisms live most of their life as biofilms encapsulated in a matrix. This lifestyle allows for prolonged survival in many environments and affects their importance in microbe-related diseases, as they can form on a variety of surfaces, including living tissue, implanted medical devices, etc. Therefore, the development and improvement of detection and monitoring methods is an important problem in microbiology. Electrochemical techniques have generally not been used for direct detection of bacteria and real-time monitoring has been limited to mostly well-characterized electroactive bacteria, with well-defined redox active metabolites. In this thesis we succeeded in monitoring bacterial growth of Salmonella Enteritidis in real time using organic electrochemical transistors, although we were unable to distinguish between biofilm-forming and non-forming strains. In addition, we were able to detect bacteria up to 10⁵ CFU/mL using this method. This new method was comparable to the classical measurement of optical density (e.g., A₆₀₀). We then focused our research on developing a method based on the detection of extracellular matrix components of biofims. To study these, various ligands are used, however, most of them are either toxic and/or non-specific, creating a need for novel ligands. In this thesis we used a series of organic fluorophores called optotracers, which allow real-time visualization of ECM components. Since optotracers have a very similar structure to PEDOT:PSS, we were able to successfully create a hybrid material from both components. This was done without the need for chemical linkage and did not affect the conductivity of the surfaces coated with the hybrid material. After the biofilm formation on the optotracer-functionalized PEDOT:PSS surfaces, the optotracers were extracted from the PEDOT:PSS and successfully bound to the ECM components. These surfaces were then used to enable successive electrochemical and optical detection, although simultaneous detection was not technically feasible in this case.

• Language: English
• Keywords: biofilm, electrochemical detection, optical detection, optotracers
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Place of publishing: Ljubljana
• Publisher: [F. Filipović]
• Year: 2022
• PID: 20.500.12556/RUL-135986
• UDC: 606:601.2:582.23:579.6:628.353.153(043.2)
• COBISS.SI-ID: 104959747
• Publication date in RUL: 06.04.2022

Secondary language

• Language: Slovenian
• Title: Kombinirana optična in elektrokemijska analiza biofilmov

Abstract

Mikroorganizmi večino svojega življenja živijo kot biofilmi, v katerih so celice ovite v zunajcelični matriks. Takšen življenski slog ščiti mikroorganizme pred neugodnimi pogoji okolja, kar pozvroča dodatni problem pri patogenih mikroorganizmih, ki lahko obraščajo tkiva gostitelja, katetre in medicinske vsadke. Nabor metod, s katerimi lahko zaznavamo in spremljamo razvoj biofilmov v realnem času, je omejen. Izjeme so elektroaktivne bakterije ki že imajo dobro definirane metabolite, ki so oksidacijsko in redukcijsko aktivni. Metod za vrednotenje rasti in razvoja mikrobnih biofilmov, ki temeljijo na elektrokemiji, še niso uporabili. V magistrskem delu smo spremljali rast patogenih bakterij Salmonella Enteritidis v realnem času, in sicer z metodo na osnovi organskih elektrokemijskih tranzistorjev. Metoda je omogočila detekcijo bakterij pri gostoti 10⁵ CFU/mL ali višji koncentraciji; ni pa omogočila ločevanja med filmotvornimi in nefilmotvornimi sevi ter spremljanja metabolne aktivnosti bakterij v odsotnosti rasti. Nova metoda je bila primerljiva s klasično metodo merjenja optične gostote (npr. pri A₆₀₀). V nadaljevanju smo raziskave usmerili v razvoj metode, ki temelji na zaznavanju zunajceličnega matriksa biofimov. Za določevanje prisotnosti matriksa raziskovalci uporabljajo različne ligande, vendar je večina le-teh toksičnih in/ali nespecifičnih, kar ustvarja potrebo po novih ligandih. V raziskavi smo zato uporabili nabor organskih fluoroforov, s katerimi smo vizualizirali komponente zunajceličnega matriksa v realnem času. Ker imajo ti optični označevaci (optotracerji) zelo podobno strukturo kot poli(3,4-etilendioksitiofen):poli(stirensulfonat) oziroma PEDOT:PSS, smo uspešno izdelali hibridni material iz obeh komponent, brez dodatnih kemičnih povezav. Prevodnost površin, prevlečenih s tem hibridnim materialom, se je ohranila in tudi optični sledilci so se uspešno ekstrahirali in vezali na zrasel biofilm. Tako smo prvič predstavili možnost za elektrokemijsko in optično spremljanje biofilma.

• Keywords: biofilm, elektrokemijska zaznava, optična zaznava, optični označevalci