Responsive nanostructured materials for bioanalyte detection and triggered antimicrobial therapy

Inorganic nanomaterials are attractive candidates for biomedicine as they can offer increased stability including light, temperature and chemical stability while also offering the benefits of high-throughput synthetic techniques such as flame spray pyrolysis. In this thesis work will be described describing novel inorganic nanoparticle systems for biomedical applications presented in four papers of which three are published in peer-reviewed scientific journals.

The first project in Paper I presents a surface treatment to address what is thought to be the major cause of implant associated infections which is biofilm formation. A near infrared (near-IR) activated plasmonic nanoparticle system with photothermal properties is developed which utilises the inter-particle coupling of spherical silver nanoparticles. This inter-particle coupling produces a strong plasmonic extinction with a wavelength dependence on the spacing between neighbouring spherical silver nanoparticles. Therefore by using a SiO2 dielectric spacer to control the average spacing between spherical silver nanoparticles in the synthesised nanoaggregates the optical and photothermal behaviour of the nanoaggregates can be tuned into the near-IR. The effectiveness of these nanoaggregates for the photothermal eradication of biofilms formed on catheter mimicking surfaces was evaluated. Nanoaggregates were directly deposited onto silicone substrates and entirely encased in a second layer of silicone. Biofilms of Escherichia coli and Staphylococcus aureus were grown on the silicone surface and near-IR light was used to activate the photothermal response of the nanoaggregates with complete eradication of biofilms achieved in a temperature dependent manner.

Applications of nanomaterials to biofilms was further continued in Paper II, a characteristic of biofilms is their dense nature as they are embedded in a glue-like self produced extracellular matrix which also attaches them to the substrate on which they grow. This can promote the formation of heterogeneous microenvironments, with concentration gradients of parameters such as oxygen, nutrients and pH readily occurring between the substrate-attached interface and the liquid in which they are grown. An understanding of the pH of these interfacial microenvironments and a high-throughput compatible measurement system is desirable to help guide the development of targeted anti-biofilm systems. In Paper II a novel all-inorganic system based on calcium phosphate nanoparticles doped with europium is presented which display pH dependent luminescence as a deposited film. Growth of bacterial biofilms of different species on these nanoparticle coatings allows the measurement of the acidic microenvironments which they produce in an ordinary well-plate luminescence spectrometer. Bacterial bioanalyte detection can be challenging due to the complex growth media which bacteria can require and the dynamic changes to the optical properties of the medium as the bacteria grow. Moreover, the developed detection mechanism must be resistant to bacterial degradation, therefore the development of robust sensors for such applications is essential.

In Paper III the development of a dual-emission ratiometric luminescence sensor for the detection of hydrogen peroxide from bacterial cultures is described. The sensing system consists of a reference emission from Y2O3:Tb3+ which are decorated with CeO2:Eu3+ nanoparticles which provide the hydrogen peroxide sensitive emission. This system was applied for the determination of hydrogen peroxide production levels by the known hydrogen peroxide producing bacterium and major human pathogen Streptococcus pneumoniae. Finally the manuscript Paper IV applies inorganic nanomaterials for the detection of ammonia (here used to refer to total NH3 and NH+ 4 levels) which is an important diagnostic bioanalyte. Ammonia production by urease producing gut bacteria is a major contributor to blood ammonia levels and must be effectively detoxified by the liver for excretion by the kidneys. Over production of ammonia or failure of the liver or kidney can lead to high levels of ammonia which are neurotoxic. However, the symptoms of this hyperammonemia are diffuse ranging from mild confusion to coma and death. Therefore a readily available system for the measurement of ammonia levels is highly desirable. In Paper IVa plasmonic silver nanoparticle based system is described for the detection of ammonia. The system relies on the reactivity of the ClO– with both ammonia and silver nanoparticles, if no ammonia is present upon addition of ClO– to the silver nanoparticles a strong decrease in their plasmonic colour is observed. However, in the presence of ammonia the ClO– is removed and therefore no colour change is observed. This system was able to detect ammonia levels at the 50 μM limit defined as hyperammonemia.

List of scientific papers

I. Padryk Merkl, Shuzhi Zhou, Apostolos Zaganiaris, Mariam Shahata, Athina Eleftheraki, Thomas Thersleff, Georgios A. Sotiriou. Plasmonic coupling in silver nanoparticle aggregates and their polymer composite films for near-infrared photothermal biofilm eradication. ACS Applied Nano Materials. 2021;4:5330-5339.
https://doi.org/10.1021/acsanm.1c00668

II. Padryk Merkl, Marie-Stéphanie Aschtgen, Birgitta Henriques-Normark. Biofilm interfacial acidity evaluation by pH-responsive luminescent nanoparticle films. Biosensors and Bioelectronics. 2021;171:112732.
https://doi.org/10.1016/j.bios.2020.112732

III. Dorian Henning, Padryk Merkl, Changhun Yun, Federico Iovino, Ling Xie, Eleftherios Mouzourakis, Constantinos Moularas,Yiannis Deligiannakis, Birgitta Henriques-Normark, Klaus Leifer and Georgios A. Sotiriou. Luminescent CeO2:Eu3+ nanocrystals for robust in situ H2O2 real-time detection in bacterial cell cultures. Biosensors and Bioelectronics. 2019;132:286-293.
https://doi.org/10.1016/j.bios.2019.03.012

IV. Padryk Merkl and Georgios A. Sotiriou. Ammonia sensing by silver nanoparticles for point of care diagnostic applications. [Manuscript]