title: Developing next generation bioassays and biotherapeutics: label-free microfluidic phage measurement by darkfield bacteria imaging
Creators: name: Donmez, Sultan Ilayda
Publisher: name: University of Reading
Year: 2025

description: Currently, antibiotic resistance is one of the biggest risks to global health and food security. Bacteriophages are recognised as a useful alternative to antibiotics and are specific to the host bacterial cell. Phage therapy is gaining popularity as a tool to tackle antibiotic resistance. In each study using bacteriophages, the determination of the bacteriophage and enumeration is critical. However, traditional methods require a long detection time (̴48 h) to determine applicable phages and their dose. In this thesis, work focused on phage detection and enumeration using microfluidic systems to overcome the shortcomings of traditional method of phage use (double layer agar method) is presented. Label-free, low effort detection of bacteria and bacterial lysis with darkfield imaging using the smartphone camera is also reported. Thus, the companion diagnostic can use the works developed in the thesis, in the next generation tests that can be used in future point of care tests, which are simple to use and give fast results. A Raspberry Pi sensor system has been integrated into the system in order to obtain quantification data and perform cell-based analyses. Thus, for low bacteria and phage concentrations, both colony forming unit/mL and plaque forming unit/mL calculations could be performed in only 4 µL liquid medium and in 5 hours, while kinetic measurements of cells could be obtained. The last research part of the thesis focused on the detection and aggregation of platelets with similar properties to bacteria, and the aggregation formed by the use of high concentrations of agonists was observed with a working principle similar to aggregometry. It was proved that platelet detection and the effects of 2 agonists on platelets can be determined depending on the light scattering intensity. Chapter 1 provides a general introduction to bacteriophages. After explaining what bacteriophages are and how they work, their effectiveness for preventing antimicrobial resistance (AMR) is critically appraised. Then, current lytic phage-based detection systems and current studies that are created by adding microfluidic to these systems are explained. Chapter 2 discusses, how, by combining the extensive analytical capability of these miniature computers in our pockets with new fields, consumer instrumentation can be advanced to realise additional positive implications for healthcare. The chapter focuses on new opportunities in three emerging areas where smartphone capabilities could be combined with biosensors and microfluidics. These are bacteriophages, aptamers and cellular measurements. Especially today, while fighting the covid virus, interest in home-based tele-diagnosis is increasing in order to reduce hospital admissions and achieve faster and more accurate results. In this chapter, the required current state of the field is critiqued. In addition, the review is based on a wide range of references to provide a balanced view of the field, and future directions in the field are summarized based on current developments in the field. Chapter 3 represents the first time in the literature that label-free microfluidic bacteria detection can be used to determine host specificity for the therapeutic bacteriophage. It has been shown that it is entirely possible to measure light scattering by bacteria with a smartphone in microcapillary film (MCF) test strips placed in the 3D printed dark field imaging system we designed, which includes a simple light source. This demonstrates the potential of microfluidics as companion diagnostic for advanced biological therapeutics in the treatment of bacterial infections. In the designed system, when bacterial target cell suspensions were taken into phage-loaded MCF capillaries, no light scattering signal was observed in the channels after incubation with the effect of lysis. To see if the specificity of the host bacteria could be determined rapidly with this system, light scattering occurred as opposed to the host strain when the loaded phage was tested on MCF test strips of other bacteria that was not the host bacteria. Thus, the potential for measuring bacteria in a number of microbiology methods, including the bacteriophage lysis of the smartphone camera plus MCF, was clearly demonstrated. Chapter 4 appraises the rapid and simple measurement of CFU/ml and PFU/ml of our automated image capture system, previously unavailable in the literature, which allows for detailed analysis of microbial growth kinetics using dipstick microfluidic strips. Bacteria and bacteriophage counting is a cornerstone for microbiological analysis, and the agar plate method has been used for over 140 years. However, this process is cumbersome and has innovation potential to make microbiology accessible for field use and point-of-care diagnostics. This section discusses a new method for counting bacteria and bacteriophages in a simple liquid assay using a dipstick microfluidic system without further labelling or coating. In our microfluidic system, it has been shown that individual bacterial colonies can be visualized in as little as 5 hours, even in liquid cultures. Removing the solid media from this process facilitates enumeration and bacteriophage counting, which requires careful control of host bacteria, especially on molten agar. Since this type of growth is not seen in bulk conventional microbiology systems such as microplates or tube cultures, the method also provides a new perspective on microbiology microsystems. In addition, accelerated darkfield imaging using the Raspberry Pi sensor system instead of a smartphone allows for detailed kinetic analysis of colony growth. Thus, while providing a new perspective to single cell-based microbial microfluidic analysis, it made it possible to establish quantitative growth kinetics simultaneously with colony counting. Chapter 5 interrogates whether platelets can be visualised in the same dark-field system used for bacteria, which would represent a significant advance give their various functions and importance in the body. As a result, we show that we can detect platelets in the dark-field imaging system in microfluidic, based on turbidity-based measurement, thanks to the light scattering feature. In addition, when determining whether we could detect platelet aggregation, significant differences were detected when two different agonists were dried in microchannels and then loaded with platelets reach plasma (PRP). Thus, in the future, a system that could not only detect platelets, but also the effects of the agonist on a particle basis platelet aggregation, is discussed. In Chapter 6, the important points of the PhD thesis are summarized and critically examined. The usage and importance of bacteriophages are emphasized, and phage-based commercial detection systems are discussed, with the benefits and drawbacks of our system being articulated. The scope for implementing our system and its important contribution to the diagnosis of companion is discussed. The potential for future work, such as new bacteriophage discovery, formulation, and production, is critically addressed.