Globally, the microbe Neisseria gonorrhoeae (NG) causes 106 million newly documented sexually transmitted infections each year. Once appropriately diagnosed, NG infections can be treated with readily available antibiotics, but patients in low-resource settings often do not return to the clinic for results. As current NG diagnostic gold standards suffer from slow turnaround time to result, a rapid, sensitive molecular diagnostic would help increase appropriate treatment at the point-of-care. Here, we report on the design and development of a minimally-instrumented paper-fluidic POC diagnostic that incorporates patient swab sample lysis, isothermal nucleic acid capture, thermophilic helicase-dependent amplification (tHDA), an internal amplification control (NGIC), and lateral flow visual detection. Limits of NG detection for the NG/NGIC multiplex tHDA assay were determined within the device, and device clinical performance was validated retroactively against qPCR-quantified patient samples in a proof-of-concept sensitivity and specificity study. This proof-of-principle paper-fluidic diagnostic, which can be completed within 80 minutes, selectively amplifies and detects NG in multiplex with NGIC. It has a clinically relevant limit of detection of 1000 NG cells per device. In urethral swab sample trials (N=20), the device approaches current gold standard NG diagnostic capabilities with 90% sensitivity and 100% specificity and vaginal swab sample trials (N=20) surpass the gold standard with 100% sensitivity and 100% specificity.

Yes / This study shows how highly branched poly(N-isopropyl acrylamide) (HB-PNIPAM) with a chain pendant solvatochromic dye (Nile red) could provide a fluorescence signal, as end groups bind to bacteria and chain segments become desolvated, indicating the presence of bacteria. Vancomycin was attached to chain ends of HB-PNIPAM or as pendant groups on linear polymers each containing Nile red. Location of the dye was varied between placement in the core of the branched polymer coil or the outer domains. Both calorimetric and fluorescence data showed that branched polymers responded to binding of both the peptide target (D-Ala-D-Aa) and bacteria in a different manner than analogous linear polymers; binding and response was more extensive in the branched variant. The fluorescence data showed that only segments located in the outer domains of branched polymers responded to binding of Gram-positive bacteria with little response when linear analogous polymer or branched polymer with the dye in the inner core was exposed to Staphylococcus aureus. / Innovate UK/Smith and Nephew Ltd. (UK) (TSB 103988) and by MRC (MR/N501888/2).

This thesis focuses on fundamental aspects of microfluidic systems and applies relevant findings to innovative designs for advanced particle manipulation applications. Computational Fluid Dynamics (CFD) is adopted for fluid modeling, based on the Finite Volume method. The accuracy of the solutions obtained is confirmed by grid sensitivity analysis and by comparisons with experimental work. Curved microchannel features and the induced Dean flow are studied through a parametric space exploration and simulations. The Lagrange-Euler coupling method – Surface Marker Point methodology – is applied to simulate large-size particles (of comparable size to the channel). Through this simulation approach, all the forces on such particles are directly derived through solving the governing equations and the influence of these particles on the flow is considered in a fully coupled manner. A new approach – the Frozen Flow & Flow Correction Coefficient method – is developed, making trans-relaxation-time simulations possible and improving computational efficiency significantly, for 3D simulations of arbitrary shape and size microparticles in complicated microfluidic channels. Detailed comparisons between simulation results and experiments involving particle sedimentation and particle equilibrium position have been conducted for methodology validation. Mechanisms of hydrodynamic particle manipulation are then studied, including hydrodynamic focusing and separation. It is found that the Tubular Pinch effect, Dean flow and the Radial Pressure Gradient effect interact to yield two distinct particle separation mechanisms. For advanced applications, particle focusing, non-magnetic and magnetic separation for neutrally buoyant particles are proposed, based on newly gained insight on the above-mentioned mechanisms. Appropriate channel designs have been proposed both for particle focusing and size-based particle separation, while the vertical-magnetic-Dean separation scheme is highlighted for magnetic separation. Finally, a new integrated system is proposed, that combines the above novel designs into a device-like ensemble. It promises to offer functionality for biomaterial separation and detection, including different types of cells, antigens and biomarkers.

During the last decades, newly developed medical devices often came along with unappropriate designs, increasing the likelihood of misuse through the operator. Part of the root cause was that no sufficient measures were applied to assimilate user needs. Consequently, usability engineering approaches are now stronger emphasized to ensure that new devices are not only safe to use but are also designed for users’ needs. Besides, testing processes in clinical microbiology laboratories are currently reshaped due to new generations of rapid testing methods. Hence, it is particularly important to apply usability engineering frameworks during the development phase to make sure devices address users’ needs and also fit into the new work- and communication flows. Based on that, this research project applies a usability engineering approach to the design process of a new rapid antibiotic susceptibility testing system of Astrego Diagnostic AB that is supposed to be used in clinical microbiology laboratories in the near future. The research questions focus on how this device can be designed to enable integration into clinical laboratories. -       How can a rapid AST testing system be integrated into the workflow of clinical microbiology laboratories? -       What are the remaining uncertainties for integrating a rapid AST system into the workflow of a clinical microbiology laboratory on the example of Astrego’s AST system? Several methods were used to address these questions, which include literature research, a competitive audit, subject matter interviews and semi-structured interviews, and observations of targeted users. The findings show that a rapid antibiotic susceptibility testing system may be used in several different ways, which also impacts its design. Process-wise, it could be used after Gram staining and bacterial identification has been conducted and, more realistically, simultaneously bacterial identification to pave the way for additional time savings further. However, uncertainties remain regarding the design of the new testing system. Depending on the number of devices that targeted laboratories need to implement to accommodate their testing volume, it makes sense to design a built-in user interface or an external one that can be accessed through a tablet or desktop. Thus, it is uncertain to what extent manual input of bacteria ID is relevant as the dRAST system fully enables manual input of Gram type and bacteria IDs while it might also be possible to avoid manual interaction by receiving this information through software interfaces.