Microfluidic blood sample preparation for rapid sepsis diagnostics
Sammanfattning: Sepsis, commonly referred to as blood poisoning, is a serious medical condition characterized by a whole-body inflammatory state caused by microbial infection. Rapid treatment is crucial, however, traditional culture-based diagnostics usually takes 2-5 days. The overall aim of the thesis is to develop microfluidic based sample preparation strategies, capable of isolating bacteria from whole blood for rapid sepsis diagnostics. Although emerging technologies, such as microfluidics and “lab-on-a-chip” (LOC) devices have the potential to spur the development of protocols and affordable instruments, most often sample preparation is performed manually with procedures that involve handling steps prone to introducing artifacts, require skilled technicians and well-equipped, expensive laboratories. Here, we propose the development of methods for fast and efficient sample preparation that can isolate bacteria from whole blood by using microfluidic techniques with potential to be incorporated in LOC systems.We have developed two means for high throughput bacteria isolation: size based sorting and selective lysis of blood cells. To process the large blood samples needed in sepsis diagnostics, we introduce novel manufacturing techniques that enable scalable parallelization for increased throughput in miniaturized devices.The novel manufacturing technique uses a flexible transfer carrier sheet, water-dissolvable release material, poly(vinyl alcohol), and a controlled polymerization inhibitor to enable highly complex polydimethylsiloxane (PDMS) structures containing thin membranes and 3D fluidic networks. The size based sorting utilizes inertial microfluidics, a novel particles focusing method that operates at extremely high flow rates. Inertial focusing in flow through a single inlet and two outlet, scalable parallel channel devices, was demonstrated with filtration efficiency of >95% and a flowrate of 3.2 mL/min. Finally, we have developed a novel microfluidic based sample preparation strategy to continuously isolate bacteria from whole blood for downstream analysis. The method takes advantage of the fact that bacteria cells have a rigid cell wall protecting the cell, while blood cells are much more susceptible to chemical lysis. Whole blood is continuously mixed with saponin for primary lysis, followed by osmotic shock in water. We obtained complete lysis of all blood cells, while more than 80% of the bacteria were readily recovered for downstream processing. Altogether, we have provided new bacteria isolation methods, and improved the manufacturing techniques and microfluidic components that, combined offer the potential for affordable and effective sample preparation for subsequent pathogen identification, all in an automated LOC format.
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