Please use this identifier to cite or link to this item:
http://hdl.handle.net/11375/16562
Title: | Design & Fabrication of a Microfluidic Device for Clinical Outcome Prediction of Severe Sepsis |
Authors: | Yang, Jun |
Advisor: | Selvaganapathy, P. Ravi |
Department: | Biomedical Engineering |
Keywords: | Microfluidic device;cell-free DNA;Severe sepsis |
Publication Date: | Jun-2015 |
Abstract: | Sepsis is an uncontrolled response to infection. Severe sepsis is associated with organ dysfunction, and has mortality rate of 30-50%. Identification of severity of sepsis and prediction on mortality is crucial in making clinical decisions. Recently, cell-free DNA (cfDNA) in blood was found to have high discriminative power in predicting ICU mortality in patients with severe sepsis. In an analysis of 80 severely septic patients, the mean cfDNA level in survivors (1.16±0.13μg/ml) was similar to that of healthy volunteers (0.93±0.76μg/ml), while that of non-survivors (4.65±0.48μg/ml) was notably higher. Therefore, rapid quantification of cfDNA concentration in blood will enable physicians to quickly predict mortality of sepsis and decide on treatment. Current methods for quantification of cfDNA involve multiple steps including centrifugation, DNA-extraction from plasma, and its quantification either through spectroscopic methods or quantitative PCR. The whole process is time consuming, thus is not suitable for immediate bedside assessment. To solve the problems, a microfluidic device is designed and fabricated in this thesis, which is potential for cfDNA quantification directly using blood in 5 minutes. The goal is to use this device for distinguishing survivors or healthy donors from non-survivors in patients with severe sepsis. The two-layer device consists of a sample channel (top) and an accumulation channel (bottom) that intersect each other. The accumulation channel is preloaded with 1% agarose gel, and the blood containing cfDNA and intercalating fluorescent dye is loaded in the sample channel. Fluorescently labeled DNA is able to be trapped and concentrated at the intersection using a DC electric field, and fluorescent intensity of the accumulated DNA is representative of its concentration in the blood. The simulated electric field in the sample channel reveals that both the magnitude and the gradient of electric field reach their maximum values at the intersection. Force analysis shows that DNA was driven into the gel by the dominate electrophoretic force, while red blood cells moved away from the gel due to a strong dielectrophoretic force. In this thesis, 4 types of samples have been used to characterize the performance of the device. It showed that DNA was efficiently accumulated at the intersection, and the fluorescent intensity could be measured using a fluorescent microscope. Samples from healthy donors were able to be distinguished from that of severely septic patients in 5 minutes. However, better resolution was needed for differentiating various cfDNA concentrations in patient samples. The discussion on the effect of applied voltage showed that 9V is an optimized setting compared with 3V and 15V. In addition, it has been proved that the fluorescent reagent could be immobilized in the device and the sample preparation could be absolutely eliminated. In summary, the device proposed in this thesis is capable of distinguishing severely septic patients from healthy donors using clinical plasma in 5 minutes, and is potential to be applied in clinical blood samples. It has low cost, and is ready to be developed into a fully functioned system. This tool can be a valuable addition to the ICU to rapidly assess the severity of sepsis for informed decision making. |
URI: | http://hdl.handle.net/11375/16562 |
Appears in Collections: | Open Access Dissertations and Theses |
Files in This Item:
File | Description | Size | Format | |
---|---|---|---|---|
Jun Yang thesis FINAL for submission.pdf | 4.54 MB | Adobe PDF | View/Open |
Items in MacSphere are protected by copyright, with all rights reserved, unless otherwise indicated.