Studies of microfluidics for cell separation and analysis



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Microfluidic separation is a promising analytical approach in biomedical and clinical research for cell and protein analysis, genetic testing, and disease diagnosis. Compared with immunological and morphological techniques, microfluidic devices have the advantages of less sample consumption, less complexity, lower cost, simple operation, and increased portability. In this dissertation, detailed fundamentals of microfluidics are discussed in Chapter I, whereas an in-depth look for microfluidic application for the sepsis diagnosis is discussed in Chapter II. Chapter III and Chapter IV present studies of biomarkers used for early sepsis diagnosis and their application when combining with a tandem affinity cell capture device. In addition, Chapter V discusses the study of the nanoparticle surface modification for the microfluidic device in order to have a better capture effectiveness for cell analysis . The conclusion and future perspectives are discussed in Chapter VI. Sepsis is a complex disorder of immune system response to infections and can be caused by a wide range of clinical contexts. Traditionally, microbiological culture was considered as the “gold standard” for the sepsis diagnosis. However, this approach requires a long analysis time, usually 3 to 10 days, and many of these analytes have negative results. The lengthy analysis time and low accuracy of detection make sepsis a life-threatening condition that affects millions of people every year. Therefore, the developments of Point-of-Care (POC) methods that can provide high accuracy and earlier detection are in unprecedented demand. In this dissertation, we present a microfluidic cell capture system that provided a high sepsis diagnosis accuracy with only 3 hours consumed for each sample analysis. Anti-CD25, anti-CD64, and anti-CD69, served as biomarkers for affinity cell capture, were analyzed by flow cytometry with septic patient samples and healthy controls. We found that anti-CD64 was the best biomarker in predicting sepsis with the area under Receiver Operating Characteristic (ROC) curve (AUC) of 0.820 regarding to white blood cells surface antigen expression. Furthermore, among all subtypes of leukocytes, including neutrophils, monocytes, and lymphocytes, both neutrophil CD64 expression and CD64+ neutrophil population demonstrated the excellent ability in diagnosing sepsis with AUC of 0.928 and 0.934, respectively. Also, significant increases of CD25+ and CD69+ lymphocyte populations were observed with p value of 0.02 and 0.042 (95% confidence interval), respectively. Therefore, all these three biomarkers are considered as effective parameters in detecting sepsis. We developed a tandem affinity microfluidic device, coupled with anti-CD25, anti-CD64, and anti-CD69, for early sepsis diagnosis. 40 septic patients and 10 healthy volunteers were involved in this study to evaluate the performance of our multi-parameter chip. Results were also compared with clinical blood culture. We found that our combined panel of anti-CD25, anti-CD64, and anti-CD69 showed a high accuracy in detecting sepsis with AUC of 0.978, whereas only 30% of patients had positive blood-culture results in clinical microbiology culture analysis. Furthermore, in our technique, all cases showed culture-negative results exceeded the threshold set by healthy controls, meaning that our approach is able to extensively reduce the false diagnosis and, therefore, assist clinicians to initiate timely antibiotic therapy. The clinical validation confirms that our multi-parameter microchip is a powerful sepsis assay for clinical point-of-care (POC) applications. Finally, in order to have a better performance of our microchip in capturing desired cells, we modified the chip surface with silica nanoparticles. The particles adhere on the channel surface via electrostatic interactions with surface coverage of 90±4%. The mean particle size was 202±6 nm. Seven different cancer cell lines that spiked into liquid biopsies were served as models to evaluate the isolation ability of our modified surface, and the results were compared with unmodified devices. The capture efficiency of our nanoparticle-modified chip exhibited an average increase of 16% compared with normal chip. Moreover, patient-derived ALL cells, COG-LL-332, were spiked into blood with concentrations ranging from 1% to 20 % of total leukocytes, and isolated with the purity of 41-65%. The enhanced ability in capturing target cells after surface modification greatly promised the POC application of our microchip in future bedside disease diagnosis.



Microfluidics, Sepsis diagnosis, CTCs isolation, Surface modification