High Efficient Capture Of Circulating Tumor Cells By Using Microfluidic Chip

Circulating tumor cells (CTCs) in the peripheral blood have shown promise as akind of biomarkers for prognosis in cancers. However, it is urgently needed todevelop efficient, reliable and high throughput platforms for CTCs enrichment anddetection, due to the rareness of CTCs (a dozen or even a few CTCs in1mL ofblood). In this thesis, we have developed a microfluidic device which separatedtumor cells (human acute lymphoblastic leukemia cells, CEM) from whole humanblood with high capture efficiency. The Ramos cells were used as control. Thecontents of the thesis are as follows:1. Investigation on the structures of the chips and their influences on the captureefficiency. The results showed that the capture efficiency could be improvedobviously by using hydrofluoric acid to roughen the surface of microchannels in themicrofluidic chips. To obtain the highest capture efficiency, we investigated theconcentration of hydrofluoric acid (HF) and the time of roughen treatment. Theoptimized condition is to treat the microchannels by using10%HF for60s. Under thesame experiment conditions,10%of capture efficiency was increased for theroughened chips, compared to the unroughened ones.2. The operation modes of aptamer probes and their influences on tumor cellscapture efficiency. For traditional cell capture technologies, the capture probes wereusually immobilized on the surface of the microchannels or other substrates. However,the cell capture efficiency would be limited due to the steric hindrance betweenprobes. In this chapter, we developed the free probe mode, i.e., the surface of themicrochannels was modified with avidin, then the free biotin-aptamer probes werebind with target cells in homogeneous phase, after that the mixture was introducedinto the chip. As the result, the target cells were captured in the microchannels due tothe binding between biotin and avidin. Under the same experiment conditions,5%ofcapture efficiency was increased for the free probe mode, compared to the fixed probemode. Various experimental conditions were optimized, such as flow rate,concentration of aptamer probes, and the incubation time between aptamer and cells.Under the optimized conditions, i.e.,3mL/h of flow rate,1μM of aptamer, and7minutes of cell-aptamer incubation time, the selectivity of cell capture and the captureefficiency of different cell concentrations in buffer were investigated. 3. Investigation of cell capture efficiency in human whole blood samples. Tocapture rare tumor cells in human blood samples is our main goal. Due to the hugedifference between blood and buffer, the influence of blood cells was investigated.Using whole human blood samples, the flow rate of samples and the concentration ofaptamer probe were optimized. Under the optimized conditions, i.e.,1mL/h of flowrate,1.5μM of aptamer, the capture efficiency for different cell concentrations inserum and blood were investigated. The capture efficiency was ca.99%for100cells/mL in human whole blood.