Isolation Of Circulating Tumor Cells Using Functionalized Nanomaterials And Microfluidics

The malignant tumor is a serious threat to the human life and health. Due to the high risk of morbidity and mortality, early diagnosis is vital in preventing and treating of such cancer. However, the low sensitivity and specialty of conventional diagnosis make it difficult to detect the tumor at the early stage of cancer progression. Once detected will be diagnosed with terminal cancer, thus the best treatment period have been missed. Circulating tumor cells (CTCs) are cells that shed from a tumor, circulate in the peripheral blood of cancer patients, and play an important role in the early diagnosis and treatment assessment of cancer. Due to the rare of CTCs, enrichment is essential for efficient detection of CTCs. Due to their controllability within nanoscale and microscale level, nanomaterials and microfluidics have developed into mature technologies in CTC detection.Functionalized nanomaterials have been widely used in optical, electrochemical and biomedical studies due to their unique features. In biomedicine, for example, the nanomaterials are used in the construction of biomarkers, drug delivery and biosensors due to their excellent biocompatibility. Along with the development of new synthesis and characterization methods, researchers have synthesized a variety of nanomaterials with different shape, size and function. The rich functional groups on the surface of nanomaterials make it feasible to be grafted with variety of biomolecules that are capable of specific recognition, thus the nanomaterials have been widely applied in CTCs isolation and detection.Microfluidics have been widely used in many fields due to their high throughput, less sample consumption, high sensitivity, high integration and high portability. The size of the microchannel matches the size of cells, which makes it capable of isolating and analyzing the single cell.In this paper, we will give a brief review of developments in CTC research. Considering the problems encountered in CTC enrichment, we carried out the studies of CTC isolation and detection based on the advantages of nanomaterials and microfluidics mentioned above.The works were summarized as follows:1. We synthesized self-assemble MnO2 nanosphere monolayer thin film. This transparent thin film was applied for capture and release cancer cells. With the surface chemical bond, epithelial cell adhesion molecule antibody (anti-EpCAM) was modified on the surface of the cell-capture substrate. We designed control assays to test the capture specificity of anti-EpCAM modified cell-capture substrate. We studied the recovery rates of cancer cells from DMEM and artificial blood cells using the MnO2 substrate. We also studied the effect of incubation time on the cell capture efficiency. We further studied the cell release efficiency and viability on the concentration of oxalic acid. We cultured the released cells and studied their acid-resistant differentiation capacity.2. We performed the study of noninvasive recovery of CTCs from cancer patients based on the synergistic effect of a microfluidic filter and the cell size amplification mediated by gelatin coated silica microbeads (SiO2@Gel MBs). We verified the capture specificity of anti-EpCAM modified SiO2@Gel MBs. We studied the recovery efficiency of cancer cells from lysed blood and whole blood using anti-EpCAM modified SiO2@Gel MBs. We also studied the efficiency of cell capture affected by the concentration of MBs used. We investigated the effect of geometric parameters of the microfilter and the rate of flow on the purification of the cancer cells. We studied the cell release efficiency and viability on the concentration of matrix metalloproteinases-9 (MMP-9) enzyme. We performed the optimized conditions on CTC isolation and detection from cancer patients.3. To improve the efficiency of CTCs captured on MBs, we applied anti-CD146 together with anti-EpCAM to target EpCAM-positive and negative CTCs in patient blood. To avoid the overlap of density of CTCs and normal blood cells and improve the purity of CTCs, we used dense MBs (with the density of 1.2g/mL) together with an improved Percoll medium (with the density of 1.15g/mL) to purify CTC-beads from normal blood cells by gradient centrifugation. To further purify CTCs from MBs while keeping high viability, we coated the MBs with biodegradable gelatin. During the research, we verified the enhanced cell-capture efficiency due to the function of dual antibodies of anti-EpCAM and anti-CD 146 using cancer cell lines. We verified the EpCAM and CD146 expression on the surface of cancer cells. We compared the purification of cell-beads using Percoll solution with different densities. Under the optimized cell capture and purification conditions, we evaluated the capability of capturing more CTCs using dual antibodies functionalized MBs than single antibody capture.