Research Of Nano-materials Based Microchip For Detecting Rare Cells In Peripheral Blood

There exist some low-abundance but really special,rare cells in peripheral blood,such as circulating tumor cells,fetal nucleated red blood cells and cells infected by a virus or parasite,and their numbers are just a few to several hundred per ml,while they have tremendous applications and value both in clinical and medical researches.These rare cells can directly or indirectly reflect the healthy condition of the body,and one of them called "liquid biopsy" is CTCs which is a key point of investigating cancer metastasis and applying for diagnosis and treatment of cancer;and another one is fetal NRBCs which can provide an alternative source of fetal genetic using for non-invasive prenatal diagnosis.However,because of the extremely low-abundance and large population of background cells in peripheral,conventional benchtop techniques have limited capabilities to isolate and analyze rare cells due to their generally low specificity and significant sample loss.Recent rapid advances in applications of nanomaterials and microfluidics have been providing robust solutions to the challenges in the separation and detection of rare cells.In addition to the apparent performance enhancements resulting in higher efficiency and sensitivity,microfluidics can bring other advanced features such as rapid handling of small sample volumes and parallel capabilities for high-throughput processing.Furthermore,biocompatible nanomaterials have been long documented that are well suited for isolating and further analyzing rare cells due to their excellent structural and biochemical support for nanoscale components of cell-surface.Therefore,because of the intensive interaction between nano-substrates and cell surface,it has been widely used in rare cells separation and in vitro identification,cell culture and in vivo long-term tracing.Considering the efficient capture of rare cell,good biocompatibility and cell identification in site,we develop some basic researches of CTCs and fetal NRBCs capture and analysis.And the thesis will be summarized as follow:1.Hydroxyapatite/Chitosan nanoparticles film using in researches of cancer cells isolation.We fabricate an excellent transparency and biocompatibility nanosubstrates made of hydroxyapatite/chitosan nano-composite films,which forms 3D nano-structure chip on glass.We study the enhanced graphic interaction between cell surface and the antibody coated nano-chip in increasing cell-capture efficiency;And we also utilize the nano-chip for capture target-cells from artificial blood samples,and evaluate the cell viability during the processes.After all,we demonstrate the capabilities in isolating and detecting CTCs from patient samples,and the potentials for further researches.2.Fetal NRBCs analysis for noninvasive prenatal diagnosis using a nanostructure microchip.We apply an affinity microchip made of HA/CTS NF and bio-conjugated with novel specific antibody anti-CD 147 for capture and analysis fetal NRBCs from peripheral blood samples;A "double positive" three-color immune-fluorescent staining method is employed to identify fetal NRBCs,and FISH technique is also applied to verify the origin of target-cells captured from peripheral blood.And next,we study the variation of fetal NRBCs number along with gestation age increasing.Furthermore,combined with in site analysis for detection of fetal chromosomal abnormalities on chip,we demonstrate the abilities of the microchip capturing and analyzing fetal NRBCs from peripheral blood and the potential applications in NIPD.3.Biotin doped conducting nano-polymer applied in fetal NRBCs capturing and releasing for noninvasive prenatal diagnosis.We prepare a nano-structure microchip of polypyrrole-biotin doping nanofilm coated with anti-CD 147 on ITO,which has an ability of controllable releasing target-cells by electrical stimulation.We set a model with TF-1 cell line to simulate fetal NRBCs for optimizing the capabilities of microchip in cell capturing and releasing.Next,we study the cell release efficiency with a stimulation of 0.8 V.After all,we apply the microchip for capturing and releasing fetal NRBCs from clinical peripheral blood samples.In additional,we analysis the recovery fetal cells for fetal genetic diagnosis with PCR,FISH and whole genome sequencing.