Researches On The Design Of Novel Interface For Circulating Tumor Cells Recognition And Application

Circulating tumor cells（CTCs）,which are about 10-20μm in diameter,are cancer cells that naturally shed from the primary or metastatic tumor sites of tumor patients and circulated in the blood.They are the main cause of cancer metastasis and death.As a new type of"liquid biopsy"technology,CTCs analysis offers the possibility of avoiding the need for invasive tissue biopsy for cancer diagnosis.Therefore,CTCs,as a non-invasive cancer detection target,have become a research hotspot in recent years.The number of CTCs and the expression level of specific surface protein markers have a certain correlation with the occurrence and development of cancer.However,due to the scarcity of CTCs and the presence of lots of background white blood cells（about 1-100 CTCs and billions of red blood cells and millions of white blood cells per milliliter of the whole blood）,high-purity enrichment and sensitive detection of CTCs are needed which would be great challenges.At the same time,the process of tumor metastasis will be accompanied by the occurrence of epithelial-mesenchymal transition（EMT）simultaneously,which leads to the heterogeneity of CTCs,namely the protein expression levels of tumor cells of different phenotypes are significantly different and researches on the identification and detection of CTCs are still limited.In addition,the realization of the intact release of the captured CTCs is conducive to the further functional analysis for the released CTCs and the comprehensive and accurate cancer research.This thesis takes the identification,capture,analysis and distinguish of CTCs as the core of the research work,and treats human breast cancer cells（MCF-7 and MDA-MB-231）and cervical cancer cells（He La）as target cells while mouse macrophages cells（RAW264.7）as control cells,where the novel CTCs capture interfaces were constructed by different strategies which successfully achieved efficient capture and sensitive detection of CTCs with further realized the efficient release of CTCs and the analysis of cell viability after release.This thesis provides novel ideas for the study on the capture,isolate and recognition of CTCs.The main researches are as follows:1.Near-Infrared Light-Switched Mo S₂ Nanoflakes@Gelatin Bioplatform for Capture,Detection,and Nondestructive Release of Circulating Tumor CellsA biological platform with near-infrared light（NIR）switches was constructed for the effective separation and downstream analysis of CTCs.First,modifying the PEG-Mo S₂ nanoflakes@gelatin nanocomposite on the surface of indium tin oxide（ITO）conductive glass,then through the amidation coupling reaction between the aptamer and gelatin,the MUC1 aptamer was modified to the interface as a recognition element,so as to achieve the specific capture of CTCs.Subsequently,Mo S₂ NFs were used as the control element of NIR regulation.Under the irradiation of NIR light（808nm）,based on the good photothermal effect of Mo S₂ NFs,the captured CTCs cells were released with high activity.The platform’s capture and release efficiencies of CTCs were 89.5%and 92.5%,respectively.In addition,the electrochemical biological platform can also achieve sensitive detection of CTCs,with a detection range of50-10⁶ cells/m L and a detection limit of 15 cells/m L.After the released CTCs were cultured continuously for 5 days,good cell morphology and proliferation ability could be observed.The biological platform designed in this work is a simple and universal system that can be used to identify,capture,release and detect different types of CTCs,which has the potential applications in cancer related research.2.Aptamer Functionalized Leukocyte Membrane Coated with UCNPs-Fe₃O₄@Si O₂ Nano-Biological Probe for Effective Separation and Fluorescence Detection of Heterogeneous Circulating Tumor CellsA nano-platform based on a magnetic upconversion（UCNPs）composite material modified by aptamers and leukocyte membranes was developed to realize the effective separation and sensitive detection of CTCs.The designed bionic immune nano-biological probe has the following characteristics:i)The UCNPs nanomaterial was effectively combined with the Fe₃O₄@Si O₂ through simple electrostatic interaction;ii)The white cell membrane was used for coating,which reduced the interference of white cells in blood samples and realized the separation of high-purity CTCs;iii)After the captured CTCs magnetically separated,the fluorescence of the UCNPs material can be used for fluorescence imaging analysis.The capture rate and purity of CTCs by the designed immunomagnetic nano-biological probe can reach96.5%and 95.3%,respectively.Due to the coating of leukocyte membranes and the functionalization of aptamers combined with magnetic separation and up-conversion fluorescence imaging,this nano-platform showed the characteristics of specific recognition,effective capture,easy separation and highly sensitive detection of CTCs.3.pH-Sensitive Dye-Based Nanobioplatform for Colorimetric Detection of Heterogeneous Circulating Tumor CellsA nano-platform for high-efficiency capture and high-sensitivity colorimetric detection of heterogeneous CTCs was designed.The platform consisted of two parts:multivalent aptamer-modified gold nanoparticles as the capture unit,and molybdenum disulfide nanoflakes（Mo S₂ NFs）loaded with aptamer and pH-responsive dyes thymolphthalein（TP）and curcumin（CUR）as a detection unit,it can detect heterogeneous CTCs at the same time.Taking MCF-7 and He La cells as CTCs models,using the high affinity of multivalent probes and the capture unit can effectively capture CTCs.Under alkaline conditions（pH=12.5）,when MCF-7 and He La cells were present,the two color-changing dyes can show obvious color changes,providing a rapid and sensitive method for the simultaneous detection of heterogeneous CTCs.Since Mo S₂ NFs can achieve efficient loading of dyes and good response performance of dyes,the nano-platform has high sensitivity for the detection of CTCs.The platform can successfully distinguish and quantitatively detect heterogeneous CTCs from whole blood samples and blood samples of cancer patients,providing a new method for the capture,identification and analysis of heterogeneous tumor cells.4.Ultra-Sensitive Electrochemical Aptamer Sensor System Based on Dual Signal Amplification for the Study of Circulating Tumor Cell Epithelial-Mesenchymal TransitionA dual-signal amplified electrochemical aptamer sensing interface based on DNA hybridization chain reaction（HCR）technology was constructed to realize the ultra-sensitive detection of heterogeneous CTCs and monitor of different stages of EMT.First,the polydopamine（p DA）was modified on the surface of the glassy carbon electrode（GCE）by electropolymerization,and then streptavidin（SA）was introduced onto the electrode through the amidation reaction.After that,using the strong interaction between biotin and SA,the biotin-AS1411 aptamer was fixed to GCE for capture heterogeneous CTCs.Finally,the aptamer can specifically recognize and capture the heterogeneous CTCs and immobilize the HCR assembly of methylene blue（MB）and ferrocene（Fc）labeled aptamers（MB-NC3S and Fc-SYL3C）to target cells surface,where high-efficiency capture and sensitive detection of target cells were achieved.At the same time,we also dynamically monitored the signal changes of MCF-7 cells during EMT,providing a new idea for phenotypic analysis of tumor cells during EMT.