High-performance Nanostructured Substrates:from Engineering To Detection Of Circulating Tumor Cells

Tumor metastasis is the major cause of cancer-related mortality,while circulating tumor cells(CTCs)propagating from primary tumor sites and entering the bloodstream,are the main factors responsible for tumor relapse and distal metastasis.Therefore,detection of CTCs with the wealth of potential information about metastatic mechanisms,is of significantly importance for early diagnosis,prognosis and personalized treatment.However,CTCs are extraordinarily rare events(a few to hundreds of CTCs per mL of blood containing approximately 8×109 cells of red blood cells and 5×106 leukocytes),and exhibit heterogeneity,thus,it is urgent ro develope high-performance methods for CTC detection with high capture efficiency and sensitivity.To date,significant efforts of researchers have been devoted to explore a variety of techniques and devices for CTCs isolation,mainly based on the physical properties(size,density,electric charges,deformability)and biological properties(cell surface protein biomarkers)distinguishing CTC from normal hematopoietic cells.However,approaches for fabricating these nanostructured substrates such as lithography,electrospinning,etching,chemical vapor deposition,and layer-by-layer assembly,often require expensive equipments,complex techniques,and sophisticated and delicate manipulation.Furthermore,it has become increasingly clear that simply identifying and counting CTCs by immunostaining or fluorescence in situ hybridization(FISH)may not provide more metastatic and clinic relevant information,it is required to develop systems enabling not only capture but also release of CTCs.Various methods such as enzyme degradation,photosensitive-induced cleavage,electrochemical desorption,thermodynamic release,chemical reagent-triggered substrate sacrifice,and competition-based ligand replacement,have been developed to detach cells from substrates.However,many of these strategies have certain limitations such as potentially destructive enzymes,cytotoxic chemical molecules,dangerous ultraviolet,and strict processing conditions,and the majority of them could not successfully realized downstream molecular anaysis and cultivation of CTCs.Therefore,devices with high-performance properties are still pursued and desired for CTC assay.According to above present situation and challenge,this paper aims at developing simple,easily prepared,and high-performance nanostructured substrates for highly efficient capture,gentle release,and analysis of CTCs.The main study works of this thesis are summarized as follows:(1)Inspired by the principle of amphiphilic copolymers-based surface modification of organic-soluble nanoparticles,ultrasonication was used to drive the 1-hexadecylamine modified magnetic nanoparticles(MNP),trioctylphosphine oxide-capped quantatum dots(QD)and dodecyl glycidol ether substituted carboxymethylchitosans(CMCHS)interacting with each other through long hydrophobic alkyl chains,and form hydrophilic fluorescent-magnetic nanobeads(FMNB)with small size,fast magnetic response and good biocompatibility.The outer surface of FMNB was convenient for biological conjugation,and human epidermal growth factor(EGF)-functionalized FMNB could specifically capture Hela cells with a high capture efficiency(92%).Furthermore,the recovered cells could be further cultured and passaged for a long time.(2)Via a low-temperature hydrothermal method,we fabricated a novel zinc phosphate-based hierarchical nanosubstrate(HZnPNS)with transparent and degradable properties.In virtue of better matched topographical interaction and high-affinity antigen-antibody interaction,HZnPNS functionalized with anti epithelial cell adhesion molecule antibody(anti-EpCAM/HZnPNS)exhibited high capture efficiency for MCF-7 cells(up to 90%).Besides,biocompatible sodium citrate colud dissolve the hierarchical architecture at room temperature,allowing that captured cells were gently released with a high viability of 92%.Furthermore,anti-EpCAM/HZnPNS was successfully applied to isolate CTCs from whole blood samples of cancer patients,as well as release CTCs for global DNA methylation analysis.(3)To engineer high-performance integtrated bioplatform for CTC detection,an overlaid polydimethylsiloxane(PDMS)chaotic mixer was incorporated onto patterned hierarchical substrates via photolithography and etching by diluted HCl,resulting in greatly enhanced cell-surface interaction benefited from herringbone micromixers,more binding sites and better matched topographical interaction.Thus,in combination with high-affinity aptamers,target cancer cells are specifically captured on the chip with high capture efficiency.Besides,as-prepared chip was endowed with DNA-responsive features through aptamer modification.Via toehold-mediated strand displacement,captured cancer cells could be nondestructively released from the chip,and futher amplifying of aptamer-containing DNA sequences on the surface of retrieved cancer cells by polymerase chain reaction(PCR)enabled detection of cell surface biomarkers.