Abstract:

Circulating tumor cells （CTC） have emerged as crucial mediators in the metastatic cascade， offering invaluable insights as real-time liquid biomarkers for cancer progression， prognosis， and treatment response. Their exceptionally low concentration in peripheral blood， which typically ranges from a handful to a few dozen cells per milliliter amidst billions of background blood cells， poses formidable challenges for isolation and molecular characterization. Despite this， the efficient and specific capture of CTC holds tremendous potential for revolutionizing early cancer detection， dynamic monitoring of therapeutic efficacy， and guiding personalized treatment strategies. Currently， the primary technologies for CTC enrichment fall into two categories： immunoaffinity-based methods that employ antibodies targeting epithelial surface markers such as epithelial cell adhesion molecule （EpCAM）， and label-free approaches that leverage physical properties including cell size， deformability， and density， exemplified by membrane filtration and centrifugal techniques. However， these conventional methods are hampered by several inherent limitations， including high operational costs， dependence on highly variable surface antigen expression， insufficient capture specificity leading to low purity， and significant interference from heterogeneous blood components such as leukocytes and platelets. Consequently， there is an urgent and growing need to develop novel functional materials and platforms that offer enhanced selectivity， robust stability in physiological conditions， excellent biocompatibility， and improved clinical applicability for the effective isolation and analysis of CTC. In this study， we innovatively integrate cell imprinting technology with a rational amino acid-based affinity strategy to develop a tryptophan-histidine-arginine （WHR） tripeptide-functionalized cell-imprinted hydrogel for highly efficient and selective capture of CTC. The design leverages the unique properties of mesoporous silica nanoparticles （MSN） as carriers， which are first synthesized and then surface-modified with epoxy groups via silane coupling agents. The WHR tripeptide is subsequently grafted onto the MSN surface through a ring-opening reaction， yielding the WHR@SiO₂ composite material. This material demonstrates strong and specific binding affinity toward sialic acid （Neu5Ac） and sialylated glycopeptides （SGP）， which are overexpressed on the surface of many cancer cells. Building on this molecular recognition capability， a three-dimensional cell-imprinted hydrogel is fabricated using poly（ethylene glycol） dimethacrylate （PEGDMA） as the cross-linking backbone via free radical polymerization. The hydrogel is molded against SMMC-7721 template cells to create cavities that complement the target cells in size， shape， and surface topology， thereby enhancing capture efficiency through both physical and biochemical matching. Experimental results demonstrate that the WHR-modified hydrogel achieves a remarkable capture efficiency of up to 94% for SMMC-7721 cells， significantly outperforming hydrogels modified with individual amino acids such as tryptophan， histidine， or arginine alone. The system also exhibits excellent hemocompatibility， with minimal adsorption of human serum albumin （HSA）， below 5%， indicating superior anti-fouling properties in biological environments. In vitro cytotoxicity assessments confirm high biocompatibility， with cell viability exceeding 90% after 48 h of co-culture. Further characterization through scanning electron microscopy （SEM） and atomic force microscopy （AFM） reveals well-defined surface imprints that mirror the morphology of template cells， confirming the successful integration of topographical cues. The synergy between the physical structure of the imprinted cavities and the biochemical affinity of the WHR tripeptide is identified as the key factor contributing to the high capture performance， even at low cell concentrations （as few as 100 cells/mL）. In conclusion， this work presents a robust and efficient platform for CTC capture that combines cell imprinting for morphological recognition with WHR-mediated affinity for sialylated glycoproteins. The hydrogel demonstrates high selectivity， stability， and biocompatibility， offering a promising tool for clinical applications in liquid biopsy and early cancer detection. The modular design of the system also allows for adaptation to other cancer types by altering the peptide sequence or template cells， highlighting its broad potential in cancer research and diagnostics.

Key words: circulating tumor cells, cell imprinting, hydrogels, interfaces, enrichment