2D carbon network arranged into high-order 3D nanotube arrays on a flexible microelectrode: integration into electrochemical microbiosensor devices for cancer detection

The study addresses the challenge of assembling low-dimensional nanocarbon materials (e.g., graphene, nanotubes) into stable higher-order 3D architectures without restacking/aggregation that diminishes active surface area and stability. It proposes a controllable, ecofriendly template strategy using ionic liquids (ILs) to synthesize mesoporous 2D N, B, and P co-doped carbon networks arranged into high-order 3D nanotube arrays on flexible carbon fiber microelectrodes. The purpose is to create robust, high-surface-area, heteroatom-doped carbon architectures with efficient charge transport and abundant active sites to enhance electrocatalytic performance for biosensing, specifically the detection of H₂O₂ as a cancer biomarker. The importance lies in enabling sensitive, real-time monitoring of H₂O₂ secretion from live cancer cells and distinguishing cancerous from normal tissues, with implications for diagnosis and therapy assessment.

Prior work highlights the diverse forms of carbon nanomaterials (graphene quantum dots, nanodiamonds, carbon nanotubes/nanohorns, 2D graphene and carbon networks) valued for catalysis, energy, biosensing, and biomedical use. These low-dimensional carbons can assemble into 3D architectures, promising for flexible/implantable electronics and sensors, but suffer from restacking/aggregation during assembly, reducing active area and stability. Template-based assembly methods are desirable for controllability and to prevent aggregation. Conventional precursors (glucose, polydopamine, heteroatom polymers) can coat templates and carbonize with good yields, yet achieving precise control of nano/microstructure, electronic properties, and multifunctionality remains challenging. Ionic liquids (ILs), with tunable anions/cations, high thermal stability, negligible vapor pressure, surface activity for coating diverse substrates, and capacity to introduce multiple heteroatoms (e.g., N, S, B, P), have emerged as versatile precursors for carbon nanomaterials. This context motivates using IL-derived, heteroatom-doped carbons structured via sacrificial templates to obtain high-order architectures for advanced biosensing.

Synthesis of NBP-CNW-NTAs on carbon fibers (CFs): - CF activation: Immerse CFs in 30% H₂O₂ at 60 °C for 24 h; rinse with deionized water (×3); dry at 60 °C. - Template growth: Grow ZnO nanorod arrays (ZnO-NRAs) on activated CFs by electrodeposition (per prior protocol; details in Supporting Information). The activated CF surface functional groups serve as nucleation sites. - IL coating: Mix ionic liquids [VEIM]BF₄ and [OMIM]PF₆ in a 4:1 volume ratio; stir to homogeneity; coat onto ZnO-NRAs. - Carbonization: Heat IL-coated ZnO-NRAs in Ar to 750 °C at 2 °C min⁻¹; hold 3 h to convert ILs into a multi-heteroatom co-doped porous carbon layer. During heating, [VEIM]BF₄ first polymerizes (around 300 °C) to P[VEIM]BF₄ (validated by GPC: Mₙ ≈ 4.74 kDa, PDI ≈ 1.012), then carbonizes to N,B-doped carbon; [OMIM]PF₆ thermally decomposes, acting as pore-former and N,P sources, generating macroporosity and additional heteroatom doping. - Template removal: Etch ZnO with 0.1 M HCl for 6 h; rinse with deionized water; dry at 80 °C to obtain NBP-CNW-NTAs/CF microelectrode. - Polymer reference: Independently synthesize P[VEIM]BF₄ by heating [VEIM]BF₄ from room temperature to 300 °C at 2 °C min⁻¹ under Ar and cooling naturally. Device fabrication and biological testing: - Microfluidic electrochemical chip: Designed and fabricated via 3D printing (details in Supporting Information). The NBP-CNW-NTAs/CF microelectrode serves as working electrode; a Pt wire is used as counter (obtained from Union Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, China). An implantable probe version was also constructed for in situ tissue measurements. - Cell culture: Cancer cell lines (breast, hepatoma, cervical) cultured in DMEM with 10% FBS and 1% penicillin–streptomycin at 37 °C, 5% CO₂. Cells harvested at ~90% confluence; counted prior to testing. Cytotoxicity of NBP-CNW-NTAs/CF assessed via CCK-8 assay. - Clinical specimens: Surgically resected human primary breast tumors (two tumor tissues) and adjacent adipose (control) obtained from Union Hospital. Samples washed and soaked in PBS at 37 °C for electrochemical testing. Characterization: - Morphology: SEM and TEM to examine CFs, ZnO-NRAs, and resultant NBP-CNW-NTAs (tooth-like morphology, interconnected pores). - Composition/structure: Evidence of IL polymerization (GPC metrics) and heteroatom (N, B, P) co-doping; discussion of porosity formation via [OMIM]PF₆ decomposition. Electrochemical evaluations for H₂O₂ sensing performance were conducted (parameters summarized in findings).

- Successfully synthesized high-order 3D nanotube arrays (NTAs) constructed from mesoporous 2D N, B, and P co-doped carbon networks (NBP-CNW) wrapped on flexible carbon fibers using IL precursors and a ZnO-NRA sacrificial template. - Structural features: Hierarchically porous architecture with macropores in 3D NTAs and nanoscale porosity in 2D CNWs; high structural stability; large electrochemically active surface area; abundant active sites; efficient charge-transport pathways. SEM/TEM show tooth-like, interconnected porous morphology. GPC of P[VEIM]BF₄: Mₙ ≈ 4.74 kDa, PDI ≈ 1.012, confirming polymerization prior to carbonization. - H₂O₂ electrocatalysis and sensing performance: Detection limit 500 nM (S/N = 3); wide linear dynamic range up to 19.52 mM; sensitivity 61.8 μA cm⁻² mM⁻¹; high anti-interference capability; good mechanical and long-term stability; excellent biocompatibility. - Biological applications: Embedded in a microfluidic electrochemical biosensor, enabling real-time tracking of H₂O₂ secretion from live cancer cells (breast, hepatoma, cervical) with/without radiotherapy, supporting discrimination among cell types and assessment of radiotherapeutic efficacy. - Clinical relevance: Integrated into an implantable probe for in situ detection on surgically resected human breast specimens, distinguishing tumor tissues from normal tissues, underscoring potential for cancer diagnosis and management.

The designed IL-derived, heteroatom-doped carbon NTAs address the central challenge of assembling low-dimensional carbon into stable, high-performance architectures by preventing restacking and providing hierarchical porosity. The 3D NTAs built from 2D CNWs yield a large ECSA and interconnected pathways that facilitate mass transport and rapid electron transfer, improving H₂O₂ redox electrocatalysis. Co-doping with N, B, and P tunes charge density and electronic structure, enhancing conductivity and wettability and increasing the density of catalytic sites. These material advantages translate to sensitive, wide-range H₂O₂ detection with robust selectivity and stability. Embedding the microelectrode into a microfluidic platform enables real-time monitoring of H₂O₂ secretion dynamics from different cancer cell lines and evaluation of radiotherapy effects, offering a practical route for cell-type discrimination and therapeutic assessment. The implantable probe results demonstrate feasibility for distinguishing malignant from normal tissues in clinical specimens, highlighting the method’s potential impact in diagnostics and intraoperative decision-making.

This work presents a facile, controllable, and sustainable IL-templated strategy to fabricate high-order 3D nanotube arrays of mesoporous 2D N, B, and P co-doped carbon networks on flexible carbon fibers. The resultant microelectrodes exhibit high structural stability, hierarchical porosity, efficient charge transport, and abundant active sites, enabling excellent H₂O₂ sensing (500 nM LOD, 19.52 mM linear range, 61.8 μA cm⁻² mM⁻¹ sensitivity) with strong anti-interference, durability, and biocompatibility. Integrated into microfluidic and implantable formats, the sensors allow real-time monitoring of H₂O₂ secretion from live cancer cells and in situ differentiation of tumor versus normal tissues in human specimens, offering a promising tool for cancer diagnosis and therapy evaluation. Future work could optimize IL chemistries and template architectures to further tune porosity and dopant configurations, extend the platform to other biomarkers and multiplexed sensing, and validate performance in larger-scale preclinical and clinical studies.