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 that low-dimensional nanocarbon materials (e.g., graphene, nanotubes) tend to restack or aggregate during assembly, reducing active surface area and stability. It proposes a controllable template strategy to arrange nanocarbon into higher-order architectures that preserve surface area and enhance functionality. The research develops a facile, ecofriendly approach to synthesize high-order 3D nanotube arrays (NTAs) from a mesoporous 2D carbon network co-doped with N, B, and P (NBP-CNW), using ionic liquids (ILs) as precursors and ZnO nanorod arrays on carbon fibers as sacrificial templates. The purpose is to create flexible microelectrodes with abundant active sites, high structural stability, and efficient charge transport to improve electrocatalytic sensing of H2O2, a key cancer biomarker. The work aims to enable real-time tracking of H2O2 from live cancer cells and in situ detection in human tissues for cancer diagnosis and therapy assessment.

The paper situates the work within advances in low-dimensional carbon allotropes (graphene quantum dots, nanodiamonds, CNTs, nanohorns, 2D graphene and carbon networks) valued for catalysis, energy, biosensing, and biomedical applications. It highlights assembly of these into 3D macroscopic architectures for flexible/implantable devices but notes issues of restacking/aggregation that reduce effective surface area. Template methods are emphasized as controllable solutions, with prior carbon precursors (glucose, polydopamine, heteroatom polymers) enabling coatings and high carbonization yields yet still falling short of delivering simultaneously controllable nano/microstructures and multifunctionality. ILs are reviewed as promising, tunable precursors due to low vapor pressure, thermal stability, surface activity for coating diverse substrates, and the ability to tailor heteroatom doping (N, S, B, P) by selecting cation/anion combinations, motivating their use here for constructing heteroatom-doped carbon with designed porosity and morphology.

Preparation of hierarchical structured microelectrode: Carbon fibers (CFs) were activated by immersion in 30% H2O2 at 60 °C for 24 h, rinsed with deionized water, and dried at 60 °C. ZnO nanorod arrays (ZnO-NRAs) were grown on CFs by electrodeposition. Two ionic liquids, [VEIM]BF4 and [OMIM]PF6, were mixed at a 4:1 volume ratio, stirred to homogeneity, and coated on ZnO-NRAs. The coated substrate was heated in Ar at 750 °C for 3 h with a ramp of 2 °C min−1 to carbonize the IL layer into a porous, multi-heteroatom-doped carbon. The ZnO template was removed by immersion in 0.1 M HCl for 6 h, followed by washing and drying at 80 °C to yield NBP-CNW-NTAs on CF (NBP-CNW-NTAs/CF). Separately, [VEIM]BF4 was polymerized thermally under Ar by heating from room temperature to 300 °C at 2 °C min−1 to obtain P[VEIM]BF4. Electrochemical microfluidic chip and implantable probe: A microfluidic electrochemical chip was assembled with the NBP-CNW-NTAs/CF as the working electrode (WE), a Pt wire (1 mm diameter) as counter electrode (CE), and an Ag/AgCl reference electrode (Ag wire coated with AgCl, 50 μm). Ten microliters of human cell sample were introduced via an inlet to the detection chamber for electrochemical measurements. For minimally invasive in situ testing, an implantable probe integrating the same three-electrode configuration was constructed and connected to a portable bipotentiostat. Cell culture and specimens: HepG2 (hepatoma), HeLa (cervical), and MCF-7 (breast) cancer cells were cultured in DMEM with 10% FBS and 1% penicillin-streptomycin at 37 °C, 5% CO2, harvested at ~90% confluence, washed, and collected by centrifugation. Cell viability and cytotoxicity of the microelectrode were evaluated using a CCK-8 assay. Surgically resected primary human breast tumor tissues (two tumor samples) and an adjacent adipose control were washed with PBS and maintained in PBS at 37 °C for electrochemical testing. Characterization notes: SEM showed ZnO-NRAs fully covering CFs; upon IL coating and carbonization, 3D high-order NBP-CNW-NTAs formed with loaf-like morphology and interconnected pores. Polymerization of [VEIM]BF4 occurred around 300 °C (GPC: Mn 4.74 kDa, PDI 1.012), which upon higher-temperature processing yielded N,B-doped carbon; [OMIM]PF6 decomposed to provide porosity and P doping.

- Successfully synthesized mesoporous 2D N,B,P co-doped carbon networks arranged into 3D nanotube arrays (NBP-CNW-NTAs) on flexible carbon fibers via an IL-derived, template-assisted route using ZnO nanorod arrays. - Structural and compositional features: hierarchically porous architecture with macropores and mesopores, high structural stability, large electrochemically active surface area, abundant active sites, and efficient charge transport pathways; homogeneous N, B, and P heteroatom doping improves electronic conductivity and wettability. - Electrochemical H2O2 sensing performance of NBP-CNW-NTAs/CF microelectrode: detection limit 500 nM (S/N = 3); wide linear dynamic range up to 19.52 μM; high sensitivity of 61.8 μA cm−2 mM−1; strong anti-interference capability; good mechanical robustness and long-term stability; excellent biocompatibility. - Real-time biosensing demonstrations: integrated into a microfluidic chip to track H2O2 secretion from live cancer cells (MCF-7, HepG2, HeLa) with and without radiotherapy, enabling differentiation of cell types and assessment of therapeutic efficacy. - Implantable application: integrated into a minimally invasive probe for in situ detection in surgically resected human breast specimens, distinguishing tumor tissue from normal adipose tissue. - Supporting polymerization/carbonization data: [VEIM]BF4 polymerizes at ~300 °C (Mn 4.74 kDa, PDI 1.012) prior to conversion to doped carbon; [OMIM]PF6 acts as pore-former and P source during high-temperature treatment.

The engineered 3D high-order nanotube arrays derived from a 2D N,B,P co-doped carbon network overcome the limitations of restacking and aggregation inherent to low-dimensional carbons by providing interconnected porosity and mechanical integrity on flexible carbon fibers. The hierarchical structure offers a large ECSA and efficient charge/ion transport, while heteroatom doping modulates the electronic structure of carbon, enhancing conductivity, wettability, and catalytic activity toward the H2O2 redox reaction. These features collectively deliver high sensitivity, low detection limit, and stability in H2O2 sensing. Embedding the microelectrode into a microfluidic system enables real-time monitoring of extracellular H2O2 from different live cancer cells and evaluating responses to radiotherapy, suggesting utility for distinguishing cancer cell types and assessing treatment efficacy. Integration into an implantable probe further demonstrates potential for minimally invasive, in situ tissue discrimination between tumor and normal samples, highlighting clinical relevance for diagnosis and management.

This work presents a facile, controllable, and sustainable strategy to fabricate high-order 3D nanotube arrays from mesoporous 2D N,B,P co-doped carbon networks on flexible carbon fibers using ionic liquid precursors and a ZnO nanorod template. The resulting microelectrode exhibits superior electrocatalytic performance for H2O2 detection, with low detection limit, wide linear range, high sensitivity, anti-interference, stability, and biocompatibility. Demonstrations in a microfluidic biosensor for live-cell monitoring and in an implantable probe for tissue discrimination underscore its promise for cancer diagnosis and therapy evaluation in clinical contexts.