A novel enzyme-free long-lasting chemiluminescence system based on a luminol functionalized β-cyclodextrin hydrogel for sensitive detection of H2O2 in urine and cells

A novel long-lasting chemiluminescent (CL) hydrogel (β-CD@luminol-Co²⁺) was synthesized by embedding luminol and cobalt ions (Co²⁺) into β-cyclodextrin (β-CD) through non-covalent interactions. Due to its porous structure and viscosity, the synthesized β-CD@luminol-Co²⁺ hydrogel exhibited long-lasting CL properties and can emit light for 12 h under both alkaline and neutral conditions. In addition, the CL intensities of β-CD@luminol-Co²⁺ were linear with the logarithm of the hydrogen peroxide (H₂O₂) concentration in the range of 1.0 × 10^-11-1.0 × 10^-7 M, and the limit of detection (LOD) was 0.63 × 10^-11 M and 0.85 × 10^-11 M under alkaline and neutral conditions, respectively. On this basis, an enzyme-free CL sensor based on β-CD@luminol-Co²⁺ was fabricated for the sensitive detection of H₂O₂ in human urine samples under alkaline conditions, and showed good accuracy and recovery. Since β-CD@luminol-Co²⁺ showed good CL properties under neutral conditions, it can be applied to detect H₂O₂ in cells. In order to prolong the emission wavelength of β-CD@luminol-Co²⁺ for better cell imaging, β-CD@luminol-FL-Co²⁺ was prepared by adding fluorescein (FL) to β-CD@luminol-Co²⁺. The as-prepared β-CD@luminol-FL-Co²⁺ also displayed long-lasting CL properties and showed a linear relationship with H₂O₂ concentrations. In addition, the maximum emission wavelength of β-CD@luminol-FL-Co²⁺ was 520 nm, which was red-shifted by 95 nm compared with β-CD@luminol-Co²⁺. The methyl thiazolyl tetrazolium (MTT) assay results and confocal microscopy images illustrated that β-CD@luminol-FL-Co²⁺ had low toxicity and can be taken up by A549 cells. Finally, β-CD@luminol-FL-Co²⁺ was successfully applied for CL imaging and detection of intracellular H₂O₂ in A549 cells under neutral conditions. This enzyme-free long-lasting CL system with high sensitivity can also be extended to real-time monitoring of H₂O₂in vivo.