Highly Sensitive Detection of Hydrogen Peroxide in Cancer Tissue Based on 3D Reduced Graphene Oxide-MXene-Multi-Walled Carbon Nanotubes Electrode

Abstract

Hydrogen peroxide (H₂O₂) is a signaling molecule that has the capacity to control a variety of biological processes in organisms. Cancer cells release more H₂O₂ during abnormal tumor growth. There has been a considerable amount of interest in utilizing H₂O₂ as a biomarker for the diagnosis of cancer tissue. In this study, an electrochemical sensor for H₂O₂ was constructed based on 3D reduced graphene oxide (rGO), MXene (Ti₃C₂), and multi-walled carbon nanotubes (MWCNTs) composite. Three-dimensional (3D) rGO-Ti₃C₂-MWCNTs sensor showed good linearity for H₂O₂ in the ranges of 1-60 μM and 60 μM-9.77 mM at a working potential of -0.25 V, with sensitivities of 235.2 µA mM^-1 cm^-2 and 103.8 µA mM^-1 cm^-2, respectively, and a detection limit of 0.3 µM (S/N = 3). The sensor exhibited long-term stability, good repeatability, and outstanding immunity to interference. In addition, the modified electrode was employed to detect real-time H₂O₂ release from cancer cells and cancer tissue ex vivo.

Keywords: H2O2; MWCNTs; MXene; cancer tissue; rGO; real-time detection.

Scheme 1

Fabrication of 3D rGO–Ti₃C₂–MWCNTs and the detection of H₂O₂ released from cancer cells and tissue.

Figure 1

SEM images of (A) 3D rGO, (B) 3D rGO–Ti₃C₂, (C) 3D rGO–MWCNTs, and (D) 3D rGO–Ti₃C₂–MWCNTs at the scale of 1 µm.

Figure 2

(A) CV of 3D rGO, rGO–Ti₃C₂, rGO–MWCNTs, and rGO–Ti₃C₂–MWCNTs electrodes in 5 mM K₃[Fe(CN)₆] and 0.1 M KCl at scan rates of 100 mV s⁻¹. (B) CV of the 3D rGO–Ti₃C₂–MWCNTs electrode in 5 mM K₃[Fe(CN)₆] and 0.1 M KCl at scan rates of 50, 100, 150, 200, 250, 300, 350, 400, 450, and 500 mV s⁻¹ (inset: plot of the oxidation and reduction peak currents vs. square root of the scan rate).

Figure 3

CV of 3D rGO, rGO–Ti₃C₂, rGO–MWCNTs, and rGO–Ti₃C₂–MWCNTs electrodes in deoxygenated 0.01 M PBS in the presence and absence of 4 mM H₂O₂.

Figure 4

(A) Amperometric response of the 3D rGO–Ti₃C₂–MWCNTs electrode upon adding H₂O₂ in deoxygenated 0.01 M PBS at a constant potential of −0.25 V under continuous stirring (inset: expand view at 1, 2, 5, and 10 µM H₂O₂). (B) Calibration curve of the 3D rGO–Ti₃C₂–MWCNTs electrode current vs. H₂O₂ concentration (inset: calibration curve showing H₂O₂ concentration from 1–60 µM).

Figure 5

(A) Reproducibility study of the 3D rGO–Ti₃C₂–MWCNTs electrode at a constant potential of −0.25 V with continuous addition of 100 μM H₂O₂ in deoxygenated 0.01 M PBS. (B) Reproducibility evaluation of five 3D rGO–Ti₃C₂–MWCNTs electrodes. (C) Long-term (4 weeks) stability of the 3D rGO–Ti₃C₂–MWCNTs electrode at a constant potential of −0.25 V with continuous addition of 100 μM H₂O₂ in deoxygenated 0.01 M PBS. (D) Stability assessment of the 3D rGO–Ti₃C₂–MWCNTs electrode for 4 weeks.

Figure 6

Selectivity of the 3D rGO–Ti₃C₂–MWCNTs electrode to 100 µM H₂O₂, 200 µM interfering species (UA, AA, DA, Glu), and 100 µM H₂O₂ in deoxygenated 0.01 M PBS at a constant potential of −0.25 V.

Figure 7

Quantitative cell viability results by CCK−8 assay for (A) 4T1 and (B) MCF−7 cells incubated with the 3D rGO–Ti₃C₂–MWCNTs electrode for 0, 1, 2, 3, 4, and 5 h. (C) Reduction current response of the 3D rGO–Ti₃C₂–MWCNTs electrode with the addition of fMLP, catalase, and fMLP-free PBS to deoxygenated 0.01 M PBS solution containing 7.0 × 10⁶ 4T1 cells, 5.0 × 10⁶ MCF−7 cells, and no cells. (D) Amperometric current response of the 3D rGO–Ti₃C₂–MWCNTs electrode after separate injections of fMLP, catalase, and fMLP-free PBS into breast cancer tissue immersed in the deoxygenated 0.01 M PBS solution.