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

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.


Introduction
The death rate from cancer increases year after year, despite advances in technology [1].The incidence of breast cancer currently accounts for 11.7% of all newly diagnosed cancer cases worldwide and has emerged as the leading cause of mortality among female cancer patients [2].The primary diagnostic modalities for cancer include marker diagnosis, pathology diagnosis, and imaging diagnosis.The Marker Diagnostics technology is capable of detecting biomarkers produced by malignant cells in various bodily fluids, including blood, tissue fluid, secretions, and tumor tissue [3].H 2 O 2 is an essential signaling molecule that regulates various biological processes, including protein synthesis, immune system function, and cell differentiation and proliferation [4][5][6].Studies demonstrate that cancer cells release more H 2 O 2 because of higher H 2 O 2 production or lower H 2 O 2 scavenging capacity during abnormal tumor growth [7,8].Therefore, measurement of H 2 O 2 levels in cells and tissue is essential for inchoate diagnosis of cancer [9].
Traditional methods for H 2 O 2 detection include fluorescence analysis [10], chemiluminescence [11], electrochemical analysis [12], and spectrophotometry [13].However, these methods have certain limitations, such as expensive equipment, cumbersome operation, stringent environmental testing requirements, and susceptibility to environmental influences [14].In contrast, electrochemical analysis methods offer the advantages of costeffectiveness, simplicity, and high selectivity [15,16].It has been discovered that specific nanomaterials, such as gold nanoclusters [17], platinum nanoparticles [18], graphene oxide [19], and its derivatives [20], possess the ability to mimic the activity of catalase.In addition, properties such as the excellent electrical, chemical, and structural properties of nanomaterials deserve special attention.Consequently, the utilization of nanomaterials in H 2 O 2 sensors can effectively enhance the sensitivity and specificity of detection, attributed to their exceptional properties [21,22].
Graphene is a two-dimensional (2D) monolayer material composed of sp 2 hybridized carbon atoms.It has garnered significant attention due to its exceptional electronic conductivity, expansive surface area, favorable biocompatibility, and impressive mechanical properties [23].However, strong van der Waals forces between individual graphene sheets can form disordered stacks or even re-stacking, affecting its excellent electrochemical properties [24].On the other hand, MXene is a novel two-dimensional transition metal carbide, nitride/carbonitrides [23].The chemical formula is Mn + 1XnTx, n = 1, 2, 3, where M is a transition metal element, X is C or N, and Tx is a surface oxygenated functional group (e.g., -F, -OH, -O-) [25].These oxygenated functional groups have promising applications for redox mechanisms in the field of electrochemistry [26,27].MXene has attracted wide interest due to its excellent metal conductivity, hydrophilicity, and high specific surface area [28].However, MXene also exhibits some limitations, such as aggregation between individual layers and weak stability [29].These reasons lead to the underutilization of oxygen-containing functional groups, which results in poor electrochemical performance.Therefore, a mixture of MXene and graphene was employed to form a 3D porous structure through hydrothermal reduction.This approach effectively mitigates the stacking issues associated with both MXene and graphene, thereby enhancing the stability of MXene while maximizing the utilization of oxygen-containing functional groups on its surface [30].In addition, the composites increase the electrochemical surface area and accelerate the electron transfer, which improves its electrochemical performance [31,32].Zhao et al. developed a novel 3D Prussian blue-doped reduced graphene oxide/MXene composite aerogel (3D PB/MGA) from rGO and MXene.The 3D PB/MGA was immobilized on a glassy carbon electrode (GCE) to construct a non-enzymatic sensor for H 2 O 2 [33].
MWCNTs possess remarkable characteristics, including a substantial specific surface area and a high electron transport capacity [34,35].The incorporation of carbon nanotubes into graphene structures effectively mitigates the aggregation of individual graphene sheets and significantly enhances their electrochemical sensor performance [24].After the incorporation of MWCNTs into MXene, they become entangled through π-π interactions, resulting in a rougher film surface and thereby enhancing the electroactive area of the composite [36].Zhang et al. developed a H 2 O 2 sensor with a wide linear range (0.05-18 mM) using MWCNTs, Ti 3 C 2 T x , and Pd [37].
The MCF−7 cell line is extensively employed in breast cancer research due to its wellestablished suitability as a model system for studying this disease [38].The 4T1 cell line was frequently utilized to establish a murine model of breast cancer.Disease progression in the 4T1 mouse model closely mimicked clinical manifestations of advanced malignant breast cancer in humans [39].N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) is a typical representative of the chemokine N-formylated oligopeptide family.The binding of fMLP to specific receptors on the cell surface can initiate the activation of intracellular signaling pathways, resulting in cellular activation and subsequent generation of H 2 O 2 [40].Catalase is capable of efficiently scavenging H 2 O 2 generated by malignant tissues.
In this study, 3D rGO immobilized MXene and MWCNTs electrode was structured for high sensitivity and selectivity detection of H 2 O 2 (Scheme 1).The stability, interference immunity, and measurement repeatability were investigated.MCF−7 and 4T1 cells, which generate H 2 O 2 endogenously under stimulation by fMLP, were chosen as model cells to demonstrate the electrochemical detection ability of the 3D rGO-Ti 3 C 2 -MWCNTs electrode.Finally, real-time continuous monitoring of H 2 O 2 in mouse breast cancer tissue was achieved.

Preparation of the 3D rGO-Ti 3 C 2 -MWCNTs Electrode
The 3D rGO-Ti 3 C 2 -MWCNTs electrode was prepared using a simple one-step hydrothermal method.First, a mixture of GO (0.4 mg mL −1 ), Ti 3 C 2 (0.4 mg mL −1 ), and MWCNTs (0.4 mg mL −1 ) was ultrasonically dispersed for 2 h.Second, the resulting suspension (2 mL) was injected into a 5 mL Teflon-lined autoclave suspended with copper wire (diameter 0.2 mm) and maintained at 180 • C for 4 h.After hydrothermal reduction, 3D rGO-Ti 3 C 2 -MWCNTs hydrogel film was modified on copper wire.The modified electrode was dried at room temperature.Finally, insulating wax was coated on the side of the electrode cylinder to form a disk electrode.The 3D rGO, rGO-Ti 3 C 2 , and rGO-MWCNTs electrodes were prepared using the same method.

Preparation of the 3D rGO-Ti3C2-MWCNTs Electrode
The 3D rGO-Ti3C2-MWCNTs electrode was prepared using a simple one-step h thermal method.First, a mixture of GO (0.4 mg mL −1 ), Ti3C2 (0.4 mg mL −1 ), and MW (0.4 mg mL −1 ) was ultrasonically dispersed for 2 h.Second, the resulting suspension ( was injected into a 5 mL Teflon-lined autoclave suspended with copper wire (diamet mm) and maintained at 180 °C for 4 h.After hydrothermal reduction, 3D rGO-T MWCNTs hydrogel film was modified on copper wire.The modified electrode was at room temperature.Finally, insulating wax was coated on the side of the electrod inder to form a disk electrode.The 3D rGO, rGO-Ti3C2, and rGO-MWCNTs elect were prepared using the same method.
Scheme 1. Fabrication of 3D rGO-Ti3C2-MWCNTs and the detection of H2O2 released from cells and tissue.

Electrochemical Characterization
Electrochemical tests were carried out on an electrochemical workstation (CH Shanghai, CH Instruments Ins.Shanghai, China).All electrochemical measurements performed using the three-electrode system.Pt electrode, Ag/AgCl electrode, an rGO-Ti3C2-MWCNTs electrode were used as the counter electrode, reference elect and working electrode, respectively.Origin 2021 software (Origin 9.1; OriginLab, N ampton, MA, USA) was used to analyze all the measurement data.Scanning electro croscopy (SEM) (Nova 450; FEI Inc., Eindhoven, Holland) was used to investigate the phology of the 3D rGO-Ti3C2-MWCNTs electrode.X-ray photoelectron spectro

Electrochemical Characterization
Electrochemical tests were carried out on an electrochemical workstation (CHI660e, Shanghai, CH Instruments Ins.Shanghai, China).All electrochemical measurements were performed using the three-electrode system.Pt electrode, Ag/AgCl electrode, and 3D rGO-Ti 3 C 2 -MWCNTs electrode were used as the counter electrode, reference electrode, and working electrode, respectively.Origin 2021 software (Origin 9.1; OriginLab, Northampton, MA, USA) was used to analyze all the measurement data.Scanning electron microscopy (SEM) (Nova 450; FEI Inc., Eindhoven, Holland) was used to investigate the morphology of the 3D rGO-Ti 3 C 2 -MWCNTs electrode.X-ray photoelectron spectroscopy (XPS, Thermo Fisher Scientific ESCALAB Xi+, Waltham, MA, USA) was used to characterize the elemental compositions of the modified electrode.The phase structure of the modified electrode was characterized via X-ray diffraction (XRD, Rigaku SmartLab, Tokyo, Japan).

Cell Culture and Animals
Human breast cancer cell line MCF−7 and mouse breast cancer cell line 4T1 were purchased from Servicebio Technology Co., Ltd.(Wuhan, China).Cells were cultured in Dulbecco's modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS) complemented with 100 U mL −1 penicillin and 100 mg mL −1 streptomycin and grown in a cell culture incubator with 5% CO 2 and 37 • C. The cells were washed with 0.01 M phosphate-buffered saline (PBS) after density reached 80−90%.Then, 1mL trypsin-EDTA (0.25%) was added to dislodge cells from the bottom of the dish.The number of cells was counted by a hemocytometer.The collected cells were resuspended in a deoxygenated 0.01 M PBS solution.
The female BALB/c mice (5-6 weeks) were purchased from Vital River Laboratory Animal Technology Co., Ltd.(Beijing, China).The experimental protocol was approved by the Animal Experimentation Committee of Xinxiang Medical College and conducted in accordance with international ethical standards for the use of experimental animals.The experimental mice were kept in an animal room with suitable temperature and humidity, alternating between light and dark for 12 h.During the breeding process, we ensured that sufficient water and food were provided for experimental mice.According to animal ethics, the tumor load of experimental mice should not exceed 4000 cubic millimeters.We cultured tumors up to 500 cubic millimeters to minimize the suffering of experimental mice.We measured the tumor volume of the experimental mice twice a week.When tumor volume reached 500 cubic millimeters, the mice were euthanized.The number of experimental mice used and the suffering experienced were minimized as much as possible throughout the experiment.All mice were kept in a specific pathogen-free (SPF) grade laboratory animal house at the Stem Cell Experiment Center of Xinxiang Medical University.All experimental animals were operated in accordance with the animal ethics and guidelines of the Laboratory Animal Center of Xinxiang Medical University.In total, 1.0 × 10 6 4T1 cells were injected subcutaneously into the lateral thigh of BALB/c mice.The experimental mice were cultured for 2−3 weeks in a pathogen-free animal house.In about two weeks, cancer tissues that increased to 500 cubic millimeters were separated.The cancer tissues were washed three times with 0.01 M PBS solution and soaked in 5 mL of deoxygenated 0.01 M PBS solution for electrochemical detection.

Detection of H 2 O 2 in Live Cells and Tissue
Amperometric detection of H 2 O 2 release from cells and tissue was performed with the 3D rGO-Ti 3 C 2 -MWCNTs electrode.After a steady background was obtained, 50 µL fMLP (1 mg mL −1 ) was injected into the buffer.In 100 s, 50 µL catalase (500 U mL −1 ) was injected.The response currents were recorded under an applied potential of −0.25 V.

Physical Characterization
The electrode surfaces of 3D rGO, rGO-Ti 3 C 2 , rGO-MWCNTs, and rGO-Ti 3 C 2 -MWCNTs were characterized by SEM.The rGO electrode surface exhibited a typical wrinkled surface morphology on the scale of 1 µm (Figure 1A).The formation of rGO with a nanosheet structure was achieved through hydrothermal reduction of GO [41].rGOs can easily aggregate because of the strong van der Waals forces and π-π stacking interactions [42].Hydrothermal reduction resulted in the formation of 3D rGO-Ti 3 C 2 with numerous surface pores and effectively prevented rGO stacking [26,27].The entanglement of MWCNTs on rGO nanosheets was achieved through the hydrothermal reduction method.Thus, the surface of 3D rGO-MWCNTs became rougher compared to that of 3D rGO (Figure 1C) [42].The introduction of Ti 3 C 2 and MWCNTs into the GO solution simultaneously resulted in an increased surface roughness and wrinkling of the 3D rGO-Ti 3 C 2 -MWCNTs electrode.The results suggested that the presence of Ti 3 C 2 and MWCNTs promoted analyte aggregation, enhanced active sites, and improved electrocatalytic activity.In addition, XRD and XPS characterizations are presented in Figure S1.
Thus, the surface of 3D rGO-MWCNTs became rougher compared to that of 3D rGO (Figure 1C) [42].The introduction of Ti3C2 and MWCNTs into the GO solution simultaneously resulted in an increased surface roughness and wrinkling of the 3D rGO-Ti3C2-MWCNTs electrode.The results suggested that the presence of Ti3C2 and MWCNTs promoted analyte aggregation, enhanced active sites, and improved electrocatalytic activity.In addition, XRD and XPS characterizations are presented in Figure S1.

Direct Electrochemical Behavior of the 3D rGO-Ti3C2-MWCNTs Electrode
Cyclic voltammetry (CV) analyses were carried out in a mixture of 5 mM K3[Fe(CN)6] and 0.1 M KCl to characterize the electron transfer process.The peak-to-peak separation (ΔEp) between oxidation peak potential and reduction peak potential responds to the degree of reversibility of the electrochemical reaction.The smaller ΔEp indicated a greater reversibility degree, higher electron transfer efficiency, and more practical application value [43].ΔEp of 3D rGO, rGO-Ti3C2, rGO-MWCNTs, and rGO-Ti3C2-MWCNTs electrodes were 192 mV, 132 mV, 140 mV, and 112 mV, respectively, at scan rates of 100 mV s −1 .Compared to the 3D rGO electrode, 3D rGO-Ti3C2 and rGO-MWCNTs electrodes had significantly increased peak current and decreased ΔEp (Figure 2A).The 3D rGO-Ti3C2-MWCNTs electrode exhibited the largest peak current and the smallest ΔEp.The results indicate that Ti3C2 and MWCNTs effectively increased the electron transfer efficiency of the modified electrode.In addition, the reaction kinetics of the 3D rGO-Ti3C2-MWCNTs electrode was investigated.The peak current gradually enhanced with an increasing scan rate (Figure 2B).Both oxidation and reduction peak currents against the square root of the scan rate showed a good linear relationship (inset of Figure 2B), indicating that the catalytic process was a diffusion-controlled process [26].

Direct Electrochemical Behavior of the 3D rGO-Ti 3 C 2 -MWCNTs Electrode
Cyclic voltammetry (CV) analyses were carried out in a mixture of 5 mM K 3 [Fe(CN) 6 ] and 0.1 M KCl to characterize the electron transfer process.The peak-to-peak separation (∆Ep) between oxidation peak potential and reduction peak potential responds to the degree of reversibility of the electrochemical reaction.The smaller ∆Ep indicated a greater reversibility degree, higher electron transfer efficiency, and more practical application value [43].∆Ep of 3D rGO, rGO-Ti 3 C 2 , rGO-MWCNTs, and rGO-Ti 3 C 2 -MWCNTs electrodes were 192 mV, 132 mV, 140 mV, and 112 mV, respectively, at scan rates of 100 mV s −1 .Compared to the 3D rGO electrode, 3D rGO-Ti 3 C 2 and rGO-MWCNTs electrodes had significantly increased peak current and decreased ∆Ep (Figure 2A).The 3D rGO-Ti 3 C 2 -MWCNTs electrode exhibited the largest peak current and the smallest ∆Ep.
The results indicate that Ti 3 C 2 and MWCNTs effectively increased the electron transfer efficiency of the modified electrode.In addition, the reaction kinetics of the 3D rGO-Ti 3 C 2 -MWCNTs electrode was investigated.The peak current gradually enhanced with an increasing scan rate (Figure 2B).Both oxidation and reduction peak currents against the square root of the scan rate showed a good linear relationship (inset of Figure 2B), indicating that the catalytic process was a diffusion-controlled process [26].
The effective surface area of 3D rGO, rGO-Ti 3 C 2 , rGO-MWCNTs, and rGO-Ti 3 C 2 -MWCNTs was calculated using the Randles-Sevcik equation as follows: where Ip represents the peak current; A eff is the effective surface area; n is the number of electrons transferred between the electrode and electrolyte, which is equal to 1; D 0 is the diffusion coefficient in the solution (D 0 = 0.673 × 10 −5 cm 2 s −1 ); v is the scan rate; and C 0 is the concentration of the analyte (C 0 = 5 × 10 −6 mol cm −3 ).Based on this equation, the electroactive surface area of 3D rGO, rGO-Ti 3 C 2 , rGO-MWCNTs, and rGO-Ti 3 C 2 -MWCNTs electrodes were calculated as 0.0096 cm 2 , 0.0143 cm 2 , 0.01205 cm 2 , and 0.0214 cm 2 , respectively.The results suggested that the simultaneous incorporation of Ti 3 C 2 and MWCNTs can significantly enhance the specific surface area of the modified electrode.
respectively.The results suggested that the simultaneous incorporation of Ti3C2 and MWCNTs can significantly enhance the specific surface area of the modified electrode.

Electrocatalytic Reduction of H2O2 by the 3D rGO-Ti3C2-MWCNTs Electrode
The electrocatalytic properties of the modified electrode toward H2O2 reduction were first investigated by CVs.With the gradual increase in H2O2, the cathodic currents of 3D rGO, rGO-Ti3C2, rGO-MWCNTs, and rGO-Ti3C2-MWCNTs electrodes showed an obvious increase, indicating that H2O2 was reduced on the modified electrode (Figure S2).The introduction of Ti3C2 and MWCNTs results in anodic steps for rGO-Ti3C2, rGO-MWCNTs, and rGO-Ti3C2-MWCNTs electrodes (Figure S4).No significant cathode reduction peaks were observed on 3D rGO and rGO-Ti3C2 electrodes.Significant H2O2 reduction peaks at −0.37 V and −0.25 V could be observed on 3D rGO-MWCNTs and rGO-Ti3C2-MWCNTs, respectively (Figure 3).Increased reduction current and reduced reduction potential indicated that the 3D rGO-Ti3C2-MWCNTs electrode could provide an ideal performance for reduction-based H2O2 detection.

Amperometric Performance of the 3D rGO-Ti3C2-MWCNTs Electrode
The amperometric response of the 3D rGO-Ti3C2-MWCNTs electrode was recorded (Figure 4A).Three to five replicate tests were performed at each experimental point to

Electrocatalytic Reduction of H 2 O 2 by the 3D rGO-Ti 3 C 2 -MWCNTs Electrode
The electrocatalytic properties of the modified electrode toward H 2 O 2 reduction were first investigated by CVs.With the gradual increase in H 2 O 2 , the cathodic currents of 3D rGO, rGO-Ti 3 C 2 , rGO-MWCNTs, and rGO-Ti 3 C 2 -MWCNTs electrodes showed an obvious increase, indicating that H 2 O 2 was reduced on the modified electrode (Figure S2).The introduction of Ti 3 C 2 and MWCNTs results in anodic steps for rGO-Ti 3 C 2 , rGO-MWCNTs, and rGO-Ti 3 C 2 -MWCNTs electrodes (Figure S4).No significant cathode reduction peaks were observed on 3D rGO and rGO-Ti 3 C 2 electrodes.Significant H 2 O 2 reduction peaks at −0.37 V and −0.25 V could be observed on 3D rGO-MWCNTs and rGO-Ti 3 C 2 -MWCNTs, respectively (Figure 3).Increased reduction current and reduced reduction potential indicated that the 3D rGO-Ti 3 C 2 -MWCNTs electrode could provide an ideal performance for reduction-based H 2 O 2 detection.
respectively.The results suggested that the simultaneous incorporation of Ti3C2 and MWCNTs can significantly enhance the specific surface area of the modified electrode.

Electrocatalytic Reduction of H2O2 by the 3D rGO-Ti3C2-MWCNTs Electrode
The electrocatalytic properties of the modified electrode toward H2O2 reduction were first investigated by CVs.With the gradual increase in H2O2, the cathodic currents of 3D rGO, rGO-Ti3C2, rGO-MWCNTs, and rGO-Ti3C2-MWCNTs electrodes showed an obvious increase, indicating that H2O2 was reduced on the modified electrode (Figure S2).The introduction of Ti3C2 and MWCNTs results in anodic steps for rGO-Ti3C2, rGO-MWCNTs, and rGO-Ti3C2-MWCNTs electrodes (Figure S4).No significant cathode reduction peaks were observed on 3D rGO and rGO-Ti3C2 electrodes.Significant H2O2 reduction peaks at −0.37 V and −0.25 V could be observed on 3D rGO-MWCNTs and rGO-Ti3C2-MWCNTs, respectively (Figure 3).Increased reduction current and reduced reduction potential indicated that the 3D rGO-Ti3C2-MWCNTs electrode could provide an ideal performance for reduction-based H2O2 detection.

Amperometric Performance of the 3D rGO-Ti3C2-MWCNTs Electrode
The amperometric response of the 3D rGO-Ti3C2-MWCNTs electrode was recorded (Figure 4A).Three to five replicate tests were performed at each experimental point to

Amperometric Performance of the 3D rGO-Ti 3 C 2 -MWCNTs Electrode
The amperometric response of the 3D rGO-Ti 3 C 2 -MWCNTs electrode was recorded (Figure 4A).Three to five replicate tests were performed at each experimental point to obtain error bars.The current response of the 3D rGO-Ti 3 C 2 -MWCNTs electrode showed good linearity in the H 2 O 2 concentration ranges of 1 µM−60 µM (R 2 = 0.998) and 60 µM−9.77mM (R 2 = 0.992), with sensitivities of 235.2 µA mM −1 cm −2 and 103.8 µA mM −1 cm −2 , respectively, and a limit of detection of 0.3 µM (S/N = 3) (Figure 4B).Further, a comparison of the 3D rGO-Ti 3 C 2 -MWCNTs electrode with other H 2 O 2 sensors is summarized in Table 1.Compared with the other literature, the 3D rGO-Ti 3 C 2 -MWCNTs electrode had the advantage of a low detection limit.

Repeatability, Stability, and Selectivity of the 3D rGO-Ti3C2-MWCNTs Electrode
The reproducibility of the 3D rGO-Ti3C2-MWCNTs electrode was also investigat The responses were very similar for all five electrodes (Figure 5A).The electrode-to-e trode reproducibility was characterized by the low relative standard deviation (RSD 3.08% (n = 5) in response to 100 µM H2O2.The RSD of 1.61% (n = 3) for 100 µM H2O2 us the same electrode demonstrated good intra-electrode reproducibility.The long-term bility of the 3D rGO-Ti3C2-MWCNTs electrode was demonstrated by periodically reco ing the amperometric response to H2O2 (Figure 5C).The reduction current to H2O2 w

Repeatability, Stability, and Selectivity of the 3D rGO-Ti 3 C 2 -MWCNTs Electrode
The reproducibility of the 3D rGO-Ti 3 C 2 -MWCNTs electrode was also investigated.The responses were very similar for all five electrodes (Figure 5A).The electrode-toelectrode reproducibility was characterized by the low relative standard deviation (RSD) of 3.08% (n = 5) in response to 100 µM H 2 O 2 .The RSD of 1.61% (n = 3) for 100 µM H 2 O 2 using the same electrode demonstrated good intra-electrode reproducibility.The longterm stability of the 3D rGO-Ti 3 C 2 -MWCNTs electrode was demonstrated by periodically recording the amperometric response to H 2 O 2 (Figure 5C).The reduction current to H 2 O 2 was maintained at 95.49% after 7 days and 88.14% after 14 days (Figure 5D).This result indicated that 3D rGO-Ti 3 C 2 -MWCNTs had excellent stability.
Immunity to interference was a very important factor for in vivo detection.Several common interferences, such as UA, AA, DA, and glucose (Glu), were added during the detection of H 2 O 2 .The current response of the 3D rGO-Ti 3 C 2 -MWCNTs electrode was recorded upon successive additions of 100 µM H 2 O 2 , 200 µM interfering species, and then 100 µM H 2 O 2 again in the deoxygenated 0.01 M PBS solution.The 3D rGO-Ti 3 C 2 -MWCNTs electrode produced almost no current response when the interfering substance was added, while the current response to H 2 O 2 was unaffected (Figure 6).The results indicated that the 3D rGO-Ti 3 C 2 -MWCNTs electrode had high interference resistance.
Biosensors 2024, 14, x 8 of 13 maintained at 95.49% after 7 days and 88.14% after 14 days (Figure 5D).This result indicated that 3D rGO-Ti3C2-MWCNTs had excellent stability.Immunity to interference was a very important factor for in vivo detection.Several common interferences, such as UA, AA, DA, and glucose (Glu), were added during the detection of H2O2.The current response of the 3D rGO-Ti3C2-MWCNTs electrode was recorded upon successive additions of 100 µM H2O2, 200 µM interfering species, and then 100 µM H2O2 again in the deoxygenated 0.01 M PBS solution.The 3D rGO-Ti3C2-MWCNTs electrode produced almost no current response when the interfering substance was added, while the current response to H2O2 was unaffected (Figure 6).The results indicated that the 3D rGO-Ti3C2-MWCNTs electrode had high interference resistance.Immunity to interference was a very important factor for in vivo detection common interferences, such as UA, AA, DA, and glucose (Glu), were added du detection of H2O2.The current response of the 3D rGO-Ti3C2-MWCNTs electr recorded upon successive additions of 100 µM H2O2, 200 µM interfering species, 100 µM H2O2 again in the deoxygenated 0.01 M PBS solution.The 3D rG MWCNTs electrode produced almost no current response when the interfering s was added, while the current response to H2O2 was unaffected (Figure 6).The r dicated that the 3D rGO-Ti3C2-MWCNTs electrode had high interference resista

Ex Vivo Experimental Analysis
The 3D rGO-Ti 3 C 2 -MWCNTs electrode was used for real-time in vitro detection of H 2 O 2 in cells.MCF−7 and 4T1 cells were used as model cells to test H 2 O 2 release upon fMLP stimulation in real time [52][53][54].Normal cells can produce H 2 O 2 at concentrations of up to 0.05~0.7 µM in a physiological environment.In breast cancer, H 2 O 2 production is several times higher than in normal cells [46,55].The deoxygenated 0.01 M PBS containing 4T1 or MCF−7 cells was supplemented with 50 µL of fMLP (1 mg mL −1 ).The amperometric current of 4T1 cells exhibited a significant increase of 20.5 nA, corresponding to the production of 2.4 µM H 2 O 2 calculated by the calibration curve in Figure 4B.Similarly, MCF−7 cells demonstrated a notable rise in amperometric current by 25.4 nA, indicating the generation of 3.4 µM H 2 O 2 .The current significantly decreased with catalase injection.As controls, the currents were also recorded following the injection of fMLP-free PBS and the injection of fMLP into PBS without cells.This phenomenon suggested that the reduction current increase was caused by H 2 O 2 released from cancer cells.The same amount of fMLP was added to EPH4−EV cells (normal cells).As shown in Figure S6, H 2 O 2 produced by normal cells was much lower than that produced by cancer cells.These results indicated that 3D rGO-Ti 3 C 2 -MWCNTs electrodes can effectively discriminate between normal cells and tumor cells in order to detect cancer.
Subsequently, the real-time monitoring of H 2 O 2 in mouse cancer tissue was successfully achieved through the utilization of the 3D rGO-Ti 3 C 2 -MWCNTs electrode.The modified electrode was inserted inside the breast cancer tissue, and a significant current increase was detected after fMLP injection.Then, the reduction current response gradually decreased with catalase injection (Figure 7D).The amperometric current of mouse cancer tissue exhibited a significant increase of 19.8 ± 3.2 nA, corresponding to the production of 2.3 µM H 2 O 2 calculated by the calibration curve in Figure 4B.As controls, the injection of fMLP-free PBS into tissue cultures did not result in any observed change.The findings indicated that breast cancer tissue produced H 2 O 2 upon fMLP stimulation and subsequently eliminated it through the addition of catalase.Therefore, the 3D rGO-Ti 3 C 2 −MWCNT electrode provided an effective idea for the ex vivo detection of tumor biomarkers with practical applications.

Conclusions
A 3D rGO-Ti3C2-MWCNTs electrode was fabricated through hydrothermal reduction and utilized for the detection of H2O2.The modified electrode was successfully utilized for real-time monitoring of the release of H2O2 from cancer cells and tissue.The experimental data demonstrated a significant enhancement in the electrocatalytic activity of  Informed Consent Statement: Not applicable.

Scheme 1 .
Scheme 1. Fabrication of 3D rGO-Ti 3 C 2 -MWCNTs and the detection of H 2 O 2 released from cancer cells and tissue.

A
3D rGO-Ti 3 C 2 -MWCNTs electrode was fabricated through hydrothermal reduction and utilized for the detection of H 2 O 2 .The modified electrode was successfully utilized for real-time monitoring of the release of H 2 O 2 from cancer cells and tissue.The experimental data demonstrated a significant enhancement in the electrocatalytic activity of the 3D rGO-Ti 3 C 2 -MWCNTs electrode to H 2 O 2 following the simultaneous incorporation of Ti 3 C 2 and MWCNTs into rGO.In addition, the rGO-Ti 3 C 2 -MWCNTs electrode exhibited excellent selectivity, stability, and reproducibility in the detection of H 2 O 2 .The ex vivo detection of H 2 O 2 in cancer tissue was conducted, which demonstrated exceptional current response.The present study introduces a novel approach to histological examination of cancerous tissue post-biopsy, thereby providing the medical community with innovative insights into early cancer detection.Supplementary Materials:The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/bios14060261/s1,FigureS1: XRD pattern (A) and XPS spectra (B) of 3D rGO, rGO-Ti 3 C 2 , rGO-MWCNTs, and rGO-Ti 3 C 2 -MWCNTs; Figure S2: CV of (A) 3D rGO, (B) 3D rGO-Ti 3 C 2 , (C) 3D rGO-MWCNTs, and (D) 3D rGO-Ti 3 C 2 -MWCNTs electrodes at different H 2 O 2 concentrations (0-10 mM) in deoxygenated 0.01 M PBS.Scan rate: 100 mV s −1 .Potential range: −0.6-0.2V; Figure S3: Calibration curves of 3D rGO, rGO-Ti 3 C 2 , rGO-MWCNTs, and rGO-Ti 3 C 2 -MWCNTs electrodes currents vs. H 2 O 2 concentration; Figure S4: CV of 3D rGO, rGO-Ti 3 C 2 , rGO-MWCNTs, and rGO-Ti 3 C 2 -MWCNTs electrodes in deoxygenated 0.01 M PBS in the presence and absence of 4 mM H 2 O 2 ; Figure S5: (A) Current response of 3D rGO-Ti 3 C 2 -MWCNTs electrode to 100 µM H 2 O 2 in deoxygenated 0.01M PBS (pH 7.0) at different potentials.(B) Influence of applied potential on amperometric response of the biosensor.(C) Change in amperometric current of the electrode against 100 µM H 2 O 2 at different pH values of PBS.(D) Dependence of the current response of 3D rGO-Ti 3 C 2 -MWCNTs electrode to 100 µM H 2 O 2 on the pH of buffer solutions at an applied potential of −0.25 V vs. Ag/AgCl; Figure S6: Reduction current response of the 3D rGO-Ti 3 C 2 -MWCNTs electrode with addition of fMLP to deoxygenated 0.01 M PBS solution containing (A) 4T1, (B) MCF−7 and (C) EPH4−EV cells.(D) EPH4−EV, 4T1 and MCF−7 cells produced hydrogen peroxide concentrations by addition of the same amount of fMLP.References [11,56-59] are cited in the supplementary materials.Author Contributions: Analysis of Data and Writing-Original Draft Preparation, S.-Q.Y.; Methodology, P.L.; Validation, H.-J.L.; Data Curation, L.-J.S.; Investigation, R.G.; Supervision, X.-M.S.; Supervision, Project Administration, and Writing-Review and Editing, Q.-Q.R.All authors have read and agreed to the published version of the manuscript.Funding: This work was supported by the Natural Science Foundation of Henan Province [grant numbers 22A416011 and 22A416010], the Open Program of Henan Key Laboratory of Biological Psychiatry [program number ZDSYS2021008], and Science and Technology Tackling of Henan [grant number 242102310006].Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of Xinxiang Medical University (XYLL-20230001, 9 February 2023).

Table 1 .
Table of comparison of the 3D rGO-Ti 3 C 2 -MWCNTs electrode with other H 2 O 2 sensors.