A Non-Enzymatic Sensor Based on Trimetallic Nanoalloy with Poly (Diallyldimethylammonium Chloride)-Capped Reduced Graphene Oxide for Dynamic Monitoring Hydrogen Peroxide Production by Cancerous Cells

Catching cancer at an early stage is necessary to make it easier to treat and to save people’s lives rather than just extending them. Reactive oxygen species (ROS) have sparked a huge interest owing to their vital role in various biological processes, especially in tumorigenesis, thus leading to the potential of ROS as prognostic biomarkers for cancer. Herein, a non-enzymatic biosensor for the dynamic monitoring of intracellular hydrogen peroxide (H2O2), the most important ROS, via an effective electrode composed of poly (diallyldimethylammonium chloride) (PDDA)-capped reduced graphene oxide (RGO) nanosheets with high loading trimetallic AuPtAg nanoalloy, is proposed. The designed biosensor was able to measure H2O2 released from different cancerous cells promptly and precisely owing to the impressive conductivity of RGO and PDDA and the excellent synergistic effect of the ternary alloy in boosting the electrocatalytic activity. Built upon the peroxidase-like activity of the nanoalloy, the developed sensor exhibited distinguished electrochemical performance, resulting in a low detection limit of 1.2 nM and a wide linear range from 0.05 μM to 5.5 mM. Our approach offers a significant contribution toward the further elucidation of the role of ROS in carcinogenesis and the effective screening of cancer at an early stage.


Introduction
Cancer is a global issue and has a major impact on public health. It is estimated that there were 18.1 million new cancer cases and 9.6 million deaths from cancer in 2018 around the world [1]. As the technology evolves, researchers are focusing on ways to detect, treat, and prevent cancer earlier to level off or decrease the incidence of and mortality due to cancer. Although cancer remains one of the leading causes of death, diagnosing it early offers the opportunity to improve survival rates [2]. The broader application of advanced techniques like X-ray imaging, computed tomography (CT), positron emission tomography (PET), and biopsy in cancer diagnosis has accelerated progress against cancer [3][4][5]. However, the accuracy of such typical methods is reduced if carcinogens are present at the very beginning. The sooner cancer is diagnosed, the higher the survival rate will be. Therefore, if identifying precancerous lesions or tumors in the early initiation phase can be accomplished, there is more chance of administering more precise therapies. Scheme 1. Schematic illustration for the preparation of poly (diallyldimethylammonium chloride) (PDDA)-AuPtAg/reduced graphene oxide (RGO)-modified glassy carbon electrode (GCE) applied for the detection of H 2 O 2 released from SKOV3, MCF-7, and A431 cells stimulated with phorbol-12-myristate-13-acetate (PMA). ROS, reactive oxygen species.
All electrochemical measurements were conducted using 283 Potentiostat-Galvanostat electrochemical workstation (EG&GPARC with M270 software) with an ordinary triple-electrode system. It consisted of an Ag/AgCl electrode saturated with KCl, a platinum wire, and a modified glassy carbon electrode as the reference, counter, and adopted working electrode, respectively. All experiments were performed at room temperature. In addition, transmission electron microscopy (TEM) image analysis was obtained from Tecnai G2 F20 instrument (Philips Holland), while an energy-dispersive X-ray spectroscopy (EDX) analyzer on the Tecnai G2 F20 carried out the EDX spectrum and mapping analysis. The X-ray diffraction (XRD) patterns were performed on Rigaku D/max-rA (Rigaku, Japan) using Cu Kα radiation (λ = 1.5418 Å).

Preparation of Modified Electrodes
The glassy carbon (GC) electrode was polished with 0.3 and 0.05 µm α-alumia powder before modification, and then washed by double distilled water and ethanol, respectively, followed by drying with high-purity nitrogen stream. Then, 10 mg of PDDA-AuPtAg/RGO was re-dispersed in 10 mL of double distilled water with sonication for 2 h, after which 6 µL of the suspension was immobilized on the surface of the GC electrode by dip-coating and dried in air.

Real-Time Detection of H 2 O 2 Released From Living Cells
The cells tested in this work were SKOV3 cells (human ovarian cancer cell line), MCF-7 cells (breast cancer cell line), and A431 cells (epidermoid carcinoma cell line) obtained from School of Basic Medical Science, Tianjin Medical University, Tianjin, China (come from ATCC). SKOV3 cells and A431 cells were maintained in McCoy's 5A (Modified) Medium and DMEM containing 10% fetal bovine serum, respectively. For MCF-7 cells, they were maintained in Dulbecco's modified essential medium (DMEM) containing 10% fetal bovine serum, 0.01 mg/ml human recombinant insulin, 100 units/mL penicillin, and 100 µg/mL streptomycin. All the cells were incubated under ambient conditions with 37 • C, 5% CO 2 , 95% humidified atmosphere, and sub-cultured after every second day.
After sub-cultured to 90% confluence, the cells were washed three times with phosphate-buffered saline (PBS) (0.1 M, pH 7.0) and collected by centrifugation. The cell number was calculated by using a cell counter. A total of 1 × 106 cells were resuspended into 10 mL 0.1 M PBS (pH 7.0) as cell samples and the control group containing catalases (300 U/mL) was also prepared. For electrochemical measurements, injection of 10 µL PMA (1 µg mL −1 ) was applied into the tested system and current responses on the PDDA-AuPtAg/RGO/GCE were recorded at the potential of 0.13 V, as shown in Scheme 1.

Characterization of PDDA-AuPtAg/RGO Nanocomposites
To identify the morphology of the as-prepared nanocomposites, transmission electron microscopy (TEM) was used for structural analysis. In Figure 1A, a typical wrinkled pattern of reduced graphene oxide sheets can be observed. Under different magnification ( Figure 1B,C), it can be clearly seen that AuPtAg alloy nanoparticles (as pointed out in Figure 2B) distributed well on the PDDA-RGO sheets with less aggregation owing to excellent dispersing performance of poly dimethyl diallyl ammonium chloride (PDDA). The PDDA-AuPtAg alloy nanocrystals displayed paralleled lattice fringes along the same orientation ( Figure 1D), indicating there were single AuPtAg nanoalloy particles locating on the PDDA-capped reduced graphene oxide layers. A TEM elemental mapping analysis (Figure 2A-F) and energy dispersive X-ray (EDX) spectroscopy ( Figure S2) were conducted to verify the components of the PDDA-AuPtAg/RGO composites. The results indicate that the obtained nanocomposites consisted of C, O, Au, Pt, and Ag. The C and O elements were evenly distributed as a film, while Au, Pt, and Ag were relatively concentrated, implying the formation of a trimetallic alloy. Figure 2G shows X-ray diffraction (XRD) spectra of GO, PDDA-Pt/RGO, PDDA-Ag/RGO, PDDA-AuAg/RGO, and PDDA-AuPtAg/RGO. GO presented a typical XRD diffraction pattern with a sharp peak at around 10° and a wider peak at 23.99°. The peak at 10° disappeared with the formation of the various nanocomposites, thus indicating that GO was reduced to RGO [38]. The diffraction peaks at around 39.8°, 46.3°, and 67.8° for PDDA-Pt/RGO were associated with the (111), (200), and (220) facets of the Pt nanoparticles, respectively [39,40]. In addition, peaks at 38.1°, 44.1°, 64.4°, and 77.3° for PDDA-Ag/RGO were attributed to the reflections on the (111) (200), (220), and (311) planes of Ag, respectively. Meanwhile, the characteristic peaks at 38.3°, 44.3°, 64.9°, and 77.69° for the bimetallic PDDA-AuAg/RGO were indexed to diffraction peaks of the (111), (200), (220), and (311) lattice planes of Au, respectively, according to Bragg's reflection with no obvious diffraction peaks of silver, indicating that the surfaces of the Ag nanoparticles were replaced with Au, which led to the formation of a core-shell structure [41]. Compared with its counterparts, all of the peaks in the diffraction pattern for PDDA- A TEM elemental mapping analysis (Figure 2A-F) and energy dispersive X-ray (EDX) spectroscopy ( Figure S2) were conducted to verify the components of the PDDA-AuPtAg/RGO composites. The results indicate that the obtained nanocomposites consisted of C, O, Au, Pt, and Ag. The C and O elements were evenly distributed as a film, while Au, Pt, and Ag were relatively concentrated, implying the formation of a trimetallic alloy. Figure 2G shows X-ray diffraction (XRD) spectra of GO, PDDA-Pt/RGO, PDDA-Ag/RGO, PDDA-AuAg/RGO, and PDDA-AuPtAg/RGO. GO presented a typical XRD diffraction pattern with a sharp peak at around 10 • and a wider peak at 23.99 • . The peak at 10 • disappeared with the formation of the various nanocomposites, thus indicating that GO was reduced to RGO [38]. The diffraction peaks at around 39. and (311) lattice planes of Au, respectively, according to Bragg's reflection with no obvious diffraction peaks of silver, indicating that the surfaces of the Ag nanoparticles were replaced with Au, which led to the formation of a core-shell structure [41]. Compared with its counterparts, all of the peaks in the diffraction pattern for PDDA-AuPtAg/RGO were broader owing to the superposition of lattice planes, which suggests the formation of a trimetallic alloy composite.
AuPtAg/RGO were broader owing to the superposition of lattice planes, which suggests the formation of a trimetallic alloy composite.

Electrochemical Behavior of Obtained Materials
Electrochemical characterization using ferro/ferricyanide as the redox probe was accomplished in cyclic voltammograms (CVs) for a bare glassy carbon electrode (GCE), PDDA-AuAg/RGO, PDDA-PtAg/RGO, PDDA-AuPt/RGO, and PDDA-AuPtAg/RGO, modified GCE electrodes ( Figure 3A). PDDA-AuPtAg/RGO/GCE revealed a pair of well-defined redox peaks at around +300 mV and +172 mV. Compared with bare GCE and the modified GCE electrodes with bimetal composites, an obvious increment in the peaks was observed for PDDA-AuPtAg/RGO/GCE owing to its fast electron-transfer kinetics. To further characterize the obtained material, microscopic electroactive areas were estimated according to the Randles-Sevcik equation [42]:

Electrochemical Behavior of Obtained Materials
Electrochemical characterization using ferro/ferricyanide as the redox probe was accomplished in cyclic voltammograms (CVs) for a bare glassy carbon electrode (GCE), PDDA-AuAg/RGO, PDDA-PtAg/RGO, PDDA-AuPt/RGO, and PDDA-AuPtAg/RGO, modified GCE electrodes ( Figure 3A). PDDA-AuPtAg/RGO/GCE revealed a pair of well-defined redox peaks at around +300 mV and +172 mV. Compared with bare GCE and the modified GCE electrodes with bimetal composites, an obvious increment in the peaks was observed for PDDA-AuPtAg/RGO/GCE owing to its fast electron-transfer kinetics. To further characterize the obtained material, microscopic electroactive areas were estimated according to the Randles-Sevcik equation [42]: where I p relates to the redox peak current, A corresponds to the electroactive surface area (cm 2 ), the diffusion coefficient (D) of the molecules in solution is (6.70 ± 0.02) × 10 −6 cm 2 /s, n represents the number of electrons participating in the redox reaction (equal to 1), v is the scan rate (V/s), and c is the bulk concentration of the redox probe (mol cm 3 ). According to Equation (1), the value of A for PDDA-AuPtAg/RGO/GCE was calculated as 0.086 cm 2 , which was 1.72, 1.45, 1.34, and 1.21 times larger than unmodified GCE, PDDA-AuAg/RGO/GCE, PDDA-PtAg/RGO/GCE, and PDDA-AuPt/RGO/GCE, respectively. These results prove that the PDDA-capped trimetallic alloy with RGO nanosheets with an enlarged surface area was more applicable for accelerating the electron transfer between K 3 [Fe (CN) 6 ] and the working electrode.

Amperometric Response towards H2O2
The constructed sensor based on PDDA-AuPtAg/RGO was used to determine the H2O2 concentration via amperometric i-t curves under optimal conditions. Figure 4A shows the amperometric response toward different concentrations of H2O2. In pace with the rising concentration of H2O2, the current response increased gradually, with 95% of the steady-state current being achieved within 5 s. The calibration curve ( Figure 4B) was fitted after five independent repetitive experiments and the PDDA-AuPtAg/RGO modified GCE revealed an exceptional linear relationship between current response and H2O2 concentration in two segments: from 0.05 M to 1 mM and from 1 mM to 5.5 mM. As shown in the insert in Figure 4B, the corresponding linear equation is I = −244.15C − 7.83 (R 2 = 0.9985) with a sensitivity of 2838.95 •mM -1 •cm -2 in the range from 0.05 M to 1 mM. In the meantime, another linear relationship, I = −66.18C − 184.12 (R 2 = 0.9978), existed from 1 mM to 5.5 mM. The calculated detection limit for H2O2 was 1.2 nM at a signal-to-noise ratio of 3. These results suggest that the PDDA-AuPtAg/RGO fabricated sensor achieved a wide detection range and a low detection limit when determining H2O2 concentration, thus showing it to be a credible and powerful

Electrochemical Response to H 2 O 2 by PDDA-AuPtAg/RGO/GCE
The electrocatalytic performance of PDDA-AuPtAg/RGO/GCE toward H 2 O 2 was investigated in the presence of 5 mM H 2 O 2 , the results of which are shown in Figure 3B. A distinctly higher current response at around 0.13 V/0.72 V was attained by fabricating PDDA-AuPtAg/RGO on the GCE. In contrast, there was no response on bare GCE and the current changes of bimetal counterparts were much lower, suggesting that the trimetallic alloy composite responded well to the reduction of H 2 O 2 . The remarkable improvements of response signals could be contributed to the outstanding peroxidase-like activity of single Au, Pt, and Ag nanoparticles and the synergistic effects after integration [43][44][45]. Besides this, PDDA-RGO nanosheets also play a compelling part in boosting the performance of the proposed electrode, offering more arching sites for trimetallic alloy nanocomposites as a supporting platform. Therefore, the electrode modified with PDDA-AuPtAg/RGO exhibited excellent catalytic activity toward the reduction of H 2 O 2 .

Optimization of the Experimental Variables
As shown in Figure 3C,D, typical CV was adopted to investigate the effects of the scan rate at PDDA-AuPtAg/RGO/GCE. It can be clearly observed that the oxidation and reduction currents for H 2 O 2 increased linearly along with the rising scan rate. In addition, there was a clear linear relationship between the redox peak current and the square root of scan rate (v 1/2 ) using the regression equation Ip (µA) = −111.17υ (mV/s) + 147.84 (R 2 = 0.997), indicating a diffusion-controlled process [46]. Other experimental parameters including concentration and deposition volume of PDDA-AuPtAg/RGO and pH were also investigated to determine the optimal sensor response ( Figure S2, S3). In summary, the PDDA-AuPtAg/RGO/GCE demonstrated the best response performance toward H 2 O 2 under the following conditions: 6 µL of PDDA-AuPtAg/RGO (1 mg/mL), phosphate-buffered saline (PBS) at PH 7.0, and a scan rate of 50 mV/s.

Amperometric Response towards H 2 O 2
The constructed sensor based on PDDA-AuPtAg/RGO was used to determine the H 2 O 2 concentration via amperometric i-t curves under optimal conditions. Figure 4A shows the amperometric response toward different concentrations of H 2 O 2 . In pace with the rising concentration of H 2 O 2 , the current response increased gradually, with 95% of the steady-state current being achieved within 5 s. The calibration curve ( Figure 4B) was fitted after five independent repetitive experiments and the PDDA-AuPtAg/RGO modified GCE revealed an exceptional linear relationship between current response and H 2 O 2 concentration in two segments: from 0.05 µM to 1 mM and from 1 mM to 5.5 mM. As shown in the insert in Figure 4B, the corresponding linear equation is I = −244.15C − 7.83 (R 2 = 0.9985) with a sensitivity of 2838.95 µA·mM −1 ·cm −2 in the range from 0.05 µM to 1 mM. In the meantime, another linear relationship, I = −66.18C − 184.12 (R 2 = 0.9978), existed from 1 mM to 5.5 mM. The calculated detection limit for H 2 O 2 was 1.2 nM at a signal-to-noise ratio of 3. These results suggest that the PDDA-AuPtAg/RGO fabricated sensor achieved a wide detection range and a low detection limit when determining H 2 O 2 concentration, thus showing it to be a credible and powerful platform for monitoring cellular H 2 O 2 levels.
In addition, a comparison on the performance of our designed sensor with previously reported H 2 O 2 biosensors is summarized in Table S1, in which it can be clearly seen that the fabricated sensor based on PDDA-AuPtAg/RGO displayed noticeably improved properties, including a wider linear range and a lower detection limit for H 2 O 2 . These improvements could be attributed to the excellent synergetic effects of the trimetallic alloy and the larger working surface provided by the PDDA-capped RGO nanosheets. synergetic effects of the trimetallic alloy and the larger working surface provided by the PDDAcapped RGO nanosheets.

Interference Immunity, Repeatability, and Stability
Specificity, reproductivity, and long-term stability are important indicators when evaluating the accuracy of the designed electrochemical sensors. An anti-interference study was performed with typical probes, including ascorbic acid, glucose, uric acid, and xanthine. As shown in Figure 5, variations compared with the current changes caused by H2O2 caused by the interference were almost non-existent (less than 2%), indicating that the constructed sensor had admirable selectivity and specificity.
To investigate the stability, the fabricated sensor was tested periodically. After storage at 4 ℃ for one month, 92% of the initial current response of the as-prepared electrodes remained, illustrating acceptable long-term stability. Five electrodes were utilized under optimal conditions for exploring the reproducibility. As shown in Figure S4, favorable reproducibility can be observed with a relative standard deviation (RSD) of 2.45%.

Interference Immunity, Repeatability, and Stability
Specificity, reproductivity, and long-term stability are important indicators when evaluating the accuracy of the designed electrochemical sensors. An anti-interference study was performed with typical probes, including ascorbic acid, glucose, uric acid, and xanthine. As shown in Figure 5, variations compared with the current changes caused by H 2 O 2 caused by the interference were almost non-existent (less than 2%), indicating that the constructed sensor had admirable selectivity and specificity.
To investigate the stability, the fabricated sensor was tested periodically. After storage at 4 • C for one month, 92% of the initial current response of the as-prepared electrodes remained, illustrating acceptable long-term stability. Five electrodes were utilized under optimal conditions for exploring the reproducibility. As shown in Figure S4, favorable reproducibility can be observed with a relative standard deviation (RSD) of 2.45%. Sensors 2019, 19, x FOR PEER REVIEW 10 of 14

In Situ Monitoring of H2O2 Released From Living Cells
The excellent performance of the PDDA-AuPtAg/RGO sensor provided the opportunity to explore its application in monitoring H2O2 in living cancer cells. It is well proven that H2O2 is a stable ROS, which is of great importance in various biological processes, and many disorders like cancer result from the unbalanced metabolism of ROS [47]. The dynamic process of releasing H2O2 upon oxidative stress was monitored with the as-prepared PDDA-AuPtAg/RGO/GCE after adding phorbol myristate acetate (PMA) to SKOV3, MCF-7, and A431 cell lines. PMA is a well-known stimulus for triggering H2O2 production to mimic oxidative metabolism in vivo. After stimulation of PMA, respiratory burst will occur and H2O2 will be the end product released from cells through fusion pores [48,49]. Determining the cytotoxicity of the obtained materials PDDA-AuPtAg/RGO was completed by MTT assays before the electrochemical measurements. As shown in Figure 6A, MCF-7, SKOV3, and A431 cells maintained 95.1%, 95.8%, and 96.7% viability, respectively, after 12 h incubation with PDDA-AuPtAg/RGO, suggesting the favorable biocompatibility of the biosensor. Figure 6B exhibits amperometric response curves for PDDA-AuPtAg/RGO/GCE in the presence of SKOV3, MCF-7, and A431 cells triggered by the addition of PMA (10 L, 1 g mL −1 ), after which the currents changed significantly, indicating H2O2 released from these cancerous cell lines was reduced on the PDDA-AuPtAg/RGO modified electrode in real-time. PBS buffer without cells and cells with catalase were used as the control. There was no current response in the absence of cells and the current remained almost constant in the presence of 300 U/ml catalase (an H2O2 scavenger) in the PBS buffer. These results reveal that the current responses can undoubtedly be ascribed to the reduction of H2O2 released from the cancerous cells upon oxidative stress [50]. After the stimulation by 10 L PMA, the current change in the SKOV3 cells was around 8.27 A, and according to the calibration curve in Figure 4B, the amount of H2O2 was calculated as ~1.98 M. Similarly, the current changes for the MCF-7 and A431 cells were 8.36 and 8.4 A, respectively, which correspond to ~2.21 and ~2.34 M H2O2 released from the cells. To confirm the accuracy, an Amplex Red Hydrogen Peroxide Assay kit (Molecular Probes) was used according to the manufacturer's instructions (Table  S2), which revealed a relative deviation between the two methods of less than 2.5%. These results suggest the excellent practicality of the proposed biosensor and its huge potential for the investigation of the mechanisms in ROS-enhanced disorders.

In Situ Monitoring of H 2 O 2 Released From Living Cells
The excellent performance of the PDDA-AuPtAg/RGO sensor provided the opportunity to explore its application in monitoring H 2 O 2 in living cancer cells. It is well proven that H 2 O 2 is a stable ROS, which is of great importance in various biological processes, and many disorders like cancer result from the unbalanced metabolism of ROS [47]. The dynamic process of releasing H 2 O 2 upon oxidative stress was monitored with the as-prepared PDDA-AuPtAg/RGO/GCE after adding phorbol myristate acetate (PMA) to SKOV3, MCF-7, and A431 cell lines. PMA is a well-known stimulus for triggering H 2 O 2 production to mimic oxidative metabolism in vivo. After stimulation of PMA, respiratory burst will occur and H 2 O 2 will be the end product released from cells through fusion pores [48,49]. Determining the cytotoxicity of the obtained materials PDDA-AuPtAg/RGO was completed by MTT assays before the electrochemical measurements. As shown in Figure 6A, MCF-7, SKOV3, and A431 cells maintained 95.1%, 95.8%, and 96.7% viability, respectively, after 12 h incubation with PDDA-AuPtAg/RGO, suggesting the favorable biocompatibility of the biosensor. Figure 6B exhibits amperometric response curves for PDDA-AuPtAg/RGO/GCE in the presence of SKOV3, MCF-7, and A431 cells triggered by the addition of PMA (10 µL, 1 µg mL −1 ), after which the currents changed significantly, indicating H 2 O 2 released from these cancerous cell lines was reduced on the PDDA-AuPtAg/RGO modified electrode in real-time. PBS buffer without cells and cells with catalase were used as the control. There was no current response in the absence of cells and the current remained almost constant in the presence of 300 U/ml catalase (an H 2 O 2 scavenger) in the PBS buffer. These results reveal that the current responses can undoubtedly be ascribed to the reduction of H 2 O 2 released from the cancerous cells upon oxidative stress [50]. After the stimulation by 10 µL PMA, the current change in the SKOV3 cells was around 8.27 µA, and according to the calibration curve in Figure 4B, the amount of H 2 O 2 was calculated as~1.98 µM. Similarly, the current changes for the MCF-7 and A431 cells were 8.36 and 8.4 µA, respectively, which correspond to~2.21 and~2.34 µM H 2 O 2 released from the cells. To confirm the accuracy, an Amplex Red Hydrogen Peroxide Assay kit (Molecular Probes) was used according to the manufacturer's instructions (Table S2), which revealed a relative deviation between the two methods of less than 2.5%. These results suggest the excellent practicality of the proposed biosensor and its huge potential for the investigation of the mechanisms in ROS-enhanced disorders.

Conclusions
An ultrasensitive electrochemical sensor based on PDDA-capped AuPtAg/RGO was designed for the dynamic monitoring of intracellular H2O2. The synergetic effects of the trimetallic alloy ensured exceptional electrochemical catalysis toward H2O2 and definitely accelerated electron transfer, while the PDDA-RGO nanosheets employed as a supporting framework effectively enlarged the active surface area. The proposed sensor demonstrated admirable performance toward H2O2 detection in the concentration range from 0.05 M to 5.5 mM with a low detection limit of 1.2 nM (signal-to-noise ratio of 3). In addition, the developed sensor was capable of real-time H2O2 measurements in three cancerous cell lines, thereby indicating its huge potential for the in-depth study of the role of ROS in tumor formation and the screening of cancer at an early stage.

Conclusions
An ultrasensitive electrochemical sensor based on PDDA-capped AuPtAg/RGO was designed for the dynamic monitoring of intracellular H 2 O 2 . The synergetic effects of the trimetallic alloy ensured exceptional electrochemical catalysis toward H 2 O 2 and definitely accelerated electron transfer, while the PDDA-RGO nanosheets employed as a supporting framework effectively enlarged the active surface area. The proposed sensor demonstrated admirable performance toward H 2 O 2 detection in the concentration range from 0.05 µM to 5.5 mM with a low detection limit of 1.2 nM (signal-to-noise ratio of 3). In addition, the developed sensor was capable of real-time H 2 O 2 measurements in three cancerous cell lines, thereby indicating its huge potential for the in-depth study of the role of ROS in tumor formation and the screening of cancer at an early stage.