Biosensor based on bimetallic/graphene composite for non‐enzymatic detection of hydrogen peroxide in living tumor cells

A highly sensitive electrochemical biosensor was manufactured with triple synergistic catalysis to detect hydrogen peroxide (H2O2). In this study, a highly sensitive biosensor based on Prussian blue‐chitosan/graphene‐hemin nanomaterial/platinum and palladium nanoparticles (PB‐CS/HGNs/Pt&Pd biosensor) was fabricated for the detection of H2O2. The materials described above were modified on the electrode surface and applied to catalyze the breakdown of hydrogen peroxide. The current response of the biosensor presented a linear relationship with H2O2 concentration from 6 × 10−2 to 20 μM (R2 = 0.9766) and with the logarithm of H2O2 concentration from 20 to 9×103 μM (R2 = 0.9782), the low detection limit of 25 nM was obtained at the signal/noise (S/N) ratio of 3. Besides, the biosensor showed an outstanding anti‐interference ability and acceptable reproducibility. PB‐CS/HGNs/Pt&Pd electrodes are effective in measuring H2O2 from living tumor cells, which implies that the biosensor has the potential to assess reactive oxygen species in various living tumor cells.


INTRODUCTION
Reactive oxygen species (ROS) is considered the essential intracellular signaling molecule that can participate in metabolism and regulate biological processes related to the development of tumors, such as DNA damage, cell growth, cell invasion, and the expression of oncogenes. 1-3 ROS mainly includes four forms, hydrogen peroxide (H 2 O 2 ), singlet oxygen ( 1 O 2 ), hydroxyl radical (•OH), and superoxide anion radical (•O 2 − ). 4 Highly sensitive determination of ROS is of great significance in chemistry, clinical, biological, and many other fields. Among ROS, H 2 O 2 is the most stable molecule under normal conditions and is commonly used as a substitute for detecting ROS. [5][6][7] At present, the primary methods for detecting H 2 O 2 include spectroscopy, 8 chromatography, 9 enzymatic, 10 and electrochemical analysis 11 ; among these, biosensors have attracted significant attention owing to their high sensitivity and low detections limit. Thus, developing highly sensitive and convenient biosensors for the measurement of H 2 O 2 is crucial in clinical diagnosis .12 Nanoparticles have improved biosensor performance by increasing the ability to load molecules and enabling electronic conduction between catalytic molecules and electrodes. 13,14 Graphene is a two-dimensional hexagonal configuration nanomaterial. It shows many advantages, for instance, high specific surface area, excellent electrical conductivity, and mechanical strength. 15, 16 Bai et al. reported a biosensor based on platinum nanoparticles/ reduced graphene oxide-chitosan-ferrocene carboxylic acid nano-hybrids . 17 Utilizing the high electrocatalytic efficiency, electrical conductivity, and reversible electrochemical behavior of the various nanoparticles, the authors integrated the benefits of each component and achieved better performance through synergistic effects. The biosensor successfully detected hydrogen peroxide from normal and cancer cells, indicating that it can be used in clinical diagnostics to measure oxidative stress in living cells.
Metal nanoparticles have critical applications in catalysis and sensing fields, which can be used as catalysts to improve redox reaction rates and efficiently increase the conduction of electrons. Compared to the corresponding single-metal counterparts, the interaction of two different elements in the bimetallic alloy generally exhibits many advantageous properties, such as high catalytic activity and better deactivation resistance . 18 Safavi and Farjami reported an amperometric biosensor based on goldplatinum alloy nanoparticles with catalytic and conductive properties due to the strong synergistic effect of gold and platinum . 19 The biosensor demonstrated high selectivity and sensitivity over a broad concentration range, allowing for a linear response to cholesterol. But the biosensor still needs to be improved, as its catalytic activity is insufficient, and its stability should be strengthened.
This work aimed to build a biosensor for the catalytic detection of H 2 O 2 . The biosensor was developed by electrodeposition of Pt and Pd nanoparticles on the gold electrode surface modified with graphene-hemin composite nanomaterial (HGNs) and Prussian blue-chitosan.

Apparatus
Electrochemical detection was completed on the CHI 660 electrochemical workstation (Shanghai CH Instruments Co., China). The experiments were accomplished with the conventional three-electrode system. The platinum wire electrode was used as the auxiliary electrode, saturated calomel electrode (SCE) was used as the reference electrode, and modified gold electrode (GE) was used as the working electrode. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were executed in PBS containing 5 mM [Fe(CN) 6 ] 3−/4− . Amperometry was fulfilled in 0.01% H 2 PtCl 6 and H 2 PdCl 6 mixed solution under −0.2 V.

Preparation of HGN nanomaterials
For the preparation of HGNs, 2 ml graphene dispersion (1 mg/ml) was mixed with 2 mg hemin and 200 μl ammonia solution, followed by the adjunction of 20 μl hydrazine hydrate solution. The mixed solution was shocked for 5 min and put in the 60 • C water bath for 6 h, and centrifuged to remove the supernatant, and the black precipitate obtained was washed twice. Finally, the HGNs were re-dispersed to form a 2 mg/ml solution for future use.

Preparation of the modified biosensor
For the construction of the detection electrode, bare gold electrodes (GE; 3 mm) were polished with 0.3 and 0.05 μm alumina powder, then rinsed with deionized water and ethanol. The prepared electrodes were placed in the mixed solution 0.5 mM K 3 [Fe(CN) 6 ], 0.5 mM FeCl 3 , and 0.01% CS (containing 0.1 mol/L KCl and 0.01 mol/L CH 3 COOH), which was carried out in 20 mV/s sweep rate potential range −0.1 to 0.45 V cyclic voltammetry scan 10 laps. The PB-CS was modified on the surface of a bare gold electrode, and then 20 μl HGNs were dropped on the electrode's surface and dried in air at room temperature. The HNGmodified electrode was dipped into 5 ml H 2 PtCl 6 (10 mM) and H 2 PdCl 6 (10 mM) solution, and then the Pt NPs and Pd NPs were electrodeposited on electrode by i-t curve at a constant potential of −0.2 V with a 100 mV/s scanning rate at room temperature with mild stirring. The electrodeposition i-t curve is shown in Figure S6; the electrode current changes significantly.

Explore the anti-interference ability of the biosensor
In order to investigate the anti-interference ability of the PB-CS/HGNs/Pt&Pd biosensor, a current response was detected with the injection of 10 μM H 2 O 2 , some possible interference substances, such as glucose (Glu), uric acid (UA), ascorbic acid (AA), dopamine (DA), and a second 10 μM H 2 O 2 into PBS.

Measurement of H 2 O 2 released by the living cells
The cells were centrifuged at 1000 rpm for 3 min and washed twice with PBS. The number of cells was counted by the counter. After current reached the stable value, the constant concentration of AA was injected into cells suspension to stimulate cells to generate H 2 O 2 . The current signal caused by catalysis of H 2 O 2 was recorded by the electrochemical station.

Statistical analysis
The results were shown as means and SD. The independent t-test was applied for comparison between groups. p-Values <0.05 were regarded as statistically significant. Least significant difference, Student-Newman-Keuls, and Bonferroni post hoc tests were used.

Principle of PB-CS/HGNs/Pt&Pd biosensor
The construction process of the biosensor is illustrated in Scheme 1. The HGNs were synthesized by π bonds, which inherit vigorous peroxidase activity and are less prone to environmental variations. PB-CS was modified on the surface of a bare gold electrode by cyclic voltammetry, HGNs were immobilized on the electrode modified with PB-CS by self-assembly. Bimetallic nanoparticles were electrodeposited on these electrodes to create the PB-CS/HGNs/Pt&Pd biosensor, which can detect H 2 O 2

Characterization of HGNs
HGNs were analyzed by the FT-IR (Bruker Tensor27, Germany), atomic force microscope (AFM, Zeiss Ultra55, Germany), Raman spectra (Thermo Fisher Scientific, USA), and UV-vis. The FT-IR spectra of the HGNs (curve a), GO (curve b), and Hemin (curve c) are labeled in Figure 1A. The GO showed two clear characteristic peaks at 3408 cm −1 (OH stretching vibration) and 1635 cm −1 (C=C stretching). In the FT-IR curve of Hemin, the peaks at 1652 and 1383 cm −1 act as the stretching vibration of the OH and antisymmetric COO−, respectively. In the curve of HGNs, the peaks at 1632 and 1464 cm −1 act as the N-H and C-O vibration, while the graphene/hemin composite nanomaterials have distinct absorption peaks at 1713, 2928, and 3443 cm −1 , indicating the synthesis of this material successfully .20 Figure 1B shows the UV-vis spectra of the GO (curve a), hemin (curve b), and HGNs (curve c). The GO (curve a) showed absorption at 233 nm, which due to the π-π* transition of aromatic C=C bonds and a shoulder at wavelength of 290-300 nm, corresponds to the n-π* transition of the C=O bond. The hemin (curve b) had a strong peak at 383 nm ascribed to the Soret band. After reduction, HGNs (curve c) had two absorption peaks at 264 and 413 nm, respectively, corresponding to the Soret bands of reduced graphene and large red-shift of hemin. This is due to π-π conjugation of graphene, and hemin made the red-shift of the Soret band of the porphyrin. Figure 1C shows the Raman spectrum of HGNs ( Figure 1C, curve a) and GO ( Figure 1C, curve b); the two characteristic bands at 1353 and 1596 cm −1 represent the D band and G band caused by defects in the first-order scattering of carbon lattice, respectively. The D-peak represents the defects of the C atomic crystal, and the G-peak represents the inplane stretching vibration of the sp 2 hybridization of the C atom. The larger the value, the more defects of the C atomic crystal. Raman spectrum of HGNs showed increased intensity ratio between D and G peaks (I D /I G ) of 1.10 compared with 1.04 of GO, which due to the reaction of GO reduced. AFM images of HGNs can be seen in Figure 1D. The image of HGNs presents a flake structure, and the thickness of the flakes is about 5 nm, which indicates that the HGNs keep a sheet-like structure similar to GO. The exchange of anions on the graphene oxide surface was assessed by X-ray photoelectron spectroscopy (XPS). Compared to the survey spectrum of graphene oxide ( Figure 1E), which contained 67.85% and 32.579% of carbon (C 1s) and oxygen (O 1s), respectively, HGNs showed extra peaks of nitrogen (N 1s) and ferrous (Fe 2p), with the carbon ratio increased to 80.46%, whereas the oxygen ratio decreased to 19.85% ( Figure 1F). Fe 2p comes from the hemin, which is formed by the complexation of protoporphyrin IX with iron(II).  Figure 2A. A pair of quasi-reversible redox peaks (curve a) appeared at the bare electrode. When the PB-CS casted on the electrode, the current of the modified electrode declined (curve b), and the non-conductive substance of CS blocked the conduction of electrons. When the HGNs were immobilized on the surface of GE/PB-CS, the current response was enhanced due to the excellent conductivity of GO (curve c). After Pt and Pd nanoparticles are deposited on the GE/PB-CS/HGNs, the current increases in the Pt and Pd NPs are parallel to a conductor (curve d).
EIS was used to explore the electronic transmission capability of the electrode in different modification stages, and the results are displayed in Figure 2B. Impedance value of gold electrode was 238 Ω (curve a), and when the PB-CS were fixed on the bare electrode surface, the electron transfer ability of the modified electrode is weakened and the impedance raise to 3062 Ω, as a result of the non-conductivity of Chitosan (curve b). After HNGs were further modified on the surface of PB-CS electrode, the resistance dropped to 2190 Ω (curve c), because graphene sheets are highly conductive. When Pt and Pd NPs were electrodeposited onto the surface of PB-CS/HGNs electrode, the resistance was decreased to 625 Ω (curve d) due to the good electric conducting properties of the Pt&Pd NPs. However, when only monometallic Pt was modified to the electrode, the resistance was 1260 Ω (curve e). These results show that the multilayers of PB-CS/HGNs/Pt&Pd can efficiently transfer electrons. Figure 3 shows the scanning electron microscope (SEM, JSM-6700) images of the developed process of the modified electrode. The bare electrode was the image of uniform dark ( Figure 3A). After the PB-CS was immobilized by CV technique, a film streak appeared on the electrode surface ( Figure 3B). Along with the modified HNGs, it can be seen that multifolded HNGs cover the surface of PB-CS and possess a more active area for loading nanoparticles ( Figure 3C). After electrochemical co-precipitation, large numbers of spherical-like structured bimetallic nanoparticles appeared on the electrode surface, providing more active sites and a larger effective surface area for catalytic reaction ( Figure 3D). This structure may be due to the high-speed electrodeposition of Pt and Pd nanoparticles on the surface of HGN-modified electrode in chloride concentration medium. Energy dispersive spectrometer (EDS) analysis was performed to analyze and verify elemental compositions. Prominent peaks of oxygen, carbon, palladium, and platinum were found on PB-CS/HGNs/Pt&Pd ( Figure 3E). Chronocoulometry was used to evaluate the apparent electrode areas of the electrodes from the reduction of KCl The slope values were slightly increased when PB-CS was covered on the electrode, because chitosan hindered the electron conduction and transfer on the electrode surface. When the HGNs were coated on the PB-CS/EG, the slope value continues to increase, owing to the specific surface area of the graphene sheet. The slope value increased sharply when the metal nanoparticles were deposited on the HGNs/PB-CS/EG due to the excellent electrical transfer ability and large specific surface area of metal nanoparticles. The above results demonstrate that PB-CS/HGNs/Pt&Pd could effectively enlarge the apparent electrode area.

Characterization of the biosensor
The CV of GE/PB-CS/HGNs/Pt&Pd NPs at different scan rates ranging from 40 to 500 mV/s is shown in Figure 4A. Both the cathodic and anodic peak currents of the modified electrode (I pa and I pc ) were proportional to the square scan rate ( Figure 4B), with R 2 0.9984 and 0.9953, respectively. These results suggested that the procedure of electrochemical reaction was predominantly diffusion-controlled, which demonstrates that the speed of the reaction depends on the diffusion speed of the substance from the solution to the electrode surface. The diffusion process is slow during the oxidation-reduction process, limiting the electrode surface reaction. So, the diffusion rate becomes a controlling factor that restricts the reaction.

Optimization of the experimental conditions
To improve the analytical function of GE/PB-CS/HGNs/Pt&Pd, some optimal conditions were carried out regarding different bimetallic, deposition time of metal nanoparticles and applied potential. The effect of different ratios of the bimetallic on the modified electrode was also investigated ( Figure 5A). As seen from Figure 5A, increased ratios of H 2 PtCl 6 and H 2 PdCl 6 could heighten the response signal, and the biosensor reached the maximal response at the ratios of H 2 PtCl 6 triple to H 2 PdCl 6 solution. However, further subjoin the proportional concentration of Pt to Pd, the response signal of the biosensor reduced, insinuating that the aggregation process of nanoparticles had almost been completed.
The deposition time of bimetallic nanoparticles was evaluated ( Figure 5B). In the electrodeposition process, the deposition sites of metal atoms raised and the particles formed gradually increased, which enables the response current signal to increase with the deposition time. When we prolong the deposition time, numerous metal atoms are enriched on the electrode surface, and the metal nanoparticles keep getting larger, resulting in a progressively smaller distance between the nanoparticles, and generating a maximum response current at 100 s. When the time was further prolonged, the response signal of the modified electrode did not change significantly. Therefore, 100 s acted as the optimum deposition time for the experiments.

Performance of the biosensor
Here we utilize the HGNs nanomaterial, PB solution, and bimetallic as the treble synergistic catalyst for catalytic detection of hydrogen peroxide. Under the optimal conditions, the amperometric (i-t curve) was used as the response signal for the detection of H 2 O 2 , the current sig-nal of the electrode for the successive injection of H 2 O 2 with a range of concentrations in 0.1 M PBS ( Figure 6A). As seen in Figure 6B,C, the current presented a linear relationship with H 2 O 2 concentration from 6 × 10 −2 to 20 μM (R 2 = 0.9766) and the logarithm of H 2 O 2 concentration from 20 to 9 × 10 3 μM (R 2 = 0.9782), LOD (low detection limit) of 25 nM at the signal/noise (S/N) ratio of 3. Compared with other H 2 O 2 biosensors listed in Table 1, the PB-CS/HGNs/Pt&Pd biosensor possessed the lowest detection limit for the detection of H 2 O 2 .

Stability, reproducibility, and selectivity study
For reproducibility testing, the modified electrodes were reused six times for H 2 O 2 measurements under the same conditions. After detections, the current signal of the sensor almost retained about 85% of its original signal. Figure S3 depicts the real electrochemical measurement signal of reproducibility of the modified electrodes. The relative standard deviation (RSD) of the sensitivities was 4.2%.These results reveal that the developed biosensors have good reproducibility. In addition, the stability of the biosensor was also investigated through storage electrodes in the refrigerator at 4 • C for 2 weeks, it kept about approximately 86.3% of the original response, demonstrat-ing the long-term storage characteristics and operational stability of the biosensor. Figure S4 depicts the actual electrochemical measurement signal of stability of the modified electrodes. To assess the linearity, we analyzed the current signal of the modified electrode in 0-500 s. Figure S5 displays the actual electrochemical signal of the modified electrodes in the linear relationship range. To evaluate the selectivity and anti-interference ability of the biosensor, the effect of some possible interfering substances, including glucose (Glu), uric acid (UA), ascorbic acid (AA), and dopamine (DA) on the response signal of H 2 O 2 was tested. As shown in Figure 6D, adding each interfering substance instead of H 2 O 2 into the PBS buffer (pH 7.0) caused a negligible current response, displaying an excellent selectivity toward H 2 O 2 and good antiinterference ability to the above interfering substances. It is significant for evaluating the oxidative stress of living tumor cells and distinguishing the oxidative ability of different living tumor cells by measuring the content of H 2 O 2 . Here, we tested the current of H 2 O 2 released by the same amount of cells (1 × 10 7 ), which was stimulated by a certain concentration of AA (1 μM). It can be seen from Figure 7B that the H 2 O 2 released by tumor cells (MCF-7, A549, BEL-7404, and Hela) is significantly higher than that of related normal cells (MDA-kb2, BEAS-2B, L02, and HUCEC), which shows that tumor cells can produce large amounts of ROS, and it is of great significance for the study of the environment of tumor cells and the therapy of cancer. The results indicate that the biosensor can detect the release of H 2 O 2 from tumor cells and has the potential for clinical diagnostic applications.

CONCLUSIONS
In summary, we developed a highly sensitive biosensor with triple synergistic catalysis to detect H 2 O 2 employing HGNs, PB solution, and bimetals as catalysts. Based on the excellent catalysis properties of the multilayer of PB-CS/HGNs/Pt&Pd, the biosensor presented a dynamic response in a linear relationship with H 2 O 2 concentration from 6 × 10 −2 to 20 μM and with a logarithm of H 2 O 2 concentration from 20 to 9 × 10 3 μM, the low detection limit of 25 nM was obtained at the signal/noise (S/N) ratio of 3.
In addition, the biosensor has shown a possible application for detecting H 2 O 2 released from tumor cells, which is significant for evaluating the oxidative stress of tumor cells in medical diagnostics.

C O N F L I C T O F I N T E R E S T
The authors declare that there is no conflict of interest.

D ATA AVA I L A B I L I T Y S TAT E M E N T
The authors do not intend to share individual identified participant data.