Colorimetric H2O2 biosensor based on reduced graphene oxide-Hemin-Pt@Pd nanocomposites

Hydrogen peroxide (H2O2) is one of the most universal and essential ingredients in distinct biological tissues. Herein, a highly sensitive TMB colorimetric H2O2 biosensor was constructed based on the good catalytic activity of Hemin-reduced graphene oxide-Platinum@Palladium nanocomposites (H-rGO-Pt@Pd NCs). Colorless 3,3’,5,5’-tetramethylbenzidine (TMB) can be oxidize to blue product (TMBox) when H-rGO-Pt@Pd NCs catalyzes H2O2 reduction. The content of H2O2 can be obtained by spectrophotometric determination of the absorbance of the solution. In the range of 0-50 μM, the absorbance is linearly related to the H2O2 concentration, the detection limit is estimated to be 0.96 μM in terms of the rule of three times standard deviation over the blank with a correlation coefficient of 0.99264. The TMB colorimetric H2O2 biosensor shows that H-rGO-Pt@Pd NCs has good enzyme-like properties, which can greatly improve the sensitivity, stability and repeatability of the enzyme-free sensor, and has a great potential in biosensors.


Introduction
Hydrogen peroxide(H 2 O 2 ) acts as a signal factor to regulate various biological processes and is a by-product of various oxidase reactions. H 2 O 2 is one of the most common but important molecules existing in different biological tissues, and is of vital significance to human health and life [1]. At present, there are many methods for measuring H 2 O 2 content, such as spectrofluorescence method, chemiluminescence method and electrochemical method [2]. Among them, colorimetric biosensor assays with exclusive advantages of visual recognition, simple operation and ultraviolet-visible spectrometer (UV-vis) quantification has the very broad application prospects in the field of clinical diagnosis [3][4]. However, for the natural enzyme are expensive, unstable, difficult to immobilization and easy to inactivation, the enzyme-based H 2 O 2 biosensors limit their further application. So it is necessary to develop the peroxidase mimics and non-enzymatic H 2 O 2 biosensors.
Recently, the materials with peroxidase-like activity have attracted great attention of researchers. Noble metal nanomaterial such as Au NPs, Pt NPs, Pd NPs have been witnessed a persistent utilization in electrochemical biosensor for their excellent conductivity, good biocompatibility, discrete electronic energy and excellent electrocatalytic activity towards the reduction of H 2 O 2 . Especially, Pt-Pd bimetallic NPs exhibits the superiority of outstanding intrinsic catalytic capacity, inherent biocompatibility and larger specific surface area over the corresponding monometallic NPs in virtue of the synergetic effect [5]. Reduced graphene oxide (rGO), as a deoxidized product of graphene oxide (GO), is also one of the most popular candidates to catalyze the electrochemical reduction of H 2 O 2 [6]. Also, rGO provides a rich site for adsorption and immobilization of substances and can increase the adsorption rate. Hemin (H) is an iron porphyrin derivative and displays electrocatalytic abilities based on the redox reaction of iron in the core [7].
In this work, a simple and sensitive TMB colorimetric H 2 O 2 biosensor based on Hemin-reduced graphene oxide-platinum@palladium nanocomposites (H-rGO-Pt@Pd NCs) was introduced. H-rGO-Pt@Pd NCs has the peroxidase-like activities by the synergistic effect of Pt@Pd NPs, rGO and Hemin. H-rGO-Pt@Pd NCs catalyzed the H 2 O 2 -mediated oxidation peroxidase substrate TMB, resulting a color change. The colorless TMB was oxidized into blue oxidized TMB in the presence of H 2 O 2 by the assistance of H-rGO-Pt@Pd NCs. The content of H 2 O 2 can be obtained by spectrophotometric determination of the absorbance of the solution.

Reagents and apparatus
GO was purchased from Xianfeng NANO Materials Tech Co., Ltd.

Synthesis of the H-rGO-Pt@Pd NCs
rGO was fabricated by using ascorbic acid and GO according to a route reported previously [8]. 30mg hemin was dissolved with 20μL NH 3 ·H 2 O to form a clear solution by intense stirring, followed by the addition of 20μL 1mg/mL rGO solution that was stirred for 30 min. then added 5μL hydrazine hydrate (N 2 H 4 ·H 2 O) and stirred at 50℃ for 2 h to obtain H-rGO solution. After-that, 2 mL PDDA (wt = 2%) and 2 mL NaCl (wt = 2%) were added to the H-rGO solution. After stirring for 12 h, a PDDA-modified H-rGO solution was obtained through centrifugation. Subsequently, 2 mL 20 mmol/L Na 2 PdCl 4 and 2 mL 20 mmol/L Na 2 PtCl 4 were added to the PDDA-modified H-rGO (0.5 mg/mL) solution. The mixture was added with 10.0 mL of glycol solution and 1.0 mol/L NaOH to adjust the pH to 12 with stirring and reacted at 140℃. After stirring for 4 h, the resulting black products were collected by centrifuging for 10 min at 12000 r/min and then washed with water several times. Subsequently, the yielded H-rGO-Pt@Pd NCs were freeze-dried for 16 h.

Detection of H2O2 with the TMB colorimetric biosensor
First, 40 μL 5.0 mg/mL of H-rGO-Pt@Pd NCs was added to 60 μL 125 mmol/LPBS buffer solution (pH 5.0), and then 20 μL 50 mmol/L of TMB was added. Third, 20 μL different concentrations of H 2 O 2 solutions was added and incubated for 30 min at 25°. Finally, the resulting reaction products solution was measured with a UH5300 ultraviolet-visible spectrophotometer. The absorbance intensity at 652 nm was record. The peak value was positively correlated with H 2 O 2 concentration

Characterization of H-rGO-Pt@Pd NCs
UV characterization of H-rGO-Pt@Pd NCs was shown in Figure 1A, the characteristic peaks of RGO and Hemin are at 260 nm and 367.5 nm, respectively. The XRD characterization of H-rGO-Pt@Pd NCs is shown in Figure 1B. There are sharp diffraction peaks at 2θ=39.76°, 46.24°, and 67.45°, which are correspond to (111), (200), (220) of Pt and Pd. rGO has a weak broad diffraction peak at 2θ=22°, which belongs to the diffraction surface of graphene material C (002). The above phenomena indicate that the H-rGO-Pt@Pd NCs was successfully prepared.  Figure 1C is the TEM image of H-rGO and Figure 1D is the TEM images of H-rGO-Pt@Pd NCs. It can be clearly seen that there are many fine particles embedded on the H-rGO composite of thin film yarn mesh. This indicates that Pt NPs and Pd NPs have been well combined with H-rGO materials, and it can be concluded that H-rGO-Pt@Pd NCs materials have been successfully prepared.

Principle of colorimetric H2O2 biosensor based on H-rGO-Pt@Pd NCs
The principle of the TMB colorimetric H 2 O 2 biosensor based on H-rGO-Pt@Pd NCs was schematically shown in Figure 2A.  Figure 2B, it can be seen that tube a contains H 2 O 2 and does not decompose in air. Therefore, there is no absorbance. Tubes b-e are light blue and have lower absorbance, this is because TMB will be partially oxidized in the presence of oxygen in the air. While tube f has the darkest blue color and has the highest absorbance. Because H-rGO-Pt@Pd NCs nanoenzyme has catalase-like catalytic activity, which can catalyze the oxidation of TMB by H 2 O 2 to make the solution produce color changes that can be observed and resolved by naked eyes.

Stability analysis of H-rGO-Pt@Pd NCs
To explore different storage temperature and time of H-rGO-Pt@Pd NCs simulation the effect, the stability of H-rGO-Pt@Pd NCs were investigated. H-rGO-Pt@Pd NCs was divided into two portions, one for under 4℃ in the refrigerator, and another at room temperature (25℃), and the catalase-like catalytic activity was tested using the TMB-H 2 O 2 chromogenic system in the time of 0 day, 1 day, 3 days and 7 days. Figure 3A showed the experimental phenomenon of the influence of different storage temperature and storage time on the stability of H-rGO-Pt@Pd NCs. It can be seen from Figure 4A that all the solutions of TMB-H 2 O 2 chromogenic system change from colorless to blue. H-rGO-Pt@Pd NCs has relatively good stability and can be stored for a long time. Through the color comparison between No. 2 and No. 3,No. 4 and No. 5,No. 6 and No. 7, we can know that H-rGO-Pt@Pd NCs is more suitable for low temperature preservation. Figure 3B and Figure 3C showed the UV-Vis absorption spectra of the effect of different storage times on the stability of H-rGO-Pt@Pd NCs under 4℃ and 25℃. The absorbance intensity of curves 1-7 at 652 nm was 1 > 3 > 2 > 5 > 4 > 7 > 6. The results showed that H-rGO-Pt@Pd nanozyme has certain stability, but its catalase-like catalytic activity will decrease with the time. By comparing curves 2 and 3, 4 and 5, 6 and 7, we can know that H-rGO-Pt@Pd NCs is more suitable for cryopreservation.

Performance analysis of H2O2 colorimetric biosensor based on H-rGO-Pt@Pd NCs
Under the optimal conditions, the H 2 O 2 colorimetric biosensor based on H-rGO-Pt@Pd NCs was used to detect H 2 O 2 . The results are shown in Figure 4. Figure 4A showed the color change of the solution at different H 2 O 2 concentrations. It can be seen that the higher of H 2 O 2 concentration was, the darker color had, which demonstrated that the colorimetric biosensor could be put to use for detecting H 2 O 2 with naked eyes. As shown in Figure 4B, the absorbance intensity at 652 nm increased gradually with the increase in the concentrations of H 2 O 2 . The absorbance intensity and the concentration of H 2 O 2 showed a linear relationship between 5-50 µM. The calibration curve for H 2 O 2 detection was presented in Figure 4C. The linear regression equation is Y=0.01228X+0.20931 with the correlation coefficient R 2 of 0.99264. The detection limit is estimated to be 0.96 µM in terms of the rule of three times standard deviation over the blank.

Conclusions
In summary, a highly sensitive TMB colorimetric H 2 O 2 biosensor was constructed based on the good catalytic activity of H-rGO-Pt@Pd NCs for H 2 O 2 and the color rendering of TMB. In the range of 5-50 µM, the absorbance intensity is linearly related to the concentration of H 2 O 2 , the regression equation was Y=0.01228X+0.20931 with a correlation coefficient of 0.99264. The H-rGO-Pt@Pd NCs has good peroxidase-like activities by the synergistic effect of Pt@Pd NPs, rGO and Hemin, which can greatly improve the sensitivity and stability of the non-enzymatic H 2 O 2 biosensors.