DNA-induced synthesis of biomimetic enzyme for sensitive detection of superoxide anions released from live cell

In this work, we successfully fabricate a rapid, sensitive sensor for the detection of superoxide anions O2˙− based on graphene/DNA/Mn3(PO4)2 biomimetic enzyme. In the design, graphene is served as excellent carrier to improve the catalysis of Mn3(PO4)2 nanoparticles; and DNA adsorbed on graphene acts as medium to assist the growth of Mn3(PO4)2 on graphene. The fabricated graphene/DNA/Mn3(PO4)2 composites exhibit excellently electrochemical activity, significantly decrease the response time and increase the sensitivity of the sensor towards O2˙−. The successful detection of O2˙− released from cancer cell demonstrated its potential applications in biology and medicine.


Introduction
Superoxide anions (O 2 c À ) are the primary type of reactive oxygen species (ROS). Under normal conditions, O 2 c À is highly reactive and unstable and its metabolism is a rapid and spontaneous process. Overproduction of O 2 c À can cause various disease, such as aging, asthma, ulcer disease, cancer, atherosclerosis, neurodegenerative diseases and other diseases. [1][2][3][4] Thus the detection of O 2 c À is very important in various biological systems. Many methods have been developed for the detection of O 2 c À including chemiluminescent, spectrophotometric, uorometric, and electrochemical technique, etc. [5][6][7][8] Among all these techniques, electrochemical method receives most attention due to its advantages of real-time assay, high sensitivity and selectivity. And most of the reported electrochemical methods for O 2 c À detection are based on enzyme catalysts such as cytochrome c and superoxide dismutase (SOD). 2,[9][10][11] Though enzyme improves the assay performance of sensors, but its high cost and poor long-term stability increase difficulty of direct monitoring of O 2 c À in biological samples. Therefore, it is a serious challenge for us to establish a fast, reliable, and sensitive non-enzyme approach for O 2 c À monitoring in physiological and pathological processes.
As early as 1982, manganese was reported possessing an effective catalytic effect in vivo protection against superoxide toxicity. 12 In recent years, further study found that manganese phosphate (Mn 3 (PO 4 ) 2 ) has the ability to catalyze the dismutation of O 2 c À compared with free Mn 2+ ion that only stoichiometrically reacts with O 2 c À . 13 Therefore Mn 3 (PO 4 ) 2 is usually selected as a substitute for enzyme to detect O 2 c À . 14 Mao et al. synthesize SiO 2 -Mn 3 (PO 4 ) 2 nanoparticles derived from phytic acid and utilize for O 2 c À detection. 15 Lan and co-workers deposit Mn 3 (PO 4 ) 2 nanoparticles on carbon nanomaterials directly for the fabrication of O 2 c À sensor. 16 These works verify the catalysis of Mn-superoxide dismutase mimics towards O 2 c À . But the synthesis and property of nanoparticles need further study to improve the catalytic performance of sensor. In this work, we synthesize Mn 3 (PO 4 ) 2 nanoparticles by DNA induction, which results in even dispersion and excellent catalytic activity towards O 2 c À disproportionation reaction.
Graphene is an increasingly important nanomaterials due to its excellent electronic conductivity, good stability, and promising catalytic performance in sensing. [17][18][19][20] However, the two dimensional structure is easy to aggregate due to the singleatom-thickness, and hydrophobic aromatic structure. To solve this problem, we functionalize graphene with deoxyribonucleic acid (DNA) which can be adsorbed on graphene through p-p stacking and don't destroy the intact structure of the carbon materials. 20 Under the induction of ssDNA, Mn 3 (PO 4 ) 2 nanoparticles was evenly deposited on the surface of graphene. The obtained graphene/DNA/Mn 3 (PO 4 ) 2 nanocomposites displayed signicant biomimetic enzyme activity, rapid and sensitive response towards O 2 c À . This approach holds a great promise for broad applications in biomedical research and clinical test.

Experimental section
Chemicals and materials MCF-7 was bought from Cell bank of the representative culture preservation committee of the Chinese Academy of Sciences, China. Manganese sulfate (MnSO 4 ), potassium phosphate tribasic (K 3 PO 4 ) and Naon were purchased from East Sichuan Chemical Industry (Group) Co., Ltd. (Chongqing, China) and used as received. Graphene was obtained from Sinocarbon Materials Technology Co., Ltd., China. Zymosan A (Zym, from Saccharomyces cerevisiae) SOD, DNA (low molecular weight extracted from salmon sperm), and potassium superoxide were purchased from Sigma-Aldrich and used without further puri-cation. The O 2 c À solutions were prepared by dissolving KO 2 in PBS solution (pH 7.0, N 2 saturated). The concentration of O 2 c À was determined by the reduction of ferri cytochrome c spectrophotometrically. 21 All the other solutions were prepared using deionized water (18 MU cm) and were degassed with high purity nitrogen before experiments. All electrochemical experiments were carried out at room temperature.

Synthesis of graphene/DNA/Mn 3 (PO 4 ) 2 nanosheets
Graphene/DNA/Mn 3 (PO 4 ) 2 nanosheets were prepared by the previously reported method. 22 Thirty milligrams of DNA was dissolved in 30 mL de-ionized water under stirring, aer which the solution was annealed at 95 C for 15 min to produce singlestranded DNA (ssDNA). Then the obtained ssDNA was mixed with 15 mL of 1 mg mL À1 graphene by mildly sonicated at 4 C for 2 h, thereaer ltration and washing were performed to remove excess ssDNA. Subsequently, the mixture was dispersed into 0.1 M MnSO 4 solution, then 0.1 M K 3 PO 4 solution were dropwisely added under stirring and kept at room temperature for 30 min. Then graphene/DNA/Mn 3 (PO 4 ) 2 nanosheets were obtained by centrifugation at 8000 rpm for 10 minutes and washing with deionized water for 3 times.
Glassy carbon electrode (GCE, d ¼ 3 mm) was polished with alumina slurry to a smooth and bright surface, washed by sonication for 30 s and dried under nitrogen. Then 5 mL of the obtained graphene/DNA/Mn 3 (PO 4 ) 2 suspension was dropped on the electrode surface and dried at room temperature. Finally, 5% Naon solution was coated to stabilize the fabricated electrode.
In situ detection of O 2 c À released form living cells MCF-7 cells were cultured in a humidied incubator (95% air with 5% CO 2 ) at 37 C. The cells were cultured in Dulbecco's Modied Eagle's Medium (DMEM) (Cellgro, USA) supplemented with 1 mol L À1 glutamine, 50 U mL À1 penicillin/ streptomycin and 10% heat inactivated fetal bovine serum. 14 For the detection of O 2 c À released from living cell, the incubation solution was removed and washed with PBS solution (pH 7.4) for three times. Before electrochemical measurement, zymosan (Zym) was added to motivate cells generation of O 2 c À . Amperometric response was recorded by CHI-660B electrochemical station at applied potential of 700 mV (versus Hg/ Hg 2 Cl 2 ).

Results and discussion
Design of the modied graphene nanosheets Scheme 1 illustrates the synthesis of graphene/DNA/Mn 3 (PO 4 ) 2 enzyme mimics by growth of Mn 3 (PO 4 ) 2 on graphene under the induction of DNA. As we all know, single-strand DNA (ssDNA) can be adsorbed on graphene by p-p interaction. 23,24 Here ssDNA was prepared by annealing double-strand DNA (dsDNA) and applied for graphene modication. In the presence of Mn 2+ , the divalent cations accumulate along the ssDNA backbone by the electrostatic interaction, which facilitate the formation of Mn 3 (PO 4 ) 2 crystal nucleus on graphene and growth of Mn 3 (PO 4 ) 2 nanoparticles upon the addition of negatively charged PO 4 3À groups.

Characterization of graphene/DNA/Mn 3 (PO 4 ) 2 nanocomposites
The morphology of the as-prepared graphene/DNA/Mn 3 (PO 4 ) 2 nanosheets is characterized with scanning electron microscope (SEM). Fig. 1A and B shows the SEM images of graphene before and aer adding MnSO 4 and K 3 PO 4 , typical wrinkle of graphene can be clearly observed and no evident Mn 3 (PO 4 ) 2 nanoparticles is found in Fig. 1B, suggesting that it is difficult to form Mn 3 (PO 4 ) 2 nanoparticles on graphene surface without DNA. However, when ssDNA is modied on graphene ( Fig. 1C and D Then FTIR spectra are determined to further verify the formation of graphene/DNA/Mn 3 (PO 4 ) 2 nanosheets. As shown in Fig. 2A, absorption peak at 1568.6 cm À1 can be assigned to the aliphatic carboxylic acid salts, the peaks at 996.0 cm À1 and 3196.3 cm À1 were attributed to inorganic phosphates. The results suggest that DNA and Mn 3 (PO 4 ) 2 have been successfully modied on the graphene. In addition, the X-ray diffraction pattern (XRD) of graphene/Mn 3 (PO 4 ) 2 in Fig. 2B shows that the diffraction peak of the nanomaterials is at around 28.2 , which is consistent with the results of Mn 3 (PO 4 ) 2 . The results further conrm that Mn 3 (PO 4 ) 2 nanoparticles are successfully modied on the surface of graphene. Then the zeta potential at various stages of the synthesis were measured to further characterize the formation of the nanocomposites. The results in Fig. S1 † indicate that the surface charge of graphene is about À0.203 mV, which decreases to À32.5 mV as DNA was adsorbed on its surface due to the negative charge of DNA backbone. When Mn 3 (PO 4 ) 2 was deposited, the zeta potential increase to À15.6 mV, indicating the formation of graphene/DNA/ Mn 3 (PO 4 ) 2 nanocomposites.  Fig. S3 †), which can be attributed to the electrochemical transformation between Mn 2+ and Mn 3+ species. However, no evident current response is observed when the mixture of O 2 c À and SOD is added, suggesting the current response attributed to the catalysis of the graphene/DNA/Mn 3 (PO 4 ) 2 nanosheets towards O 2 c À . These results verify the biomimetic enzyme activity of graphene/ DNA/Mn 3 (PO 4 ) 2 to catalyze the dismutation of O 2 c À . 25 In order to further study the electrochemical performance of graphene/DNA/Mn 3 (PO 4 ) 2 nanosheets, graphene/Mn 3 (PO 4 ) 2 were synthesized by direct depositing Mn 3 (PO 4 ) 2 nanoparticles on graphene in the absence of DNA, DNA/Mn 3 (PO 4 ) 2   nanoparticles were by prepared depositing Mn 3 (PO 4 ) 2 on dsDNA template and graphene/DNA/Mn 2+ was synthesized by adsorbing DNA on graphene and subsequent depositing Mn 2+ on DNA. Electrochemical performance of these materials were explored with electrodes modied with the above three nanomaterials ( Fig. 3A-C). As shown in Fig. 3A, the electrode modi-ed with graphene/Mn 3 (PO 4 ) 2 (graphene/Mn 3 (PO 4 ) 2 /GCE) possesses excellent electrical conductivity but the current response towards O 2 c À is weak, verifying the good conductivity of graphene and poor modication of Mn 3 (PO 4 ) 2 (Fig. 3A). Similarly, the electrodes modied with Mn 3 (PO 4 ) 2 /DNA and graphene/DNA/Mn 2+ display evident current response to O 2 c À , but the current response is lower than that on graphene/DNA/ Mn 3 (PO 4 ) 2 /GCE (as shown in Fig. 3B and C). The phenomena further prove that DNA plays important roles in the synthesis of graphene/DNA/Mn 3 (PO 4 ) 2 nanosheets. In addition, in comparison with the Mn 3 (PO 4 ) 2 /DNA/GCE (Fig. 3B), we can see that the graphene/DNA/Mn 3 (PO 4 ) 2 /GCE (Fig. 3D) displays stronger current signal due to the excellent conductivity of graphene.

Electrochemical properties of graphene/DNA/Mn 3 (PO 4 ) 2 nanosheets
The reason for DNA facilitating the growth of Mn 3 (PO 4 ) 2 nanoparticles on graphene can be ascribed to two factors. First, the p-p interaction between DNA and graphene induces ordered assembly of DNA on the surface of graphene, which is a critical factor to the synthesis of Mn 3 (PO 4 ) 2 nanoparticles. Next, Mn 2+ is adsorbed on DNA backbone to assist the nucleation of Mn 3 (PO 4 ) 2 nanoparticles. In addition, in comparison with other conventional method, the DNA induced synthesis is easier to control the morphology and dispersion of products by adjusting temperature, pH, or DNA concentration.

Electrochemical response of O 2 c À at the present electrode
Chronoamperometry is utilized to investigate the electrochemical response of graphene/DNA/Mn 3 (PO 4 ) 2 /GCE towards O 2 c À in 10 mM PBS (pH ¼ 7.4). Fig. 4A shows the stepwise current response of the present electrode with successive increasing O 2 c À concentration from 5 nM to 400 nM. From the results in Fig. 4B we can see that the prepared sensor displays signicant current response to the O 2 c À concentrations in the range of 5 nM to 400 nM with a regression equation expressed as I (mA) ¼ 0.00354 c (nM) + 0.1504 (R 2 ¼ 0.999). The detection limit of the assay is calculated to be about 1.67 nM (S/N ¼ 3) with a sensitivity of 3.54 mA mM À1 , which is much more superior to most of previous reports. 14,23,[26][27][28] The response time of the electrode to O 2 c À is as short as 5 s, which is an advantage for the detection of O 2 c À due to its short lifetime. These results demonstrate that the present sensor may meet the requirement of O 2 c À detection in normal physiological conditions. 29 The selectivity and stability of the present electrode The selectivity experience was carried out in the presence of various interfering species, such as K + , Na + , Cl À , NO 3   response. The result suggests that the proposed sensor possesses excellent specicity to O 2 c À detection in biological systems. In addition, the reproducibility and stability of the electrode were explored. The electrochemical response of the sensor was performed using ve different electrodes fabricated at the same time, the standard derivations of current response was lower than 5.2%. When the electrode was stored at 4 C for one month, the current response towards O 2 c À decrease 6.8% of original value (as shown in Fig. S4 †). These results indicate that the excellent reproducibility and stability of the sensor.

Detection of O 2 c À released from cancer cell
In order to further investigate the potential application of the method in biological systems, the detection of O 2 c À released from MCF-7 cells was performed at ambient temperature. As shown in Fig. 6, upon the addition of different concentration (0.5 g L À1 , 1.0 g L À1 or 2.0 g L À1 ) of zymosan (Zym, a drug was used to stimulate living cells to release O 2 c À ), a signicant current response was observed (corresponding to O 2 c À oxidation). According to the calibration curve in Fig. 5B, the concentration of O 2 c À released from MCF-7 cells is calculated to be around 156.38 nM, 315.71 nM and 631.81 nM, respectively (the concentration of O 2 c À released by per cell and total cells are calculated and listed on Table 1). The results indicate that current response of O 2 c À released form cancer cell under Zym stimulation is a concentration-dependent behavior. In addition, the results of control experiments (curve d and e shown in Fig. 6) indicate that no current was obtained upon the addition of either Zym or SOD-Zym mixture, conrming that the current responses were attributed to the drug-induced O 2 c À release from cells.

Conclusions
A novel O 2 c À biomimetic enzyme sensor based on graphene/ DNA/Mn 3 (PO 4 ) 2 nanosheets is successfully developed. The synthesized nanosheets display strong electrocatalytic activity towards O 2 c À with high sensitivity, excellent selectivity and fast current response. The successful determination of O 2 c À released from cancer cells demonstrates the great potential for application in biological system. This work provides a new method for the fabrication of biomimetic enzyme sensor and a great promise for biosensing and biomedical application.

Conflicts of interest
There are no conicts to declare