Sensitive Molybdenum Disulfide Based Field Effect Transistor Sensor for Real-time Monitoring of Hydrogen Peroxide

A reliable and highly sensitive hydrogen peroxide (H2O2) field effect transistor (FET) sensor is reported, which was constructed by using molybdenum disulfide (MoS2)/reduced graphene oxide (RGO). In this work, we prepared MoS2 nanosheets by a simple liquid ultrasonication exfoliation method. After the RGO-based FET device was fabricated, MoS2 was assembled onto the RGO surface for constructing MoS2/RGO FET sensor. The as-prepared FET sensor showed an ultrahigh sensitivity and fast response toward H2O2 in a real-time monitoring manner with a limit of detection down to 1 pM. In addition, the constructed sensor also exhibited a high specificity toward H2O2 in complex biological matrix. More importantly, this novel biosensor was capable of monitoring of H2O2 released from HeLa cells in real-time. So far, this is the first report of MoS2/RGO based FET sensor for electrical detection of signal molecules directly from cancer cells. Hence it is promising as a new platform for the clinical diagnosis of H2O2-related diseases.

Reactive oxygen species (ROS) play crucial roles in regulating DNA damage, signal transduction, cell proliferation and apotosis, etc. [1][2][3][4] . Hydrogen peroxide (H 2 O 2 ), as a most common representative of ROS, is not only involved in several bodily disorders such as atherosclerosis, cancer, and Alzheimer's disease, but also acts as an essential component in the physiological signaling pathways of healthy organisms, which is essential for cell growth, differentiation, migration, and immune system function [5][6][7] . Therefore, fast and accurate detection of H 2 O 2 released from living cells is important for biological and clinical diagnostics application.
For detecting H 2 O 2 , there are many analytical methods, such as fluorometry 8 , spectrophotometry 9 , colorimetry 10 , electrochemical methods 11,12 , etc. Among these, the electrochemical methods for sensing H 2 O 2 have been widely used due to their high sensitivity, fast response, and easy miniaturization. Most of electrochemical sensors involve functionalization of enzymes or proteins on the sensing interface [13][14][15] . Enzyme-based methods have been widely studied due to their remarkable sensitivity and specificity. However, the immobilization procedure of preparing the enzyme electrode has great influence on the biocatalytic activities of enzymes, leading to a limited stability and complicated immobilization procedure. Compared with enzymatic methods, the sensors based on nanomaterials (such as metal nanoparticles, carbon nanomaterials and metallic oxide nanostructures,) with high sensitivity and good stability brings H 2 O 2 sensing to non-enzymatic era. Nanomaterials' intrinsic catalytic characteristics (extremely small size and a large surface area per unit of volume) and their ability in scavenging reactive oxygen species in general can be used to mimic the catalytic activity of natural enzymes 16,17 . For example, graphene with large specific surface area, excellent electronic conductivity, and good chemical stability has been Scientific RepORtS | (2019) 9:759 | DOI: 10.1038/s41598-018-36752-y frequently reported to construct H 2 O 2 sensing devices 18 . However, there are flaws in some ways including sensitivity, selectivity, and so on.
Recently, 2D sheet-like structure of transition metal dichalcogenides (TMDCs) has attained significant amount of interest due to their potential applications in nanoelectronics, sensing, and energy harvesting. Among these 2D TMDCs nanosheets, molybdenum disulfide (MoS 2 ) is a naturally lamellar material with three atom layers (S-Mo-S) stacked together to form a sandwich structure. The unique feature renders thin MoS 2 nanosheets considerable interest and application in catalysis, transistors, lithium ion batteries, and sensors, etc. [19][20][21] . Recently, MoS 2 has been reported to directly detecting H 2 O 2 without using enzyme and has shown intrinsic peroxidase-like activity. Lei et al. 22 utilized excellent peroxidase-like activity of few layer MoS 2 for the colorimetric detection of H 2 O 2 with high sensitivity. Wang et al. 23 fabricated a sensor for electrochemical detection of H 2 O 2 released from cells based on MoS 2 nanoparticles, and discovered the electrocatalytical activity of MoS 2 nanoparticles toward the reduction of H 2 O 2 . Owing to the high sensitivity, rapid measurement, label-free detection, and compatibility with large-scale integrated circuit, field effect transistors (FETs) biosensors have attracted considerable interests [24][25][26][27][28] . Similarly, MoS 2 based FET biosensor have also been applied for detecting biological molecules. Sarkar et al. 29 has shown impressive sensitivity of the MoS 2 based biosensor for detecting pH and proteins. Lee et al. 30 and Jiang et al. 31 utilized MoS 2 as the sensing material for highly sensitive detection of DNA and mercury ion, respectively. Compared with zero band gap graphene, the advantage of MoS 2 FET sensor is ascribed to the suitable band gap and high on/off ratio of MoS 2 . Although MoS 2 can be used as the prospective candidate for the sensing channel material of FET and catalyst for the hydrogen evolution reaction (HER) 32 , so far little research is focused on utilization of MoS 2 in the FET biosensor as a perfect catalyst toward H 2 O 2 .
In this work, we have prepared a high performance FET sensor with the introduction of MoS 2 and reduced graphene oxide (RGO) for highly sensitive and specific detection of H 2 O 2 from cancer cell, in which the MoS 2 nanosheets is employed as the catalytic layer and RGO is used as the conductive layer. As illustrated in Fig. 1, a RGO sheet is drop-casted on the FET sensor surface between source and drain channel as a highly conductive bridge to facilitate rapid transport of electrons. Then the well-exfoliated MoS 2 nanosheets are assembled on the surface of RGO. The MoS 2 nanosheets act as an excellent catalyst and show highly catalytic activity toward H 2 O 2 . The as-prepared FET sensor responds fast and is extremely sensitive to H 2 O 2 with the detection limit down to 1 pM. Furthermore, the MoS 2 /RGO FET biosensor is applied to monitor trace amount of H 2 O 2 released from cancer cells.

Results and Discussion
Characterization of MoS 2 nanosheets and MoS 2 /RGO FET device. Ultrasonication has been proved to be a simple but an effective way to exfoliate graphite, bulk MoS 2 , and some other layered materials, because ultrasonic waves generate cavitation bubbles capable of breaking up the MoS 2 crystalline and producing MoS 2 nanosheets 33 . As known, N-methyl-2-pyrrolidone (NMP) is an excellent solvent for exfoliating 2D layered  34 . So we explored NMP as the solvent to prepare MoS 2 nanosheets by utilization of ultrasonication in the experiment. The structure and morphology of the as-exfoliated MoS 2 nanosheets were characterized by TEM. The TEM images in Fig. 2a clearly show that the well-exfoliated nanosheets were very thin, and a histogram of measured nanosheets size in Fig. 2b indicates that the average lateral sizes of MoS 2 were 200-250 nm. Then, XRD was employed to characterize the MoS 2 nanosheets. As shown in Fig. S1, the XRD spectra of bulk MoS 2 crystals matched with the previously reported literature 35 . The typical diffraction peaks centered at 14.3° is attributed to the (002), which belongs to the bulk MoS 2 . After exfoliated, the characteristic peak disappeared, indicating the existence of lamellar form in the exfoliated MoS 2 . The results demonstrate the successful fabrication of MoS 2 nanosheets.
As known, the MoS 2 nanosheets contain stable hexagonal semiconducting phase (2H phase) and metastable metallic phase(1T). XPS was employed (Fig. 2c,d) to survey the spectrum of Mo and S and the surface chemical properties of the as-prepared MoS 2 nanosheets. The peaks at 232.6, 229.4 and 226.5 eV, corresponded to Mo 4+ 3d 3/2 , Mo 4+ 3d 5/2 and S 2s , respectively. In the S2p spectrum, S 2p 1/2 and S 2p 3/2 peaks also appeared at 163.5 eV and 162.3 eV, which is consistent with previously published papers [35][36][37] . These results show that the dominant 2H phase in the MoS 2 nanosheets have been obtained from sonication-assisted exfoliation of MoS 2 powder. A fluorescence experiment was also conducted to verify that the prepared MoS 2 was a structure of nanosheet. As is well known, the oligonucleotides of DNA can adsorb on the surface of the layered 2D TMDCs including MoS 2 , WS 2, etc, via van der Waals interactions, and subsequently the layered 2D TMDCs could quench the fluorescence of single-stranded DNA due to fluorescence resonance energy transfer (FRET). However, pristine TMDCs powder can't quench the fluorescence of single-stranded DNA 40,41 . Fig. S2b shows strong fluorescent emission at the wavelength of approximately 520 nm for the FAM fluorophore-labeled DNA (FAM-DNA). With the addition of the as-prepared MoS 2 , up to 97% quenching of the fluorescent emission was observed, showing that MoS 2 could effectively quench the fluorescence of FAM-DNA. The result further indicates that the obtained MoS 2 nanosheet is layered nanomaterial of high quality. Figure S3a represents typical Raman spectra of RGO, in which the D band at 1350 cm −1 and the G band at 1600 cm −1 were displayed, respectively. The Raman spectra results of MoS 2 nanosheets show two characteristic peaks at 383 and 407 cm −1 , respectively. The strong Raman peaks of the MoS 2 nanosheets suggest that the exfoliated MoS 2 nanosheets are of high quality.
The SEM image was employed to characterize the prepared MoS 2 FET device. As shown in Fig. 2e, it was seen that a few layer RGO sheet was laid flat in the channel and was connected to a pair of Au electrodes. After MoS 2 nanosheets were drop-casted on the surface of RGO, the dense MoS 2 nanosheets were well-proportionally distributed on the sensing channel (Fig. 2f). All the SEM results demonstrate the successful fabrication of MoS 2 / RGO FET device.
Electrical properties of MoS 2 /RGO FET device. Current-voltage (I-V) curves were used to characterize the electrical properties of the MoS 2 /RGO-based FET device. As shown in Fig. 3, the transfer characteristic curves of the device were measured with RGO and MoS 2 /RGO FET devices, respectively. The ambipolar characteristics could be clearly observed at a small range of gate voltage (from −0.1 to 0.3 V) under ambient conditions. The dirac point of RGO transfer curve was found to be 0.1 V (Fig. 3a). After drop-casting MoS 2 onto the surface of RGO, the Dirac point of the device shifted to left (0.08 V). When gate bias of Vg = 0.1 V was applied, the device showed n-type doping, indicating that the electronic conduction is dominant in graphene channels. This phenomenon is consistent with those reported by the literatures [42][43][44] . The I ds -V ds curve was obtained to further examine the electrical characteristics of the MoS 2 /RGO FET device in Fig. 3b. The drain-source current decreased with a slight reduction of the gate voltage, indicating that the device response was sensitive to the gate voltage.

Real-time electrical detection of H 2 O 2 .
The sensitivity of the MoS 2 /RGO FET sensor was investigated by applying the freshly prepared H 2 O 2 solutions of increasing concentrations ranging from 1 pM to 100 nM to the sensor, and the real time measurements were recorded. The changes of I SD were used to monitor the responses of the MoS 2 /RGO FET sensor upon addition of H 2 O 2 at various concentrations in real time. The sensor response to H 2 O 2 was quantified using the normalized current changes (ΔI/I 0 = (I SD -I 0 )/I 0 ), where I 0 is the initial current and I SD is the stabilized current after changing the concentration of H 2 O 2 . As shown in Fig. 4a, the I SD of FET sensor showed a gradual decrease as the concentration of H 2 O 2 increased. The sensing mechanism may be attributed to generation of the positive charges upon addition of H 2 O 2 , leading to a conductance decrease of the MoS 2 / RGO FET sensor. As reported, MoS 2 can play as peroxidase mimics 22 47 . Furthermore, the applied gate bias of Vg = 0.1 V was less than This means that the reaction of catalytic decomposition of H 2 O 2 occurred on the surface of MoS 2 /RGO. As discussed above, since MoS 2 is a sandwich structure composed of two sulfur atoms and one molybdenum atom, protons can be penetrated to the middle layer, and the improvement in catalytic performance is probably due to the activity enhancement of the active sites in MoS 2 by the intercalated protons 48 . Because of its intrinsic structural characteristics, MoS 2 can act as peroxidase mimics for decomposing H 2 O 2 , wherein it produces positive charges in the process of catalysis. The positive charges were then bound to the surface of RGO, thereby attracting their counterions in graphene and inducing n-type doping. As the concentration of H 2 O 2 rose, more positive charges were generated and bound to the surface of the graphene, and the carrier concentration decreased correspondingly, leading to the decreased current. The mechanistic scheme is displayed as Fig. S4. Figure 4b shows Fig. 4a). This further implies that MoS 2 is able to decompose H 2 O 2 effectively, thereby producing a positive charge. Furthermore, more charge carrier density is formed on the MoS 2 /RGO FET device than the RGO FET device, making the larger readable signal in current change at low H 2 O 2 concentration range. The specificity of the MoS 2 /RGO FET sensor towards H 2 O 2 was further investigated by real-time recording I SD upon addition of a series of interfering species in 1 × PBS solution, including ascorbic acid (AA), uric acid (UA), glutamate (Glu), glycine (GLY), Noradrenaline hydrochloride (NE), L-glutamine (L-GA). As show in Fig. 4c, However, when 100 nM H 2 O 2 was injected, a remarkable current response was observed, even in the case that interfering species of high concentration coexisted in the analyte. To directly demonstrate the response difference, current changes of the various substances were summarized (Fig. 4d). These results firmly exhibit high specificity of the MoS 2 /RGO FET sensor toward H 2 O 2 . Then, the repeatability and stability tests were also conducted to illustrate the excellent property of the MoS 2 /RGO FET sensors by using more than 3 sensors, respectively. Firstly, for stability test, the as-prepared MoS 2 /RGO FET device was stored in a vacuum oven for 3, 7, 10 and 14 days, respectively, then used for the detection of 100 nM H 2 O 2 . As described in the Fig. S5a, the shift of the dirac voltage was only changed 12.5% compared with its original value over 2 weeks. This signal decrease may be caused by the nonspecific surface adsorptions. Secondly, the repeatability of the MoS 2 /RGO FET sensors was also evaluated. One MoS 2 /RGO FET sensor was chosen to repeatedly detect 100 nM H 2 O 2 concentration for 7 times, as shown in the Figure S5b. The dirac voltage change of the sensor remained nearly the same after 7 measurements and a relative standard deviation (RSD) was 2.1%. The results demonstrate high repeatability of the sensor.

Real-time monitoring of H 2 O 2 released from HeLa cells. H 2 O 2 plays a significant role in many cell
functions and can be used as potential marker for tumor cells. Consequently, it is very meaningful to sensitively detect H 2 O 2 from living cell because of its diverse biological functions. For this experiment, we first investigated the influence of weak acid environment on sensor's performance from pH 6.4 to 7.4 (These different pH values were made by adding HCl or NaOH to PBS solutions, which were finally adjusted by a commercial pH meter).
The results show that the weak acid environment did not have significant influence on the change of Dirac point after different pH values were applied ( Figure S6). Then, real-time detection of extracellular H 2 O 2 released from HeLa cells was performed by the MoS 2 /RGO FET sensor. As known, the HeLa cells can generate H 2 O 2 when stimulated by phorbol 12-myristate 13-acetate (PMA, PMA is a potent activator of protein kinase C (PKC), which can activate PKC to produce H 2 O 2 ). On the contrary, H 2 O 2 can be decomposed by catalase. Before cell level measurement, HeLa cells were cultured for 24 h on the MoS 2 /RGO FET sensor surface by using a self-made plastic culture chamber. After cell culture, the cells were found to be in good condition, as seen in Fig. 5, Inset. Afterwards, the culture medium was replaced with the same amount of PBS solution. As shown in Fig. 5, when PMA (with the final concentration of 1 μg/mL) was added into the cell chamber, the current declined immediately and then slowly stabilized in a short time (red line). Based on the current change generated in Fig. 5, we could semi-quantify the released H 2 O 2 from cells by the working curve in Fig. 3b    Fabrication of the MoS 2 /RGO sensor. The RGO-based FET device was produced by the previously reported method 25 . Firstly, 10 mg of GO was added to 10 mL of 98% hydrazine followed by sonication for 45 min to produce a black suspension of hydrazinium graphene, and the suspension was placed for 1 week to obtain the thorough reduction of GO. liquid reservoir was mountained on the sensing channel (Fig. S7). After that, the total device was sterilized for 30 min via ultraviolet in a biosafety cabinet. Then the sensor was used for cell culture experiments. HeLa cells were seeded on the MoS 2 /RGO sensors confined in a self-made liquid reservoir at a density of ~1 × 10 4 cell/cm 2 . After 10 h of incubation, cells were used for stimulation and detection. Upon detection, the culture medium was then changed by the 1 × PBS. After reaching a steady-state baseline, PMA (1 μg/mL) as the H 2 O 2 stimulant was introduced into the self-made liquid reservoir and H 2 O 2 released from HeLa cells was detected by the real-time working mode present in the form of changes in current. Then catalase (300 U/mL) was injected into the liquid reservoir for the purpose of degrading H 2 O 2 , as the control experiment. The electrical measurement condition was the same as above described.

Materials
Scientific RepORtS | (2019) 9:759 | DOI:10.1038/s41598-018-36752-y Instrumentation. The morphology of the as-prepared MoS 2 was characterized by TEM (JEOL JEM-2100, Japan) operated at 200 kV. The MoS 2 dispersion was further diluted with ethanol and dropped on a carbon-coated film copper grid for subsequent TEM observation. The X-ray photoelectron spectroscopy (XPS) analysis was conducted using an ESCALAB 250 Xi XPS system (Thermo Fisher Scientific, American). UV-visible spectra were measured by UV-2550 (Shimadzu Co. Ltd., Japan). Raman spectra were taken by using Invia Renishaw spectrometer (RM 1000, England) equipped with 514.5 nm laser line. X-ray diffraction (XRD) analysis was conducted on PANalytical X'Pert Pro diffractometer (PANalytical, Holland). The fluorescence spectra were obtained by a Hitachi F-4600 spectrophotometer (Hitachi Co. Ltd., Japan). Scanning electron microscopy (SEM) images were obtained on a field-emission scanning electron microscope (Zeiss, Germany). All electrical measurements were recorded with a Keithley 4200 semiconductor characterization system and a shield probe station (Everbeing BD-6, Taiwan).