In situ synthesis of MoS2 on a polymer based gold electrode platform and its application in electrochemical biosensing

Two-dimensional (2D) nanomaterials have attracted increasing attention because of their advantageous physical and chemical properties. In particular, 2D transition metal dichalcogenides (TMD) show a wide range of electronic, optical, mechanical, chemical, and thermal properties. In contrast to zero-band gap graphene, TMD molybdenum disulde (MoS2) has an indirect band gap of 1.29 eV in the bulk state and it can be tuned from indirect to direct band gap (1.90 eV) through layer control. Moreover, in some cases MoS2 is utilized for the hydrodesulfurization reaction and for the hydrogen evolution reaction (HER) as a catalyst. Similarly, other 2D materials, such as graphene, epimeric monosaccharide-quinone hybrid on gold electrode have been utilized for electrochemical biosensors. Substantial efforts have been devoted to prepare thin layers of MoS2, by methods including adhesive tape-based micromechanical exfoliation, physical and chemical vapor deposition (CVD) and hydrothermal synthesis. Previous reports showed that an exfoliated single-layer MoS2 has a high


Introduction
Two-dimensional (2D) nanomaterials have attracted increasing attention because of their advantageous physical and chemical properties. 1 In particular, 2D transition metal dichalcogenides (TMD) show a wide range of electronic, optical, mechanical, chemical, and thermal properties.In contrast to zero-band gap graphene, TMD molybdenum disulde (MoS 2 ) has an indirect band gap of 1.29 eV in the bulk state and it can be tuned from indirect to direct band gap (1.90 eV) through layer control.Moreover, in some cases MoS 2 is utilized for the hydrodesulfurization reaction 2 and for the hydrogen evolution reaction (HER) as a catalyst. 1,3,4Similarly, other 2D materials, such as graphene, 5,6 epimeric monosaccharide-quinone hybrid 7 on gold electrode have been utilized for electrochemical biosensors.Substantial efforts have been devoted to prepare thin layers of MoS 2 , by methods including adhesive tape-based micromechanical exfoliation, 8 physical and chemical vapor deposition (CVD) [9][10][11] and hydrothermal synthesis. 12,13Previous reports showed that an exfoliated single-layer MoS 2 has a high mobility (200 cm 2 V À1 s À1 ) with a hafnium oxide (HfO 2 ) gate dielectric, and the on/off ratio of over 10 8 at room temperature, 14,15 however MoS 2 shows much low mobility (30-60 cm 2 V À1 s À1 ) on Si/SiO 2 . 16It is difficult to achieve large grain size with high uniformity.While thermal chemical vapor deposition (CVD) method is the most extensive studied methods for synthesizing large and thin lms of MoS 2 . 10Its applicability to exible devices is limited because of the high (>600 C) temperatures this method requires during the synthesis process for decomposition of carrier gases.Techniques that overcome this temperature issue to allow applications to exible devices including growing MoS 2 on a polymer substrate, which can be carried out at a process temperature below 300 C or transfer of the MoS 2 lm from a Si/SiO 2 substrate.The transfer step causes cracks or wrinkles in devices, so both methods have the potential to deteriorate MoS 2 properties during the transfer process.Hence, there is a need to develop a new low temperature synthesis techniques that allow the direct synthesis of (in situ) MoS 2 on the polymer substrate are needed to achieve high yield and performance.
Plasma enhanced chemical vapor deposition (PECVD) is well known for low temperature synthesis.During plasma synthesis, nanomaterials are subjected to various forces such as electrostatic forces, neutral drag, thermophoresis, ion drag in the sheath region, and photophoresis.These forces can be controlled by optimizing PECVD process parameters.In the past, our group succeeded in synthesizing mono-dispersed silicon nanoparticles with a uniform size and controlled morphology. 17Previous reports demonstrate synthesis of nanomaterials, such as carbon nanotubes, 18 graphene, 19 silicon nanoparticles and silicon nanowires, 20 at temperatures below 500 C using PECVD.The synthesis of nanomaterials at low temperatures is helped by the presence of radicals and dense sheaths (carrier gas and source gas) that facilitate the dissociation and decomposition of the gases.In this paper, we present an effective route for the in situ synthesis of MoS 2 on a polymeric printed circuit boards (PCB).The in situ synthesis of MoS 2 using PECVD is discussed in terms of the results of X-ray photoelectron spectroscopy (XPS), high resolution transmission electron microscopy (HR-TEM) and Raman scattering spectroscopy.Then the as-prepared MoS 2 modied on Au electrode was used to immobilize IgG-HRP, and catalytic activity of the resultant HRP electrode to H 2 O 2 reduction was observed with help of hydroquinone (HQ).

Chemicals and materials
Phosphate buffer solution (pH ¼ 7.0) was purchased from Sigma Aldrich (P4417, USA).IgG-HRP (NCl1430KR) was purchased from Thermoscientic.Hydroquinone (h3660) and hydrogen peroxide solution were purchased from Sigma Aldrich.The deionized water (DI water, >18 MU cm) used to prepare all solutions in this study was obtained from a water purication system.All chemicals were of reagent grade.

Fabrication of the Au electrode on PCB
We fabricated a layer of gold for all three working, counter, and reference electrodes to simplify fabrication process.Each gold (Au) electrode chip was fabricated by the semiconducting processes including positive photoresist (PR) coating, patterning, li off and passivation in Scheme 1a.Approximately 250 three-electrode Au chips were fabricated on a glass wafer (4 inches in diameter).Au electrode chip with sensing PCB was fabricated by an electroplating method and used in further experiment.Each Au electrode chip was diced and glued on PCB. 21The center of Au electrode (diameter, 2 mm) was used further for the in situ synthesis of MoS 2 .The other two side Au electrodes were masked during synthesis.The centre Au electrode with MoS 2 (Au-MoS 2 electrode) was used as a working electrode during electrochemical cyclic voltammetry (CV) measurements.
In situ synthesis of layered MoS 2 on the Au electrode PCB Due to low carrier mobility, MoS 2 itself is not suitable as an electrode material for device applications.There have been some studies on MoS 2 FET based biosensor by transfer of MoS 2 onto a metal electrode, which may be susceptible to contact resistance related issue.Thus, in the present research, was deposited by implementing PECVD in situ synthesis.MoS 2 was synthesized using PECVD at 300 C with H 2 S gas as a precursor on a prefabricated gold (Au) electrode on a glass wafer.The synthesis of MoS 2 on a Si/SiO 2 wafer under various process parameter conditions was reported previously. 22The process for the in situ synthesis of MoS 2 is shown in Scheme 1b.The synthesis of MoS 2 begins with the deposition of Mo metal on Au(111) herringbone surface by e-beam evaporation.The thickness of the molybdenum metal layer was 1 nm using a deposition rate below $0.1 Å s À1 under high vacuum conditions.During the deposition process, some of the Mo atoms were deposited on the surface of the Au electrode and some penetrated into the Au, forming an Au-Mo composite structure.This also facilitated a reduction of the contact resistance, which was opposite of that seen with the transfer of Mo on Au electrodes. 23,24During the PECVD process, the Au-Mo components reacted with the combination of H 2 S (source gas) and Ar (carrier gas) plasma in an ultra-high vacuum (UHV) at 300 C. The synthesized MoS 2 thin lms were characterized by HR-TEM (JEOL-JEM ARM 200F), Raman spectroscopy (Alpha300 M+, WITec GmbH, Germany) wavelength and power of laser are 532 nm and 2 mW respectively and X-ray photoelectron spectroscopy (XPS, ESCA2000 (VG Microtech Inc.)).hydroquinone worked as a diffusional mediator.Eqn (1)-( 4) shows the overall electron relay mechanism for hydrogen peroxide reduction via the immobilized HRP enzyme.

Evaluation of H 2 O 2 with IgG-HRP by cyclic voltammetry
HQ oxidation + 2H + 2e À / HQ reduction (3) In the presence of HRP and hydroquinone (HQ), the addition of H 2 O 2 triggered the oxidation of HQ to benzoquinone (BQ) which is subsequently reduced giving a current response of the electrode.

Results and discussion
Preparation and characterization of in situ Au-MoS 2 In the plasma phase, which is an ionized gas phase, the H 2 S source gas is easily dissociated at low temperature.By taking advantage of this property, we synthesized wafer-scale layered MoS 2 at 300 C on the Au electrode deposited PCB, which is a polymeric substrate.
XPS was used to determine the chemical bonding composition of the surface.Fig. 1a and b show the XPS spectra the MoS 2 nanomaterial on Au electrodes.The Mo 3d spectrum (Fig. 1a) exhibited two well-separated peaks at binding energy levels of 229.3 eV and 232.6 eV respectively.The S 2p spectrum (Fig. 1b) shows two merged peaks at 162.5 eV and 163.4 eV.The XPS scans for other synthesis temperature conditions conrmed the chemical bonding states of the MoS 2 layers.The calculated atomic concentration of Mo and S from XPS was 34.4% and 65.6%, yielding a ratio of 1 : 1.90.The ratio from our PECVD-produced MoS 2 is the same as the ratio of the CVDsynthesized MoS 2 . 26ig. 1c shows the morphology of the MoS 2 thin lm as obtained by analysing HR-TEM, transferred to the TEM grid (TED PELLA.INC, 200 nm silicon nitride membrane).The measured grain size at 300 C ranged from 5-7 nm and was of a crystalline nature.Raman spectra (Fig. 1d) show that the MoS 2 synthesized at 300 C had E 1 2g and A 1g modes with observed Raman peaks at 380.15 and 405.15 cm À1 .The 15 cm À1 difference between these peaks indicates the presence of about 6 to 7 layers of MoS 2 . 27

Electrochemical investigation
The effect of scan rate on the anodic and cathodic peak currents at the Au-MoS 2 electrode/HRP was investigated in 10 mM PBS (pH 7.0) using cyclic voltammetry (CV).As shown in Fig. 2a, it depicts the CV response of the Au-MoS 2 electrode with different scan rate.The current value was observed in the absence of  H 2 O 2 which resulted from surface defects at 10 mV s À1 (curve 1).However, aer the addition of 5 mM H 2 O 2 at 10 mV s À1 , the center of the reduction peak slightly increased to À0.25 V (curve 2).The redox peaks were clearly observed with a scan rate ranging from 10 to 100 mV s À1 demonstrating that the reduction and oxidation peak currents Increased as the scan rate increased.When the scan rate was changed 10 to 100 mV s À1 , the reduction peak was proportional to the voltage from À0.25 to À0.29 V (curves 2, 3, and 4).The shi of the potential and the increase of the current indicated a notable enhancement of the electrocatalytic activity of the Au-MoS 2 electrode toward the reduction of H 2 O 2 .Fig. 2b  As shown in Fig. 3, well-characterized redox peak currents were observed at an IgG-HRP concentration ranging from 0 to 20 ng mL À1 with a scan rate of 100 mV s À1 .The reduction current increased noticeably with the increase of IgG-HRP concentration.The inset indicated the linearity of the current level measured against the concentration of IgG-HRP from 0 to 20 ng mL À1 at a corresponding potential of À0.8 V. Moreover it was found that reduction peak currents were proportional to the concentrations of HRP, indicating typical electrochemical behaviour of immunoassays as well as a much larger peak current, which was observed because of the direct electron transfer between the HRP and underlying electrode, which was enhanced by the MoS 2 thin lm.In comparison with previous reported work the presented H 2 O 2 sensor has lower detection limit 28 and linear response. 29From these observations, the sensing mechanism of this sensor is the conjugation of the IgG target molecules and antibodies to IgG immobilized on Au-MoS 2 /immuno-substances by adsorption of HRP-conjugated IgG.By utilizing well-known sandwich based immunoassay, the detection efficiency of the targeted sample can be enhanced by the competitive reaction.Furthermore, the current level caused by antibody-target-detection can be increased by reducing the possibility of non-specic binding of the antibodies aer pre-treatment of the bovine serum albumin (BSA) blocking step, this setup is able to detect nothing but targeted conjugation.

Conclusions
In summary, an in situ synthesis method for bulk layer of MoS 2 was developed to fabricate H 2 O 2 sensors.MoS 2 was successfully synthesized at 300 C on a polymeric PCB substrate.The resultant Au-MoS 2 /IgG-HRP electrode shows good electrocatalytic performance toward the reduction of H 2 O 2 when HQ is used as mediator.The cyclic voltammetry results showed that the sensor of Au-MoS 2 conjugated with IgG-HRP thus exhibited excellent analytical responses to H 2 O 2 with a wide linear range.In particular, the linear range of IgG-HRP was measured to be 0-20 ng mL À1 with an R 2 value of 0.998.The aim of this study to immobilize on Au-MoS 2 /immuno-substances, which can be used as an application in electrochemical biosensor.Our ndings show that in situ synthesis process of MoS 2 can be used as biocompatible matrix for the enzyme immobilization and construction of electrochemical biosensor.Moreover it can be expected to have the potential for commercial applications because of its low temperature in situ synthesis process and the ease at which other chemical and biosensors could be developed using this sensing platform.

For the H 2
Scheme 1 Schematic of Au-MoS 2 and IgG-HRP conjugated Au-MoS 2 (a) Semiconductor processes for gold electrode fabrication (b) (1) 1 nm thick Mo is deposited on an Au electrode using an e-beam evaporator, (2) H 2 S + Ar plasma is generated and reacts with the Mo thin film in the PECVD chamber, (3) H 2 S+ combines or penetrates into the Mo thin film to form MoS 2 .(c) Schematic of the MoS 2 biosensor device.For biosensing, the MoS 2 is functionalized with receptors for specifically capturing target biomolecules.

Fig. 1
Fig. 1 Chemical composition analysis of as-prepared MoS 2 thin films using PECVD by X-ray photoemission spectroscopy (XPS) (a) Mo 3d and (b) S 2p region.(c) A typical HR-TEM image taken from the center of MoS 2 at 300 C. The insets are the selected area electron diffraction (SAED) (d) the Raman spectra, which is the E 1 2g and A 1g vibrational modes of MoS 2 on an Au electrode.
depicts the cyclic voltammetry (CV) response of the Au-MoS 2 electrode conjugated with IgG-HRP with a scan rate of 100 mV s À1 .Fig. 2b, curve 1, shows the CV response of the Au-MoS 2 electrode in 0.1 M PBS, while curve 2 is response with the addition of 5 mM H 2 O 2 , and curve 3 shows the response of Au-MoS 2 /HRP (1 ng mL À1 ) in the presence of H 2 O 2 .The H 2 O 2 reduction was promoted by the electrocatalysis from IgG-HRP immobilized on the Au-MoS 2 electrode.