CVD graphene as an electrochemical sensing platform for simultaneous detection of biomolecules

The development of electrochemical biosensors for the simultaneous detection of ascorbic acid (AA), dopamine (DA), uric acid (UA), tryptophan (Trp), and nitrite (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\rm{NO}}}_{2}^{-}$$\end{document}NO2−) in human serum is reported in this work. Free-standing graphene nanosheets were fabricated on Ta wire using the chemical vapor deposition (CVD) method. CVD graphene, which here served as a sensing platform, provided a highly sensitive and selective option, with detection limits of AA, DA, UA, Trp, and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\rm{NO}}}_{2}^{-}$$\end{document}NO2− of 1.58, 0.06, 0.09, 0.10, and 6.45 μM (S/N = 3), respectively. The high selectivity of the electrode is here explained by a relationship between the bandgap energy of analyte and the Fermi level of graphene. The high sensitivity in the oxidation current was determined by analyzing the influence of the high surface area and chemical structure of free-standing graphene nanosheets on analyte adsorption capacity. This finding strongly indicates that the CVD graphene electrode can be used as a biosensor to detect five analytes in human serum.

to the references, the GO composite-modified-electrodes were detected simultaneously with the DA and 5-hydroxytryptamine 28 , DA and UA 29 , guanine and adenine 30 , AA, DA, and UA 31,32 , AA, DA, UA, and folic acid 33 , and l-tyrosine and l-tyrosine 34 . However, these graphene-based materials use of drip coating, electrodeposition, and screen printing method, these graphene based-materials are modified to the basis electrodes. Weak adhesion between the sensitive film and the based electrode can shorten the life of the sensitive layer. They also require special storage conditions. CVD graphene has higher electrical conductivity and fewer defects than graphene prepared using wet-chemical methods 35 . CVD graphene is grown directly on the conductor, and the strong binding between the two prevents them separating from each other 36,37 . These give the electrodes strong stability and long life. The CVD graphene-based sensor has been used to detect levodopa 38 and glucose 39,40 . Further increases in the surface area of graphene electrodes have been found to enhance the adsorption capacity of graphene layer for the analyte 41,42 , thereby increasing the sensitivity of the graphene electrode. To achieve this, the vertical growth of graphene must be fostered to form a graphene wall on a metal substrate. Moreover, these graphene walls would ideally be highly dense, and the high edge density increases the charge storage ability 43 , thereby increasing adsorption and accelerating the electron transfer rate. More importantly, free-standing graphene walls are regularly exposed to different edge structures, which indicates the presence of different band-gap energy levels on graphene layer 44 . This renders the electronic transition process different from that of other graphene 45 , then the sensitive layer may have a better potential selectivity. Recently, a method of directly synthesizing graphene electrodes using CVD has been developed. This made the production of high-quality graphene with vertically oriented features possible 46 .
Based on these above considerations, a biosensing electrode with CVD graphene as sensitive layer was here developed, and Ta wire served as the basis electrode. The present work focuses on evaluating the simultaneous detection of AA, DA, UA, Trp, and − NO 2 , and response mechanism of this graphene designed for the simultaneous detection of multiple analytes is evaluated.

Results
Characterization of graphene/Ta wire electrodes. Figure 1a shows the three-electrode testing system with the graphene/Ta wire as the sensing electrode. Figure 1b and c clearly indicate that graphene layers with pores between the graphene sheets formed an open network. The graphene sheets were thin and transparent, and the thinner sheet was divided from the surface of the nanosheet. The graphene layer contained many curved nanosheets with vertical growth trends. Network structures of graphene nanosheets provide more channels between the graphene sheets to facilitate the entrance of electrolyte ions or electrons into the inter-space. The effective area of graphene layers calculated using electrochemical kinetics is 2.24 cm 2 (Fig. S1), which is ten times the actual area (0.2262 cm 2 ) of graphene/Ta wire electrode. The thickness of graphene nanosheets can be confirmed using high-resolution transmission electron microscopy (TEM) (Fig. 1d) and atomic force microscopy (AFM) (Fig. 1e) images. The thickness of these graphene sheets ranged from 0.34 nm to 3 nm, corresponding to stacking of approximately 1-10 layers of the monoatomic graphene sheets. The conductivity of graphene affects the sensitivity of the sensing electrode. The conductivity of multi-layer graphene is lower than that of single-layer graphene. Compared with the low-layer graphene prepared by the liquid-phase oxidation method, the vertical multi-layer graphene sheets prepared using the CVD method have high orientation, low contact resistance (between the Ta wire and graphene layer), and less oxygen-containing groups, which impart high electrical properties to the prepared graphene/Ta wire. Of course, if we can prepare a graphene electrode with vertically arranged single-layer graphene sheets, then the sensitivity of the graphene electrode will be higher than the current graphene/Ta wire.
The Raman spectrum of graphene layer is shown in Fig. 1f. The spectrum feature include a D band at approximately 1355.5 cm −1 , G band at around 1592.7 cm −1 , and 2D band at approximately 2702.5 cm −1 . The D band is a characteristic feature, indicating defects. It can be caused by excitation of the edges of graphene sheets and their random orientation 47,48 . The G band shows sp 2 vibrations in the graphitic plane, which confirms the presence of graphene sheets. Intense, well-defined 2D bands with a narrow width of full width at half maximum is peculiar for few layer graphene. The X-ray diffraction (XRD) pattern of the graphene/Ta wire is shown in Fig. 1g. A sharp peak at about 2θ = 26° corresponding to the (002) reflection of graphite (JCPDS 75-1621) was observed with interlay space (d-spacing) of 0.34 nm, indicating the graphene layer formed on Ta wire. In addition, the TaC phases were observed, which indicated that the carbon atoms first dissolved into the interior of Ta which formed the TaC transition layer and then formed graphene nanosheets through further deposition of carbon atoms 46 . Electrochemical behaviors of graphene electrode. Figure 6 ], respectively. For the TaC/Ta wire, the Nyquist plot consisted of two semicircles in high-and medium-frequency regions followed by an inclined line in low-frequency region. For the graphene/ Ta wire, the Nyquist plots consisted of a semicircle in high-frequency region followed by an inclined line in a medium-low-frequency region. According to the Randles-type circuit in Fig. 2b, the series resistance (R s ) is created by the electrolyte. The semicircle indicates the charge-transfer resistance (R ct ) at the external graphene/ electrolyte interface; the interface resistance (R int ) is contributed from the bottom graphene layer. As shown, the diameter of the semicircle for the TaC/Ta wire (R ct = 200 Ω cm 2 ) is much larger than that of the graphene/Ta wire (R ct = 37.2 Ω cm 2 ), indicating the graphene/Ta wire electrode exhibited better charge transfer performance than the TaC/Ta wire and had excellent conductivity.
The individual electrochemical response of these analytes needs to be studied before the sensitivity of graphene/Ta wire electrodes toward the simultaneous detection of AA, DA, UA, Trp, and  Fig. S2. The oxidation potential is generally governed by electron transfer from the analyte to the sensing electrode and hole transfer from the analyte to the counter electrode 49 . The graphene/Ta wire for use as a sensing electrode, the oxidation potential is dominated by both the difference in energy between the Fermi level of graphene (E F ≈ −4.6 eV) 50 and the highest occupied molecular orbital (HOMO) of analytes, and energy gaps between the HOMO and lowest unoccupied molecular orbital (LUMO) of the analytes. HOMO and LUMO energies approximately represent the a ability of molecules to donate and accept electrons 51 . H. Y. et al. 49 , reported that the oxidation potential increases with the increase of LUMO-HOMO energy gap. In this study, molecules and MO orbitals were prepared using GaussView 5.0 and all the calculations were performed using Gaussian V9.5. Moreover, all the molecules were fully optimized using the density functional theory (DFT)/B3LYP method with 6-31 G basis sets in water. The MO data was calculated at the B3LYP/6-311++G (d, p) level for AA and DA, while that of UA, Trp, and − NO 2 was at the RHF/ AM1 level. Frontier molecular orbital analyses of the HOMO and the LUMO are summarized in Fig. 3b. The LUMO-HOMO energy gaps were, from highest to lowest, The sensing mechanism of the graphene/Ta wire is shown in Fig. 3c. The anodic peaks correspond to the oxidation of hydroxyl groups to carbonyl groups of the furan ring in AA, oxidation of catechol to o-quinone in DA, oxidation of bridging double bonds to hydroxyl groups in UA, oxidation of the phenyl ring in Trp, and oxidation of the − NO 2 to − NO 3 . The oxidation peak current is determined using the sum of the electron flow to the graphene and the hole flow to the Pt electrode. The oxidation current is affected by the adsorption strength between the graphene and the analytes. DA, UA, and Trp contain aromatic rings in their molecular structures (Fig. 3c). Graphene nanosheets have strong adsorption affinities to aromatic rings because strong π-π interactions between the free π-bonds of sp 2 atoms from the six-benzene-ring graphene and the aromatic rings from to DA, UA, and Trp. Hence, high oxidation currents translate to higher sensitivity in the detection of DA, UA, and Trp than in the detection of AA and − NO 2 . Due to the availability of both large peak separations and enhanced currents, the graphene/Ta wire electrode is the best electrode for sensing the simultaneous detection of AA, DA, UA, Trp, and − NO 2 . Freestanding graphene nanosheets are exposed to a lot of edges defects (Raman spectrum in Fig. 1f) and to the active sites from edge defects of graphene [53][54][55] .    Table S1.
When the five analytes were detected simultaneously, i.e. when the concentrations of all five were changed, the calibration curves were linear and within the concentration ranges  49 . The interface interaction of monolayer adsorption is strong, while that of multilayer adsorption is weak; hence, the slope of the working curve changed with increase in the concentration of analytes. Further, since the DA molecule contains an aromatic ring, a well-ordered organic layer was formed between the graphene and the aromatic ring of DA through π-π stacking interactions, which resulted in strong adsorption; therefor, the slope did not change within this concentration range. Compared with the flat graphene composite electrode, the sensitivity of the graphene/Ta wire electrode reached the same level, and the detection limits of DA, UA, and Trp are very advantageous 5,26,56 .
The LOD for AA, DA, UA, Trp, and − NO 2 was found to be 1.58, 0.06, 0.09, 0.10, and 6.45 μM, respectively. The lowest LODs obtained here for simultaneous determination of AA, DA, UA, Trp, and − NO 2 were comparable to or in some cases better than previously reported values. In Table S3, some of the analytical characteristics obtained in this work were compared to values previously reported in the literature 5,21,49,[56][57][58][59] . All this evidence supports the idea that the graphene/Ta wire electrode has an excellent ability to determine AA, DA, UA, Trp, and − NO 2 . The stability of the graphene/Ta wire electrode was also studied in this work. When the electrode was cyclically swept for 100 cycles, there was a 4.8% decrease in the initial response of the graphene electrode, indicating that the graphene electrode had excellent stability (Fig. S4). The reproducibility of the graphene electrode was determined using six different electrodes. The relative standard deviations (RSD) of the current responses for AA, DA, UA, Trp, and − NO 2 were 3.4%, 5%, 4%, 5%, and 4.5%, respectively. Several forms of inorganic, organic, and biological interference were tested to determine whether they could affect the detection of AA, DA, UA, Trp, and  Table S4. The recovery was in the range of 94-104% for all analytes. We compared the results of various methods for the determination of concentration of the five analytes in serum (Table S5). In general, the concentrations of AA, DA, UA, and Trp are closer to those obtained by CL 60 , HPLC-CL 61 , GC/MS 62 , and UV methods 63 ; however, the concentration of NO 2ˉ is significantly higher than that measured by the spectrophotometric methods in the ref. 64. Since the sensitivities of various detection methods are different, and so are the serum samples, the results vary. However, the results show the presence these substances in human serum. These results indicated that graphene/Ta wire could produce results accurate enough for clinical diagnosis.

Conclusions
Free-standing graphene nanosheets were here fabricated on Ta wire, which was used to selectively and simultaneously detect AA, DA, UA, Trp, and − NO 2 using a DPV method. The graphene electrode provided highly selective, sensitive results, with detection limits of AA, DA, UA, Trp, and − NO 2 of 1.58, 0.06, 0.09, 0.10, and 6.45 μM (S/N = 3), respectively. The key features of the graphene electrode responsible for its sensitivity improvement were found to be as follows: (i) free-standing nanosheets interlaced to form a network with mesoporous structures, which increased the surface area of the electrode, thereby facilitating ion adsorption on the surface of the electrode; (ii) high conductivity from CVD graphene nanosheets accelerated the ion diffusion and electron transfer. Free-standing graphene nanosheets are exposed to a lot of edges defects and to the active sites from edge defects of graphene. The Fermi level of graphene, HOMO, and LUMO of analytes affected the oxidation potential of the analytes. The two factors endowed the graphene electrode with high selectivity.

Methods
Reagents and materials. AA  Preparation of grapheme. Ta wires with a diameter of 0.6 mm were cut from 5 cm length wire-like substrates. The free-standing graphene nanosheets covered Ta wires, i.e. the graphene/Ta wire electrodes were prepared using a high-power direct current (DC) arc plasma jet CVD operating at gas recycling mode. The Ta wires were cleaned and attached to catalysts as follows before being placed in the deposition chamber: ultrasonic cleaning in ethanol and ultra-pure water, drying, immersion in the catalyst precursor solution (0.6 M Ni(NO 3 ) 2 ), and drying. The main deposition parameters are shown in Table S2.
Characterization. The morphology of the CVD graphene obtained by scanning electron microscopy (SEM; Carl Zeiss MERLIN Compact, Germany), transmission electron microscopy (TEM; JEOL JEM-2100, Japan) and atomic force microscopy (AFM; Agilent 5500, U.S.) measurements, respectively. Sample treatment for AFM measurement: The graphene/Ta wire electrode was immersed in ethanol and sonicated in an ultrasonic bath for 48 h (600 W/40 kHz). During this time, a small amount of graphene sheet was peeled off and dispersed in ethanol. The graphene/ethanol solution was then sprayed onto the Si wafer and dried. The Raman spectrum of graphene was recorded on a Renishaw Raman microscope with an excitation laser wavelength at 532 nm. X-ray diffraction (XRD) spectra were created using a Rigaku Ultima IV x-ray diffractometer with CuK α radiation (wavelength of 0.15406 nm).
The electrochemical performance was investigated using cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS) with a CHI 660E electrochemical workstation (Shanghai Chenhua Instrument Co., Ltd.). In a three-electrode system, a graphene/Ta wire electrode/or other sample electrodes, a platinum (Pt) sheet and a saturated calomel electrode (SCE) served as the working electrode, counter electrode, and reference electrode, respectively (Fig. 1a).