Scalable Preparation of Low-Defect Graphene by Urea-Assisted Liquid-Phase Shear Exfoliation of Graphite and Its Application in Doxorubicin Analysis

The mass production of graphene is of great interest for commercialization and industrial applications. Here, we demonstrate that high-quality graphene nanosheets can be produced in large quantities by liquid-phase shear exfoliation under ambient conditions in organic solvents, such as 1-methyl-2-pyrrolidinone (NMP), with the assistance of urea as a stabilizer. We can achieve low-defect graphene (LDG) using this approach, which is relatively simple and easily available, thereby rendering it to be an efficient route for the mass production of graphene. We also demonstrate the electrochemical sensing of an LDG-modified electrode for the determination of doxorubicin (DOX). The sensor shows an enhanced electrocatalytic property towards DOX, leading to a high sensitivity (7.23 × 10−1 μM/μA) with a detection limit of 39.3 nM (S/N = 3).


Introduction
Graphene can be applied to everything from electronics and pharmaceuticals to sensing, biomedical and energy devices [1,2]. With exceptional properties derived from the atom-thick layer of graphite, it is becoming more prevalent and more critical in industry and scientific research. Although there is a myriad of uses for this wonder material, production can often be expensive and time-consuming. Graphene can be prepared using epitaxial growth and chemical vapor deposition (CVD) [3,4]. Even with the production of high-quality graphene, however, these techniques suffer from the drawbacks of complicated procedures, the need for toxic chemicals, expensive production or limited quantity, making it difficult to scale up to mass production for commercialization and industrial-scale application. With respect to mass production, chemical exfoliation is the facile and cost-effective technique in which the graphene preparation begins with the oxidation of graphite to graphene oxide (GO) and its subsequent reduction to get reduced graphene oxide (rGO) [5,6]. However, the resulting rGO is low-quality graphene with many of the defects of oxygen-containing functional groups.
To circumvent these issues, one of the most promising techniques is the physical exfoliation of layered graphite to graphene, which can lead to the mass production of low-defect graphene (LDG) [7]. The mechanical forces in physical exfoliation, such as sonication [8,9], ball milling [10,11] and shear exfoliation [12,13] can overcome the van der Waals forces that hold the graphite layers together, enabling graphene production on a large scale at a low cost. Graphene prepared from sonication and the ball-milling method, however, has more fragmentation and defects than expected, which is attributed to the sonication-induced cavitation, or collisions among the grinding media during the milling process. On the other hand, liquid-phase shear exfoliation with a high-shear mixer is easily available and makes it relatively simple to mass-produce defectless graphene. The size and amount of graphene

Instrumentation and Measurements
The shear exfoliation was performed with an L5M-A mixer homogenizer (Silverson, Co., East Longmeadow, MA, USA) with a Square Hole High Shear Screen ™ workhead. The absorbance of the exfoliated LDG was measured by UV-vis spectroscopy (SINCO Co., Ltd., Seoul, Korea). The concentrations of the LDG were calculated from the standard curve for the absorbance at 660 nm of the commercial graphene solution. The Raman spectroscopy was performed using an EnSpectr R532 Raman spectrometer (Enhanced Spectrometry, Inc., San Jose, CA, USA). Transmission electron microscopy (TEM) was carried out on a JEOL 2010 device (JEOL, Inc., Peabody, MA, USA) with an accelerating voltage 200 keV. The X-Ray diffraction (XRD) patterns were recorded using a Rigaku SmartLab X-ray diffractometer (Rigaku Co., Tokyo, Japan) with Cu Kα source. Fourier transform infrared spectroscopy (FTIR) was performed with a NICOLET iS10 (Thermo Scientific Korea Ltd., Seoul, Korea). The X-ray photoelectron spectroscopy (XPS) signals were recorded using a Thermo Scientific K-Alpha XPS system (Thermo Fisher Scientific, Paisley, UK) equipped with a micro-focused monochromatic Al Kα X-ray source (1486.6 eV). The electrochemical measurements were performed with a CHI 660D electrochemical workstation (CH Instruments, Inc., Austin, TX, USA). A conventional three-electrode cell was used with a Pt wire as a counter electrode, an Ag/AgCl electrode was used as a Nanomaterials 2020, 10, 267 3 of 11 reference electrode and a bare glassy carbon electrode (GCE, diameter = 3 mm), or modified electrode, was used as a as working electrode at room temperature.

Shear Exfoliation of Graphite by Using a Homogenizer
The LDG samples were prepared by the liquid-phase shear exfoliation method using a high-speed mixer homogenizer, as shown in Scheme 1. In a typical procedure, 1 g graphite was added into the 100 mL of aqueous urea solution (100 g/100 mL). The mixture solution was sonicated in a sonication bath (4 W) for 20 min at room temperature to dissolve the urea. After sonication, the mixture solution was exfoliated by the mixer homogenizer at a speed of 3000 rpm for 4 h. Then, to remove the urea, the exfoliated few-layered graphene was collected by centrifugation at 3000 rpm for 20 min, twice. The pellet of few-layered graphene was re-dispersed in 100 mL NMP. The resultant dispersion was mixed by the mixer homogenizer at a speed of 3000 rpm for 5 h. Finally, to obtain the exfoliated LDG, the mixture solution was centrifuged at 3000 rpm for 20 min and the supernatant was collected for the LDG solution. The LDG solution was evaporated under a vacuum to discard the solvent at 50 • C.

Preparation of Graphene Electrode for Electrochemical Sensor
Before the electrode fabrication, the GCE was polished with 1.0, 0.3, and 0.05 µm Al 2 O 3 powder and washed in an ultrasonic bath for 10 min, then rinsed with DI water thoroughly. After that, 5 µL of 1 mg/mL LDG in ethanol was drop-casted onto the GCE and dried with an infrared spectroscopy (IR) lamp for a few minutes, then rinsed with DI water several times. Before the electrochemical sensing experiments, the LDG-modified GCE was activated electrochemically by cyclic voltammetry (CV) in 10 mM PBS buffer solution (pH 7.4); the potential was scanned from 0 to 1.0 V (vs. Ag/AgCl) three times with a 5 mV/s scan rate. Nanomaterials 2020, 10, x FOR PEER REVIEW 3 of 11 conventional three-electrode cell was used with a Pt wire as a counter electrode, an Ag/AgCl electrode was used as a reference electrode and a bare glassy carbon electrode (GCE, diameter = 3 mm), or modified electrode, was used as a as working electrode at room temperature.

Shear Exfoliation of Graphite by Using a Homogenizer
The LDG samples were prepared by the liquid-phase shear exfoliation method using a highspeed mixer homogenizer, as shown in Scheme 1. In a typical procedure, 1 g graphite was added into the 100 mL of aqueous urea solution (100 g/100 mL). The mixture solution was sonicated in a sonication bath (4 W) for 20 min at room temperature to dissolve the urea. After sonication, the mixture solution was exfoliated by the mixer homogenizer at a speed of 3000 rpm for 4 h. Then, to remove the urea, the exfoliated few-layered graphene was collected by centrifugation at 3000 rpm for 20 min, twice. The pellet of few-layered graphene was re-dispersed in 100 mL NMP. The resultant dispersion was mixed by the mixer homogenizer at a speed of 3000 rpm for 5 h. Finally, to obtain the exfoliated LDG, the mixture solution was centrifuged at 3000 rpm for 20 min and the supernatant was collected for the LDG solution. The LDG solution was evaporated under a vacuum to discard the solvent at 50 °C.

Preparation of Graphene Electrode for Electrochemical Sensor
Before the electrode fabrication, the GCE was polished with 1.0, 0.3, and 0.05 μm Al2O3 powder and washed in an ultrasonic bath for 10 min, then rinsed with DI water thoroughly. After that, 5 μL of 1 mg/mL LDG in ethanol was drop-casted onto the GCE and dried with an infrared spectroscopy (IR) lamp for a few minutes, then rinsed with DI water several times. Before the electrochemical sensing experiments, the LDG-modified GCE was activated electrochemically by cyclic voltammetry (CV) in 10 mM PBS buffer solution (pH 7.4); the potential was scanned from 0 to 1.0 V (vs. Ag/AgCl) three times with a 5 mV/s scan rate. Scheme 1. Illustration of mass production of low-defect graphene (LDG) by the urea-assisted liquidphase shear exfoliation of graphite.

Effect of Processing Parameters on Graphene Concentration
To confirm the production of the LDG sheets, as described in scheme 1, Raman spectroscopy was first employed to monitor the exfoliation of graphite into graphene sheets. As shown in Figure  1A, three peaks were observed at 1339 (D band), 1573 (G band) and 2688 cm −1 (2D band) for both graphite and LDG. Using the ratio of peak intensities ID/IG in Raman spectra, the level of disorder in graphene can be characterized. As the disorder in the graphene increases, ID/IG displays two different behaviors, as follows: (i) at a low-defect concentration, like pure graphite materials, ID/IG ≃ 1/Ld 2 (Stage Scheme 1. Illustration of mass production of low-defect graphene (LDG) by the urea-assisted liquid-phase shear exfoliation of graphite.

Effect of Processing Parameters on Graphene Concentration
To confirm the production of the LDG sheets, as described in Scheme 1, Raman spectroscopy was first employed to monitor the exfoliation of graphite into graphene sheets. As shown in Figure 1A, three peaks were observed at 1339 (D band), 1573 (G band) and 2688 cm −1 (2D band) for both graphite and LDG. Using the ratio of peak intensities I D /I G in Raman spectra, the level of disorder in graphene can be characterized. As the disorder in the graphene increases, I D /I G displays two different behaviors, as follows: (i) at a low-defect concentration, like pure graphite materials, I D /I G 1/L d 2 (Stage 1); (ii) at a relatively high defect concentration, like produced graphene by chemical or physical exfoliation, I D /I G L d 2 (Stage 2), where L d represents the mean distance between two defects [22,23]. The transition between Stage 1 and 2 is usually observed at I D /I G 3 [22]. So, in the present case, the I D /I G value increased as the level of defects decreased. The exfoliation caused an increase in the I D /I G value to 0.39 in the LDG, which is higher than that of graphite (0.16), but considerably lower than that of GO reported previously [24]. This should be attributed to the edge defects of the exfoliated samples rather than the lattice destruction, meaning that very few defects existed in the as-prepared LDG [25].
In addition, the 2D band of LDG turned out to be a relatively symmetrical shape and appeared at lower position (~10 cm −1 ) when compared to the 2D band of graphite. A shoulder peak on G band, along with the D, G and 2D band, appeared at 1616 cm −1 in the LDG, which is namely D band and is evidence of few-layer graphene. These results successfully demonstrate the reduction in the number of graphene layers and the subsequent formation of few-layer graphene with a low density of defects [26].
To optimize the preparation procedure, we investigated the effects of the processing parameters, such as the shearing speed, urea concentration, shearing time in urea and shearing time in NMP on the concentration of LDG solutions and the intensity ratio of the D band and G band (I D /I G ) using Raman spectroscopy ( Figure 1B-E). The processing parameters were optimized with the maximal concentration and the highest I D /I G value of LDG, indicating the lowest level of defects. To test the influence of the processing parameters, only one parameter was varied, while the other parameters were kept constant. Figure 1B shows the effect of the shearing speed in the urea solution on the concentration and the I D /I G value of LDG dispersions. As the shearing speed increased in the range of 1000 to 5000 rpm, the concentration of LDG also increased. However, the I D /I G of LDG dispersions reached its maximum at 3000 rpm and decreased after that. This indicates that the highest LDG concentration with the lowest level of defects was obtained at a shearing speed of 3000 rpm. Therefore, 3000 rpm was selected as the optimal shearing speed for the preparation of LDG dispersion. A similar trend was observed in the test for the effect of the shearing speed in NMP, showing the highest I D /I G value of LDG at 3000 rpm, although the concentration increased with increasing shearing speed ( Figure 1C). This is because the graphene size was reduced with the increase of edge defects and agglomeration, as the shear force increased with the shearing speed. Thus, 3000 rpm in NMP was enough to achieve a high yield of good quality. The effect of the urea concentration on the exfoliation efficiency of graphite was also examined ( Figure 1D). The concentration and the I D /I G value of LDG dispersions increased with increasing concentrations of urea. This result suggests that the urea molecules could expand the interlayer space of graphite to overcome the van der Waals force between graphite layers and, thus, LDG concentration significantly increased due to the presence and adsorption of more concentrated urea molecules. A suitable concentration of urea was chosen to be 100 g/100 mL, due to the solubility of urea (107.9 g/100 mL in 20 • C). The effect of the shearing times in aqueous urea solution and NMP, respectively, were further evaluated in the range of 1 to 5 h ( Figure 1E,F). The highest concentrations and I D /I G values of LDG dispersions were achieved at 4 h in urea and 5 h in NMP and, thus, they were chosen as the optimal shearing times. Interestingly, the graphene prepared without urea assistance showed a lower concentration and I D /I G value than those of LDG ( Figure S1, Supplementary Materials), proving the role of urea as a stabilizer and a molecular wedge in shear exfoliation. Taken together, these results show that the highest concentration of LDG with high quality can be prepared under the following processing parameters: (i) the shearing speed in urea solution is 3000 rpm; (ii) the shearing speed in NMP is 3000 rpm; (iii) the urea concentration is 100 g/100 mL; (iv) the shearing time in urea is 4 h; (v) the shearing time in NMP is 5 h. Nanomaterials 2020, 10, x FOR PEER REVIEW 5 of 11

Characterization of Low-Defect Graphene
The XRD patterns of the LDG and graphite powder were utilized to confirm the exfoliation of the graphite to graphene sheets (Figure 2A). While the XRD pattern of the graphite showed a sharp diffraction peak at 2θ = 26.5°, corresponding to a d-spacing of 0.34 nm, the LDG showed a broad peak around 24° and very weak diffraction peak around 26.5°, which indicates the exfoliation of graphite. The LDG also exhibited a broad peak around 10°, demonstrating that the interlayer spacing was expanded during the exfoliation [27,28]. As shown in Figures 2B and 2C, both TEM and SEM images also revealed the microstructures of the exfoliated graphene sheets in the LDG dispersions. The reduction in the thickness of the graphene sheets (mono-, bi-and tri-layer, Figure 2B) and their lateral size (~1000 nm) is evident compared with the graphite flakes, confirming the high degree of exfoliation of the process. The SEM image of the LDG clearly shows a thin planar structure with curled and irregular edges ( Figure 2C). According to statistical analysis based on the SEM and TEM images of the LDG sheets, lateral size and layer distribution were measured in Figure S2. By

Characterization of Low-Defect Graphene
The XRD patterns of the LDG and graphite powder were utilized to confirm the exfoliation of the graphite to graphene sheets (Figure 2A). While the XRD pattern of the graphite showed a sharp diffraction peak at 2θ = 26.5 • , corresponding to a d-spacing of 0.34 nm, the LDG showed a broad peak around 24 • and very weak diffraction peak around 26.5 • , which indicates the exfoliation of graphite. The LDG also exhibited a broad peak around 10 • , demonstrating that the interlayer spacing was expanded during the exfoliation [27,28]. As shown in Figure 2B,C, both TEM and SEM images also revealed the microstructures of the exfoliated graphene sheets in the LDG dispersions. The reduction in the thickness of the graphene sheets (mono-, bi-and tri-layer, Figure 2B) and their lateral size (~1000 nm) is evident compared with the graphite flakes, confirming the high degree of exfoliation of the process. The SEM image of the LDG clearly shows a thin planar structure with curled and irregular edges ( Figure 2C). According to statistical analysis based on the SEM and TEM images of the LDG sheets, lateral size and layer distribution were measured in Figure S2. By measuring~90 graphene sheets, a lateral size histogram ( Figure S2A) displays that more than 84% of the graphene sheets are larger than 3 µm, which is much higher than that of liquid phase exfoliated graphene sheets by sonication [14,29]. The layer distribution ( Figure S2B) shows that monolayer graphene accounts for 48.8% of the graphene sheets in the dispersion. We believe that the large sheet size and high exfoliation efficiency are related to the intercalation of urea molecules into the graphite and the liquid phase shear exfoliation process. These results suggest the successful preparation of well-exfoliated and non-oxidized graphene sheets with high quality, verifying that this approach is a nondestructive and efficient method to produce LDG sheets on a large scale.
Nanomaterials 2020, 10, x FOR PEER REVIEW 6 of 11 measuring ~90 graphene sheets, a lateral size histogram ( Figure S2A) displays that more than 84% of the graphene sheets are larger than 3 μm, which is much higher than that of liquid phase exfoliated graphene sheets by sonication [14,29]. The layer distribution ( Figure S2B) shows that monolayer graphene accounts for 48.8% of the graphene sheets in the dispersion. We believe that the large sheet size and high exfoliation efficiency are related to the intercalation of urea molecules into the graphite and the liquid phase shear exfoliation process. These results suggest the successful preparation of well-exfoliated and non-oxidized graphene sheets with high quality, verifying that this approach is a nondestructive and efficient method to produce LDG sheets on a large scale. XPS was performed to probe the chemical composition and binding states of the exfoliated LDG (Figures 3 and S3 and Table S1). The XPS survey curves of LDG, commercially available graphene and graphite show a pronounced peak at 285 eV and a weak peak at 533 eV, except for graphene oxide (GO), which displayed considerable peak heights at both 292 and 538 eV, due to the oxygen functionalities derived from chemical exfoliation ( Figure 3A). Based on the results from XPS, the oxygen content of the LDG powder was estimated to be 6.4 atomic %, thus being higher than that of the graphite precursor (3.0%), but considerably lower than that of GO (32.5%) and even that of commercially available graphene (11.1%), as summarized in Table S1. The overall oxygen of LDG is associated with the edge defects of LDG in the form of oxide groups, which is in agreement with the results from Raman and XRD spectra, and consistent with the aforementioned higher crystalline size of graphene sheets [30]. The calculated C/O ratio of the LDG (14.6) is significantly higher than that of commercially available graphene (8.0) and that of GO (2.1). Figure 3B-D show the C 1s core-level spectra of graphite, LDG, and commercially available graphene, respectively. The pattern of LDG ( Figure 3C) exhibits a similar structure to that of graphite ( Figure 3B), revealing that the intrinsic inplane crystal structure of graphite remains intact during the shear exfoliation treatment. The XPS data of the C 1s profiles also prove that the LDG ( Figure 3C has fewer oxygen-containing functional groups (such as C-O, C=O, and COO-) than GO ( Figure S1) and even commercially available graphene ( Figure 3D). XPS was performed to probe the chemical composition and binding states of the exfoliated LDG (Figure 3, Figure S3 and Table S1). The XPS survey curves of LDG, commercially available graphene and graphite show a pronounced peak at 285 eV and a weak peak at 533 eV, except for graphene oxide (GO), which displayed considerable peak heights at both 292 and 538 eV, due to the oxygen functionalities derived from chemical exfoliation ( Figure 3A). Based on the results from XPS, the oxygen content of the LDG powder was estimated to be 6.4 atomic %, thus being higher than that of the graphite precursor (3.0%), but considerably lower than that of GO (32.5%) and even that of commercially available graphene (11.1%), as summarized in Table S1. The overall oxygen of LDG is associated with the edge defects of LDG in the form of oxide groups, which is in agreement with the results from Raman and XRD spectra, and consistent with the aforementioned higher crystalline size of graphene sheets [30]. The calculated C/O ratio of the LDG (14.6) is significantly higher than that of commercially available graphene (8.0) and that of GO (2.1). Figure 3B-D show the C 1s core-level spectra of graphite, LDG, and commercially available graphene, respectively. The pattern of LDG ( Figure 3C) exhibits a similar structure to that of graphite ( Figure 3B), revealing that the intrinsic in-plane crystal structure of graphite remains intact during the shear exfoliation treatment. The XPS data of the C 1s profiles also prove that the LDG ( Figure 3C has fewer oxygen-containing functional groups (such as C-O, C=O, and COO-) than GO ( Figure S1) and even commercially available graphene ( Figure 3D).

Electrochemical Detection of Doxorubicin with LDG Electrode
To assess the performance of LDG as a sensing material for practical applications, we fabricated the LDG-modified GCE (LDG-GCE) for the detection of DOX. DOX, as one of the clinically important anti-cancer drugs, has been extensively used in the treatment of various cancers. However, long-term clinical use of DOX is limited due to its side effects, such as systemic toxicity, drug resistance, and life-threatening heart damage. Therefore, the development of highly sensitive analytical techniques for DOX detection is of great significance in clinics and pharmaceutics.
Before the electrochemical sensing experiments, CV and electrochemical impedance spectroscopy (EIS) were performed to investigate the charge transfer ability of LDG-GCE in 0.1 M KCl solution containing 3 mM of K3Fe(CN)6. As shown in Figure 4A, both LDG-GCE and GCE show a pair of well-defined redox peaks. However, the peak potential difference (ΔEp = 70 mV) of LDG-GCE is smaller than that of bare GCE (119 mV), while the peak currents for LDG-GCE are significantly higher, indicating that the LDG-GCE electrode exhibited a better electrochemical performance. The EIS experiments also showed better electron-transfer characteristics for LDG-GCE. The charge transfer resistance (Rct) of LDG-GCE was estimated to be 25.9 Ω from the diameter of a semicircle in the high frequency region of a Nyquist plot ( Figure 4B), which was much smaller than that of the GCE (Rct = 168.8 Ω). This suggests that rapid electron transfer occurred on the LDG-GCE surface.

Electrochemical Detection of Doxorubicin with LDG Electrode
To assess the performance of LDG as a sensing material for practical applications, we fabricated the LDG-modified GCE (LDG-GCE) for the detection of DOX. DOX, as one of the clinically important anti-cancer drugs, has been extensively used in the treatment of various cancers. However, long-term clinical use of DOX is limited due to its side effects, such as systemic toxicity, drug resistance, and life-threatening heart damage. Therefore, the development of highly sensitive analytical techniques for DOX detection is of great significance in clinics and pharmaceutics.
Before the electrochemical sensing experiments, CV and electrochemical impedance spectroscopy (EIS) were performed to investigate the charge transfer ability of LDG-GCE in 0.1 M KCl solution containing 3 mM of K 3 Fe(CN) 6 . As shown in Figure 4A, both LDG-GCE and GCE show a pair of well-defined redox peaks. However, the peak potential difference (∆E p = 70 mV) of LDG-GCE is smaller than that of bare GCE (119 mV), while the peak currents for LDG-GCE are significantly higher, indicating that the LDG-GCE electrode exhibited a better electrochemical performance. The EIS experiments also showed better electron-transfer characteristics for LDG-GCE. The charge transfer resistance (R ct ) of LDG-GCE was estimated to be 25.9 Ω from the diameter of a semicircle in the high frequency region of a Nyquist plot ( Figure 4B), which was much smaller than that of the GCE (R ct = 168.8 Ω). This suggests that rapid electron transfer occurred on the LDG-GCE surface.  The electrochemical detection of DOX was performed with differential pulse voltammetry (DPV) and chronoamperometry in 10 mM PBS solution (pH = 7.4) containing various concentrations of DOX, using LDG-GCE. Figure 5A shows the typical DPV curves in which only one broad peak was observed at 0.366 V. Upon the addition of DOX in the range of 0.3 to 5.0 μM, the LDG-GCE exhibited an increase in the oxidation peak currents. The corresponding calibration plots for DOX showed a good linear relationship between the DPV peak currents and the concentration of DOX in the range of 0.3-3.0 μM. As is to be expected from the electrochemical performance of LDG-GCE, the sensor shows better sensitivity when compared with bare GCE ( Figure 5B). The calculated sensitivity for the electrochemical detection of DOX was 7.23 × 10 −1 μM/μA with LDG-GCE, which is higher than bare GCE (3.54 × 10 −1 μM/μA). Moreover, the limit of detection (LOD) for LDG-GCE (39.3 nM) at an S/N ratio of 3 was also much better than that of bare GCE with LDG-GCE (254 nM) and bare GCE. These results suggest that the LDG-GCE exhibited an excellent performance with respect to the detection of DOX. To evaluate the practical applications in DOX analysis, chronoamperometric measurements were carried out in 0.1 M PBS (pH 7.4). Figure 5C shows the amperometric current-time (i-t) plots of the LDG-GCE at 0.366 V in response to the successive addition of DOX the PBS solution. The corresponding calibration curves are displayed as i (nA) = 1.06 × 10 −8 + 0.00828 C (μM) in Figure 5C inset. The relationship between DOX concentration and the corresponding current is a linear one in the range of 0.3-2.7 μM with a LOD of 653 nM (S/N = 3). Table S2 summarizes and compares the analytical parameters of the LDG-GCE sensor with other electrodes, revealing that the proposed sensor shows a high sensitivity with a low LOD and suitable linear range. The selectivity experiments were performed by comparing the amperometric responses to DOX with potential interfering compounds found in biological fluids, such as glucose, urea, tryptophan, KCl, and NaCl. As shown in Figure 5D, no significant interference was observed during the amperometric detection of 3 μM DOX in the presence of 30 μM glucose, urea, KCl, NaCl, and tryptophan. This result underlines the suitable selectivity of LDG-GCE. The electrochemical detection of DOX was performed with differential pulse voltammetry (DPV) and chronoamperometry in 10 mM PBS solution (pH = 7.4) containing various concentrations of DOX, using LDG-GCE. Figure 5A shows the typical DPV curves in which only one broad peak was observed at 0.366 V. Upon the addition of DOX in the range of 0.3 to 5.0 µM, the LDG-GCE exhibited an increase in the oxidation peak currents. The corresponding calibration plots for DOX showed a good linear relationship between the DPV peak currents and the concentration of DOX in the range of 0.3-3.0 µM. As is to be expected from the electrochemical performance of LDG-GCE, the sensor shows better sensitivity when compared with bare GCE ( Figure 5B). The calculated sensitivity for the electrochemical detection of DOX was 7.23 × 10 −1 µM/µA with LDG-GCE, which is higher than bare GCE (3.54 × 10 −1 µM/µA). Moreover, the limit of detection (LOD) for LDG-GCE (39.3 nM) at an S/N ratio of 3 was also much better than that of bare GCE with LDG-GCE (254 nM) and bare GCE. These results suggest that the LDG-GCE exhibited an excellent performance with respect to the detection of DOX. To evaluate the practical applications in DOX analysis, chronoamperometric measurements were carried out in 0.1 M PBS (pH 7.4). Figure 5C shows the amperometric current-time (i-t) plots of the LDG-GCE at 0.366 V in response to the successive addition of DOX the PBS solution. The corresponding calibration curves are displayed as i (nA) = 1.06 × 10 −8 + 0.00828 C (µM) in Figure 5C inset. The relationship between DOX concentration and the corresponding current is a linear one in the range of 0.3-2.7 µM with a LOD of 653 nM (S/N = 3). Table S2 summarizes and compares the analytical parameters of the LDG-GCE sensor with other electrodes, revealing that the proposed sensor shows a high sensitivity with a low LOD and suitable linear range. The selectivity experiments were performed by comparing the amperometric responses to DOX with potential interfering compounds found in biological fluids, such as glucose, urea, tryptophan, KCl, and NaCl. As shown in Figure 5D, no significant interference was observed during the amperometric detection of 3 µM DOX in the presence of 30 µM glucose, urea, KCl, NaCl, and tryptophan. This result underlines the suitable selectivity of LDG-GCE.

Conclusions
We have demonstrated the scalable production of graphene sheets with high quality by a simple and facile method, via NMP-mediated shear exfoliation of graphite with the assistance of urea. The successful production of LDG on a large scale was possible using a high speed mixer homogenizer by optimizing the processing parameters for the shear exfoliation, such as shearing speed, urea concentration, and shearing time by comparing the produced graphene concentration and ID/IG value from the Raman spectra. The characterization experiments confirmed that the graphene sheets, produced by shear exfoliation with optimized parameters, are single layered, or few-layered at least, with negligible defects. In addition, using the LDG as a modification material for an electrode, we have fabricated an LDG-GCE electrochemical sensor for DOX, demonstrating sensitive and selective DOX analysis. Therefore, the proposed approach is promising for the industrial production of graphene for numerous applications.
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figure S1: Comparison of (A) the concentration and (B) the ID/IG ratio in Raman spectroscopy in graphene solution prepared with (black square dot) and without (red square dot) assistance of urea., Figure S2: (A) lateral size and (B) layer distribution of LDG calculated from 95 isolated sheets in SEM and TEM images., Figure S3: High resolution C1s spectra of GO., Table S1: XPS data of graphene oxide (GO), graphite, commercially available graphene and LDG., Table S2: Comparison of the analytical performance of LDG-GCE with other electrodes for DOX detection.

Conclusions
We have demonstrated the scalable production of graphene sheets with high quality by a simple and facile method, via NMP-mediated shear exfoliation of graphite with the assistance of urea. The successful production of LDG on a large scale was possible using a high speed mixer homogenizer by optimizing the processing parameters for the shear exfoliation, such as shearing speed, urea concentration, and shearing time by comparing the produced graphene concentration and I D /I G value from the Raman spectra. The characterization experiments confirmed that the graphene sheets, produced by shear exfoliation with optimized parameters, are single layered, or few-layered at least, with negligible defects. In addition, using the LDG as a modification material for an electrode, we have fabricated an LDG-GCE electrochemical sensor for DOX, demonstrating sensitive and selective DOX analysis. Therefore, the proposed approach is promising for the industrial production of graphene for numerous applications.
Supplementary Materials: The following are available online at http://www.mdpi.com/2079-4991/10/2/267/s1, Figure S1: Comparison of (A) the concentration and (B) the I D /I G ratio in Raman spectroscopy in graphene solution prepared with (black square dot) and without (red square dot) assistance of urea., Figure S2: (A) lateral size and (B) layer distribution of LDG calculated from 95 isolated sheets in SEM and TEM images., Figure S3: High resolution C1s spectra of GO., Table S1: XPS data of graphene oxide (GO), graphite, commercially available graphene and LDG., Table S2: Comparison of the analytical performance of LDG-GCE with other electrodes for DOX detection.

Conflicts of Interest:
The authors declare no conflict of interest.