New Approach for Porous Chitosan–Graphene Matrix Preparation

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Article http://pubs.acs.org/journal/acsodf

New Approach for Porous Chitosan−Graphene Matrix Preparation through Enhanced Amidation for Synergic Detection of Dopamine and Uric Acid Halima Begum, Mohammad Shamsuddin Ahmed, and Seungwon Jeon* Department of Chemistry and Institute of Basic Science, Chonnam National University, Gwangju 61186, Republic of Korea S Supporting Information *

ABSTRACT: Amide-functionalized materials have emerged as promising nonprecious catalysts for electrochemical sensing and catalysis. The covalent immobilization of chitosan (CS) onto graphene sheet (GS) (denoted as CS−GS) has been done via higher degree of amidation reaction to develop an electrochemical sensing matrix for simultaneous determination of dopamine (DA) and uric acid (UA). The enhanced amidation between CS and GS has not been reported previously. However, electrochemical results have revealed that the CS−GS enhances the electrocatalytic performance in terms of the oxidation potential and peak current due to the higher degree of amide functionalization compared to that of CS/GS, which has a lower amidation. Differential pulse voltammetry-based studies have indicated that the CS−GS matrix works at a lower detection limit (0.14 and 0.17 μM) (S/N = 3) and over a longer linear range (1−700 and 1−800 μM), with a comparatively higher sensitivity (2.5 and 2.0 μA μM−1 cm−2), for DA and UA, respectively. In addition, the CS−GS matrix demonstrates good selectivity toward the detection of DA and UA in the presence of a 10-fold higher concentration of AA and glucose. The as-prepared three-dimensional porous CS−GS also endows selective determination toward DA and UA in various real samples.

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Graphene oxide (GO)13 is the oxidized and exfoliated sheet of an sp2-hybridized graphene sheet (GS) that carries various oxygenated groups, such as epoxide, hydroxyl, carbonyl, and carboxyl. Among all of the oxygenated groups, the carboxyl group of GO is the most suitable site for reacting with an amine group to form an amide bond through the condensation reaction.14−17 However, GS itself has zero band gap, a fact that poses a considerable barrier to its application in digital electronic devices, such as semiconductors, sensors, and electrocatalysts.14 Several approaches, including implanting heteroatoms (e.g., N atoms) into a GS plane, have been applied to overcome this disadvantage.14,18,19 The N atom is considered the best candidate among various heteroatoms (i.e., B, S, P, and O) for carbon substitution because of its five valence electrons that are capable of strong interactions with adjacent carbon atoms. N functionalization creates a net positive charge on adjacent carbon atoms in the GS lattice to facilitate electrocatalysis by attracting electrons from the anodic reaction. This phenomenon has been proven through quantum mechanics calculations and subsequent experimental observations.9,12,20−22 Also, an sp3-carbon-rich polymer matrix might influence the electro-

INTRODUCTION Dopamine (DA) and uric acid (UA) are important biospecies that play significant roles in the physiological function of metabolism, the central nervous system, and the circulatory system of the human body. Deficiency or maladjustment in their levels may lead to many diseases, such as cancer, Parkinson’s disease, and hyperuricemia.1−4 Therefore, simultaneous determination of DA and UA is of critical importance for developing nerve physiology, making diagnoses, and controlling medicine. However, conventional electrodes normally result in poor selectivity and low reproducibility. Moreover, accurate determination of DA and UA often proves difficult due to the concentration of ascorbic acid (AA) being at a millimolar level in the human body and their capability of oxidation at a similar potential to that of the bare electrode.5,6 Because of the high concentration of AA in the humoral fluids, the development of a simple and rapid method for the determination of much lower concentrations of DA and UA (being in lower concentrations in humoral fluids than those of AA) with a high sensitivity and selectivity is desirable for diagnostic applications. For overcoming these shortcomings, various carbon-based materials have been proposed as electrode modifiers;7−12 unfortunately, most of them are limited by a narrow linear range and failure to detect at a high level.7−12 Among them, nitrogen (N)-rich porus materials are viewed as one of the promising electrode materials.12 © 2017 American Chemical Society

Received: March 20, 2017 Accepted: June 16, 2017 Published: June 29, 2017 3043

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Scheme 1. Schematic Diagram of GS, CS−GS, and CS/GS Preparation

catalytic behavior of sp2-carbon-rich GS because of its effective charge polarization, as reported by Tao et al.23 Chitosan (CS) with sp3 carbon is a natural polysaccharide polymer that has attracted significant attention due to its excellent biological compatibility, good hydrophilicity, and filmforming ability.24 However, due to its relatively poor conductivity, CS is often associated with conductive materials, such as carbon materials and metal nanomaterials, to form composite film for immobilization.21 However, −NH2 group is among the many functional groups in CS that could be easily applied in chemical modification (i.e., covalent functionalization) with −COOH via the amidation reaction.21,24 In the meantime, many researchers have made nanocomposites with GS and CS via amidation.24−28 However, unfortunately, the abundance of amidation may be hampered in those nanocomposites due to the lower amount of the −COOH group in GO (compared to that in other groups) normally.29−33 Therefore, to overcome this shortcoming, GO needs special chemical pretreatment to increase the amount of −COOH on its surface to increase the degree of amidation between GS and CS. In this article, we explore for the first time the utilization of an enhanced amidation reaction between CS and GS for preparing a potential electrocatalyst for the detection of DA and UA. However, CS was immobilized with GS (CS−GS) to construct a three-dimensional (3D) porous structured interface through a covalent amidation reaction (Scheme 1). As a result, this film showed notable effects on simultaneous electrocatalytic detection of DA and UA in the presence of high concentrations of AA and glucose within a long linear range.

Figure 1. SEM (a, b) and TEM (c, d) images of CS/GS and CS−GS; insets: HRTEM images of CS−GS and CS/GS, with their N element mapping.

the synthesized CS−GS. These crumpled structures were due to the attachment of a N atom to the GS lattice as a result of the covalent functionalization via amidation.34 The crumpled CS−GS films effectively prevented the aggregation of graphitic sheets. Similarly, the TEM image of CS/GS (Figure 1c) shows an aggregated structure of CS on GS and an inhomogeneous thickness of the CS/GS film. The TEM image of CS−GS (Figure 1d) shows a random distribution of a considerably crumpled structure with a large amount of wrinkles and a multilayered structure due to covalent functionalization through amidation.35,36 The substantially crumpled CS−GS films were interconnected with one another to form a porous structure, which contributed to the enhancement of the electromechanical and mechanical properties.37,38 However, high-resolution TEM images were also recorded and they

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RESULTS AND DISCUSSION Instrumental Characterization. The surface morphology of CS−GS and CS/GS was observed using the scanning electron microscopy (SEM) and transmission electron microscopy (TEM) techniques, and both consisted of a filmlike structure plane, as shown in Figure 1. The SEM image of CS/ GS (Figure 1a) shows a wrinkled and sheetlike structure of GS films and smooth and comparatively thick CS films on GS. The SEM image of CS−GS (Figure 1b) shows randomly distributed, considerably higher crumpled structures within 3044

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Figure 2. BET surface area calculated from nitrogen adsorption−desorption isotherms of CS−GS and CS/GS (a), and XRD patterns of GS, CS− GS, CS/GS, and CS (b).

Figure 3. XPS spectra (a) and core-level C1s XPS spectra (b) of GO and GO−COOH; core-level C1s XPS spectra of GS, CS, CS−GS, and CS/GS (c); and core-level N1s XPS spectra of CS, CS−GS, and CS/GS (d).

edge; thus, a huge −COOH group was placed at the edge of GO during conversion from GO to GO−COOH. Figure 2a shows the results of N2 absorption/desorption analysis to characterize porous structure of the as-prepared CS−GS and CS/GS samples. On the basis of the Brunauer− Emmett−Teller (BET) method, the specific surface area (SSA) of CS−GS and CS/GS samples was found to be 381.3 and 102.8 m2 g−1, respectively. The SSA is an important parameter to understand the electrochemical properties of CS−GS and CS/GS.39 However, the porous CS−GS had 3.7 times higher SSA than the nonfunctionalized CS/GS, thereby confirming the hierarchical 3D porous structure of the CS−GS sample. This finding agreed well with the morphological analysis through SEM.

showed that CS coexisted on the GS film in CS/GS (Figure 1c, inset). On the other hand, both CS and GS were embedded on each other to form a porous structure in the CS−GS (Figure 1d, inset). N elemental mapping revealed that N was distributed homogeneously in the CS−GS sample but not in the CS/GS sample (inset of the corresponding figures), which also proved better homogeneous film formation in CS−GS. For comparison, bare GS and CS films were analyzed through TEM (Figures S1a and 2b), which showed a slightly wrinkled sheetlike structure and a comparatively thin filmlike structure for GS and CS film, respectively. The average size of the three samples was measured to be 720, 690, and 480 nm for GS, CS/ GS, and CS−GS, respectively (Figure S1c). The size of CS−GS was significantly reduced, which is an indication of an increased 3045

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Figure 4. CVs at 50 mV s−1 (a) and Nyquist plots from 105 to 10−2 Hz frequencies (b) on bare GCE, CS/GS/GCE, and CS−GS/GCE in an Ar. saturated 0.1 M KCl solution containing 5.0 mM Fe(CN)3−/4− 6

and/or GO−COOH samples. These results indicate an efficient reduction of the oxygen-containing functional groups by NaBH4 (for GS) and CS (for CS−GS and CS/GS), although CS itself is a good reductant, as reported earlier.26 However, the intensity of the O−CO peak was reduced significantly by up to 1.22% in the CS−GS sample relative to that in the CS/GS sample (3.9%), probably because of the CS grafting via amide functionalization. Importantly, it was observed that the C−O peak intensity in CS−GS was significantly stronger than that in the GS and CS/GS samples, probably because of the influence of the C−N bond over the C−O bond in the CS−GS sample.49,50 Similarly, a strong C−O peak intensity was observed in the CS sample, while it also contents huge C−O bond along with C−N bond. This scenario indicated a higher degree of amidation between GS and CS in CS−GS than that in the CS/GS sample. Figure 3d shows the N1s XPS spectra of bare CS, CS−GS, and CS/GS with two absorbance peaks. For CS, the absorbance peaks at 401.0 and 398.5 eV were attributed to the −NH2 and C−N bonds, respectively,51,52 and these were positively shifted at 401.05 and 398.7 eV, respectively, for the CS−GS sample. After amidation, the C−N peak intensity increased substantially up to 89.5% (for CS−GS) from 52.7% (for CS), thereby confirming again the superior amide functionalization between GS and CS in the CS−GS sample (Figure 1). However, we found that a lower degree of amidation between GS and CS occurred spontaneously in the CS/GS (peak intensity was 66.3%), as reported without 1-(3-dimethylaminopropyl)-3ethylcarbodiimide (EDC)/N-hydroxysuccinimide (NHS) treatment in the previous literature.24−28 Therefore, CS is grafted into GS through the enhanced amidation reaction to form the N-rich CS−GS matrix. Further covalent functionalization was characterized by the FTIR spectra (Figure S2). The spectrum of GO displayed several characteristic peaks, such as those for the CO group at 1722 cm−1, CC group at 1622 cm−1, and C−O group at 1035 cm−1. These results are consistent with the reported values.15,31 All of the mentioned peaks were observed in the GO−COOH spectrum. Significantly, the CO peak intensity was increased compared to that in the GO spectrum, which indicated −COOH formation on the GO sheet. Compared to those of the oxygen functional groups in the GO and GO− COOH, the signals of the oxygen functional groups in CS−GS and GS were very weak, indicating the presence of less oxygeneous functional groups attached to the surface. For CS−

The XRD patterns of CS, GS, CS−GS, and CS/GS are shown in Figure 2b. In the GS XRD pattern, the diffraction peaks appearing at ∼25.5 and 41.3° could be ascribed to the (002) and (100) planes of the graphitic carbon (ICSD ref 98005-2230), suggesting that the GO has been reduced to GS.40 In the CS pattern, the diffraction peak appearing at ∼20.6° in the XRD pattern can be ascribed to the (200) plane of CS.41 For the CS−GS and CS/GS samples, the diffraction peaks for the (002) plane appeared at 23.6 and 23.5°, respectively, which indicates that the CS−GS matrix was better ordered, with the absence of free nanosheets in this sample, and vice versa.42,43 In addition, the better covalent functionalization between CS and GS was verified by the disappearance of the CS characteristic diffraction peak in the CS−GS XRD pattern, whereas the characteristic peaks of both CS and GS appeared in the CS/GS XRD pattern (Figure S1b). Details of the chemical structure and bonding state changes during various reactions were elucidated through X-ray photoelectron spectroscopy (XPS) analysis. First, we observed the conversion reaction of GO to GO−COOH, and the XPS survey spectra of GO and GO−COOH are shown in Figure 3a. Both XPS survey spectra exhibit several distinct peaks at approximately 285.6 and 533.4 eV, which were attributed to the C and O elements, respectively.44,45 The C/O atomic ratio was similar for both the GO (1.95) and GO−COOH (2.01) samples. The high-resolution C1s XPS spectra of all aforementioned samples were recorded (Figure 3b), and they showed the defective sp3-carbon along with the basal-plane sp2carbon of the graphitic structure.34,46 The C1s spectrum of GO in Figure 3b shows four absorbance peaks of oxygenated sp3-C at 286.6, 287.8, and 289.4 eV, which were attributed to C−O, CO, and O−CO, respectively, and a distinct oxygen-free sp2-C (CC) at 285.0 eV.47,48 Compared with GO, the GO− COOH sample showed a stronger absorbance peak for O−C O and a comparatively weaker absorbance peak for CO. This observation indicates that conversion to the carboxyl group on the GO−COOH sheets was successful. Schematic diagrams of GO and GO−COOH are presented in the corresponding figure insets. Afterward, the amidation between CS and GO−COOH was elucidated further through the XPS analysis. Figure 3c shows the core-level of C1s XPS spectra of GS, CS, CS−GS, and CS/ GS. By comparison, all of the above-mentioned samples showed a stronger suppression for the oxygen-containing components of their C1s XPS spectra than that by the GO 3046

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Figure 5. CVs for 0.5 mM DA, 0.5 mM UA, and 0.5 mM DA + UA recorded on bare GCE (a), CS/GS/GCE (b), and CS−GS/GCE (c) in Arsaturated 0.1 M PBS (pH 7.4) at a 50 mV s−1 scan rate.

GS, the peak at 1199 cm−1 was attributed to the C−N group.15 These results also confirm the conversion of GO to GO− COOH and the subsequent enhancement of amidation between CS and GS. Electrochemical Measurements. The vital interfacial properties of the modified electrodes, such as electrical conductivity and electrocatalytic features, were first analyzed by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurements. Both the experiments were carried out in argon (Ar)-saturated 0.1 M KCl containing 5 mM [Fe(CN)6]3−/4− redox probe, and the EIS was done within the frequency of 105−10−2 Hz, as shown in Figure 4. Figure 4a shows the CVs of bare GCE, CS/GS, and CS−GS. As can be seen, the redox peak current of [Fe(CN)6]3−/4− from CS−GS was 4 and 2 times larger than that from the GCE and CS/GS. Briefly, the CV indicated that the bare GCE impeded the electron transfer of [Fe(CN)6 ]3−/4− due to its lower conductivity. Compared with bare GCE, CS/GS displayed a higher redox peak current because the introduction of conductive GS led to an increase in the redox probe on the electrode surface. However, the peak separation (ΔEp = Epa − Epa) was much higher (ΔEp = 0.24 V) than that of bare GCE (ΔEp = 0.08 V), probably due to the presence of free CS nanosheets with a lower degree of amidation, which allows staying CS/GS interface with negatively charged3 while higher intensity of O−CO was observed from C1s XPS spectrum. A higher redox peak current and smaller peak separation (ΔEp = 0.057 V) of [Fe(CN)6]3−/4− were then observed on the CS− GS-modified electrode, indicating faster electron transfer behavior, probably due to the large active SSA with a positively charged interface originating from a higher degree of amidation; as a result, the electron transfer between Fe(CN)63−/4− and CS−GS was accelerated. The electron transfer characteristics were interpreted using Randle’s circuit in the EIS experiment in Figure 4b. Randle’s circuit consists of electrolyte resistance (Rs), electron-transfer resistance (Rct), double-layer capacitance (Cdl), and Warburg impedance (W) (Figure 4b, inset). The Nyquist plot includes a semicircular portion and a linear portion; the semicircular portion corresponds to the electron transfer limited process, and the linear part corresponds to the diffusion process at higher and lower frequencies. The semicircle diameters correspond to the Rct.53 As can be seen, the Nyquist plot of bare GCE displayed a well-defined, enlarged semicircle, with an Rct of about 855 Ω at a high frequency. Compared to that from

bare GCE, the diameter of the semicircle in the Nyquist plot from CS/GS was decreased, with an Rct of 788 Ω. However, the smallest semicircle in the Nyquist plot was observed for CS− GS, with the lowest Rct of 260 Ω, which is 3 times lower than that of the CS/GS. The superior conductivity is probably due to the presence of N in the CS−GS matrix.54 Also, the porous structure with a higher SSA may enhance the conductivity because of better chemisorption of the redox probe onto the CS−GS surface.55 Interestingly, the Z″ value increased with a 30° angle in the bare GCE curve but increased with a 20° angle in the CS/GS plot, indicating that the diffusion process was probably weak at the CS/GS-modified electrode. On the other hand, the Z″ value increased much more strongly with a higher angle, 60°, at the CS−GS-modified electrode, indicating that the diffusion process was much better at the CS−GS-modified electrode. These results indicate that CS−GS could act as a conductive surface to promote faster electron transfer. Electrochemical Sensing of DA and UA. To probe the potential application of CS/CS and CS−GS, the modified electrodes were utilized for the simultaneous detection of DA and UA by CVs in Ar-saturated 0.1 M phosphate buffered saline (PBS) (pH 7.4) at a 50 mV s−1 scan rate, as shown in Figure 5. Figure 5a depicts the CVs on bare GCE of 0.1 M PBS in the presence of 0.5 mM DA, UA, and a mixture of DA and UA (DA + UA). As can be seen, the oxidation and reduction peak potentials (Epa and Epc, respectively) appeared in the DA curve (0.20 and 0.12 V) and only an oxidation peak appeared in the UA curve (Epa = 0.29 V). These peaks appeared with a variation in peak current and potential in the mixed solution, indicating an irregular detection of DA (Epa = 0.29 V and Epc = 0.07 V) and UA (Epa = 0.49 V) in bare GCE. This was due to the sluggish electron transfer kinetics at the bare GCE that occurred because of weak conductivity and electrode fouling caused by the deposition of DA and UA and their oxidative products on the electrode surface.56 According to Figure 5b, the Epa and Epc peaks (0.25 and 0.13 V, respectively) appeared in the presence of DA, with an improved current intensity at the CS/GS-modified GCE. In the UA curve, only the oxidation peak appeared at 0.33 V, with a better peak current intensity. In mixed solution, however, all of the peaks appeared at approximately the same position, signifying the presence of DA and UA, but the UA peak was unrecognizable and the current intensity was weaker in the mixed solution compared to those in the UA only solution. Moreover, the background current was much stronger than that in bare GCE. These 3047

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Figure 6. CVs recorded at different scan rates on CS−GS/GCE in Ar-saturated 0.1 M PBS at pH 7.4, containing 0.5 mM DA (a) and 0.5 mM UA (b); plots of peak currents vs square root of scan rate (ν) (c, d); and peak potential (Ep) vs ln v (e, f).

scan rate in Figure 6c,d by obeying the following regression equations

results suggest that still sluggish electron transfer kinetics and fouling occur at the CS/GS/GCE. However, well-defined and sharp oxidation peaks of DA (Epa = 0.17 V) and UA (Epa = 0.14 V) at CS−GS/GCE were observed due to the presence of DA and UA in the solo solution (Figure 5c). The peak currents for these two molecules were also higher than those for other electrodes. The cathodic peak of DA appeared at Epc = 0.12 V, suggesting that the reversibility of DA at the CS−GS/GCE was remarkably improved. All of the above peaks appeared at approximately same position in a mixed solution of DA and UA, with approximately the same current intensity and peak sharpness. The background current was also much lower than that of the CS/GS-modified electrode, whereas the lower background current leads to less fouling and improved electrocatalysis.57 This improvement could be attributed to the higher SSA and better conductivity resulting from the porous structure and higher degree of covalent amidation in the CS−GS matrix. Electrochemical Kinetics of DA and UA at CS−GS. To understand the electrochemical kinetics in the CS−GS matrix, we recorded the CVs of 0.5 mM DA and UA on the CS−GSmodified electrode at various scan rates (ν, mV s−1), as shown in Figure 6. As shown in Figure 6a, the redox peak current of DA enhances gradually with an increase in the scan rate. The same phenomenon was observed in the CVs of UA, wherein the irreversible oxidation peak current (Ipa) of UA also enhanced gradually with an increase in the scan rate (Figure 6b). Meanwhile, the redox peak current (Ipa and Ipc) of DA and the Ipa of UA showed good linear relationships with good correlation coefficients (R2) as a function of the square root of

Ipa (μ A) = 9.662v (mV s−1) − 10.193 (R2 = 0.998)

Ipc (μ A) = 11.536v (mV s−1) + 44.553 (R2 = 0.998) Ipa (μ A) = 13.006v (mV s−1) + 15.755 (R2 = 0.998)

These results indicate that all of the electrochemical reactions of DA and UA on the modified electrode were diffusioncontrolled processes.56,58 Also, Figure 6e,f shows that both Epa and Epc of DA and the Epa of UA are linearly proportional as a function of ln ν and follow regression equations below Epa (mV) = 0.038ν (mV s−1) + 0.075 (R2 = 0.998) Epc (mV) = −0.021ν (mV s−1) + 0.181 (R2 = 0.998) Epa (mV) = 0.011ν (mV s−1) + 0.027 (R2 = 0.998)

However, an important electrochemical parameter, the standard electron transfer rate constant (ks), was then calculated according to Laviron’s equations for DA (eq 1) and UA (eq 2);28 whereas DA is a reversible oxidation process, UA is an irreversible one. Thus, the value of ks was calculated as 7.95 and 7.44 s−1 for DA and UA, respectively, which are comparable to those obtained from other carbon-based sensors,59,60 suggesting that DA has fast electron transfer kinetics on the developed sensing interface. These values could remain unchanged with 3048

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E Pa(V) = −0.055pH + 0.8388 (R2 = 0.995)

varying pH, as the electron transfer step of the heterogeneous reaction may not be affected by pH.61

However, the plot of EPa versus pH derived slopes of 59 and 55 mV pH−1, which are close to the theoretical Nernstian value of 59 mV pH−1. These results indicate that an equal number of electrons and protons took part in the electrode reactions, which is in agreement with the literature reported.62,63 Therefore, considering the effect of pH on Ipa, 0.1 M PBS of pH 7.4 was chosen as the electrolyte for all of the experiments in this study. Simultaneous Detection of DA and UA at CS−GS. The differential pulse voltammetry (DPV) technique offers a higher sensitivity and a better resolution than those of the CV technique for quantitative analysis because it applies a small voltage pulse superimposed on the linear voltage sweep and samples the differential current shortly after the pulse.64 So, DPV was utilized to evaluate the performance of the CS−GS matrix for the quantitative determination of DA and UA. Figure 8a shows the DPVs of DA with increasing amounts from 1 to 700 μM in Ar-saturated 0.1 M PBS at pH 7.4. The oxidation peaks of DA enhanced gradually with an increase in the DA concentration (CDA), and Ipa showed a good linear relationship with the CDA over the range tested (Figure 8b), according to the regression equation given below

log ks = α log(1 − α) + (1 − α) log α − log(RT /nFv) − [α(1 − α)nF ΔEp/2.3RT ]

(1)

Epa = E°′ − (RT /αnF ) ln(RTks/αnF ) + (RT /αnF ) ln v (2)

where α is the electron transfer coefficient (0.72); n is the number of electron; F is the Faraday constant (96 485 C mol−1); R and T are the universal gas constant and temperature (K), respectively; and E°′ is the formal potential. Optimization of pH for DA and UA Detection at CS− GS. The pH of the detection medium can affect the rate of mass transport to the electrode surface, as in the present study. The effect of pH on the current response of the CS−GSmodified electrode toward 100 μM DA and UA was investigated separately within the pH range of 3.0−8.0. Figure 7a,b shows that the Ipa of DA and UA gradually increased with

Ipa (μ A) = 0.179C DA (μ M) + 1.206 (R2 = 0.998)

On the basis of the signal-to-noise (S/N = 3) characteristic, the limit of detection (LOD) was estimated to be 0.14 μM, with a sensitivity of 2.5 μA μM−1 cm−2. Similarly, Figure 8c shows the DPV curves with increasing concentration of UA (CUA) from 1 to 800 μM. The peak currents of UA increased linearly with an increase in CUA (Figure 8d), according to the linear function Ipa (μ A) = 0.140C UA (μ M) + 2.175 (R2 = 0.998)

Also, on the basis of S/N = 3, the LOD was estimated to be 0.17 μM, with a sensitivity of 2.0 μA μM−1 cm−2. Figure 8e shows the DPV curves for different concentrations of CDA (30−140 μM) in Ar-saturated 0.1 M PBS at pH 7.4 coexisting with 40 μM UA for synergic determination. Both DA- and UA-signifying peaks were in the same Epa, and the Ipa was proportional to the CDA (Figure 8f), which suggests that their oxidation products do not adsorb on the surface of CS− GS/GCE to hinder the synergic detection of DA and UA. The regression equation of the Ipa versus CDA curve was calculated as below

Figure 7. CVs recorded on CS−GS/GCE in Ar-saturated 0.1 M PBS at different pH values, containing 100 μM DA (a) and 100 μM UA (b), and plots of the corresponding peak current (c) and peak potential (d) as the function of pH value for DA.

Ipa (μ A) = 0.177C DA (μ M) + 1.756 (R2 = 0.998)

Similarly, Figure 8g shows DPV curves with varied CUA from 10 to 130 μM coexisting with 40 μM DA for synergic determination. The Ipa of UA increased linearly with an increase in CUA (Figure 8h), according to the linear function

an increase in the pH up to 7.4, which is nearly neutral and the pH of humoral fluid. However, further increase in the pH caused a decrease in Ipa for DA and UA, probably because the hydroxyl anion prevented the access of DA or UA to the adsorption sites on the CS−GS/GCE surface. Plots of Ipa and EPa with respect to the pH value are shown in Figure 7c,d for DA and UA, respectively. The Ipa of DA and UA increased with an increase in pH. It reached a maximum at pH 7.4 and then decreased rapidly with a further increase in pH. In addition, their EPa values shifted toward negative values with an increase in the pH of 0.1 M PBS. The linear relationships of DA and UA are expressed as follows

Ipa (μ A) = 0.148C UA (μ M) + 0.577 (R2 = 0.998)

Therefore, all of these assays indicate that the proposed biosensor, CS−GS/GCE, allows synergic and sensitive detection of DA and UA without significant interference from each other. Table 1 shows a comparison of the analytical performance of CS−GS/GCE to that of other sensors. We can see that the proposed biosensor has a better performance for the detection of DA and UA particularly over that of other GS with CS-based biosensors in terms of a long detection range.

E Pa(V) = −0.059pH + 0.691 (R2 = 0.996) 3049

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results show that the common inorganic ions, such as Na+, K+, Ca2+, Mg2+, Zn2+, Cl−, NO3−, and SO42−, in 100-fold excess do not show interference with DA and UA detection (Figure S3b). These results suggest that this biosensor has a satisfactory selectivity for the synergic determination of DA and UA in the presence of various interfering bioorganic and/or inorganic ions. Real Sample Analysis, Reproducibility, and Stability. Real-time monitoring of the performance of the fabricated biosensor was carried out by detecting DA and UA in different real samples [urine (Ur) and cow milk (Milk)], as shown in Figure S3c. The results are shown in Figure 9b and Table 2. The recoveries were in the range of 99.05−101.6%, suggesting good feasibility and reliability of the proposed electrode for synergic detection of DA and UA in urine and/or milk samples. The reproducibility of CS−GS/GCE was evaluated by preparing five parallel electrodes for the determination of 100 μM DA and UA. The relative standard deviations of the five electrodes were 2.7 and 3.1% for DA and UA, respectively. The stability of the modified electrode was also investigated. After the modified electrode was kept at ambient temperature for 21 days, repeatable sensing performance was achieved for the detection of DA and UA, suggesting that the sensor has a good stability.

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CONCLUSIONS Finally, an electroactive and covalently functionalized porous CS−GS matrix was prepared via enhanced amidation for the first time. Owing to its good electrochemical activity, high SSA, and 3D porous film structure through enhanced amide functionalization, the CS−GS matrix has received our attention for application as an electrochemical biosensor for synergic determination of DA and UA in the presence of AA and glucose. On the basis of the electrochemical performances of CS−GS, the DA and UA can be determined simultaneously within a larger detection range than that reported for other GS/ CS-based biosensors. Meanwhile, satisfactory results were obtained for the determination of DA and UA in real samples with a higher selectivity for the prepared biosensor matrix.

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EXPERIMENTAL SECTION Enhanced Amidation Reaction between CS and GS. GO was prepared from graphite powder (Aldrich, 325 mesh, 99.999%) via the modified Hummer’s method.32,33 GO− COOH was prepared with a modified procedure,17,34 as shown in Scheme 1. Briefly, 50 mg of GO was loaded in a 50 mL round-bottom flask with 15 mL of dimethylformamide (DMF). Then, 0.5 g of chloroacetic acid (Aldrich, Korea) and 0.6 g of NaOH were added to the aforementioned solution; this solution was maintained under ultrasonication for an hour and was subsequently stirred for 3 h at room temperature (RT, ∼25 °C). The solid was collected through filtration. Thereafter, 20 mg of CS, 20 mg of EDC (Aldrich, Korea), and 80 mg of NHS (Aldrich, Korea) were mixed in 5 mL of DMF and added into 5 mL of the GO−COOH suspension (5 mg mL−1 in DMF); again, the mixture maintained under sonication for 3 h. As the final step, the above solution was refluxed at 120 °C for a day. The as-obtained CS−GS was filtered, washed several times with distilled water, and dried under vacuum at 50 °C for 12 h. For comparison, GS and CS/GS were prepared by the same procedure with little change. The GS was prepared with the

Figure 8. DPVs on CS−GS/GCE at various concentrations of DA (1−700 μM) (a) and of UA (1−800 μM) (c) in a 0.1 M PBS solution (pH 7.4); the corresponding plot of Ipa vs CDA (b) and Ipa vs CUA (d); DPV profile for synergic determination of DA (30−140 μM) with 40 μM UA (e) and UA (10−130 μM) with 40 μM DA (g); the corresponding plots of Ipa vs CDA (f) and Ipa vs CUA (h).

This also demonstrates that this biosensor holds great application prospect for synergic determination of DA and UA. Interference Studies. One important issue for the feasibility of a biosensor is its capability to distinguish the analytes selectively from the interferents. In this work, the electrochemical responses of 100 μM DA and UA were measured at CS−GS/GCE in the presence of different interferents. The effects of some bioorganic molecules, such as AA (10-fold) and glucose (Glu, 10-fold), on the electrochemical responses of DA and UA were investigated (Figure S3a), and all response variations were lower than 2.5% from the blank response in 0.1 M PBS (Figure 9a). In addition, the 3050

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Table 1. Comparison of the Proposed Biosensing Performance to That of Other Reported Sensors LOD (μM) electrode materials a

CS−GS PPGE PAA-MWNTs AgNP/Zeo-Y PANI-AuNP GNP/Ch PAM/rGO N−S−PC CNT/GO/sCSa GR-AuNPs-CD-CSa SGNF/IL/CSa CS-graphenea PdNPs/GR/CSa GR-CSa Pt NS/C60 PdAg NFs/rGO PGE a

linear range (μM)

DA

UA

DA

UA

main interferents

references

0.14 0.03 0.02 0.016 3 0.12 0.1 0.02 0.06 0.08 0.04 1 0.1 0.0045 0.07 0.048 0.2

0.17 0.12 0.11 0.025 20 0.6 0.5 0.06 0.05 0.04 0.10 2 0.17

1−700 0.15−15 0.04−3 0.02−0.18 7−148 0.2−80 0.3−50 0.1−50 1.25−357 0.1−120 0.05−240 1−24 0.5−200 0.03−20.06 0.5−211.5 0.4−96 0.2−8

1−800 0.3−150 0.3−10 0.05−0.7 29−720 1.2−100 1−50 0.1−50 1.25−533 0.05−70 0.12−260 2−45 0.5−200

AA, glucose AA AA AA

this work 4 6 7 8 9 11 12 27 35 37 65 66 67 68 69 70

0.63 0.081 1

9.5−1187 1−150 1−60

glucose glucose

AA, UA AA glucose AA

Graphene-CS-modified electrode.

XPSPEAK41 system software. Dynamic light scattering analysis of the samples was performed using Malvern Zetasizer Nano. Electrochemical Characterization. All of the electrochemical measurements, including CV and DPV, were conducted using a three-electrode potentiostat (CHI 700C electrochemical workstation). A Pt wire and Ag/AgCl electrodes were used as the auxiliary and reference electrodes, respectively. EIS was performed on Versa State 3, manufactured by Metek. The 1 mg mL−1 suspension of CS−GS was prepared in water via ultrasonication. A 10 μL portion of CS−GS ink was dropped onto the prepolished glassy carbon electrode (GCE, 3 mm in diameter). CS/GS-coated GCE was also prepared by following the same protocol. All electrochemical experiments were performed in high-purity Ar-saturated 0.1 M PBS (at pH 7.4) electrolyte at RT (∼25 °C). Real Sample Preparation. For the synergic determination of DA and UA in cow milk samples, 1 L of cow milk was bought from the local market and used directly at RT. The urine sample was simply treated by centrifugation for 10 min at 5000 rpm to remove precipitated proteins and other particulate matter. Finally, the spiked concentrations of UA and DA was added into the as-received cow milk and urine samples containing Ar-saturated 0.1 M PBS and was calculated from the calibrated linear regression equations.

Figure 9. Bar diagram of Ipa derived from DPV responses of 100 μM DA and UA on CS−GS/GCE in the presence of 1 mM glucose (Glu) and AA (a), urine (Ur), and milk (b) in an Ar-saturated 0.1 M PBS solution (pH 7.4).

addition of NaBH4 instead of CS, and CS/GS was prepared without chloroacetic acid treatment. Physical Characterization. TEM images and energydispersive X-ray spectroscopy images were obtained using the TECNAI model FI-20 (FEI, Netherland). SEM images were obtained on a JSM-7500F field emission scanning electron microanalyzer (JEOL). The BET surface area and pore-size distribution were obtained through the Barrett−Joyner− Halenda method using the adsorption and desorption of the nitrogen isotherm (BelsorpII mini; BEL Japan Inc.). XPS images were obtained using a MultiLab 2000 with a 14.9 keV Al K X-ray source. Curve fitting was conducted using an

Table 2. Synergic Determination of DA and UA in Real Samples at the CS−GS/GCE added (μM)

found (μM)

samples

DA

UA

milk

10 50 200 10 50 200

10 50 200 10 50 200

urine

DA 10.11 50.02 199.2 10.07 49.95 198.4

± ± ± ± ± ±

recovery % UA

0.6 0.5 1.5 0.7 0.3 1.2 3051

10.14 49.97 198.6 10.16 49.93 198.1

± ± ± ± ± ±

0.5 0.5 1.5 0.7 0.5 1.3

DA

UA

101.1 100.04 99.6 100.7 99.9 99.2

101.4 99.94 99.3 101.6 99.86 99.05 DOI: 10.1021/acsomega.7b00331 ACS Omega 2017, 2, 3043−3054

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.7b00331. TEM images of GS, CS, the average size of GS, CS/GS, and CS−GS and the enlarged XRD pattern; FTIR spectra of GO, GO−COOH, GS, and CS−GS; the response of 100 μM DA on CS−GS/GCE in the presence of common inorganic ions (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +82 62 530 0064. Fax: +82 62 530 3389. ORCID

Seungwon Jeon: 0000-0002-4588-1361 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (NRF2015R1D1A1A09059344).

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