Biosensors and Bioelectronics 70 (2015) 310–317
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Shape-controlled ceria-reduced graphene oxide nanocomposites toward high-sensitive in situ detection of nitric oxide Fang Xin Hu a,b,c, Jia Le Xie a,b,c, Shu Juan Bao a,b,c, Ling Yu a,b,c, Chang Ming Li a,b,c,n a
Institute for Clean Energy & Advanced Materials, Southwest University, Chongqing 400715, China Faculty of Materials and Energy, Southwest University, Chongqing 400715, China c Chongqing Key Laboratory for Advanced Materials and Technologies of Clean Energies, Chongqing 400715, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 10 February 2015 Received in revised form 22 March 2015 Accepted 23 March 2015 Available online 31 March 2015
Nitric oxide (NO) is an important signal molecule released by most cancer cells under drug stimulation or/and disease development but it is extremely challenging to in situ while real-time sensitively detect NO due to its large diffusivity, low concentration and fast decay. Herein, shape-controlled reduced graphene oxide nanocomposing with ceria (rGO–CeO2) was synthesized via hydrothermal reaction to construct a highly sensitive real-time sensing platform for NO detection. The crystal shape of CeO2 nanoparticles in rGO–CeO2 composites significantly affects the sensing performance of rGO–CeO2, of which the regular hexagonal nanocrystal CeO2 achieves the highest sensitivity (1676.06 mA cm 2 M 1), a wide dynamic range (18.0 nM to 5.6 mM) and a low detection limit (9.6 nM). This attributes to a synergical effect from high catalytic activity of the specifically shaped CeO2 nanocrystal and good conductivity/high surface area of rGO. This work demonstrates a way by rationally compose individual merit components while well control the nanostructure for a superior synergistic effect to build a smart sensing platform, while offering a great application potential to sensitively real-time detect NO released from living cells for diagnosis or/and studies of complicated biological processes. & 2015 Elsevier B.V. All rights reserved.
Keywords: Ceria-reduced graphene oxide nanocomposites Nitric oxide Real-time living cell detection Electrochemical sensor
1. Introduction Real-time detection of bio-interesting molecules released from living cells are very critical to understand mechanisms of cellular functions and pathology while offering important applications in diseases diagnosis and drug discovery (Guo et al. 2012; Ma et al. 2014). Among these biomolecules, nitric oxide (NO) is an important bio-regulatory and signaling molecule associated with physiological and pathophysiological pathways (Lim et al. 2006; Malinski and Taha 1992), for example, regulating many biological processes including the central nervous system, cardiovascular tone, gastrointestinal tract, genitourinary system, immune process (Friedemann et al. 1996; Yang et al. 2010), antimicrobial agent, tumoricidal factor (Shin et al. 2007), Parkinson's disease (Ng et al. 2011) as well as asthma (Li et al. 2011). Nevertheless, the high diffusivity, low concentration and high reactivity of NO toward oxygen and metal-containing proteins in biological milieu make the real-time, quantitative detection very difficult (Kim et al. 2009; Woldman et al. 2009). Different strategies have been tried to ease n Corresponding author at: Institute for Clean Energy and Advanced Materials, Southwest University, Chongqing 400715, China. Fax: þ 86 23 68254969. E-mail address: [email protected] (C.M. Li).
http://dx.doi.org/10.1016/j.bios.2015.03.056 0956-5663/& 2015 Elsevier B.V. All rights reserved.
bottlenecks of the real-time measurements of NO (Peng et al. 2008). Compared with chemiluminescence, an electrochemical sensing is able to real-time detect NO with simpleness, low-cost and miniaturization (Hu et al. 2012), and thus is very promising to real-time detect NO while avoiding damage of the cell metabolism and associated regulatory pathways of living cells. To real time electrochemically detect low concentration of NO, a highly sensitive sensing platform is critical. Variety of materials have been employed to construct NO sensors for improvement of the sensitivity, such as graphene (Guo et al. 2012), carbon nanotubes (Chng and Pumera 2012), Pt (Park et al. 2012) and functionalized zinc oxide (Liu et al. 2009). Recently, ceria, CeO2 containing a rare earth element has drawn research attention owing to its unique 4f shell electronic structure involved numerous transition modes for remarkable redox properties (Tang et al. 2005). Its different nanocomposites such as Au–CeO2 (Si and Flytzani-Stephanopoulos 2008), mixed CeO2–ZrO2 oxides (Varez et al. 2006), Cu– CeO2 (Bera et al. 2002), graphene–CeO2 (Wang et al. 2011) and Pt– CeO2 (Goguet et al. 2004) have been developed for broad applications. CeO2 supported materials also exhibit excellent adsorption and reactivity toward NO (Dowding et al. 2012) and could be expected to be used for NO detection. However, it has not been reported yet.
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Graphene, a two-dimensional (2D) sheet of covalently bonded single-layer carbon atoms with superior physico-chemical properties for good conductivity and functionality (Lee et al. 2008; Huang et al. 2012). A rGO–CeO2 nanocomposites could provide an ideal platform for real-time sensing of NO. However, the morphologies of rGO–CeO2 have not been delicated tailored through the synthesis and its application for NO sensing. We synthesized different types of rGO–CeO2 nanocomposites from GO and cerous nitrate via an alkaline hydrothermal process with controlling of the Ce3 þ /OH molar ratio in two stages: initial nucleation of CeO2 nuclei on rGO nanosheets and subsequent growth. TEM characterization shows that rGO–CeO2 nanocomposite obtained with a Ce3 þ /OH molar ratio of 1:3 has well-dispersed regular hexagonal nanocrystal CeO2, and delivers the highest real-time sensitivity toward electrochemical detection of NO. The excellent performance could be ascribed to a synergistic effect from the composed CeO2 and rGO, of which the former contributes to the high catalytic activity toward NO oxidation and the latter offers good conductivity and high surface area to greatly increase absorption sites for NO while enhancing electron transfer rate as well as high biocompatibility for cell attachment to in situ detect NO released from cells.
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under stirring. The mixture was heated at 35 °C for 6 h, and then slowly diluted with deionized water under an ice-cooling environment. After continuously stirred for 2 h at room temperature, the solution was mixed with hydrogen peroxide (30%) slowly and stirred for 30 min. Later, the mixture was diluted with deionized water and centrifuged. Graphite oxide was obtained through washing the centrifuged product with 10% aqueous HCl solution. At last, exfoliation was performed by sonicating 0.1 mg mL 1 graphite oxide aqueous solution under ambient condition for 60 min. To obtain the reduced graphene oxide and ceria nanocomposites (rGO–CeO2), a conventional alkaline hydrothermal method was performed. Briefly, 0.900 g of PVP, 0.434 g or 0.868 g of Ce (NO3)3 6H2O and 500 mL GO solution (15.0 mg mL 1) were dissolved in 30 mL of deionized water. Then NaOH were added into the mixture as precipitant salt with its amount ranging from 1 to 10 mmol. After stirred for 30 min under room temperature, the mixture was transferred into a 50 mL Teflon lined autoclave, which was heated at 180 °C for 24 h. The obtained precipitate was harvested by centrifuge and then washed several times with deionized water. Finally, the resultant products (rGO–CeO2) were obtained by drying precipitates in an oven at 70 °C for 3 h. Besides, CeO2 nanocrystals were synthesized with the same method except adding GO solution.
2. Material and methods 2.4. Fabrication of the modified electrode 2.1. Reagents and chemicals Cerium nitrate hexahydrate (Ce(NO3)3 6H2O), sodium hydroxide (NaOH), polyvinylpyrrolidone (PVP, molecular weight 30,000), Acetylcholine (Ach), Hemoglobin (Hb), natural graphite, sulfuric acid (H2SO4), sodium nitrite (NaNO2), hydrogen peroxide (30%), hydrochloric acid (HCl), potassium peroxydisulfate (K2S2O8), phosphorus pentoxide (P2O5), potassium hypermanganate (KMnO4) and potassium hydroxide (KOH) were obtained from Sigma-Aldrich. All other chemicals were purchased from Sigma and used as obtained. Deionized water was used throughout the experiments. 2.2. Apparatus The electrochemical measurements were performed on a CHI660D electro-chemical work station (Shanghai, China). The conventional three-electrode system included a modified electrode as working electrode, a saturated calomel as reference electrode (SCE), and a platinum wire as counter electrode. Transmission electron microscopy was carried out on a JEM-2100 (TEM, Jeol, Japan). Scanning electron micrographs were studied with a scanning electron microscope (SEM, Hitachi, S-4800, Japan). The elemental compositions of the products were determined by energy dispersive X-ray spectroscopy (EDS, INCAX-Max250). X-ray powder diffraction (XRD) patterns were obtained using a XRD7000 with Cu Kα1 radiation (λ ¼ 1.5406 Å). 2.3. Synthesis of rGO–CeO2 nanocomposite In this strategy, graphite oxide was synthesized from natural graphite according to the Hummers’ method with some modification (Guo et al. 2010). A mixed solution was prepared containing natural graphite (1 g) and H2SO4, K2S2O8, P2O5 (weight ratio of 5:1:1), and the mixture was heated at 80 °C for 6 h. Then, the mixture was stirred overnight at room temperature and diluted with deionized water and filtered. The obtained filtered powder was dried at 80 °C under vacuum. After that it was added into H2SO4 (ice-cooling) solution and stirred for 15 min. Subsequently, KMnO4 was slowly put into the mixture with ice-cooling
NO sensing platform was prepared according to following procedure. Prior to use, glass carbon electrode (GCE, ؼ3 mm) was carefully polished by 0.3 and 0.05 mm alumina, followed by successive ultrasonic with distilled water and ethanol for 2 min until a mirror like surface was obtained. Then, rGO–CeO2 suspension (10.0 mg mL 1) was prepared by dispersing rGO–CeO2 power in deionized water with aid of ultrasonic. Subsequently, 5 mL rGO–CeO2 suspensions were dropped on clean GCE surface and dried in room temperature to obtain rGO–CeO2/GCE. The final electrode was applied to detect NO dissolved in PBS. For comparison, GO/GCE and CeO2/GCE were also prepared with same procedure for preparation of rGO–CeO2/GCE by replacing nanomaterials with GO or CeO2, respectively. Scheme 1a and b illuminates the preparation process of rGO–CeO2 composite and its electrocatalysis toward NO oxidation as well as real time monitoring of NO released from the living cells, respectively. 2.5. Cell culture and real time monitoring cell released NO molecules Human lung carcinoma cells (A549) were cultured in a humidified incubator (95% air with 5% CO2) at 37 °C with culture medium, which was prepared by mixing 1640 medium, 1% antibiotic and 10% fetal bovine serum in autoclaved deionized water and filtered. A549 cells were cultured in a petri dish (ؼ3 cm) with asprepared culture medium for 24 h and controlled with different cell densities such as 5.0 104 mL 1and 1.0 105 mL 1. Real time monitoring NO molecules released from cells was performed by chronoamperometry. In order to ensure accuracy of measured NO concentrations, cell culture medium inside device was mildly stirred during cell released NO measurement.
3. Results and discussion 3.1. Properties of rGO–CeO2 nanocomposite The measured TEM images reveal that plain CeO2 nanocrystals (Fig. 1a) have an average diameter of 14–16 nm with
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Scheme 1. (a) Illustration of the preparation process of rGO–CeO2, insert was HRTEM image of CeO2. (b)Schematic display of the electro-catalytic oxidation of NO on the rGO–CeO2/GCE and real time monitoring of NO released from the living cells on rGO–CeO2/GCE.
accumulation tendency while rGO–CeO2 (Fig. 1b) shows well-dispersed CeO2 nanocrystals grown on the rGO sheet with an average size of 10–12 nm, indicating that the addition of GO plays a critical role in nucleation and growth of CeO2 nanocrystals to restrict the crystal size and effectively improve the crystal
nanoparticle distribution uniformity. The surface morphologies of nanomaterials prepared with different Ce3 þ /OH molar ratios are investigated by TEM. As in Fig. 1c, CeO2 nanocrystals in rGO–CeO2 synthesized with a Ce3 þ / OH molar ratio of 2:1 are mainly round-shaped (5–8 nm) with
Fig. 1. TEM images of (a) CeO2 and (b) rGO–CeO2 synthesized at Ce3 þ /OH ¼1:3. TEM images of rGO–CeO2 synthesized at different Ce3 þ /OH molar ratios, (c) 2:1; (d) 1:1; (e) 1:2; (f) 1:3; (h) 1:4; (i) 1:5; and (j) 1:10. (g) HRTEM image of rGO–CeO2 synthesized at Ce3 þ /OH ¼ 1:3.White arrows indicate pores inside the crystal.
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certain accumulation. With decreasing Ce3 þ /OH molar ratio to 1:1, CeO2 nanocrystals (Fig. 1d) are mainly quadrilateral with uniform despersion of 8–10 nm-sized nanocrystals. Fig. 1e displays irregular CeO2 nanocrystals (6–12 nm) of rGO–CeO2 obtained at a Ce3 þ /OH molar ratio of 1:2, indicating a transitional growth process from the quadrilateral to hexagonal shaped ones. The inset image of Fig. 1e shows a typical irregular CeO2 nanocrystal. Fig. 1f displays regular hexagonally shaped CeO2 nanocrystals of rGO–CeO2 obtained with a Ce3 þ /OH molar ratio of 1:3 with inconspicuous corners, edges and pores in nanocrystals as indicating by arrows. The crystal size finally enlarges to 10–12 nm with uniform dispersion. Fig. 1g shows HRTEM images of CeO2, from which, facets of CeO2 NPs are determined by directing the incident electron beam perpendicular to the hexagon facet of the crystal. The inter-fringe distances of dominant 2D lattice fringes of HRTEM image are 0.26 nm or 0.28 nm, close to the {200} lattice spacing of cubic phase of CeO2 at 0.271 nm (Yang and Gao 2006). CeO2 crystals sizes are around 10 nm in rGO–CeO2 synthesized at Ce3 þ /OH molar ratios of 1:4 (Fig. 1h) and most of them have irregular hexagonal or quadrilateral shapes with tendency of agglomeration. When molar ratios are further decreased to 1:5 (Fig. 1i) and 1:10 (Fig. 1j), heavily agglomerated CeO2 crystals are obtained with sizes of 16–20 nm and 10–12 nm, respectively. Morphologies are not as regular as those prepared with higher molar ratios. TEM images display that irregular hexagon or quadrilateral nanocrystals fuse with some neighboring nuclei through sharing the common crystallographic orientation, resulting in the assembled single-crystalline particles. SEM image in Fig. S1 (supporting information) reveals that rGO is covered by CeO2 nanocrystals. Layered rGO sheets exhibit high specific surface area for CeO2 germinating, indicating successful nucleation and growth of CeO2 nanocrystals on rGO surface. However, with Ce3 þ /OH molar ratio of 2:1, rGO–CeO2 nanocomposite is agglomerated (Fig. S1a). At Ce3 þ /OH molar ratio of 1:1 and 1:2 (Fig. S1b and c), the prepared rGO–CeO2 nanocomposites are uniform. Compared to Fig. S1b–d (Ce3 þ /OH ¼1:3) displays more uniformly distributed CeO2 nanocrystals with larger sizes. At Ce3 þ /OH molar ratio of 1:4, the size of CeO2 nanocrystal starts to decreases (Fig. S1e), while when decreasing Ce3 þ /OH molar ratio to 1:5 (Fig. S1f) and 1:10 (Fig. S1g), agglomerations of particles occur. XRD analysis of rGO–CeO2 nanocomposites
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prepared with all Ce3 þ /OH molar ratios shows a typical pattern (Fig. S2A), of which peaks can be assigned to diffraction of (111), (200), (220), (311), (222), (400), (331) and (420) respectively according to JCPDS Card no. 34-0394, thus confirming a cubic structure of ceria. The EDS analysis of materials prepared with Ce3 þ /OH molar ratio of 1:3 (Fig. S2B) reveals that the main chemical elements in nanocomposites are C, O and Ce, clearly evidencing a successful synthesis of rGO–CeO2 composite. In terms of the morphology evolvement of rGO–CeO2 composite, a formation mechanism could be proposed as in Scheme 2. At the beginning, Ce3 þ and OH are reacted to form Ce(OH)3 precipitates on GO sheets followed by conversion to 2–3 nm CeO2 nuclei via oxidation and rapid dehydration. The reported growth of CeO2 nanocrystal during a hydrothermal process although not on graphene but could be applicable has been reported (Lin et al. 2012) to follow two processes: a dominant process to attach 2– 3 nm nuclei (dispersion of nanoparticles) on a lattice matched surface and an assistant process for Ostwald ripening (dissolution precipitation). With different Ce3 þ /OH molar ratios, the reaction rate and amount of CeO2 nuclei formed should be different to result in different shapes, sizes and uniformity of CeO2 nanocrystals. An effective collision and rate of reaction species to form uniformly dispersed nuclei play a critical role in synthesis of uniformly distributed nuclei and followed growth of CeO2 nanocrystals for certain sizes and uniformity, which can be apparently affected by pH of solution since pH can cause different density of OH on CeO2 nuclei surface and solubility of Ce4 þ from the CeO2 nuclei and thus for various collision rates, GO actually provides an excellent substrate for nucleation and crystal growth while PVP prevents particles aggregation in hydrothermal reaction as well as promotes the orientated growth through an effective collision. 3.2. Electrocatalytic behaviors of rGO–CeO2 toward NO oxidation The electrocatalytic behavior of rGO–CeO2/GCE toward NO oxidation was investigated by cyclic voltammetry (CV) in 0.01 M pH 7.4 PBS with and without 90 mM NO. The results in Fig. 2A show that there is no obvious oxidation peak in blank PBS (curve a), while a large peak with peak potential at 790 mV vs. saturated calomel electrode (SCE) is observed (curve b) for rGO– CeO2 electrode after addition of NO, obviously due to NO
Scheme 2. Illustration of different morphology evolvement mechanism of rGO–CeO2 composites.
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Fig. 2. (A) CV response of rGO–CeO2/GCE in 0.01 M pH 7.4 PBS with (curve b) and without (curve a) 90 mM NO. (B) CV response of different modified sensors to 90 mM NO in 0.01 M pH 7.4 PBS, (a) rGO/GCE, (b) CeO2/GCE, and (c) rGO–CeO2/GCE. Crosses sign start potential and arrows indicate the scan direction.
oxidation. CVs were also measured at different modified electrodes in the same NO solution (Fig. 2B), showing that no well-defined NO oxidation peaks were observed at rGO/GCE (curve a), evidencing that plain rGO have no electrocatalytic activity toward NO oxidation. However, rGO has a large double layer capacitance and high oxidation current possibly from rGO oxidation, which significantly interfere the signal from NO reduction. In contrast, both CeO2/GCE and rGO–CeO2/GCE exhibit well-defined, sharp oxidation peaks at 895 mV (curve b) and 790 mV (curve c), respectively, revealing only CeO2 possesses good electrocatalytic activity toward NO oxidation. Further, the much higher peak current and more negative peak potential of rGO–CeO2 than CeO2 for NO oxidation (Fig. 2A and B) show that the former has much larger reaction surface area and better electrocatalytic activity than latter, clearly indicating that the composing of rGO and CeO2 is not a simple sum of both merits but generates great synergistic effect to significantly boost both reaction surface and catalytic activity. In addition, apparently the growth CeO2 on rGO also greatly suppresses the interference reactions of rGO for higher signal to noise (S/N) ratio. Thus, it can be concluded that the superior electrocatalytic behavior of rGO–CeO2 can be attributed to a strong synergistic effect of both CeO2 and rGO. It is very likely that the oxygen vacancies in crystal structures of CeO2 (Cui et al. 2009) could enhance the NO adsorption as that reported (Niwa. et al.
1982). A scheme of an electrochemical oxidation of NO was studied (Kosminsky et al. 2001). The mechanism of NO oxidation on rGO–CeO2 electrode could be proposed that NO is firstly adsorbed on CeO2, followed by transferring an electron to CeO2 as
NO → NO+ + e−
(1)
and NO þ is a relatively strong Lewis acid (Kosminsky et al. 2001) that can react with water according to the following equation:
NO+ + H2 O→ NO2− + 2H+
(2)
3.3. Effect of rGO–CeO2 composite nanostructures on electrocatalytic behavior toward NO oxidation The nanostructure of rGO–CeO2 composite was delicately tailored as discussed above and its effect on electrocatalytic behavior toward NO oxidation was further investigated. Fig. 3A shows that the NO sensors made from CeO2 round, quadrilateral, irregular and hexagonal CeO2 nanocrystals produce NO oxidation peak potentials of 894 mV (curve a), 863 mV (curve b), 823 mV (curve c) and 790 mV (curve d), respectively, indicating that the regular hexagonal CeO2 nanocrystal has more negative oxidation peak potential and higher electrocatalytic peak current than that of
Fig. 3. (A) CV response of rGO–CeO2/GCE in 0.01 M pH 7.4 PBS to 180 mM NO with rGO–CeO2 synthesized at different Ce3 þ /OH molar ratios (a) 2:1; (b) 1:1; (c) 1:2; (d) 1:3; (e) 1:4; (f) 1:5; and (g) 1:10. (B) Performance comparison of rGO–CeO2/GCE in 0.01 M pH 7.4 PBS to 180 mM NO between different Ce3 þ /OH molar ratios and CV response peak currents or peak potentials. Cross signs start potential and arrows indicate the scan direction.
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quadrilateral and round ones. This is very possibly attributed to the different crystal nanostructures possessing different reaction surface areas and different number of active reaction centers at a same steric packing size. For example, at 10 nm packing size, the hexagonal CeO2 nanocrystal provides much larger surface area (150 3 nm2) than quadrilateral (100 nm2) and round (78.5 nm2) CeO2 nanocrystals for much larger reaction area to result in higher peak current toward NO oxidation. The more negative NO oxidation peak potential indicates that the hexagonal nanostructure also induces higher intrinsic electrocatalytic activity than the
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quadrilateral one. This is very likely that the former structure can expose more oxygen vacancies to NO for stronger NO adsorption to enhance electrocatalytic process. However, with Ce3 þ /OH molar ratio decreases to 1:4 (curve e), the composite has both irregular hexagonal and quadrilateral structures and the NO oxidation peak current decreases and peak potentials shift to more positive (818 mV), indicating decayed electrocatalytic performance. With decrease of Ce3 þ /OH molar ratios to 1:5 (curve f) and 1:10 (curve g), the formed nanocrystals are aggregated and oxidation peaks of NO shift further to more
Fig. 4. (A) and (B) Selectivity performance of the rGO–CeO2/GCE. (C) Chronoamperometric response of the rGO–CeO2/GCE upon continuous injection of different concentration NO at an applied potential of 0.8 V in 0.01 M pH 7.4 PBS. (D) Linear calibration curve of rGO–CeO2/GCE to NO. (E) Real time monitoring of NO released from A549 cells stimulated by different amount of ACh in cell culture medium, (a) 0.5 mM ACh; (b) 0.2 mM ACh; (c) 1 mM ACh and 1 mM Hb. (F) Real time monitoring of NO released from A549 cells in cell culture medium with controlled cell density. The insets are microscope photographs of A549 cells with different cell densities. Error bars represent the standard deviations of five independent measurements.
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positive (865 mV and 876 mV, respectively). It is clear that the electrocatalytic performance decayed when Ce3 þ /OH molar ratio is less than 1:3. This is probably that highly concentrated OH results in high collision rate with Ce3 þ for a much faster nucleation rate in hydrothermal reaction, leading to irregular crystal structure and even serious aggregation, which apparently decreases the exposure of reaction sites to NO for decayed electrocatalytic performance. Fig. 3B clearly shows volcano relations of peak current and peak potential of NO oxidation versus Ce3 þ /OH molar ratio. Surprisingly, the highest electrocatalytic activity (the most negative potential) and the highest catalytic current (peak current) are well in agreement to point to a Ce3 þ /OH molar ratio of 1:3, which is used for the following investigations. 3.4. Real-time detection of NO molecules released from living cells The stability of the rGO–CeO2/GCE sensor toward NO molecule was investigated by intermittently detecting chronoamperometrically in a 1200 nM NO solution. The rGO–CeO2/GCE loses about 10% of its original response after its storage in PBS for 20 days. Further, after 20 continuous CV measurements, current responses of the sensor were nearly unchanged, indicating a good repeatability. The selectivity and anti-interference ability of the sensor in real-time detection of NO released from cells were studied by adding various interfering species. Results show that 60 mM Ca2 þ , K þ , Na þ , CO32–, NO3–, Cl– and 1 mM uric acid (UA) do not cause any observable interference in detection of 0.6 mM NO (Fig. 4A). Besides, selectivity of the biosensor against ascorbic acid (AA) and dopamine (DA), commonly existing biological species was also investigated. In evaluation, biological concentrations of DA ((0.01– 1 mM) (Wightman et al. 1988) and AA ( 2.3 mM) (Kutnink et al. 1987) have been reported. Results show that 2.3 mM AA and 1 mM DA don't cause obvious interference for released 4.3 mM NO (equal to cell density of 1 105 mL 1) as displayed in Fig. 4B. The good selectivity of the sensor toward NO molecule could be contributed from the catalytic activity of rGO–CeO2 specifically toward NO oxidation. The chronoamperometric current-time response of rGO–CeO2/ GCE upon successive addition NO to a continuous stirred PBS (0.01 MpH 7.4) at 0.8 V vs. SCE was used to measure a calibration curve (Fig. 4C). The sensor exhibits a rapid stepped increase response with the injection of NO in less than 4 s to achieve 95% of steady-state current. The short response time could warrent a realtime detection of NO released from living cells, which has half-life around 5 seconds. Fig. 4D pictures the calibration curve of rGO– CeO2/GCE for NO detection with a linear equation as I (nA) ¼ 198.1 þ0.119 CNO (nM), which gives a correlation coefficient of 0.997 (n ¼31) for a wide linear range of 18.0 nM to 5.6 mM, a sensitivity of 1676.06 mA cm 2 M 1 and a low detection limit of 9.6 nM in terms of 3 S/K. Besides, a comparison between reported and this works is listed in Table 1 S in the supporting information, showing that this work delivers wider linear range and lower detection limit for NO detection. The rGO–CeO2/GCE sensor was used to real-time in situ detect NO molecules released from living A549 cells. ACh is chose as a function drug to stimulate living cells to release NO molecular. It has been reported (Asakawa et al. 2008) that ACh signal is transmitted to intercellular signaling cascades via some receptors, which can mediate rapid increment of intercellular Ca2 þ concentration. Then, Ca2 þ -calmodulin complex activates endothelial NO synthase to promote generation of NO. Detailed investigation of dynamic responses of A549 cells with a cell density of 5 104 mL 1 on rGO–CeO2 sensor is presented in Fig. 4E. The stimulating agents is continuously injected under mild stirring as indicated by arrow. As soon as 0.5 mM (curve a) or
0.2 mM (curve b) ACh is added into cell culture medium, a significant current response appears corresponding to NO oxidation. It is worthy of a note that the sensing is an in situ detection since the GCE sensor directly detects NO released from a number of cells contacted with the electrode surface. Such in situ detections with large electrodes have been widely reported such as Guo et al. 2011 and Ma et al. 2014. In contrast, mixture of 1 mM ACh and 1 mM Hb (curve c) don't cause obvious step increase indicating that released NO molecules is consumed by Hb. Furthermore, the calculated concentrations of NO released from A549 cells are around 4478.18 nM or 1591.18 nM for addition of 0.5 mM or 0.2 mM ACh, respectively, according to drug stimulated current response and calibration equation for rGO–CeO2/GCE. Current responses caused by adding 0.5 mM ACh drug is about 2.8 times than that of 0.2 mM ACh suggesting the response under ACh stimulation is a concentration-dependent behavior as shown in Fig. 4E. Moreover, responses of rGO–CeO2 sensor to A549 with different cell densities are investigated as shown in Fig. 4F. The rGO– CeO2/GCE don't exhibit obvious response to blank culture dish without A549 cell (curve a). After cell densities are controlled as 5.0 104 mL 1 (curve b) and 1.0 105 mL 1 (curve c), obvious responses generate on rGO–CeO2/GCE with stimulation of 0.2 mM ACh drug. From the drug stimulated current response and calibration equation the calculated concentrations of NO released from A549 cells are around 4304.62 nM or 1769.33 nM for 1.0 105 mL 1 and 5.0 104 mL 1 cell densities, respectively. The current responses caused by adding 0.2 mM Ach drug to A549 with 1.0 105 mL 1cell density are about 2.4 times than that of 5.0 104 mL 1 cell density.
4. Conclusions rGO–CeO2 composites nanostructures were delicately tailored by regulating ratio of Ce3 þ /OH during preparation process and the formation mechanism is proposed. Results show clearly that a Ce3 þ /OH molar ratio of 1:3 produces a regular hexagonal nanocrystal CeO2 for the best electrocatalytic performance toward NO oxidation, which is mainly contributed by a remarkable synergistic effect between rGO and CeO2. Real-time in situ detection of NO molecules released from living cells was investigated by controlling cell densities and adding stimulation drugs, revealing that NO released by living cells under Ach stimulation is a concentration-dependent behavior. This work demonstrates how we tailor a composite nanostructure to produce strong synergical effect on electrocatalytic performance while offering a facile and reliable platform to in situ real-time detect bio-signal molecules released by living cells.
Acknowledgements We would like to gratefully acknowledge financial support from the 973 program of China (No. 2013CB127804), National Natural Science Foundation of China, China (Nos. 21205098, and 21273173), Fundamental Research Funds for the Central Universities (XDJK2012C049), Chongqing Key Laboratory for Advanced Materials and Technologies of Clean Energies, start-up Grant under SWU111071 from the Southwest University, Chongqing Engineering Research Center for Rapid Diagnosis of Dread Disease and Chongqing Development and Reform Commission. Appendix A. Supplementary information Supplementary data associated with this article can be found in
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the online version at http://dx.doi.org/10.1016/j.bios.2015.03.056.
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Shape-controlled ceria-reduced graphene oxide nanocomposites toward high-sensitive in situ detection of nitric oxide Fang Xin Hu a,b,c, Jia Le Xie a,b,c, Shu Juan Bao a,b,c, Ling Yu a,b,c, Chang Ming Li a,b,c,n a
Institute for Clean Energy & Advanced Materials, Southwest University, Chongqing 400715, China Faculty of Materials and Energy, Southwest University, Chongqing 400715, China c Chongqing Key Laboratory for Advanced Materials and Technologies of Clean Energies, Chongqing 400715, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 10 February 2015 Received in revised form 22 March 2015 Accepted 23 March 2015 Available online 31 March 2015
Nitric oxide (NO) is an important signal molecule released by most cancer cells under drug stimulation or/and disease development but it is extremely challenging to in situ while real-time sensitively detect NO due to its large diffusivity, low concentration and fast decay. Herein, shape-controlled reduced graphene oxide nanocomposing with ceria (rGO–CeO2) was synthesized via hydrothermal reaction to construct a highly sensitive real-time sensing platform for NO detection. The crystal shape of CeO2 nanoparticles in rGO–CeO2 composites significantly affects the sensing performance of rGO–CeO2, of which the regular hexagonal nanocrystal CeO2 achieves the highest sensitivity (1676.06 mA cm 2 M 1), a wide dynamic range (18.0 nM to 5.6 mM) and a low detection limit (9.6 nM). This attributes to a synergical effect from high catalytic activity of the specifically shaped CeO2 nanocrystal and good conductivity/high surface area of rGO. This work demonstrates a way by rationally compose individual merit components while well control the nanostructure for a superior synergistic effect to build a smart sensing platform, while offering a great application potential to sensitively real-time detect NO released from living cells for diagnosis or/and studies of complicated biological processes. & 2015 Elsevier B.V. All rights reserved.
Keywords: Ceria-reduced graphene oxide nanocomposites Nitric oxide Real-time living cell detection Electrochemical sensor
1. Introduction Real-time detection of bio-interesting molecules released from living cells are very critical to understand mechanisms of cellular functions and pathology while offering important applications in diseases diagnosis and drug discovery (Guo et al. 2012; Ma et al. 2014). Among these biomolecules, nitric oxide (NO) is an important bio-regulatory and signaling molecule associated with physiological and pathophysiological pathways (Lim et al. 2006; Malinski and Taha 1992), for example, regulating many biological processes including the central nervous system, cardiovascular tone, gastrointestinal tract, genitourinary system, immune process (Friedemann et al. 1996; Yang et al. 2010), antimicrobial agent, tumoricidal factor (Shin et al. 2007), Parkinson's disease (Ng et al. 2011) as well as asthma (Li et al. 2011). Nevertheless, the high diffusivity, low concentration and high reactivity of NO toward oxygen and metal-containing proteins in biological milieu make the real-time, quantitative detection very difficult (Kim et al. 2009; Woldman et al. 2009). Different strategies have been tried to ease n Corresponding author at: Institute for Clean Energy and Advanced Materials, Southwest University, Chongqing 400715, China. Fax: þ 86 23 68254969. E-mail address: [email protected] (C.M. Li).
http://dx.doi.org/10.1016/j.bios.2015.03.056 0956-5663/& 2015 Elsevier B.V. All rights reserved.
bottlenecks of the real-time measurements of NO (Peng et al. 2008). Compared with chemiluminescence, an electrochemical sensing is able to real-time detect NO with simpleness, low-cost and miniaturization (Hu et al. 2012), and thus is very promising to real-time detect NO while avoiding damage of the cell metabolism and associated regulatory pathways of living cells. To real time electrochemically detect low concentration of NO, a highly sensitive sensing platform is critical. Variety of materials have been employed to construct NO sensors for improvement of the sensitivity, such as graphene (Guo et al. 2012), carbon nanotubes (Chng and Pumera 2012), Pt (Park et al. 2012) and functionalized zinc oxide (Liu et al. 2009). Recently, ceria, CeO2 containing a rare earth element has drawn research attention owing to its unique 4f shell electronic structure involved numerous transition modes for remarkable redox properties (Tang et al. 2005). Its different nanocomposites such as Au–CeO2 (Si and Flytzani-Stephanopoulos 2008), mixed CeO2–ZrO2 oxides (Varez et al. 2006), Cu– CeO2 (Bera et al. 2002), graphene–CeO2 (Wang et al. 2011) and Pt– CeO2 (Goguet et al. 2004) have been developed for broad applications. CeO2 supported materials also exhibit excellent adsorption and reactivity toward NO (Dowding et al. 2012) and could be expected to be used for NO detection. However, it has not been reported yet.
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Graphene, a two-dimensional (2D) sheet of covalently bonded single-layer carbon atoms with superior physico-chemical properties for good conductivity and functionality (Lee et al. 2008; Huang et al. 2012). A rGO–CeO2 nanocomposites could provide an ideal platform for real-time sensing of NO. However, the morphologies of rGO–CeO2 have not been delicated tailored through the synthesis and its application for NO sensing. We synthesized different types of rGO–CeO2 nanocomposites from GO and cerous nitrate via an alkaline hydrothermal process with controlling of the Ce3 þ /OH molar ratio in two stages: initial nucleation of CeO2 nuclei on rGO nanosheets and subsequent growth. TEM characterization shows that rGO–CeO2 nanocomposite obtained with a Ce3 þ /OH molar ratio of 1:3 has well-dispersed regular hexagonal nanocrystal CeO2, and delivers the highest real-time sensitivity toward electrochemical detection of NO. The excellent performance could be ascribed to a synergistic effect from the composed CeO2 and rGO, of which the former contributes to the high catalytic activity toward NO oxidation and the latter offers good conductivity and high surface area to greatly increase absorption sites for NO while enhancing electron transfer rate as well as high biocompatibility for cell attachment to in situ detect NO released from cells.
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under stirring. The mixture was heated at 35 °C for 6 h, and then slowly diluted with deionized water under an ice-cooling environment. After continuously stirred for 2 h at room temperature, the solution was mixed with hydrogen peroxide (30%) slowly and stirred for 30 min. Later, the mixture was diluted with deionized water and centrifuged. Graphite oxide was obtained through washing the centrifuged product with 10% aqueous HCl solution. At last, exfoliation was performed by sonicating 0.1 mg mL 1 graphite oxide aqueous solution under ambient condition for 60 min. To obtain the reduced graphene oxide and ceria nanocomposites (rGO–CeO2), a conventional alkaline hydrothermal method was performed. Briefly, 0.900 g of PVP, 0.434 g or 0.868 g of Ce (NO3)3 6H2O and 500 mL GO solution (15.0 mg mL 1) were dissolved in 30 mL of deionized water. Then NaOH were added into the mixture as precipitant salt with its amount ranging from 1 to 10 mmol. After stirred for 30 min under room temperature, the mixture was transferred into a 50 mL Teflon lined autoclave, which was heated at 180 °C for 24 h. The obtained precipitate was harvested by centrifuge and then washed several times with deionized water. Finally, the resultant products (rGO–CeO2) were obtained by drying precipitates in an oven at 70 °C for 3 h. Besides, CeO2 nanocrystals were synthesized with the same method except adding GO solution.
2. Material and methods 2.4. Fabrication of the modified electrode 2.1. Reagents and chemicals Cerium nitrate hexahydrate (Ce(NO3)3 6H2O), sodium hydroxide (NaOH), polyvinylpyrrolidone (PVP, molecular weight 30,000), Acetylcholine (Ach), Hemoglobin (Hb), natural graphite, sulfuric acid (H2SO4), sodium nitrite (NaNO2), hydrogen peroxide (30%), hydrochloric acid (HCl), potassium peroxydisulfate (K2S2O8), phosphorus pentoxide (P2O5), potassium hypermanganate (KMnO4) and potassium hydroxide (KOH) were obtained from Sigma-Aldrich. All other chemicals were purchased from Sigma and used as obtained. Deionized water was used throughout the experiments. 2.2. Apparatus The electrochemical measurements were performed on a CHI660D electro-chemical work station (Shanghai, China). The conventional three-electrode system included a modified electrode as working electrode, a saturated calomel as reference electrode (SCE), and a platinum wire as counter electrode. Transmission electron microscopy was carried out on a JEM-2100 (TEM, Jeol, Japan). Scanning electron micrographs were studied with a scanning electron microscope (SEM, Hitachi, S-4800, Japan). The elemental compositions of the products were determined by energy dispersive X-ray spectroscopy (EDS, INCAX-Max250). X-ray powder diffraction (XRD) patterns were obtained using a XRD7000 with Cu Kα1 radiation (λ ¼ 1.5406 Å). 2.3. Synthesis of rGO–CeO2 nanocomposite In this strategy, graphite oxide was synthesized from natural graphite according to the Hummers’ method with some modification (Guo et al. 2010). A mixed solution was prepared containing natural graphite (1 g) and H2SO4, K2S2O8, P2O5 (weight ratio of 5:1:1), and the mixture was heated at 80 °C for 6 h. Then, the mixture was stirred overnight at room temperature and diluted with deionized water and filtered. The obtained filtered powder was dried at 80 °C under vacuum. After that it was added into H2SO4 (ice-cooling) solution and stirred for 15 min. Subsequently, KMnO4 was slowly put into the mixture with ice-cooling
NO sensing platform was prepared according to following procedure. Prior to use, glass carbon electrode (GCE, ؼ3 mm) was carefully polished by 0.3 and 0.05 mm alumina, followed by successive ultrasonic with distilled water and ethanol for 2 min until a mirror like surface was obtained. Then, rGO–CeO2 suspension (10.0 mg mL 1) was prepared by dispersing rGO–CeO2 power in deionized water with aid of ultrasonic. Subsequently, 5 mL rGO–CeO2 suspensions were dropped on clean GCE surface and dried in room temperature to obtain rGO–CeO2/GCE. The final electrode was applied to detect NO dissolved in PBS. For comparison, GO/GCE and CeO2/GCE were also prepared with same procedure for preparation of rGO–CeO2/GCE by replacing nanomaterials with GO or CeO2, respectively. Scheme 1a and b illuminates the preparation process of rGO–CeO2 composite and its electrocatalysis toward NO oxidation as well as real time monitoring of NO released from the living cells, respectively. 2.5. Cell culture and real time monitoring cell released NO molecules Human lung carcinoma cells (A549) were cultured in a humidified incubator (95% air with 5% CO2) at 37 °C with culture medium, which was prepared by mixing 1640 medium, 1% antibiotic and 10% fetal bovine serum in autoclaved deionized water and filtered. A549 cells were cultured in a petri dish (ؼ3 cm) with asprepared culture medium for 24 h and controlled with different cell densities such as 5.0 104 mL 1and 1.0 105 mL 1. Real time monitoring NO molecules released from cells was performed by chronoamperometry. In order to ensure accuracy of measured NO concentrations, cell culture medium inside device was mildly stirred during cell released NO measurement.
3. Results and discussion 3.1. Properties of rGO–CeO2 nanocomposite The measured TEM images reveal that plain CeO2 nanocrystals (Fig. 1a) have an average diameter of 14–16 nm with
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Scheme 1. (a) Illustration of the preparation process of rGO–CeO2, insert was HRTEM image of CeO2. (b)Schematic display of the electro-catalytic oxidation of NO on the rGO–CeO2/GCE and real time monitoring of NO released from the living cells on rGO–CeO2/GCE.
accumulation tendency while rGO–CeO2 (Fig. 1b) shows well-dispersed CeO2 nanocrystals grown on the rGO sheet with an average size of 10–12 nm, indicating that the addition of GO plays a critical role in nucleation and growth of CeO2 nanocrystals to restrict the crystal size and effectively improve the crystal
nanoparticle distribution uniformity. The surface morphologies of nanomaterials prepared with different Ce3 þ /OH molar ratios are investigated by TEM. As in Fig. 1c, CeO2 nanocrystals in rGO–CeO2 synthesized with a Ce3 þ / OH molar ratio of 2:1 are mainly round-shaped (5–8 nm) with
Fig. 1. TEM images of (a) CeO2 and (b) rGO–CeO2 synthesized at Ce3 þ /OH ¼1:3. TEM images of rGO–CeO2 synthesized at different Ce3 þ /OH molar ratios, (c) 2:1; (d) 1:1; (e) 1:2; (f) 1:3; (h) 1:4; (i) 1:5; and (j) 1:10. (g) HRTEM image of rGO–CeO2 synthesized at Ce3 þ /OH ¼ 1:3.White arrows indicate pores inside the crystal.
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certain accumulation. With decreasing Ce3 þ /OH molar ratio to 1:1, CeO2 nanocrystals (Fig. 1d) are mainly quadrilateral with uniform despersion of 8–10 nm-sized nanocrystals. Fig. 1e displays irregular CeO2 nanocrystals (6–12 nm) of rGO–CeO2 obtained at a Ce3 þ /OH molar ratio of 1:2, indicating a transitional growth process from the quadrilateral to hexagonal shaped ones. The inset image of Fig. 1e shows a typical irregular CeO2 nanocrystal. Fig. 1f displays regular hexagonally shaped CeO2 nanocrystals of rGO–CeO2 obtained with a Ce3 þ /OH molar ratio of 1:3 with inconspicuous corners, edges and pores in nanocrystals as indicating by arrows. The crystal size finally enlarges to 10–12 nm with uniform dispersion. Fig. 1g shows HRTEM images of CeO2, from which, facets of CeO2 NPs are determined by directing the incident electron beam perpendicular to the hexagon facet of the crystal. The inter-fringe distances of dominant 2D lattice fringes of HRTEM image are 0.26 nm or 0.28 nm, close to the {200} lattice spacing of cubic phase of CeO2 at 0.271 nm (Yang and Gao 2006). CeO2 crystals sizes are around 10 nm in rGO–CeO2 synthesized at Ce3 þ /OH molar ratios of 1:4 (Fig. 1h) and most of them have irregular hexagonal or quadrilateral shapes with tendency of agglomeration. When molar ratios are further decreased to 1:5 (Fig. 1i) and 1:10 (Fig. 1j), heavily agglomerated CeO2 crystals are obtained with sizes of 16–20 nm and 10–12 nm, respectively. Morphologies are not as regular as those prepared with higher molar ratios. TEM images display that irregular hexagon or quadrilateral nanocrystals fuse with some neighboring nuclei through sharing the common crystallographic orientation, resulting in the assembled single-crystalline particles. SEM image in Fig. S1 (supporting information) reveals that rGO is covered by CeO2 nanocrystals. Layered rGO sheets exhibit high specific surface area for CeO2 germinating, indicating successful nucleation and growth of CeO2 nanocrystals on rGO surface. However, with Ce3 þ /OH molar ratio of 2:1, rGO–CeO2 nanocomposite is agglomerated (Fig. S1a). At Ce3 þ /OH molar ratio of 1:1 and 1:2 (Fig. S1b and c), the prepared rGO–CeO2 nanocomposites are uniform. Compared to Fig. S1b–d (Ce3 þ /OH ¼1:3) displays more uniformly distributed CeO2 nanocrystals with larger sizes. At Ce3 þ /OH molar ratio of 1:4, the size of CeO2 nanocrystal starts to decreases (Fig. S1e), while when decreasing Ce3 þ /OH molar ratio to 1:5 (Fig. S1f) and 1:10 (Fig. S1g), agglomerations of particles occur. XRD analysis of rGO–CeO2 nanocomposites
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prepared with all Ce3 þ /OH molar ratios shows a typical pattern (Fig. S2A), of which peaks can be assigned to diffraction of (111), (200), (220), (311), (222), (400), (331) and (420) respectively according to JCPDS Card no. 34-0394, thus confirming a cubic structure of ceria. The EDS analysis of materials prepared with Ce3 þ /OH molar ratio of 1:3 (Fig. S2B) reveals that the main chemical elements in nanocomposites are C, O and Ce, clearly evidencing a successful synthesis of rGO–CeO2 composite. In terms of the morphology evolvement of rGO–CeO2 composite, a formation mechanism could be proposed as in Scheme 2. At the beginning, Ce3 þ and OH are reacted to form Ce(OH)3 precipitates on GO sheets followed by conversion to 2–3 nm CeO2 nuclei via oxidation and rapid dehydration. The reported growth of CeO2 nanocrystal during a hydrothermal process although not on graphene but could be applicable has been reported (Lin et al. 2012) to follow two processes: a dominant process to attach 2– 3 nm nuclei (dispersion of nanoparticles) on a lattice matched surface and an assistant process for Ostwald ripening (dissolution precipitation). With different Ce3 þ /OH molar ratios, the reaction rate and amount of CeO2 nuclei formed should be different to result in different shapes, sizes and uniformity of CeO2 nanocrystals. An effective collision and rate of reaction species to form uniformly dispersed nuclei play a critical role in synthesis of uniformly distributed nuclei and followed growth of CeO2 nanocrystals for certain sizes and uniformity, which can be apparently affected by pH of solution since pH can cause different density of OH on CeO2 nuclei surface and solubility of Ce4 þ from the CeO2 nuclei and thus for various collision rates, GO actually provides an excellent substrate for nucleation and crystal growth while PVP prevents particles aggregation in hydrothermal reaction as well as promotes the orientated growth through an effective collision. 3.2. Electrocatalytic behaviors of rGO–CeO2 toward NO oxidation The electrocatalytic behavior of rGO–CeO2/GCE toward NO oxidation was investigated by cyclic voltammetry (CV) in 0.01 M pH 7.4 PBS with and without 90 mM NO. The results in Fig. 2A show that there is no obvious oxidation peak in blank PBS (curve a), while a large peak with peak potential at 790 mV vs. saturated calomel electrode (SCE) is observed (curve b) for rGO– CeO2 electrode after addition of NO, obviously due to NO
Scheme 2. Illustration of different morphology evolvement mechanism of rGO–CeO2 composites.
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Fig. 2. (A) CV response of rGO–CeO2/GCE in 0.01 M pH 7.4 PBS with (curve b) and without (curve a) 90 mM NO. (B) CV response of different modified sensors to 90 mM NO in 0.01 M pH 7.4 PBS, (a) rGO/GCE, (b) CeO2/GCE, and (c) rGO–CeO2/GCE. Crosses sign start potential and arrows indicate the scan direction.
oxidation. CVs were also measured at different modified electrodes in the same NO solution (Fig. 2B), showing that no well-defined NO oxidation peaks were observed at rGO/GCE (curve a), evidencing that plain rGO have no electrocatalytic activity toward NO oxidation. However, rGO has a large double layer capacitance and high oxidation current possibly from rGO oxidation, which significantly interfere the signal from NO reduction. In contrast, both CeO2/GCE and rGO–CeO2/GCE exhibit well-defined, sharp oxidation peaks at 895 mV (curve b) and 790 mV (curve c), respectively, revealing only CeO2 possesses good electrocatalytic activity toward NO oxidation. Further, the much higher peak current and more negative peak potential of rGO–CeO2 than CeO2 for NO oxidation (Fig. 2A and B) show that the former has much larger reaction surface area and better electrocatalytic activity than latter, clearly indicating that the composing of rGO and CeO2 is not a simple sum of both merits but generates great synergistic effect to significantly boost both reaction surface and catalytic activity. In addition, apparently the growth CeO2 on rGO also greatly suppresses the interference reactions of rGO for higher signal to noise (S/N) ratio. Thus, it can be concluded that the superior electrocatalytic behavior of rGO–CeO2 can be attributed to a strong synergistic effect of both CeO2 and rGO. It is very likely that the oxygen vacancies in crystal structures of CeO2 (Cui et al. 2009) could enhance the NO adsorption as that reported (Niwa. et al.
1982). A scheme of an electrochemical oxidation of NO was studied (Kosminsky et al. 2001). The mechanism of NO oxidation on rGO–CeO2 electrode could be proposed that NO is firstly adsorbed on CeO2, followed by transferring an electron to CeO2 as
NO → NO+ + e−
(1)
and NO þ is a relatively strong Lewis acid (Kosminsky et al. 2001) that can react with water according to the following equation:
NO+ + H2 O→ NO2− + 2H+
(2)
3.3. Effect of rGO–CeO2 composite nanostructures on electrocatalytic behavior toward NO oxidation The nanostructure of rGO–CeO2 composite was delicately tailored as discussed above and its effect on electrocatalytic behavior toward NO oxidation was further investigated. Fig. 3A shows that the NO sensors made from CeO2 round, quadrilateral, irregular and hexagonal CeO2 nanocrystals produce NO oxidation peak potentials of 894 mV (curve a), 863 mV (curve b), 823 mV (curve c) and 790 mV (curve d), respectively, indicating that the regular hexagonal CeO2 nanocrystal has more negative oxidation peak potential and higher electrocatalytic peak current than that of
Fig. 3. (A) CV response of rGO–CeO2/GCE in 0.01 M pH 7.4 PBS to 180 mM NO with rGO–CeO2 synthesized at different Ce3 þ /OH molar ratios (a) 2:1; (b) 1:1; (c) 1:2; (d) 1:3; (e) 1:4; (f) 1:5; and (g) 1:10. (B) Performance comparison of rGO–CeO2/GCE in 0.01 M pH 7.4 PBS to 180 mM NO between different Ce3 þ /OH molar ratios and CV response peak currents or peak potentials. Cross signs start potential and arrows indicate the scan direction.
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quadrilateral and round ones. This is very possibly attributed to the different crystal nanostructures possessing different reaction surface areas and different number of active reaction centers at a same steric packing size. For example, at 10 nm packing size, the hexagonal CeO2 nanocrystal provides much larger surface area (150 3 nm2) than quadrilateral (100 nm2) and round (78.5 nm2) CeO2 nanocrystals for much larger reaction area to result in higher peak current toward NO oxidation. The more negative NO oxidation peak potential indicates that the hexagonal nanostructure also induces higher intrinsic electrocatalytic activity than the
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quadrilateral one. This is very likely that the former structure can expose more oxygen vacancies to NO for stronger NO adsorption to enhance electrocatalytic process. However, with Ce3 þ /OH molar ratio decreases to 1:4 (curve e), the composite has both irregular hexagonal and quadrilateral structures and the NO oxidation peak current decreases and peak potentials shift to more positive (818 mV), indicating decayed electrocatalytic performance. With decrease of Ce3 þ /OH molar ratios to 1:5 (curve f) and 1:10 (curve g), the formed nanocrystals are aggregated and oxidation peaks of NO shift further to more
Fig. 4. (A) and (B) Selectivity performance of the rGO–CeO2/GCE. (C) Chronoamperometric response of the rGO–CeO2/GCE upon continuous injection of different concentration NO at an applied potential of 0.8 V in 0.01 M pH 7.4 PBS. (D) Linear calibration curve of rGO–CeO2/GCE to NO. (E) Real time monitoring of NO released from A549 cells stimulated by different amount of ACh in cell culture medium, (a) 0.5 mM ACh; (b) 0.2 mM ACh; (c) 1 mM ACh and 1 mM Hb. (F) Real time monitoring of NO released from A549 cells in cell culture medium with controlled cell density. The insets are microscope photographs of A549 cells with different cell densities. Error bars represent the standard deviations of five independent measurements.
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positive (865 mV and 876 mV, respectively). It is clear that the electrocatalytic performance decayed when Ce3 þ /OH molar ratio is less than 1:3. This is probably that highly concentrated OH results in high collision rate with Ce3 þ for a much faster nucleation rate in hydrothermal reaction, leading to irregular crystal structure and even serious aggregation, which apparently decreases the exposure of reaction sites to NO for decayed electrocatalytic performance. Fig. 3B clearly shows volcano relations of peak current and peak potential of NO oxidation versus Ce3 þ /OH molar ratio. Surprisingly, the highest electrocatalytic activity (the most negative potential) and the highest catalytic current (peak current) are well in agreement to point to a Ce3 þ /OH molar ratio of 1:3, which is used for the following investigations. 3.4. Real-time detection of NO molecules released from living cells The stability of the rGO–CeO2/GCE sensor toward NO molecule was investigated by intermittently detecting chronoamperometrically in a 1200 nM NO solution. The rGO–CeO2/GCE loses about 10% of its original response after its storage in PBS for 20 days. Further, after 20 continuous CV measurements, current responses of the sensor were nearly unchanged, indicating a good repeatability. The selectivity and anti-interference ability of the sensor in real-time detection of NO released from cells were studied by adding various interfering species. Results show that 60 mM Ca2 þ , K þ , Na þ , CO32–, NO3–, Cl– and 1 mM uric acid (UA) do not cause any observable interference in detection of 0.6 mM NO (Fig. 4A). Besides, selectivity of the biosensor against ascorbic acid (AA) and dopamine (DA), commonly existing biological species was also investigated. In evaluation, biological concentrations of DA ((0.01– 1 mM) (Wightman et al. 1988) and AA ( 2.3 mM) (Kutnink et al. 1987) have been reported. Results show that 2.3 mM AA and 1 mM DA don't cause obvious interference for released 4.3 mM NO (equal to cell density of 1 105 mL 1) as displayed in Fig. 4B. The good selectivity of the sensor toward NO molecule could be contributed from the catalytic activity of rGO–CeO2 specifically toward NO oxidation. The chronoamperometric current-time response of rGO–CeO2/ GCE upon successive addition NO to a continuous stirred PBS (0.01 MpH 7.4) at 0.8 V vs. SCE was used to measure a calibration curve (Fig. 4C). The sensor exhibits a rapid stepped increase response with the injection of NO in less than 4 s to achieve 95% of steady-state current. The short response time could warrent a realtime detection of NO released from living cells, which has half-life around 5 seconds. Fig. 4D pictures the calibration curve of rGO– CeO2/GCE for NO detection with a linear equation as I (nA) ¼ 198.1 þ0.119 CNO (nM), which gives a correlation coefficient of 0.997 (n ¼31) for a wide linear range of 18.0 nM to 5.6 mM, a sensitivity of 1676.06 mA cm 2 M 1 and a low detection limit of 9.6 nM in terms of 3 S/K. Besides, a comparison between reported and this works is listed in Table 1 S in the supporting information, showing that this work delivers wider linear range and lower detection limit for NO detection. The rGO–CeO2/GCE sensor was used to real-time in situ detect NO molecules released from living A549 cells. ACh is chose as a function drug to stimulate living cells to release NO molecular. It has been reported (Asakawa et al. 2008) that ACh signal is transmitted to intercellular signaling cascades via some receptors, which can mediate rapid increment of intercellular Ca2 þ concentration. Then, Ca2 þ -calmodulin complex activates endothelial NO synthase to promote generation of NO. Detailed investigation of dynamic responses of A549 cells with a cell density of 5 104 mL 1 on rGO–CeO2 sensor is presented in Fig. 4E. The stimulating agents is continuously injected under mild stirring as indicated by arrow. As soon as 0.5 mM (curve a) or
0.2 mM (curve b) ACh is added into cell culture medium, a significant current response appears corresponding to NO oxidation. It is worthy of a note that the sensing is an in situ detection since the GCE sensor directly detects NO released from a number of cells contacted with the electrode surface. Such in situ detections with large electrodes have been widely reported such as Guo et al. 2011 and Ma et al. 2014. In contrast, mixture of 1 mM ACh and 1 mM Hb (curve c) don't cause obvious step increase indicating that released NO molecules is consumed by Hb. Furthermore, the calculated concentrations of NO released from A549 cells are around 4478.18 nM or 1591.18 nM for addition of 0.5 mM or 0.2 mM ACh, respectively, according to drug stimulated current response and calibration equation for rGO–CeO2/GCE. Current responses caused by adding 0.5 mM ACh drug is about 2.8 times than that of 0.2 mM ACh suggesting the response under ACh stimulation is a concentration-dependent behavior as shown in Fig. 4E. Moreover, responses of rGO–CeO2 sensor to A549 with different cell densities are investigated as shown in Fig. 4F. The rGO– CeO2/GCE don't exhibit obvious response to blank culture dish without A549 cell (curve a). After cell densities are controlled as 5.0 104 mL 1 (curve b) and 1.0 105 mL 1 (curve c), obvious responses generate on rGO–CeO2/GCE with stimulation of 0.2 mM ACh drug. From the drug stimulated current response and calibration equation the calculated concentrations of NO released from A549 cells are around 4304.62 nM or 1769.33 nM for 1.0 105 mL 1 and 5.0 104 mL 1 cell densities, respectively. The current responses caused by adding 0.2 mM Ach drug to A549 with 1.0 105 mL 1cell density are about 2.4 times than that of 5.0 104 mL 1 cell density.
4. Conclusions rGO–CeO2 composites nanostructures were delicately tailored by regulating ratio of Ce3 þ /OH during preparation process and the formation mechanism is proposed. Results show clearly that a Ce3 þ /OH molar ratio of 1:3 produces a regular hexagonal nanocrystal CeO2 for the best electrocatalytic performance toward NO oxidation, which is mainly contributed by a remarkable synergistic effect between rGO and CeO2. Real-time in situ detection of NO molecules released from living cells was investigated by controlling cell densities and adding stimulation drugs, revealing that NO released by living cells under Ach stimulation is a concentration-dependent behavior. This work demonstrates how we tailor a composite nanostructure to produce strong synergical effect on electrocatalytic performance while offering a facile and reliable platform to in situ real-time detect bio-signal molecules released by living cells.
Acknowledgements We would like to gratefully acknowledge financial support from the 973 program of China (No. 2013CB127804), National Natural Science Foundation of China, China (Nos. 21205098, and 21273173), Fundamental Research Funds for the Central Universities (XDJK2012C049), Chongqing Key Laboratory for Advanced Materials and Technologies of Clean Energies, start-up Grant under SWU111071 from the Southwest University, Chongqing Engineering Research Center for Rapid Diagnosis of Dread Disease and Chongqing Development and Reform Commission. Appendix A. Supplementary information Supplementary data associated with this article can be found in
F.X. Hu et al. / Biosensors and Bioelectronics 70 (2015) 310–317
the online version at http://dx.doi.org/10.1016/j.bios.2015.03.056.
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