Biosensors and Bioelectronics 61 (2014) 374–378
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Fluorescent hydrogen peroxide sensor based on cupric oxide nanoparticles and its application for glucose and L-lactate detection Ai-Ling Hu a,b, Yin-Huan Liu c, Hao-Hua Deng a,b, Guo-Lin Hong c, Ai-Lin Liu a,b, Xin-Hua Lin a,b, Xing-Hua Xia d, Wei Chen a,b,n a
Department of Pharmaceutical Analysis, Fujian Medical University, Fuzhou 350004, China Nano Medical Technology Research Institute, Fujian Medical University, Fuzhou 350004, China c Department of Laboratory Medicine, Second Hospital of Fuzhou, Fuzhou 350007, China d State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 18 February 2014 Received in revised form 15 April 2014 Accepted 3 May 2014 Available online 27 May 2014
A novel fluorescent hydrogen peroxide sensor was developed based on the peroxidase-like activity of cupric oxide nanoparticles. Cupric oxide nanoparticles effectively catalyzed the decomposition of hydrogen peroxide into hydroxyl radicals. Then terephthalic acid was oxidized by hydroxyl radical to form a highly fluorescent product. The linear range of hydrogen peroxide estimated to be 5.0 10 6– 2.0 10 4 M with a detection limit of 3.4 10 7 M. Moreover, this detection system enabled the sensing of analytes which can enzymatically generate hydrogen peroxide. By coupling the oxidation of glucose or L-lactate catalyzed by their corresponding oxidase enzymes with terephthalic acid oxidation catalyzed by cupric oxide nanoparticles, sensitive assays of glucose and L-lactate with detection limits of 1.0 10 6 and 4.5 10 8 M were realized. The successful applications of this approach in human serum samples have also been demonstrated. & 2014 Elsevier B.V. All rights reserved.
Keywords: Hydrogen peroxide Glucose L-lactate Cupric oxide nanoparticles Terephthalic acid Fluorimetry
1. Introduction Glucose is a source of energy of the living cells and metabolic intermediate in biological systems. It can also provide significant information of many diseases such as hypoglycemia or diabetes. The disease is associated with long-term damage, dysfunction, and failure of different organs, especially the eyes, kidneys, nerves, heart, and blood vessels. As another important metabolite, L-lactate also plays a key role in clinical analysis. The increase of L-lactate has been widely used as an excellent indicator of hypoxia, poor perfusion of tissue, acute circulatory shock, liver failure, congestive heart failure and diabetic ketoacidosis (Ander et al., 1998; Gajovic et al., 2000). Failure of arterial serum lactate to achieve normal levels has been associated with an increased mortality among medical and trauma patients (McNelis et al., 2001). Therefore, the detection of L-lactate serves as an aid for diagnosing heart disease, exercise physiology, neonatology and neurology studies (Shen et al., 2012; Wyss et al., 2011).
n Corresponding author at: Department of Pharmaceutical Analysis, Fujian Medical University, Fuzhou 350004, China. Tel./fax: þ86 591 22862016. E-mail address: [email protected] (W. Chen).
http://dx.doi.org/10.1016/j.bios.2014.05.048 0956-5663/& 2014 Elsevier B.V. All rights reserved.
Up to now, various methods such as chemiluminescence (BallestaClaver et al., 2008; Lan et al., 2008), fluorometry (Chang et al., 2009; Groegel et al., 2011), electrochemistry (Ghamouss et al., 2006; Hu et al., 2005; Manesh et al., 2010), and spectrophotometry (Sanz et al., 2005; Tumang et al., 2001) have been reported for glucose and L-lactate detection. Among them, horseradish peroxidase (HRP) has been widely used to fabricate sensors for detection of the products from reactions catalyzed by corresponding oxidase. Owing to many disadvantages of natural enzymes like difficult and high-cost purification processes as well as inherent instability, more and more attention has been paid to constructing enzyme mimics with similar functions to natural enzymes in recent years (Wei and Wang 2013; Wiester et al., 2011). A variety of inorganic nanomaterials including ferromagnetic nanoparticles (Gao et al., 2007), carboxyl-modified graphene oxide (Song et al., 2010), gold nanoparticles (Jv et al., 2010; Wang et al., 2012), V2O5 nanowires (Andre et al., 2011), AgM bimetallic alloy nanostructures (He et al., 2010), CoFe2O4 magnetic nanoparticles (Shi et al., 2011b), Au@Pt nanostructures (He et al., 2011; Liu et al., 2012), BiFeO3 (Luo et al., 2010), Co3O4 nanoparticles (Mu et al., 2012), helical carbon nanotubes (Cui et al., 2011), and carbon nanodots (Shi et al., 2011a), have been evaluated to possess intrinsic enzyme mimetic activity similar to that found in natural peroxidase. In comparison with HRP, peroxidase nano-mimics demonstrate great catalytic property and high-stability. In our recent studies, we found that cupric oxide
A.-L. Hu et al. / Biosensors and Bioelectronics 61 (2014) 374–378
nanoparticles (CuO NPs) are not only highly effective catalysts to peroxidase substrates but also considerably more stable and possess an almost unchanged catalytic activity over a wide range of pH and temperatures (Chen et al., 2011; Hong et al., 2013). Since fluorescence spectroscopy is one of the most useful analytical tools in bioanalysis, many fluorescent probes, such as europium coordination complexes (Wolfbeis et al., 2003), dichlorofluorescin (Sanchez Ferrer et al., 1990), Amplex Red (Lien et al., 2012), quantum dots (Yuan et al., 2009), gold nanoclusters (Jin et al., 2011), and cationic conjugated polymers (He et al., 2006), have been developed for the detection of H2O2. As has already been discovered, terephthalic acid (TA) is a specific fluorescence dosimeter for dOH in a variety of physical and chemical systems (Barreto et al., 1995; Charbouillot et al., 2011; Dutta et al., 2013; Ishibashi et al., 2000). In comparison with other peroxidase substrates like TMB and ABTS, TA has some unique properties such as low cost, pure oxidation products easily obtained, more stability for storage and less vulnerability to degradation. In the present work, therefore, TA is used as a fluorescent peroxidase substrate to establish a TA-CuO NPs system for the determination of H2O2. By coupling the oxidation of glucose or L-lactate catalyzed by their corresponding oxidase enzymes with the TA oxidation catalyzed by CuO NPs, fluorometric methods were further developed for quantitative analysis of glucose and L-lactate in human serum.
2. Experimental 2.1. Chemicals and materials All chemicals and reagents were of analytical grade and used without further purification. Cupric acetate, sodium hydroxide, terephthalic acid, glucose, maltose, D-fructose and 30% (v/v) H2O2 were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Glucose oxidase, L-lactate and α-lactose were purchased from Aladdin Reagent Company (Shanghai, China). Lactate oxidase was purchased from Sigma Co. Ltd. Terephthalic acid was neutralized with NaOH and used as 100 mM stock solution in water. The stock solution of glucose was allowed to incubate at room temperature overnight before use. The water used throughout all experiments was purified by a Milli-Q system (Millipore, USA). Clinical serum samples were provided by the Second Hospital of Fuzhou. The cupric oxide nanoparticles were prepared via a previously reported quick-precipitation method (Chen et al., 2012a). First, 150 mL of 0.02 M copper acetate aqueous solution was mixed with 0.5 mL glacial acetic acid in a round-bottomed flask equipped with a refluxing device. The solution was heated to boiling with vigorous stirring. Then 10 mL of 0.04 g/mL NaOH aqueous solution was rapidly added into the above boiling solution, where a large amount of black precipitate was simultaneously produced. The precipitate was centrifuged, washed three times with absolute ethanol, and dried in air at room temperature. The as-prepared CuO nanoparticles, which are without any surface modification, can well disperse in distilled water and form a transparent brown solution. The appearance of the solution remains unchanged even after 6 months, which performs a perfect stability. 2.2. H2O2 sensing In a typical experiment, (a) 0.8 mL of 18.75 mM TA, 50 μL of the CuO nanoparticles stock solution (0.04 mg/mL), and 500 μL H2O2 of different concentrations were added into 3.65 mL of 200 mM phosphate buffer (pH 7.0); (b) the mixed solution was incubated in a 45 1C water bath for 20 min; (c) the resulting reaction solution
375
was measured by using a Cary Eclipse fluorescence spectrometer under the excitation wavelength of 315 nm. 2.3. Glucose detection 62.5 mL of 1 mg/mL GOx and 500 mL glucose of different concentrations were added into 62.5 mL of 200 mM phosphate buffer solution (pH 7.0), and incubated at 37 1C for 20 min. 3.525 mL phosphate buffer solution, 0.8 mL of 18.75 mM TA, and 50 mL of the CuO NPs (0.04 mg/mL) were added to the above glucose reaction solution for another 2 h at 45 1C. The resulting reaction solution was measured by using a Cary Eclipse fluorescence spectrometer under the excitation wavelength of 315 nm. Glucose detection by oxidase endpoint method was carried out by adding 100 mL of 1 mg/mL GOx, 100 mL HRP, 200 mL TMB, and 500 mL glucose of different concentrations into 3.1 mL of 200 mM phosphate buffer solution (pH 7.0). The mixture was then incubated at 37 1C for 10 min. The resulting reaction solution was measured by using a Shimadzu UV-2450 spectrophotometer. 2.4.
L-lactate
detection
50 mL of 0.01 mg/mL LOx and 400 mL L-lactate of different concentrations were added into 50 mL of 200 mM phosphate buffer solution (pH 7.0), and incubated at 37 1C for 10 min. 3.65 mL phosphate buffer solution, 0.8 mL of 18.75 mM TA, and 50 mL of the CuO NPs (0.04 mg/mL) were added to the above glucose reaction solution for another 2 h at 45 1C. The resulting reaction solution was measured by using a Cary Eclipse fluorescence spectrometer under the excitation wavelength of 315 nm. L-lactate detection by oxidase endpoint method was carried out by adding 50 mL of 0.01 mg/mL LOx, 50 mL HRP, 100 mL TMB, and 400 mL lactate of different concentrations into 3.4 mL of 200 mM phosphate buffer solution (pH 7.0). The mixture was then incubated at 37 1C for 10 min. The resulting reaction solution was measured by using a Shimadzu UV-2450 spectrophotometer. 2.5. Serum samples detection For glucose and L-lactate determination in serum, the samples were first pretreated by ultrafiltration to eliminate the possible interference of proteins. The pretreated samples were diluted by a phosphate buffer solution and determined according to the processes mentioned above for glucose and L-lactate. The results of proposed method were compared with that of the glucose or lactate oxidase endpoint method, which is a widely used clinical method in hospitals for glucose and lactate assay. Standard addition experiments were further conducted by adding three different concentrations of glucose or L-lactate in the real serum samples.
3. Results and discussion 3.1. Sensing protocol Fig. 1A depicts the principle of the cupric oxide nanoparticlesbased fluorescent sensor for hydrogen peroxide. As a peroxidase mimetic, cupric oxide nanoparticles can break up the O–O bond of H2O2 into two hydroxyl radicals (Chen et al., 2012b). Then the resulting hydroxyl radicals react with TA, a non-fluorescent molecule, to form highly fluorescent hydroxyterephthalate (TAOH), which shows emission maximum at 422 nm when excited at 315 nm. The fluorescence intensity of TAOH is found to be proportional to the concentration of H2O2, which lead to develop a fluorometric sensor for hydrogen peroxide determination.
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Fig. 2. Emission spectra of the TA-CuO NPs system in the presence of varying concentration of H2O2 (10 6 M): (a) 0; (b) 5; (c) 10; (d) 20; (e) 40; (f) 60; (g) 80; (h) 100; (i) 150; (j) 200.
fluorescent. Thus, the excess of TA will not influence the measurement of the fluorescence intensity of the reaction solution. In order to expand the linear range for the determination of H2O2, we selected a moderately higher TA concentration of 3 mM for the further experiments. As shown in Fig. S1D, the generation of TAOH is significantly promoted initially with an increased CuO NPs load, and then kept almost constant beyond 0.4 mg/L of CuO NPs. Thus, the load of CuO NPs is selected at 0.4 mg/L for the determination of H2O2. Fig. 1. (A) Schematic illustration of the cupric oxide nanoparticles-based fluorescent sensor for hydrogen peroxide. (B) The excitation and emission fluorescence spectra of (a) TA, (b) TA þ CuO NPs, (c) TA þ H2O2, and (d) TA þH2O2 þCuO NPs. The inset photograph displays the luminescence upon excitation (302 nm) under a hand-held UV lamp.
To confirm the sensing protocol, fluorescent spectra under different conditions were studied. From Fig. 1B, it was observed that no fluorescence produced in TA or the TA–CuO NPs system. Although slow oxidation of TA can be induced by hydrogen peroxide in the absence of catalyst, very weak fluorescence was observed. In presence of cupric oxide nanoparticles, the fluorescence intensity could be greatly enhanced up to about 40 folds. 3.2. Optimization of reaction conditions In order to optimize the possible analytical method for the hydrogen peroxide determination, the effects of experimental conditions including solution pH, reaction temperature, TA concentration, and CuO NPs concentration were investigated. The solution pH is an important factor that affects the catalytic activity of CuO NPs in the sensing system. It is clear from Fig. S1A that the catalytic oxidation of TA by H2O2 with CuO NPs as the catalyst is much faster in neutral solutions than in acidic and basic solutions. The maximum fluorescence intensity was obtained at pH 7.0. The effect of reaction temperature on the fluorescence intensity was studied in the range of 20–55 1C (Fig. S1B). The fluorescence intensity of the sensing system increased with an increasing reaction temperature in the range of 20–45 1C and decreased when the temperature was higher than 45 1C. Fig. S1C illustrates the influence of TA concentration on the fluorescence intensity of the reaction solution. It can be seen that the fluorescence intensity is enhanced with increasing of TA concentration up to 2.8 mM, and then keeps almost constant beyond 2.8 mM of TA. The reactant TA is non-fluorescent, but the product TAOH is highly
3.3. Hydrogen peroxide sensing The analytical determination of hydrogen peroxide is of considerable importance for medical diagnosis since hydrogen peroxide is formed as an intermediate product in the case of a large number of important detection processes. The possibility of using the proposed method for the determination of hydrogen peroxide was investigated. Fig. 2 depicted the typical fluorescence response in the presence of different concentrations H2O2. Under the optimal reaction conditions as described above, the fluorescence intensity versus hydrogen peroxide concentration was linear over the range from 5.0 10 6 M to 2.0 10 4 M range with the correlation coefficient of 0.996. The limit of detection for hydrogen peroxide was 3.4 10 7 M (S/N ¼3). The RSD was 1.8% for 2.0 10 4 M hydrogen peroxide (n ¼6). 3.4. Analytical application in determination of glucose Reliable and fast determination of glucose is of considerable importance in biotechnology, clinical diagnostics and food industry. As H2O2 is the product of GOx-catalyzed reaction of glucose oxidation, fluorescent detection of glucose could be realized by coupling the TA–CuO colloids system with the glucose catalytic reaction by GOx. As shown in Fig. 3, the measured fluorescence intensity increased markedly upon increasing the concentration of glucose. The controlled experimental results show that O2, glucose oxidase, and glucose were all essential to the producing of fluorescence, since the exclusion of either component would yield no H2O2. Good linear relationship was obtained in the range of 3.0 10 6–1.0 10 4 M. The correlation coefficient was 0.999, and the relative standard deviation for six repeated measurements of 1.0 10 4 M glucose was 1.5%. The detection limit value obtained for the experiment was 1.0 10 6 M (S/N ¼3), which was lower than that of other methods (Table S1).
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Fig. 3. (A) Emission spectra represent the effect of the glucose–GOx system with different glucose concentrations on the fluorescence intensity. Glucose concentration from (a) to (i) (10 6 M): (a) 0; (b) 5; (c) 10; (d) 20; (e) 40; (f) 60; (g) 80; (h) 100; (i) 120. (B) Calibration plot for glucose determination.
Fig. 4. (A) Emission spectra represent the effect of the lactate–LOx system with different lactate concentrations on the fluorescence intensity. Lactate concentration from (a) to (i) (10 6 M): (a) 0; (b) 1.6; (c) 4; (d) 8; (e) 16; (f) 32; (g) 48; (h) 64; (i) 80. (B) Calibration plot for L-lactate determination.
Table 1 Analytical results of glucose in human serum.
samples ranging from 90.7% to 105.8%. The results demonstrate that this new method is suitable and satisfactory for glucose analysis of real samples.
Sample Proposed method (mM) 1 2 3
4.21 70.07 4.17 70.13 37.68 70.27
Glucose oxidase endpoint method (mM)
Relative deviation (%)
3.977 0.20 4.39 7 0.24 41.247 0.56
6.05 5.01 8.63
For testing if the detection of glucose is specific, control experiments were taken using lactose, maltose, and fructose. Because of the high substrate specificity of GOx, no significant signals were obtained for the control samples with concentrations of 1 mM (Fig. S2). Therefore, a sensitive and selective glucose biosensor can be fabricated in a facile approach. To evaluate the feasibility of the sensing system for analysis of glucose in biological samples, the developed method was applied to the determination of glucose in human serum samples. From Table 1, it can be seen that the results obtained by the proposed method were in good agreement with those measured by the glucose oxidase endpoint method. The practical applicability of the proposed method was further verified through standard addition experiments, with the recoveries of glucose in three serum
3.5. Analytical application in determination of L-lactate It is well known that lactate oxidase (LOx) catalyzes the oxidation of L-lactate to pyruvic acid and H2O2. Therefore, fluorescent detection of L-lactate could be realized by coupling the TA– CuO NPs system with the L-lactate catalytic reaction by LOx. Typical L-lactate response curves are shown in Fig. 4. The linear range of lactate is from 8.0 10 7 to 8.0 10 5 M with a correlation coefficient of 0.998. The limit of detection was calculated to be 4.5 10 8 M (S/N¼3), which was lower than that of other methods (Table S2) (Ballesta-Claver et al., 2008; Claver et al., 2009; Goran et al., 2011; Groegel et al., 2011; Romero et al., 2010; Zheng et al., 2010). The relative standard deviation for six repeated measurements of 8.0 10 5 M L-lactate was 1.9%. To investigate the selectivity of the proposed L-lactate sensor, fluorescent responses in the presence of ethanol, urea, glucose, lactose, maltose, uric acid, citric acid, and pyruvic acid were studied. As can be seen from Fig. S3, the presence of foreign substances (10 mM) individually has no significant effect on the L-lactate sensor.
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Table 2 Analytical results of L-lactate in human serum.
Sample
Proposed method (mM)
Lactate oxidase endpoint method (mM)
Relative deviation (%)
1 2 3
3.09 70.03 3.47 70.14 2.41 70.02
3.187 0.04 3.23 7 0.09 2.40 7 0.05
2.83 7.43 0.42
In order to explore the applicability and feasibility of the method, L-lactate content in human serum samples was determined. From Table 2, it can be seen that the results obtained by the proposed method agree well with those measured by the lactate oxidase endpoint method. Furthermore, the recoveries for lactate in human serum samples were observed to be ranged from 94.0 to 115.6%. Thus the proposed method shows satisfactory results for the analysis of lactate in human serum samples.
4. Conclusion In summary, a simple, highly selective and inexpensive strategy for H2O2 sensing has been successfully developed by using terephthalic acid as substrate and cupric oxide nanoparticles as catalyst. In comparison with traditional peroxide substrate TMB, TA exhibits several advantages such as low cost, pure oxidation products easily obtained, more stability for storage. As a mimic peroxidase, cupric oxide nanoparticles are considerably more stable and possess an almost unchanged catalytic activity over a wide range of pH and temperatures. Coupling the oxidation of glucose or L-lactate catalyzed by their corresponding oxidase enzymes, sensitive assays of glucose and L-lactate were realized with detection limits of 1.0 10 6 and 4.5 10 8 M. In addition, we demonstrated the successful application of the present approach in real serum samples, which suggests its great potential for diagnostic purpose.
Acknowledgments The authors gratefully acknowledge the financial support of the National Natural Science Foundation of China (21175023), the Program for New Century Excellent Talents in University (NCET12–0618), the Natural Science Foundation of Fujian Province (2011J01034, 2012J06019), and the Medical Elite Cultivation Program of Fujian, P.R.C (2013-ZQN-ZD-25).
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Fluorescent hydrogen peroxide sensor based on cupric oxide nanoparticles and its application for glucose and L-lactate detection Ai-Ling Hu a,b, Yin-Huan Liu c, Hao-Hua Deng a,b, Guo-Lin Hong c, Ai-Lin Liu a,b, Xin-Hua Lin a,b, Xing-Hua Xia d, Wei Chen a,b,n a
Department of Pharmaceutical Analysis, Fujian Medical University, Fuzhou 350004, China Nano Medical Technology Research Institute, Fujian Medical University, Fuzhou 350004, China c Department of Laboratory Medicine, Second Hospital of Fuzhou, Fuzhou 350007, China d State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 18 February 2014 Received in revised form 15 April 2014 Accepted 3 May 2014 Available online 27 May 2014
A novel fluorescent hydrogen peroxide sensor was developed based on the peroxidase-like activity of cupric oxide nanoparticles. Cupric oxide nanoparticles effectively catalyzed the decomposition of hydrogen peroxide into hydroxyl radicals. Then terephthalic acid was oxidized by hydroxyl radical to form a highly fluorescent product. The linear range of hydrogen peroxide estimated to be 5.0 10 6– 2.0 10 4 M with a detection limit of 3.4 10 7 M. Moreover, this detection system enabled the sensing of analytes which can enzymatically generate hydrogen peroxide. By coupling the oxidation of glucose or L-lactate catalyzed by their corresponding oxidase enzymes with terephthalic acid oxidation catalyzed by cupric oxide nanoparticles, sensitive assays of glucose and L-lactate with detection limits of 1.0 10 6 and 4.5 10 8 M were realized. The successful applications of this approach in human serum samples have also been demonstrated. & 2014 Elsevier B.V. All rights reserved.
Keywords: Hydrogen peroxide Glucose L-lactate Cupric oxide nanoparticles Terephthalic acid Fluorimetry
1. Introduction Glucose is a source of energy of the living cells and metabolic intermediate in biological systems. It can also provide significant information of many diseases such as hypoglycemia or diabetes. The disease is associated with long-term damage, dysfunction, and failure of different organs, especially the eyes, kidneys, nerves, heart, and blood vessels. As another important metabolite, L-lactate also plays a key role in clinical analysis. The increase of L-lactate has been widely used as an excellent indicator of hypoxia, poor perfusion of tissue, acute circulatory shock, liver failure, congestive heart failure and diabetic ketoacidosis (Ander et al., 1998; Gajovic et al., 2000). Failure of arterial serum lactate to achieve normal levels has been associated with an increased mortality among medical and trauma patients (McNelis et al., 2001). Therefore, the detection of L-lactate serves as an aid for diagnosing heart disease, exercise physiology, neonatology and neurology studies (Shen et al., 2012; Wyss et al., 2011).
n Corresponding author at: Department of Pharmaceutical Analysis, Fujian Medical University, Fuzhou 350004, China. Tel./fax: þ86 591 22862016. E-mail address: [email protected] (W. Chen).
http://dx.doi.org/10.1016/j.bios.2014.05.048 0956-5663/& 2014 Elsevier B.V. All rights reserved.
Up to now, various methods such as chemiluminescence (BallestaClaver et al., 2008; Lan et al., 2008), fluorometry (Chang et al., 2009; Groegel et al., 2011), electrochemistry (Ghamouss et al., 2006; Hu et al., 2005; Manesh et al., 2010), and spectrophotometry (Sanz et al., 2005; Tumang et al., 2001) have been reported for glucose and L-lactate detection. Among them, horseradish peroxidase (HRP) has been widely used to fabricate sensors for detection of the products from reactions catalyzed by corresponding oxidase. Owing to many disadvantages of natural enzymes like difficult and high-cost purification processes as well as inherent instability, more and more attention has been paid to constructing enzyme mimics with similar functions to natural enzymes in recent years (Wei and Wang 2013; Wiester et al., 2011). A variety of inorganic nanomaterials including ferromagnetic nanoparticles (Gao et al., 2007), carboxyl-modified graphene oxide (Song et al., 2010), gold nanoparticles (Jv et al., 2010; Wang et al., 2012), V2O5 nanowires (Andre et al., 2011), AgM bimetallic alloy nanostructures (He et al., 2010), CoFe2O4 magnetic nanoparticles (Shi et al., 2011b), Au@Pt nanostructures (He et al., 2011; Liu et al., 2012), BiFeO3 (Luo et al., 2010), Co3O4 nanoparticles (Mu et al., 2012), helical carbon nanotubes (Cui et al., 2011), and carbon nanodots (Shi et al., 2011a), have been evaluated to possess intrinsic enzyme mimetic activity similar to that found in natural peroxidase. In comparison with HRP, peroxidase nano-mimics demonstrate great catalytic property and high-stability. In our recent studies, we found that cupric oxide
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nanoparticles (CuO NPs) are not only highly effective catalysts to peroxidase substrates but also considerably more stable and possess an almost unchanged catalytic activity over a wide range of pH and temperatures (Chen et al., 2011; Hong et al., 2013). Since fluorescence spectroscopy is one of the most useful analytical tools in bioanalysis, many fluorescent probes, such as europium coordination complexes (Wolfbeis et al., 2003), dichlorofluorescin (Sanchez Ferrer et al., 1990), Amplex Red (Lien et al., 2012), quantum dots (Yuan et al., 2009), gold nanoclusters (Jin et al., 2011), and cationic conjugated polymers (He et al., 2006), have been developed for the detection of H2O2. As has already been discovered, terephthalic acid (TA) is a specific fluorescence dosimeter for dOH in a variety of physical and chemical systems (Barreto et al., 1995; Charbouillot et al., 2011; Dutta et al., 2013; Ishibashi et al., 2000). In comparison with other peroxidase substrates like TMB and ABTS, TA has some unique properties such as low cost, pure oxidation products easily obtained, more stability for storage and less vulnerability to degradation. In the present work, therefore, TA is used as a fluorescent peroxidase substrate to establish a TA-CuO NPs system for the determination of H2O2. By coupling the oxidation of glucose or L-lactate catalyzed by their corresponding oxidase enzymes with the TA oxidation catalyzed by CuO NPs, fluorometric methods were further developed for quantitative analysis of glucose and L-lactate in human serum.
2. Experimental 2.1. Chemicals and materials All chemicals and reagents were of analytical grade and used without further purification. Cupric acetate, sodium hydroxide, terephthalic acid, glucose, maltose, D-fructose and 30% (v/v) H2O2 were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Glucose oxidase, L-lactate and α-lactose were purchased from Aladdin Reagent Company (Shanghai, China). Lactate oxidase was purchased from Sigma Co. Ltd. Terephthalic acid was neutralized with NaOH and used as 100 mM stock solution in water. The stock solution of glucose was allowed to incubate at room temperature overnight before use. The water used throughout all experiments was purified by a Milli-Q system (Millipore, USA). Clinical serum samples were provided by the Second Hospital of Fuzhou. The cupric oxide nanoparticles were prepared via a previously reported quick-precipitation method (Chen et al., 2012a). First, 150 mL of 0.02 M copper acetate aqueous solution was mixed with 0.5 mL glacial acetic acid in a round-bottomed flask equipped with a refluxing device. The solution was heated to boiling with vigorous stirring. Then 10 mL of 0.04 g/mL NaOH aqueous solution was rapidly added into the above boiling solution, where a large amount of black precipitate was simultaneously produced. The precipitate was centrifuged, washed three times with absolute ethanol, and dried in air at room temperature. The as-prepared CuO nanoparticles, which are without any surface modification, can well disperse in distilled water and form a transparent brown solution. The appearance of the solution remains unchanged even after 6 months, which performs a perfect stability. 2.2. H2O2 sensing In a typical experiment, (a) 0.8 mL of 18.75 mM TA, 50 μL of the CuO nanoparticles stock solution (0.04 mg/mL), and 500 μL H2O2 of different concentrations were added into 3.65 mL of 200 mM phosphate buffer (pH 7.0); (b) the mixed solution was incubated in a 45 1C water bath for 20 min; (c) the resulting reaction solution
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was measured by using a Cary Eclipse fluorescence spectrometer under the excitation wavelength of 315 nm. 2.3. Glucose detection 62.5 mL of 1 mg/mL GOx and 500 mL glucose of different concentrations were added into 62.5 mL of 200 mM phosphate buffer solution (pH 7.0), and incubated at 37 1C for 20 min. 3.525 mL phosphate buffer solution, 0.8 mL of 18.75 mM TA, and 50 mL of the CuO NPs (0.04 mg/mL) were added to the above glucose reaction solution for another 2 h at 45 1C. The resulting reaction solution was measured by using a Cary Eclipse fluorescence spectrometer under the excitation wavelength of 315 nm. Glucose detection by oxidase endpoint method was carried out by adding 100 mL of 1 mg/mL GOx, 100 mL HRP, 200 mL TMB, and 500 mL glucose of different concentrations into 3.1 mL of 200 mM phosphate buffer solution (pH 7.0). The mixture was then incubated at 37 1C for 10 min. The resulting reaction solution was measured by using a Shimadzu UV-2450 spectrophotometer. 2.4.
L-lactate
detection
50 mL of 0.01 mg/mL LOx and 400 mL L-lactate of different concentrations were added into 50 mL of 200 mM phosphate buffer solution (pH 7.0), and incubated at 37 1C for 10 min. 3.65 mL phosphate buffer solution, 0.8 mL of 18.75 mM TA, and 50 mL of the CuO NPs (0.04 mg/mL) were added to the above glucose reaction solution for another 2 h at 45 1C. The resulting reaction solution was measured by using a Cary Eclipse fluorescence spectrometer under the excitation wavelength of 315 nm. L-lactate detection by oxidase endpoint method was carried out by adding 50 mL of 0.01 mg/mL LOx, 50 mL HRP, 100 mL TMB, and 400 mL lactate of different concentrations into 3.4 mL of 200 mM phosphate buffer solution (pH 7.0). The mixture was then incubated at 37 1C for 10 min. The resulting reaction solution was measured by using a Shimadzu UV-2450 spectrophotometer. 2.5. Serum samples detection For glucose and L-lactate determination in serum, the samples were first pretreated by ultrafiltration to eliminate the possible interference of proteins. The pretreated samples were diluted by a phosphate buffer solution and determined according to the processes mentioned above for glucose and L-lactate. The results of proposed method were compared with that of the glucose or lactate oxidase endpoint method, which is a widely used clinical method in hospitals for glucose and lactate assay. Standard addition experiments were further conducted by adding three different concentrations of glucose or L-lactate in the real serum samples.
3. Results and discussion 3.1. Sensing protocol Fig. 1A depicts the principle of the cupric oxide nanoparticlesbased fluorescent sensor for hydrogen peroxide. As a peroxidase mimetic, cupric oxide nanoparticles can break up the O–O bond of H2O2 into two hydroxyl radicals (Chen et al., 2012b). Then the resulting hydroxyl radicals react with TA, a non-fluorescent molecule, to form highly fluorescent hydroxyterephthalate (TAOH), which shows emission maximum at 422 nm when excited at 315 nm. The fluorescence intensity of TAOH is found to be proportional to the concentration of H2O2, which lead to develop a fluorometric sensor for hydrogen peroxide determination.
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Fig. 2. Emission spectra of the TA-CuO NPs system in the presence of varying concentration of H2O2 (10 6 M): (a) 0; (b) 5; (c) 10; (d) 20; (e) 40; (f) 60; (g) 80; (h) 100; (i) 150; (j) 200.
fluorescent. Thus, the excess of TA will not influence the measurement of the fluorescence intensity of the reaction solution. In order to expand the linear range for the determination of H2O2, we selected a moderately higher TA concentration of 3 mM for the further experiments. As shown in Fig. S1D, the generation of TAOH is significantly promoted initially with an increased CuO NPs load, and then kept almost constant beyond 0.4 mg/L of CuO NPs. Thus, the load of CuO NPs is selected at 0.4 mg/L for the determination of H2O2. Fig. 1. (A) Schematic illustration of the cupric oxide nanoparticles-based fluorescent sensor for hydrogen peroxide. (B) The excitation and emission fluorescence spectra of (a) TA, (b) TA þ CuO NPs, (c) TA þ H2O2, and (d) TA þH2O2 þCuO NPs. The inset photograph displays the luminescence upon excitation (302 nm) under a hand-held UV lamp.
To confirm the sensing protocol, fluorescent spectra under different conditions were studied. From Fig. 1B, it was observed that no fluorescence produced in TA or the TA–CuO NPs system. Although slow oxidation of TA can be induced by hydrogen peroxide in the absence of catalyst, very weak fluorescence was observed. In presence of cupric oxide nanoparticles, the fluorescence intensity could be greatly enhanced up to about 40 folds. 3.2. Optimization of reaction conditions In order to optimize the possible analytical method for the hydrogen peroxide determination, the effects of experimental conditions including solution pH, reaction temperature, TA concentration, and CuO NPs concentration were investigated. The solution pH is an important factor that affects the catalytic activity of CuO NPs in the sensing system. It is clear from Fig. S1A that the catalytic oxidation of TA by H2O2 with CuO NPs as the catalyst is much faster in neutral solutions than in acidic and basic solutions. The maximum fluorescence intensity was obtained at pH 7.0. The effect of reaction temperature on the fluorescence intensity was studied in the range of 20–55 1C (Fig. S1B). The fluorescence intensity of the sensing system increased with an increasing reaction temperature in the range of 20–45 1C and decreased when the temperature was higher than 45 1C. Fig. S1C illustrates the influence of TA concentration on the fluorescence intensity of the reaction solution. It can be seen that the fluorescence intensity is enhanced with increasing of TA concentration up to 2.8 mM, and then keeps almost constant beyond 2.8 mM of TA. The reactant TA is non-fluorescent, but the product TAOH is highly
3.3. Hydrogen peroxide sensing The analytical determination of hydrogen peroxide is of considerable importance for medical diagnosis since hydrogen peroxide is formed as an intermediate product in the case of a large number of important detection processes. The possibility of using the proposed method for the determination of hydrogen peroxide was investigated. Fig. 2 depicted the typical fluorescence response in the presence of different concentrations H2O2. Under the optimal reaction conditions as described above, the fluorescence intensity versus hydrogen peroxide concentration was linear over the range from 5.0 10 6 M to 2.0 10 4 M range with the correlation coefficient of 0.996. The limit of detection for hydrogen peroxide was 3.4 10 7 M (S/N ¼3). The RSD was 1.8% for 2.0 10 4 M hydrogen peroxide (n ¼6). 3.4. Analytical application in determination of glucose Reliable and fast determination of glucose is of considerable importance in biotechnology, clinical diagnostics and food industry. As H2O2 is the product of GOx-catalyzed reaction of glucose oxidation, fluorescent detection of glucose could be realized by coupling the TA–CuO colloids system with the glucose catalytic reaction by GOx. As shown in Fig. 3, the measured fluorescence intensity increased markedly upon increasing the concentration of glucose. The controlled experimental results show that O2, glucose oxidase, and glucose were all essential to the producing of fluorescence, since the exclusion of either component would yield no H2O2. Good linear relationship was obtained in the range of 3.0 10 6–1.0 10 4 M. The correlation coefficient was 0.999, and the relative standard deviation for six repeated measurements of 1.0 10 4 M glucose was 1.5%. The detection limit value obtained for the experiment was 1.0 10 6 M (S/N ¼3), which was lower than that of other methods (Table S1).
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Fig. 3. (A) Emission spectra represent the effect of the glucose–GOx system with different glucose concentrations on the fluorescence intensity. Glucose concentration from (a) to (i) (10 6 M): (a) 0; (b) 5; (c) 10; (d) 20; (e) 40; (f) 60; (g) 80; (h) 100; (i) 120. (B) Calibration plot for glucose determination.
Fig. 4. (A) Emission spectra represent the effect of the lactate–LOx system with different lactate concentrations on the fluorescence intensity. Lactate concentration from (a) to (i) (10 6 M): (a) 0; (b) 1.6; (c) 4; (d) 8; (e) 16; (f) 32; (g) 48; (h) 64; (i) 80. (B) Calibration plot for L-lactate determination.
Table 1 Analytical results of glucose in human serum.
samples ranging from 90.7% to 105.8%. The results demonstrate that this new method is suitable and satisfactory for glucose analysis of real samples.
Sample Proposed method (mM) 1 2 3
4.21 70.07 4.17 70.13 37.68 70.27
Glucose oxidase endpoint method (mM)
Relative deviation (%)
3.977 0.20 4.39 7 0.24 41.247 0.56
6.05 5.01 8.63
For testing if the detection of glucose is specific, control experiments were taken using lactose, maltose, and fructose. Because of the high substrate specificity of GOx, no significant signals were obtained for the control samples with concentrations of 1 mM (Fig. S2). Therefore, a sensitive and selective glucose biosensor can be fabricated in a facile approach. To evaluate the feasibility of the sensing system for analysis of glucose in biological samples, the developed method was applied to the determination of glucose in human serum samples. From Table 1, it can be seen that the results obtained by the proposed method were in good agreement with those measured by the glucose oxidase endpoint method. The practical applicability of the proposed method was further verified through standard addition experiments, with the recoveries of glucose in three serum
3.5. Analytical application in determination of L-lactate It is well known that lactate oxidase (LOx) catalyzes the oxidation of L-lactate to pyruvic acid and H2O2. Therefore, fluorescent detection of L-lactate could be realized by coupling the TA– CuO NPs system with the L-lactate catalytic reaction by LOx. Typical L-lactate response curves are shown in Fig. 4. The linear range of lactate is from 8.0 10 7 to 8.0 10 5 M with a correlation coefficient of 0.998. The limit of detection was calculated to be 4.5 10 8 M (S/N¼3), which was lower than that of other methods (Table S2) (Ballesta-Claver et al., 2008; Claver et al., 2009; Goran et al., 2011; Groegel et al., 2011; Romero et al., 2010; Zheng et al., 2010). The relative standard deviation for six repeated measurements of 8.0 10 5 M L-lactate was 1.9%. To investigate the selectivity of the proposed L-lactate sensor, fluorescent responses in the presence of ethanol, urea, glucose, lactose, maltose, uric acid, citric acid, and pyruvic acid were studied. As can be seen from Fig. S3, the presence of foreign substances (10 mM) individually has no significant effect on the L-lactate sensor.
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Table 2 Analytical results of L-lactate in human serum.
Sample
Proposed method (mM)
Lactate oxidase endpoint method (mM)
Relative deviation (%)
1 2 3
3.09 70.03 3.47 70.14 2.41 70.02
3.187 0.04 3.23 7 0.09 2.40 7 0.05
2.83 7.43 0.42
In order to explore the applicability and feasibility of the method, L-lactate content in human serum samples was determined. From Table 2, it can be seen that the results obtained by the proposed method agree well with those measured by the lactate oxidase endpoint method. Furthermore, the recoveries for lactate in human serum samples were observed to be ranged from 94.0 to 115.6%. Thus the proposed method shows satisfactory results for the analysis of lactate in human serum samples.
4. Conclusion In summary, a simple, highly selective and inexpensive strategy for H2O2 sensing has been successfully developed by using terephthalic acid as substrate and cupric oxide nanoparticles as catalyst. In comparison with traditional peroxide substrate TMB, TA exhibits several advantages such as low cost, pure oxidation products easily obtained, more stability for storage. As a mimic peroxidase, cupric oxide nanoparticles are considerably more stable and possess an almost unchanged catalytic activity over a wide range of pH and temperatures. Coupling the oxidation of glucose or L-lactate catalyzed by their corresponding oxidase enzymes, sensitive assays of glucose and L-lactate were realized with detection limits of 1.0 10 6 and 4.5 10 8 M. In addition, we demonstrated the successful application of the present approach in real serum samples, which suggests its great potential for diagnostic purpose.
Acknowledgments The authors gratefully acknowledge the financial support of the National Natural Science Foundation of China (21175023), the Program for New Century Excellent Talents in University (NCET12–0618), the Natural Science Foundation of Fujian Province (2011J01034, 2012J06019), and the Medical Elite Cultivation Program of Fujian, P.R.C (2013-ZQN-ZD-25).
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