Fluorescein isothiocyanate-capped gold nanoparticles for fluorescent detection of reactive oxygen species based on thiol oxidation and their application for sensing glucose in serum

Abstract

This study reports a simple and sensitive method for fluorescent detection of reactive oxygen species (ROS) based on the oxidation of 2-mercaptoethanol (2-ME) and 2-ME-induced increase in fluorescence of fluorescein isothiocyanate-capped gold nanoparticles (FITC-AuNPs). FITC molecules can be readily attached to the surface of citrate-capped AuNPs through their isothiocyanate group. FITC-AuNPs fluoresce weakly mainly due to the efficient fluorescence resonance energy transfer from FITC to AuNPs. It is found that the presence of 2-ME enables FITC to be removed from the surface of the AuNPs through the formation of Au–S bonds, thereby restoring the fluorescence of FITC. In contrast, when 2-ME is oxidized with ROS under alkaline condition, the generated 2-ME disulfide is unable to remove FITC from the NP surface. As a result, the weak fluorescence of FITC-AuNPs increased gradually after adding increasing concentrations of ROS. The sensitivity of this method toward ROS was significantly improved after removal of the AuNPs by centrifugation. Because the glucose oxidase-catalyzed oxidation of glucose yielded gluconic acid and H₂O₂, this method was also utilized to detect glucose. Under optimum conditions, the minimum detectable concentrations for H₂O₂, superoxide anion, hydroxyl radical, and glucose were found to be 1, 0.6, 0.6 and 1 μM, respectively. This method has been successfully applied to the determination of glucose in serum.

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1. Introduction

Reactive oxygen species (ROS) include not only free radicals, such as superoxide radical and hydroxyl radical, but also non-radicals, such as hydrogen peroxide (H₂O₂) and hypochlorous acid. The production of ROS results from pollutants, UV light, cigarette smoke and radiation.¹ Peroxidase-catalyzed reaction also generates ROS. In normal aerobic cells, ROS can be scavenged in the presence of biochemical antioxidant, such as glutathione.² Oxidative stress occurs when the balance between ROS and antioxidant is disrupted by excessive ROS or deficient antioxidants. The overproduction of ROS induces DNA damage, protein degradation, lipid peroxidation, and enzyme inactivation.^2,3 Thus, ROS is implicated with the pathogenesis of various human diseases and conditions, including aging, mutagenesis, chronic inflammation, and certain types of cancer.⁴

Because of the biological and clinical significance of ROS, researchers have proposed numerous chemosensors for selective and sensitive detection of ROS based on fluorophores,⁵ chromophores,⁶ luminophores,^7,8 conjugated polymer-DNA composites,⁹ and quantum dots.¹⁰ Recently, gold nanoparticles (AuNPs) with novel optical and electrical properties are appealing in diverse fields, such as biosensor, catalyst, and separation science.^11–13 This material is a promising alternative for the detection of ROS. In the presence of AuCl₄⁻ and surfactant, the H₂O₂-induced growth of the AuNPs enhanced spectral absorbance features^14,15 and resonance light scattering.¹⁶ Because the glucose oxidase (GOx)-catalyzed oxidation of glucose generates H₂O₂, this method was further exploited for the determination of glucose. The hydroxyl radical-induced cleavage of single-stranded DNA (ssDNA) could stabilize citrate-capped AuNPs against conditions of high ionic strength. However, under identical conditions, the aggregation of the AuNPs occurred in the presence of ssDNA.¹⁷ Although the methods mentioned above all provided satisfactory sensitivity, they were only used to detect one kind of ROS. Lee et al. reported that fluorescein-labeled hyaluronic acids were severely quenched by fluorescence resonance energy transfer (FRET) after they were attached to the surface of AuNPs.¹⁸ After fluorescein-labeled hyaluronic acids were degraded by ROS, fluorescein-labeled hyaluronic acid fragments were released from the Au surface. As a result, fluorescein was restored to their original fluorescence intensity. However, the preparation of this kind of NPs is time-consuming and sophisticated.

This study presents a simple and inexpensive method for sensitive detection of H₂O₂, superoxide anion, and hydroxyl radical using fluorescein isothiocyanate-modified AuNPs (FITC-AuNPs) and 2-mercaptoethanol (2-ME) (Scheme 1). The fluorescence of FITC molecules is severely quenched by FRET while they are attached to the surface of the AuNPs.¹⁹ The same quenching mechanism was observed in the case of Nile red- and rhodamine 6G-adsorbed AuNPs.^20,21 Upon the addition of 2-ME to a solution of FITC-AuNPs, FITC molecules are released from the Au surface due to the displacement of isothiocyanate group of FITC by thiol group of 2-ME. The released FITC fluoresce strongly after removal of the AuNPs by centrifugation. Upon the addition of ROS to 2-ME,^22,23 the produced 2-ME disulfide is incapable of displacing FITC from the Au surface; thus, FITC molecules do not release into a solution. After centrifugation of the AuNPs, the supernatant containing only 2-ME disulfide does not exhibit a fluorescence response. We investigated the effect of the molar ratio of 2-ME to H₂O₂ on the sensitivity of this method toward ROS. Because glucose is oxidized, with GOx as catalyst, to gluconic acid and H₂O₂, this method was also applied to the determination of glucose in serum.

Scheme 1 Schematic illustration of the strategy of ROS detection based on the oxidation of 2-ME and 2-ME-induced increase fluorescence of FITC-AuNPs.

2. Experimental

2.1 Chemicals and preparation

Hydrogen tetrachloroaurate (III) dehydrate, Na₂HPO₄, and Na₃PO₄ were purchased from Alfa Aesar (Ward Hill, MA). 2-ME, trisodium citrate, arabinose, xylose, glucose, galactose, cellobiose, lactose, maltose, raffinose, cyclodextrin, GOx (EC1.1.3.4 from Aspergillus niger, 210 000 units g⁻¹), FITC, KO₂, and FeCl₂ were obtained from Sigma-Aldrich (Louis, MO, USA). A solution of H₂O₂ (30%) was obtained from Showa (Tokyo, Japan). The mixture of Fe²⁺ and H₂O₂ at a molar ratio of 1 : 1 generates hydroxyl radical.¹⁷ The pH of a solution of 20 mM Na₃PO₄ was adjusted to 12.0 with Na₂HPO₄. Water used in all experiments was doubly distilled and purified by a Milli-Q system (Millipore, Milford, MA, USA).

2. 2 Synthesis of FITC-AuNPs

We prepared citrate-capped AuNPs by means of the chemical reduction of a metal salt precursor (hydrogen tetrachloroaurate, HAuCl₄) in the liquid phase. To achieve this, trisodium citrate (38.8 mM, 25 mL) was rapidly added to a solution of HAuCl₄ (1 mM, 250 mL) that was heated under reflux. This heating continued for an additional 15 min, during which time the color of the solution changed to a deep red. A H7100 Transmission Electron Microscopy (TEM) (Hitachi High-Technologies Corp., Tokyo, Japan) operating at 75 keV was used to collect TEM images of the AuNPs. TEM images show that the average size of as-prepared AuNPs is 13 ± 1 nm. The absorption spectra of the AuNPs were collected using a double-beam UV-visible spectrophotometer (Cintra 10e, GBC Scientific Equipment Pty Ltd., Dandenong, Victoria, Australia). The surface plasmon resonance (SPR) peak of the AuNPs was 520 nm. The concentration of the AuNPs was estimated to be 8.0 nM according to Beer's law (A = εbc). The cuvette had a path length (b) of 1 cm. The extinction coefficient (ε) of 13 nm AuNPs at 520 nm is 2.78 × 10⁸ M⁻¹cm⁻¹. We added FITC (1 mM, 20 μL) to a solution of citrate-capped AuNPs (8 nM, 20 mL) and equilibrated the resulting mixture at 4 °C overnight.

2.3 Sample preparation

Different concentrations of 2-ME were prepared in 20 mM phosphate at pH 12.0. For ROS sensing, 2-ME (0.1 μM–10 mM, 500 μL) was reacted with ROS (0–1000 μM, 100 μL) for 20 min. After that, the mixture was incubated with FITC-AuNPs (2 nM, 400 μL) for 0–30 min. The resulting solution was centrifuged at 14 000 rpm for 10 min. The fluorescence spectrum of the obtained supernatant was recorded using a Hitachi F-4500 fluorometer (Hitachi, Tokyo, Japan) while the excitation wavelength was set at 488 nm.

For glucose sensing, glucose (0–1000 μM, 100 μL) was reacted with GOx (20 mg mL⁻¹, 20 μL) at 37 °C for 30 min while they were prepared in 10 mM phosphate at pH 7.0.²⁴ At pH 12.0, the generated H₂O₂ was reacted with 2-ME (2 μM, 500 μL) for 20 min. The mixture was incubated with FITC-AuNPs (8 nM, 100 μL) for 15 min. The following steps, including centrifugation and fluorescence measurement, were the same as those for the detection of ROS. To evaluate the selectivity of this method, we replaced glucose with arabinose, xylose, galactose, cellobiose, lactose, maltose, raffinose, or cyclodextrin, one at a time.

2.4 Analysis of glucose in serum

Blood samples were collected from a healthy adult male with the age of 24 years. To obtain serum samples, the collected whole blood samples were immediately centrifuged at 3000 rpm for 10 min at 4 °C. Serum samples (100 μL) were spiked with standard glucose (100 μL, 0–5 mM). The resulting solutions were mixed with 800 μL of 10 mM phosphate (pH 7.0). The 10-diluted serum samples were filtered using the 3 kDa Nanosep centrifugal device (Pall Co., East Hills, NY, USA) at 14000 rpm for 10 min. The obtained solutions (100 μL) were incubated with GOx (30 μL, 20 mg mL⁻¹) at 37 °C for 30 min. The produced H₂O₂ was incubated with 2-ME (2 μM, 500 μL) for 20 min. The mixture was reacted with FITC-AuNPs (8 nM, 100 μL) for 15 min. The following steps, including centrifugation and fluorescence measurement, were the same as those for the detection of ROS.

3. Result and discussion

3.1 Fluorescence turn-on assay of H₂O₂

We examine whether or not 2-ME and 2-ME disulfide can displace FITC from the surface of the AuNPs. After centrifugation of a solution of 0.8 nM FITC-AuNPs, the obtained supernatant had an extremely low fluorescence (Spectrum a in Fig. 1A). This result indicates that FITC molecules were not released into a solution. In other words, FITC molecules were still adsorbed on the surface of the AuNPs through their isothiocyanate group.²⁵ In contrast, the supernatant, obtained from the centrifugation of a mixture of 1 μM 2-ME and 0.8 nM FITC-AuNPs, caused a ca. 20.5-fold increase in fluorescence intensity (I_F0) at 520 nm (Spectrum b in Fig. 1A). Obviously, 2-ME had stronger binding to AuNPs than FITC. A previous study has shown that H₂O₂ can oxidize thiols to the corresponding disulfide under alkaline conditions.²³ Thus, in this study, the oxidation of 1 μM 2-ME with 100 μM H₂O₂ was conducted at pH 12.0. A solution containing H₂O₂-treated 2-ME and 0.8 nM FITC-AuNPs was centrifuged before measuring their fluorescence spectrum. As compared to spectrum a in Fig. 1A, the obtained supernatant resulted in a ca. 5.2-fold increase in fluorescence intensity (I_F) at 520 nm (Spectrum c in Fig. 1). This result supports our notion that oxidized 2-ME corresponding to 2-ME disulfide is incapable of displacing FITC from NP surface. Moreover, an incomplete reaction between 2-ME and 100 μM H₂O₂ yields coexistence of 2-ME and 2-ME disulfide; therefore, a small increase in fluorescence was still caused by unreacted 2-ME. Fig. S1 (ESI†) shows that the fluorescence intensity of FITC remained almost constant in the presence of H₂O₂, 2-ME, and H₂O₂-treated 2-ME, suggesting that they did not interfere with the fluorescence intensity of FITC. Based on these findings, we point out that H₂O₂ can be detected based on the oxidation of 2-ME and 2-ME-induced fluorescence increase of FITC-AuNPs. To support that the centrifugation is a crucial step, the fluorescence spectra of FITC-AuNPs were measured in the absence and presence of 2-ME or H₂O₂-treated 2-ME. As compared to the fluorescence intensity of FITC-AuNPs (spectrum a in Fig. 1B), the addition of 2-ME and H₂O₂-treated 2-ME led to a ca. 18.5- and 4.0-fold increase in fluorescence intensity at 520 nm, respectively (spectrum b and c in Fig. 1B). Obviously, due to collisional quenching of the fluorescence,²⁶ the fluorescence intensity of the released FITC before centrifugation was weaker than that after centrifugation. Moreover, the difference (△I_F = I_F0 − I_F) in fluorescence intensity between spectrum b and c in Fig. 1A was larger than that between spectrum b and c in Fig. 1B. This finding provides clear evidence that the sensitivity of this method toward H₂O₂ can be improved by removing the AuNPs.

Fig. 1 (A) Fluorescence spectra of the supernatants from the centrifugation of (a) FITC-AuNPs, (b) FITC-AuNPs and 2-ME, and (c) FITC-AuNPs and H₂O₂-treated 2-ME. (B) Fluorescence spectra of (a) FITC-AuNPs, (b) FITC-AuNPs and 2-ME, and (c) FITC-AuNPs and H₂O₂-treated 2-ME. (c in A and B) A solution of 1 μM 2-ME was reacted with 100 μM H₂O₂ in 20 mM phosphate at pH 12.0 for 20 min. (b and c in A and B) The H₂O₂-treated 2-ME or 2-ME was incubated with 0.8 nM FITC-AuNPs for 20 min. The excitation wavelength was set at 488 nm. The fluorescence intensities (I_F) are plotted in arbitrary units (a.u.).

To further demonstrate that H₂O₂ is capable of oxidizing 2-ME, absorption spectra and TEM images of FITC-AuNPs were examined in the absence and presence of 2-ME or H₂O₂-treated 2-ME. Previous studies reported that 2-ME induced the aggregation of the AuNPs.²⁷ If the oxidation of 2-ME by H₂O₂ occurred, 2-ME-induced aggregation of the AuNPs should be suppressed. In contrast to the absorption spectrum of FITC-AuNPs (Spectrum a in Fig. 2A), the addition of 1 μM 2-ME to a solution of FITC-AuNPs led to a decrease in the SPR peak at 520 nm and the formation of a new red shifted band (Spectrum b in Fig. 2A). When H₂O₂-treated 2-ME was present in an identical solution, a slight degree of aggregation of the AuNPs was observed (Spectrum c in Fig. 2A). These results were confirmed by TEM images, revealing dispersed AuNPs in the absence of 2-ME, aggregated AuNPs in the presence of 2-ME, and slightly aggregated AuNPs in the presence of H₂O₂-treated 2-ME (Fig. 2B).

Fig. 2 (A) Absorption spectra and (B) TEM images of solutions of (a) FITC-AuNPs, (b) FITC-AuNPs and 2-ME, and (c) FITC-AuNPs and H₂O₂-treated 2-ME. The other conditions are the same as those in Fig. 1.

Next, we proved that 2-ME disulfide (oxidized 2-ME) can not remove FITC from the surface of the AuNPs. Before adding FITC to citrate-capped AuNPs, we mixed them with 2-ME disulfide or 2-ME. After the centrifugation of a solution containing 2-ME disulfide, the AuNPs, and FITC, the obtained supernatant fluoresced weakly (Spectrum a in Fig. S2, ESI†). Apparently, FITC molecules were attached to the NP surface even though 2-ME disulfide was first added to a solution of citrate-capped AuNPs. This finding clearly indicates that the FITC has stronger binding to the surface of that AuNPs than 2-ME disulfide. However, when 2-ME was used in place of 2-ME disulfide, the supernatant showed a strong fluorescence (Spectrum b in Fig. S2, ESI†). This is attributed to that FITC can not remove 2-ME from the surface of the AuNPs. Taken together, we suggest that the detection of H₂O₂ can be conducted by the oxidation of 2-ME, followed by the addition of FITC-AuNPs.

3.2 Effect of the molar ratio of 2-ME to H₂O₂

We next investigated the effect of the molar ratio of 2-ME to H₂O₂ on the detection of H₂O₂. The concentration of H₂O₂ was fixed at 10⁻⁴ M while the concentration of 2-ME varied from 0.1 μM to 10 mM. Fig. 3 presents that the value of △I_F was maximized when the molar ratio of H₂O₂ to 2-ME changed to 100 : 1. The value of △I_F gradually decreased with an increase in the concentration of 2-ME from 1 μM to 10 mM. This finding implies that the oxidation of 2-ME to 2-ME disulfide by H₂O₂ was extremely slow. If this oxidation reaction proceeded fast, a large △I_F should be found at the molar ratio of 1 : 1. However, a small △I_F was observed in this case, suggesting that a large number of unreacted 2-ME molecules were still present in an aqueous solution. Luo et al. reported that the time for the oxidation of 4 mM cysteine by 2 mM H₂O₂ exceeded 900 min.²² Kirihara et al. demonstrated that the oxidation of thiols to the corresponding disulfides by H₂O₂ required a longer reaction time (>24 h) in the absence of catalysts.²³ Based on these results and previous studies, we suggest that the high molar ratio of H₂O₂ to 2-ME can accelerate the oxidation reaction of 2-ME to the corresponding disulfide. Meanwhile, the value of △I_F dramatically decreased when the molar ratio of H₂O₂ to 2-ME exceeded 100 : 1. As compared to 1 μM 2-ME, fewer FITC molecules were liberated upon the addition of <1 μM 2-ME to a solution of FITC-AuNPs. Thus, the value of I_F0 was close to that of I_F, leading to a small △I_F.

Fig. 3 Effect of the concentration of 2-ME on the value of △I_F (△I_F = I_F0 − I_F). I_F0 is the fluorescence intensity (520 nm) of the supernatant from the centrifugation of a solution of FITC-AuNPs and 2-ME, while I_F is the fluorescence intensity (520 nm) of the supernatant from the centrifugation of a solution of FITC-AuNPs and H₂O₂-treated 2-ME. A solution of 2-ME (0.1 μM–10 mM) was reacted with H₂O₂ (100 μM) in 20 mM phosphate at pH 12.0 for 20 min. The resulting solution was incubated with 0.8 nM FITC-AuNPs for 20 min. After removal of the AuNPs by centrifugation, the supernatant was detected by exciting at 488 nm.

3.3 Quantification of ROS and glucose

As shown in Fig. S3 (ESI†), the fluorescence intensity (I_F) at 520 nm of the supernatant reached a plateau at 15 min after we added H₂O₂-treated 2-ME to a solution of FITC-AuNPs and centrifuged the resulting mixture. A 15-min incubation time between H₂O₂-treated 2-ME and FITC-AuNPs was chosen in this study. Fig. 4A displays that the fluorescence intensity at 520 nm of the supernatant gradually decreased with increasing the H₂O₂ concentration. Similar results were seen in the case of the superoxide anion and hydroxyl radical (Fig. 4B and 4C). This reveals that the superoxide anion and hydroxyl radical also reacted with 2-ME. At the concentrations of 5, 10, 50, and 100 μM H₂O₂, the relative standard deviation of fluorescence measurements varied from 1.6 to 4.4% (Fig. S4, ESI†). Fig. 4D shows that three linear calibration curves were established by plotting △I_F against the concentrations of H₂O₂, superoxide anion, and hydroxyl radical. The correlation coefficients (R²) were 0.9971 (y = 13.7x + 1.0), 0.9999 (y = 5.0x + 0.6), and 0.9999 (y = 1.1x + 0.4) for the quantification of H₂O₂, superoxide anion, and hydroxyl radical in the concentration ranges of 4–100, 1–100, and 1–100 μM, respectively. The minimum detectable concentrations for H₂O₂, superoxide anion, and hydroxyl radical were found to be 1, 0.6, and 0.6 μM, respectively. The detection sensitivity of ROS obtained by this method is comparable to that obtained by hyaluronic acid-capped AuNPs.¹⁸ Moreover, this method offers superior ROS-detection capability compared to commercial available fluorescent probes, 2,7-dichlorodihydrofluorescein and 2-[6-(40-amino)phenoxy-3H-xanthen-3-on-9-yl] benzoic acid.¹⁸ Although H₂O₂ can be detected by catalyzing the growth of Au seeds, the growth rate of AuNP seeds is susceptible to changes in the environment.^14–16 We suggest that the distinct advantages of this method are its sensitivity, low-cost, and easy preparation. To further confirm that the centrifugation is an important step for quantification of H₂O₂, the present method was utilized for the quantification of H₂O₂ without centrifugation. Fig. S5 (ESI†) shows that the correlation coefficients (R²) were 0.9810 for the quantification of H₂O₂ in the concentration range of 10–60 μM and the minimum detectable concentration of H₂O₂ was 10 μM. Moreover, the present method without centrifugation had poor reproducibility of signal intensity and short linear range. Based on these results, we point out that the centrifugation step is needed to avoid the collisional quenching between FITC and AuNPs.

Fig. 4 Fluorescence response of the supernatants from the centrifugation of solutions of 0–100 μM ROS, 1 μM 2-ME, 0.8 nM FITC-AuNPs: (A) H₂O₂, (B) superoxide anion, and (C) hydroxyl radical. (D) Calibration curves for the quantification of ROS. The error bars represent standard deviations based on three independent measurements. ROS was reacted with 2-ME at pH 12.0 for 20 min. The resulting mixture was incubated with FITC-AuNPs for 15 min. The other conditions are the same as those in Fig. 3.

Because H₂O₂ could be generated from the GOx-catalyzed oxidation of glucose, we applied a two-step analysis to detect glucose in the presence of GOx. First, the reaction of glucose and GOx were performed at 37 °C for 30 min while they were prepared in 10 mM phosphate at pH 7.0.^24,28 Then, the produced H₂O₂ was incubated with 2-ME at pH 12.0 for 20 min and the resulting product was added to a solution of FITC-AuNPs. After removal of the AuNPs by centrifugation, Fig. 5A displays that the fluorescence intensity of the supernatant gradually decreased with increases in the glucose concentration. Fig. S6 (ESI†) shows that the presence of only glucose did not have such effect on the fluorescence intensity of the supernatant. Although the presence of only GOx induced a decrease in fluorescence intensity of the supernatant, the concentration of GOx remained constant in our detection system. Inset in Fig. 5A shows a linear correlation between △I_F and the concentration of glucose over the range of 6–100 μM (y = 39.4x + 0.5; R² = 0.9989). This method was capable of detecting glucose at concentration as low as 1.0 μM and thus can detect glucose at physiologically relevant concentration. To assess the selectivity of this method for glucose, other carbohdyrates—including arabinose, xylose, galactose, cellobiose, lactose, maltose, raffinose, cyclodextrin—were used instead of glucose under identical conditions. Fig. 5B shows that this method is highly selective for the detection of glucose. We note that the concentration of glucose in blood samples is approximately 10–20-fold larger than that of all other carbohydrates.²⁹

Fig. 5 (A) Quantification of glucose by a two-step analysis. GOx (0.4 mg mL⁻¹) catalyzed the oxidation of glucose (0–100 μM) to gluconic acid and H₂O₂. The produced H₂O₂ was reacted with 1 μM 2-ME at pH 12.0 for 20 min. The resulting mixture was incubated with FITC-AuNPs for 15 min. After removal of the AuNPs by centrifugation, the supernatant was detected by exciting at 488 nm. The arrows indicate the signal changes with increases in glucose concentrations (0, 6, 8, 40, 70, 100 μM). Inset: Plot of △I_F as a function of glucose concentration. The error bars represent standard deviations based on three independent measurements. (B) Selectivity of a two-step analysis. The concentration of each carbohydrate is 1 mM.

3.4 Analysis of Glucose in serum

The present method was used for the practical analysis of glucose in serum samples. The normal glucose concentration in human serum ranges from 70 to 125 mg dL⁻¹ (3.8–6.9 mM).³⁰ Due to variations in chemical composition of biological fluids, standard addition method was used to correct possible matrix effects on the analyte uptake. Thus, serum samples were spiked with 0–5 mM standard glucose prior to analysis of glucose in serum. As shown in Fig. S7 (ESI†) shows, a gradual decrease in fluorescence intensity at 520 nm of the supernatant was observed after a 100-fold dilution of the spiked sample was analyzed by our proposed method. A linear correlation (y = 39.8x + 0.6; R² = 0.9978) was obtained by plotting △I_Fversus the spiked concentration of glucose (Inset in Fig. S7, ESI†). The recoveries of these measurements were in the range of 90.2% to 112.8%. The concentration of glucose in serum was determined to be 6.10 ± 0.48 mM (n = 3). This result is in agreement with the concentration (5.25 ± 0.22 mM; n = 3) of glucose in serum determined by a commercially available glucometer (Model Accu-Chek Advantage). Based on a t-test (95% confidence level, 4 degrees of freedom) and F-test (95% confidence level), the result obtained from our method was shown to be in accordance with that obtained from a commercially available glucometer.

4. Conclusions

We have demonstrated that the fluorescence turn-on detection of ROS has been successfully achieved based on the oxidation of 2-ME to its disulfide, 2-ME-induced fluorescence increase of FITC-AuNPs, and removal of the AuNPs. Because the GOx-catalyzed oxidation of glucose generated H₂O₂, the sensing of glucose in serum has been accomplished by our proposed method. Under the optimum reaction conditions, this method enabled analyses of H₂O₂, superoxide anion, and hydroxyl radical, and glucose, with the minimum detectable concentrations corresponding to 1, 0.6, 0.6, and 1 μM, respectively. Our proposed method provides the advantages of simplicity, sensitivity, and low cost. We believe that this method may serve as a fluorescent reporter for the activities of oxidases and for the detection of their substrates.

Acknowledgements

We would like to thank National Science Council (NSC 97-2113-M-110-001-) and National Sun Yat-sen University-Kaohsiung Medical University Joint Research Center for the financial support of this study. We also thank National Sun Yat-sen University and Center for Nanoscience & Nanotechnology for the measurement of fluorescence spectrum.

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Footnote

† Electronic supplementary information (ESI) available: Supplementary figures. See DOI: 10.1039/c0ay00428f

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