FRET-Based Biofriendly Apo-GOx-Modified Gold Nanoprobe for

Article pubs.acs.org/ac

FRET-Based Biofriendly Apo-GOx‑Modified Gold Nanoprobe for Specific and Sensitive Glucose Sensing and Cellular Imaging Lu Li, Feifei Gao, Jian Ye, Zhenzhen Chen, Qingling Li, Wen Gao, Lifei Ji, Ruirui Zhang, and Bo Tang* College of Chemistry, Chemical Engineering and Materials Science, Engineering Research Center of Pesticide and Medicine Intermediate Clean Production, Ministry of Education, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Normal University, Jinan, Shandong Province 250014, China S Supporting Information *

ABSTRACT: In this paper, we have developed a biofriendly and high sensitive apo-GOx (inactive form of glucose oxidase)-modified gold nanoprobe for quantitative analysis of glucose and imaging of glucose consumption in living cells. This detection system is based on fluorescence resonance energy transfer between apo-GOx modified AuNPs (Au nanoparticles) and dextran-FITC (dextran labeled with fluorescein isothiocyanate). Once glucose is present, quenched fluorescence of FITC recovers due to the higher affinity of apo-GOx for glucose over dextran. The nanoprobe shows excellent selectivity toward glucose over other monosaccharides and most biological species present in living cells. A detection limit as low as 5 nM demonstrates the high sensitivity of the nanoprobe. Introduction of apo-GOx, instead of GOx, can avoid the consumption of O2 and production of H2O2 during the interaction with glucose, which may exert effects on normal physiological events in living cells and even lead to cellular damage. Due to the low toxicity of this detection system and reliable cellular uptake ability of AuNPs, imaging of intracellular glucose consumption was successfully realized in cancer cells.

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utilizing nanoprobes based on a CdTe QDs (quantum dots)− GOx (glucose oxidase) complex or a FRET (fluorescence resonance energy transfer) system assembled with ConA (concanavalin) conjugated CdTe QDs and AuNPs (Au nanoparticles).35,36 Zhou et al. developed a glucose probe based on glucose-mediated assembly of phenylboronic acid modified CdTe/ZnTe/ZnS QDs and performed the glucose imaging in mouse melanoma B16F10 cells.37 Although, QDs, ConA, and GOx are usually used for glucose sensing, they may be problematic as a result of toxicity in living cells. The toxicity of QDs has been researched extensively, and the biocompatibility of QDs remains questionable.38−40 Con A is also reported to associate with a variety of toxicological effects in cell cultures.41−46 When GOx is used in glucose sensing, H2O2 (hydrogen peroxide) produced during the GOx-catalyzed oxidation of glucose may cause oxidative stress through the oxidation of biomolecules, leading to cellular damage.47 As a consequence of these deficiencies, a great incentive still exists for the development of a new, biofriendly nanoprobe to meet the rising need for highly sensitive glucose imaging in living cells.

s an important bioactive substance, glucose plays an essential role in all processes related to living cells. Optimal glucose concentrations are crucial in the natural growth of cells. Usually free cytosolic glucose concentration is at millimolar levels. However, the decrease of external glucose and the presence of some transport inhibitors (e.g., cytochalasin B), indicating rapid metabolism, lead to reduced glucose levels in the cell; cytosolic glucose concentrations can vary by several orders of magnitude.1 The deprivation of glucose can induce oxidative stress and affect signal transduction and gene expression in human cells.2,3 So, a highly sensitive determination of cellular glucose is very significant for understanding its chemical and biological function in certain physiological and pathological events and may afford a useful tool for the diagnosis and therapy of diseases.4 Various methods have been proposed for glucose sensing.5−14 Among these methods, fluorescence imaging analysis offers an appealing approach for the visualization of glucose at the cellular level.15−17 Although many fluorescence probes have been synthesized for imaging of glucose in living cells, some of them are still complicated in molecular structure and require elaborate biochemical synthesis or the sensitivity and selectivity of them should be improved.1,18−21 Recent advances in the application of nanomaterials in biosensing have attracted great attention.22−26 A large number of nanomaterials have been fabricated as nanoprobes for glucose determination.27−34 For example, we have achieved highly sensitive glucose detection © XXXX American Chemical Society

Received: July 12, 2013 Accepted: September 16, 2013

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dx.doi.org/10.1021/ac4021227 | Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

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(EDC), dextran-FITC and N-hydroxysuccinimide (NHS) were obtained from Sigma-Aldrich. D-Glucose was purchased from Amresco. Chloroauric acid and hexadecyl trimethyl ammonium bromide (CTAB) were obtained from the China pharmaceutical group, Shanghai chemical reagent company. All other chemicals and solvents used were of analytical grade. Water was purified with a Sartorius Arium 611 VF system (Sartorius AG, Germany) to a resistivity of 18.2 MΩ cm. Instrumentation. Optical absorption spectroscopy measurements were performed with a UV-1901 PC dual-beam spectrophotometer (Shimadzu Corp., Kyoto, Japan), using 1.0 cm path-length quartz cuvettes. Fluorescence spectra and fluorescence lifetimes were measured using an Edinburgh FLS920 spectrofluorimeter (Edinburgh Instruments Ltd., England) equipped with xenon and hydrogen lamps. Transmission electron microscopy (TEM) images were obtained by using a Hitachi model H-800 instrument (Japan). Confocal fluorescence imaging studies were performed with a TCS SP5 confocal laser scanning microscopy (Leica Company, Ltd., Germany) with an objective lens (40×). Centrifugation was performed with a Sigma 3K15 refrigerated centrifuge. Cells were disrupted using a VC 130PB ultrasonic disintegrater (Sonics & Materials Inc., Newtown, CT). Preparation of AuNPs. AuNPs were prepared by the classical citrate reduction route.56 Briefly, 150 mL of an aqueous chlorauric acid solution (0.5 mM) was brought to a boil, and 8.5 mL of 2% sodium citrate solution was added. The solution first changed to a bluish color, then to a purplish color, and eventually to ruby-red. The solution was further boiled for 15 min and then left to cool to room temperature. The concentration of the as-prepared AuNPs was measured by absorbance to be 15 nM approximately and denoted to be 1×. Preparation of Alkanethiol-Capped AuNPs. The surface modification of AuNPs using 16-MHDA was done as a description by Radhakumary.57 The synthesized AuNPs solutions were degassed with nitrogen before use to prevent the oxidation of the alkanethiol. Equal volumes of AuNPs and Tween 20 (2 mg/mL) in phosphate buffer at pH 7.4 were gently mixed and allowed to stand for a minimum of 20 min to allow for the physisorption of Tween 20 to AuNPs. Then 16MHDA (0.5 mM, 0.6 mL) solution was added into the above AuNPs solution (10 mL), and the final mixture was allowed to stand overnight in order to allow for 16-MHDA to be chemisorbed onto the AuNPs. Unreacted 16-MHDA was removed by repeated centrifugation for 15 min at 14000 rpm followed by decantation of supernatants and resuspension in a PBS buffer (pH = 7.4, 10 mL). Conjugation of Apo-GOx on Alkanethiol-Capped AuNPs (AuNPs-apo-GOx). The preparation and activity measurement of apo-GOx are provided in the Supporting Information. Immobilization of apo-GOx with 16-MHDAcapped gold nanoparticles was done on the basis of the EDC/ NHS chemistry via the formation of an amide linkage between the carboxyl groups of the 16-MHDA and the primary amine groups of the apo-GOx, following procedures similar to those given in the literature.35 Briefly, 0.5 mg EDC and 0.25 mg NHS were added to 2 mL of the stock 16-MHDA-capped AuNPs solutions with continuous gentle mixing for 30 min at room temperature. Then, centrifugation (13000 rpm, 15 min) and resuspension in PBS buffer (pH = 7.4) was repeated to a final volume of 2 mL. After that, apo-GOx (0.5 mg/mL, 0.2 mL) was added and was continuously stirred at room temperature for 2 h. Then the reaction continued overnight at 4 °C. The apo-

In this article, a biofriendly apo-GOx (inactive form of glucose oxidase)-modified nanoprobe based on a FRET system utilizing AuNPs as an acceptor and FITC (fluorescein isothiocyanate) as a donor is constructed to realize the highly sensitive detection and imaging of glucose in living cells. The glucose sensing principle is illustrated in Scheme 1. Compared Scheme 1. Glucose Sensing Principle Based on the Assembled AuNPs-apo-GOx-Dextran-FITC Nanoprobe

with a small molecule, the gold nanoparticle possesses a higher extinction coefficient and was chosen as the acceptor of FRET.48−50 It also acted as a carrier for the biomacromolecules in the proposed probe due to its reliable cellular uptake ability.51,52 Apo-GOx, the catalytically inactive form of glucose oxidase, is highly specific to glucose.53 On the other hand, apoGOx still preserves the ability of interaction with carbohydrates containing the glucopyranosyl subunits such as dextran.54 Hence, dextran-FITC was selected to link apo-GOx-modified AuNPs to form the assembled nanoprobe for glucose. An efficient FRET from FITC to AuNPs exists within the nanoprobe in the absence of glucose, thus no fluorescence could be observed. Upon introduction of glucose into the sensing system, glucose competes with dextran-FITC on the binding sites of apo-GOx and displaces the dextran−FITC segment in the nanoprobe,55 which results in the fluorescence recovery of FITC. In this system, exogenous H2O2, which may exert effects on normal physiological events in cells, is not produced as a result of the suppressed glucose oxidation when apo-GOx is used. The larger formation constant of glucose and apo-GOx could make sure the high selectivity of this sensor and ultrasensitive quantitative detection with a detection limit of 5.0 nM could be realized due to the high fluorescence quantum yield of FITC and the high extinction coefficient of AuNPs. Because of the low toxicity of this detection system and reliable cellular uptake ability of the nanoprobe when AuNP was chosen as a carrier, the method was successfully used for the imaging of glucose consumption in cancer cells. This high sensitivity and specificity of the nanoprobe make it possible to visualize glucose at low concentration when cells experience glucose deprivation. The new, simple, and turn-on assay may supply a potential method in biomedical studies and diagnosis of various diseases, especially metabolic diseases.

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EXPERIMENTAL SECTION Materials and Apparatus. Glucose oxidase (GOx), trisodium citrate, Tween 20, 16-mercaptohexadecanoic acid (16-MHDA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide B



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were acquired on a confocal laser scanning microscope (LEICA TCS SPE) using a 488 nm laser excitation source.

GOx-modified AuNPs were separated from the solution by removing free apo-GOx through ultrafiltration under centrifugation at 12000 rpm for 10 min with a Nanosep filter (Millipore YM-30, USA). The final AuNPs-apo-GOx products were dissolved in PBS to give a fixed concentration (2 mL, 1×), ready to be used in the following fluorescence assays. Glucose Sensing by Fluorescence Detection. DextranFITC solution (1 mg/mL, 0.6 mL) was added to the prepared AuNPs-apo-GOx solution (3 mL) for an equilibration period of 2.5 h. Then excess dextran-FITC was removed by repeated centrifugation (13000 rpm, 10 min) followed by decantation of supernatants and resuspension in PBS buffer (3 mL). Under this condition, the nanoprobe AuNPs-apo-GOx-dextran-FITC was formed and could be used for the subsequent glucose sensing. Afterward, the above sensing solution (200 μL), PBS buffer solution (0.1 mL, 10 mM, pH 7.4) and different concentrations of glucose were added to the 1.5 mL microtube, and each solution was diluted with ultrapure water to a final volume of 1.00 mL. After reaction at 37 °C for 25 min, the fluorescence spectra were obtained in the spectral range from 500 to 600 nm by use of the maximal excitation wavelength at 492 nm. Cell Culture. The human hepatoma cell line (HepG2) was obtained from American Type Culture Collection, Manassas. Cells were cultured in RPMI 1640 (Hyclone, penicillin 100 units/mL, streptomycin 100 μg/mL) plus 10% fetal bovine serum (FBS, Gibco) and maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Cell Lysate Preparation Using Ultrasonic Treatment. The cell suspension (1 mL) was washed five times by centrifugation for 5 min at 1000 rpm, and the pellet was resuspended in 1 mL PBS. The number of cells in the final cell suspension was counted using a hemocytometer. Then the cells were disrupted for 10 min in a VC 130PB ultrasonic disintegrater (Sonics & Materials Inc.). During sonic disruption, the temperature was maintained below 4 °C with circulating ice water. The broken cell suspension was centrifuged at 14000 rpm at 4 °C for 30 min and the pellet was discarded. The cleared lysate was carefully transferred to a fresh tube and stored at −80 °C until use. MTT Assay. To investigate the cytotoxicity of the nanoprobe, a MTT assay was carried out when the nanoprobe existed. HepG2 cells (1 × 106 cells/well) were dispersed within replicate 96-well microtiter plates to a total volume of 200 μL well−1. Plates were maintained at 37 °C in a 5% CO2/95% air incubator for 24 h. The nanoprobe was added to each well after the original medium was removed. HepG2 cells were incubated with the probe for 0, 12, 24, and 48 h or with different concentrations of probe for 24 h with or without glucose. Then, 100 μL MTT solutions (0.5 mg mL−1 in PBS) were added to each well. After 4 h, the remaining MTT solution was removed, and 150 μL of DMSO was added to each well to dissolve the formazan crystals. The absorbance was measured at 490 nm with a RT 6000 microplate reader. Imaging Intracelluar Glucose. First, the cells were cultured in RPMI 1640 for 2 days and then incubated with glucose-free RPMI 1640 medium for different time periods. Next, 30 μL of glucose-free RPMI 1640 medium containing 0.5% CTAB was added to the cells and incubated for 10 min to enhance membrane permeability. After the CTAB was washed three times with glucose-free RPMI 1640 medium, cells were incubated with 0.2× the nanoprobe for 30 min and then the cells were washed three times with PBS. Fluorescence images

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RESULTS AND DISCUSSION Characterization and Evaluation of the Glucose Nanoprobe. To assemble the nanoprobe, AuNPs were synthesized with citrate as the stabilizer and exhibited a typical plasmon absorption peak at 520 nm (line a in Figure 1A). The

Figure 1. (A) Normalized optical absorption spectra of AuNPs (curve a), 16-MHDA-Au (curve b), AuNPs-apo-GOx (curve c), and the AuNPs-apo-GOx-dextran-FITC nanoprobe (curve d). (B) Fluorescence spectra of AuNPs-apo-GOx (curve a), AuNPs-apo-GOx-dextranFITC nanoprobe in the absence (curve b) and presence (curve c) of glucose. (C) TEM image of AuNPs. (D) TEM image of AuNPs-apoGOx. Solutions were prepared in the PBS buffer (10 mM, pH 7.4). The concentration of glucose was 0.12 μM.

TEM image (Figure 1C) indicated an average size of 10 nm and verified the formation of spherical AuNPs. The absorption maxima further shifted to 524 nm upon modification with 16MHDA (line b in Figure 1A), indicating an increase in the size of AuNPs. After conjugation with apo-GOx, the maximum absorption peak of AuNPs-apo-GOx red shifted to 535 nm (line c in Figure 1A); formation of a thicker monolayer around the nanoparticles could be observed from the TEM image (Figure 1D), demonstrating the successful surfactant modification of AuNPs with apo-GOx. After the reaction between apo-GOx and C



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dextran, the final probe AuNPs-apo-GOx-dextran-FITC was formed. It exhibited two absorption peaks; one was at 535 nm, indicating the existence of apo-GOx-modified AuNPs, and the other absorption peak at 499 nm (line d in Figure 1A) was consistent with FITC, verifying the combination of AuNPs-apoGOx and dextran-FITC. On the basis of the previously established method,58 each AuNP was calculated to carry 4 ± 1 dextran-FITCs. Details of the characterization are provided in Figure S2 of the Supporting Information. The fluorescence emission of the AuNPs-apo-GOx, AuNPs-apo-GOx-dextranFITC nanoprobe in the absence and presence of glucose was also measured and illustrated in Figure 1B. There was no fluorescence emission from AuNPs-apo-GOx. After incubation with AuNPs-apo-GOx and repeated centrifugation to remove excess dextran-FITC, the fluorescence intensity of dextranFITC was faint as a result of quenching by AuNPs with high extinction coefficients, which also indicated the formation of AuNP-apo-GOx-dextran-FITC. After glucose was added, significant fluorescence recovery at the wavelength of 514 nm was observed and can be explained that glucose dissociated the combination of AuNPs-apo-GOx and dextran-FITC due to stronger binding ability between apo-GOx and glucose. The FRET mechanism of this nanoprobe was confirmed, and fluorescence quenching is described by the well-known Stern− Volmer equation (Figure S3 of the Supporting Information);59 the Stern−Volmer quenching constant KSV = 3.33× 108 M−1 demonstrates highly efficient quenching of FITC by gold nanoparticles, which is several orders of magnitude more efficient than typical small molecule dye−quencher pairs.50 Furthermore, the FRET efficiency of the nanoprobe was estimated from time-resolved fluorescence measurements.60 Using the average lifetime values of the free dextran-FITC and dextran-FITC after conjugation to AuNPs-apo-GOx, we estimated the FRET efficiency to be 0.46 for the nanoprobe. Sensitivity of the Nanoprobe for Glucose. Experimental conditions, including volume of probe solution and time response of the assay, were optimized to get an excellent performance of the nanoprobe (Figure S5 of the Supporting Information). Under optimal experimental conditions, we monitored changes of fluorescence spectra of the probe in the presence of different concentrations of glucose (Figure 2). As expected, there is a low background fluorescence before the

addition of glucose due to the high quenching efficiency of AuNPs-apo-GOx. In the presence of glucose, glucose occupies the combination sites on apo-GOx followed by the fluorescence recovery of FITC. The increase of fluorescence intensity is proportional to the increase of glucose concentration in the range from 20 to 200 nM. A calibration curve of fluorescence signal versus concentration of glucose was obtained (inset in Figure 2) with a linear coefficient R = 0.9953 and a detection limit of glucose concentration as low as 5.0 nM (3σ, n = 10). The proposed nanoprobe has achieved one of the most sensitive approaches for glucose detection compared with other reported methods (Table 1). The low detection limit allows Table 1. Comparison for Glucose Sensing Methods detection methods

detection limit

sample type

references

chemiluminescent electrochemistry phosphorescence silica nanoparticles-based fluorescence QD-based fluorescence AuNPs-based fluorescence

4 μM 0.3 ± 0.1 μM 0.12 nM 4.4 μM

none urine serum serum

5 6 12 32

50 μM 5.0 nM

cell cell

38 this work

ultrasensitive accurate quantitation of glucose at low concentrations, which is of great significance in further glucose studies, such as when organisms experience glucose deprivation. Selectivity of the Nanoprobe for Glucose. The selectivity of the nanoprobe is fundamental to conduct the bioanalysis. Potential interference substances such as monosaccharides (fructose, galactose, etc.) and other biological species (such as metal ions, BSA, biothiols, and other amino acids, etc.) that exist inside cells were examined to assess the selectivity of the assembled nanoprobe for glucose. In the selectivity determination, the fluorescence responses of 0.10 μM glucose and interferential species with different concentrations in the range from 0.10 μM to 1000 μM were determined. When the error was controlled within ±5.0% in the relative fluorescence intensity, the tolerable maximum concentrations of interferential species were shown in Figure 3. It can be seen that the concentrations of the most serious interferential species, such as Al3+, Cu2+, Zn2+, and Fe3+, that did not influence glucose determination, were still 10-fold higher than that of glucose. Actually, in living cells, the concentrations of these interferential species are much lower than that of glucose. So, the nanoprobe could exhibit excellent selectivity in the determination of glucose in cells. Determination of Glucose in Cell Lysate. The nanoprobe was applied to quantify the glucose in cell lysate, which generally contains other carbohydrates, metal ions, and other biological substances. The HepG2 (human liver hepatocellular carcinoma) cells (1.6 × 106 /mL, 1 mL) were lysed in the ice bath using a VC 130PB ultrasonic disintegrator. The treated cell lysate samples were diluted 800 times with PBS buffer to ensure the concentration of glucose in the linear range and to obtain quantitative recovery of the spiked glucose. The concentration of glucose in the cell lysate was determined by the standard addition method using commercial glucose as the standard (Figure S6 of the Supporting Information). The results are listed in Table 2. The amount of glucose in HepG2 cells was calculated to be 16.5 fmol/cell, which was in good agreement with those reported in literature.1 The good recoveries of known amounts of glucose in the cell lysate

Figure 2. Fluorescence responses of the probe toward different concentrations of glucose. The inset displays the linear relationship between the fluorescence intensity and the concentrations of glucose. Concentrations of glucose from bottom to top are 0, 0.02, 0.05, 0.08, 0.10, 0.12, 0.14, 0.18, and 0.20 μM. D



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Figure 3. Fluorescence responses of the nanoprobe to various species: glucose (0.10 μM); sucrose, D-fructose, lactose, maltose, L-cystenine, glutathione, glycine, lysine, L-phenylalanine, tyrosine, tryptophan, BSA, cholesterol, ascorbic acid, mannitol (10 μM); K+ (1000 μM); Na+ (100 μM); Ca2+, Mg2+, Co2+, Fe2+ (10 μM); Al3+, Cu2+, Zn2+, and Fe3+ (1.0 μM). All spectra were acquired in 10 mM PBS with pH 7.4 at 37 °C.

Table 2. Determination of Glucose in Cell Lysate (n = 3) determined glucose in diluted sample (nM)

added glucose (nM)

measured glucose (nM)

recovery (%)

33

70.0 150

74 ± 2.6 156 ± 3.5

106 104

sample definitely demonstrate the accuracy and reliability of the present nanoprobe for glucose determination in practical applications. Evaluation of Cytotoxicity of the Nanoprobe. To evaluate the cytotoxicity of the nanoprobe, we performed a MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay in HepG2 cells as an example. The absorbance of MTT at 490 nm is dependent upon the degree of activation of the cells. The cell viability is then expressed by the ratio of the absorbance of the cells incubated with the nanoprobe to that of the cells incubated with the culture medium only. The results indicated that the nanoprobe showed almost no cytotoxicity or side effects in living cells within 12 h when the appropriate amount of probe was used (Figure S7 of the Supporting Information) and confirmed that the nanoprobe could be applied to intracellular glucose imaging. Confocal Imaging of Glucose in HepG2 Cells. To explore the potential of the assembled nanoprobe for real-world applications, fluorescence imaging for the glucose consumption in cancer cells was performed. HepG2 cells cultured in the medium without glucose was chosen as model samples. First, the cells were cultured in RPMI 1640 for 2 days and then incubated with glucose-free RPMI 1640 medium for 0, 4, and 12 h, respectively. Next, 10 μL of 0.5% CTAB was added to the cells to enhance membrane permeability. After the CTAB was washed away, the cells were incubated with the assembled nanoprobe for 30 min, and then the confocal imaging was performed, and the mean fluorescence intensity of cells was recorded. As shown in Figure 4, the HepG2 cells which were not treated by glucose-free medium (represented as 0 h)

Figure 4. (A) Confocal fluorescence images and (B) normalized fluorescence intensity of HepG2 cells incubated with nanoprobe after treatment with glucose-free RPMI 1640 medium for 0, 4, and 12 h, respectively. The control represents cells without a nanoprobe. Scale bar = 10 μm.

showed bright fluorescence, indicating the high glucose concentration in the cells. A time-dependent decreased fluorescence was observed in cells treated with glucose-free medium for 4 to 12 h, which could be explained that the removal of external glucose affected the glucose uptake normally and the constant metabolism in cells reduced the glucose concentration. The faint fluorescence from cells in glucose-free medium for 12 h demonstrated that the assembled nanoprobe could monitor the glucose changes, even glucose concentration at very low level. Besides, no fluorescence emission was observed in control cells without a nanoprobe, proving that the low fluorescence from cells in glucose-free medium for 12 h did not come from auto fluorescence of the cells. Additionally, the confocal fluorescence images showed the glucose predominantly localized in the cytosol of HepG2, E



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which was in good agreement with those obtained in the literature.18

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CONCLUSIONS In summary, we have developed a new glucose-specific nanoprobe by taking advantage of competitive binding and modulation in FRET efficiency. The nanoprobe exhibits high sensitivity with a detection limit as low as 5.0 nM and excellent selectivity toward glucose over other monosaccharides and most biological species present in living cells. Because of the low toxicity of this detection system and reliable cellular uptake ability of AuNPs, consumption of cellular glucose has been successfully imaged with a fluorescence confocal microscopy. It is anticipated that the nanoprobe can be applied to the imaging of glucose at low level in living cells and may supply a potential tool for biomedicine research and diagnosis of various diseases, especially metabolic diseases.

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ASSOCIATED CONTENT

S Supporting Information *

Preparation of apo-GOx and its activity measurements, quantitation of dextran-FITC loaded on the nanoprobe, calculation of the FRET efficiency by fluorescence lifetime, confirmation of the FRET mechanism, optimal experimental conditions for glucose sensing, detection of glucose in cell lysate and evaluation of the cell viability. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: (86)531 86180010. Fax: (86)531 86180017. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the 973 Program (Grant 2013CB933800), the National Natural Science Foundation of China (Grants 21035003, 21227005, and 21205074), the Specialized Research Fund for the Doctoral Program of Higher Education of China (Grants 20113704130001 and 20123704120006), the Program for Changjiang Scholars and Innovative Research Team in University and Key Project of Chinese Ministry of Education (Grant 212102).

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