Quantitative Analysis of Glucose Metabolic Cleavage in Glucose

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Quantitative Analysis of Glucose Metabolic Cleavage in Glucose Transporters Overexpressed Cancer Cells by Target-Specific Fluorescent Gold Nanoclusters Tsai-Mu Cheng, Hsueh-Liang Chu, Yi-Cheng Lee, Di-Yan Wang, Che-Chang Chang, KuanLan Chung, Hung-Chi Yen, Chu-Wen Hsiao, Xi-Yu Pan, Tsung-Rong Kuo, and Chia-Chun Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04961 • Publication Date (Web): 21 Feb 2018 Downloaded from http://pubs.acs.org on February 27, 2018

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Analytical Chemistry

Quantitative Analysis of Glucose Metabolic Cleavage in Glucose Transporters Overexpressed Cancer Cells by Target-Specific Fluorescent Gold Nanoclusters

Tsai-Mu Cheng1, Hsueh-Liang Chu1, Yi-Cheng Lee2, Di-Yan Wang3, Che-Chang Chang1, Kuan-Lan Chung4, Hung-Chi Yen4, Chu-Wen Hsiao4, Xi-Yu Pan5, Tsung-Rong Kuo5,6*, Chia-Chun Chen4*

1

Graduate Institute of Translational Medicine, College of Medicine and Technology,

Taipei Medical University, Taipei 11031, Taiwan 2

Green Energy and Environment Research Laboratories, Industrial Technology

Research Institute, Hsinchu 31040, Taiwan 3

Department of Chemistry, Tunghai University, Taichung 40704, Taiwan

4

Department of Chemistry, National Taiwan Normal University, Taipei 11677, Taiwan

5

Graduate Institute of Nanomedicine and Medical Engineering, College of

Biomedical Engineering, Taipei Medical University, Taipei 11031, Taiwan 6

International Ph.D. Program in Biomedical Engineering, College of Biomedical

Engineering, Taipei Medical University, Taipei 11031, Taiwan

Corresponding Authors E-mail: [email protected] and [email protected]

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Abstract The glucose metabolism rate in cancer cells is a crucial piece of information for the cancer aggressiveness. A feasible method to monitor processes of oncogenic mutations has been demonstrated in this work. The fluorescent gold nanoclusters conjugated with glucose (glucose-AuNCs) were successfully synthesized as a cancer-targeting probe for glucose transporters (Gluts) overexpressed by U-87 MG cancer cells, which can be observed under confocal microscopy. The structural and optical characterizations of fluorescent glucose-AuNCs were confirmed by transmission electron microscope (TEM) and Fourier transform infrared spectroscopy (FTIR). The MTT assay exhibited the high biocompatibility of water-soluble glucose-AuNCs for further biomedical applications. The glucose metabolic cleavage of glucose-AuNCs by glycolytic enzymes from U-87 MG cancer cell was measured by fluorescence change of glucose-AuNCs. The fluorescence change based on the integrated area under fluorescence spectra (At) of glucose-AuNCs was plotted as a function of different reaction time (t) with glycolytic enzymes. The fitted curve of At versus t showed the first-order kinetics to explain the mechanism of glucose metabolic cleavage rate of glucose-AuNCs by glycolytic enzymes. The rate constant k could be utilized to determine the glucose metabolism rate of glucose-AuNCs for the quantitative analysis of cancer aggressiveness. Our work provides a practical application of target-specific glucose-AuNCs as a fluorescence probe to analyze the glucose metabolism in Gluts overexpressed cancer cells.


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Introduction In the oncogenic mutations, the uptake of glucose has been dramatically increased to meet the bioenergetic demands of cell growth and proliferation.1-6 The Warburg effect has shown that even in the presence of oxygen, cancer cells metabolize glucose by aerobic glycolysis that is different from that of normal cells.7-13 Recently, the increase of glucose metabolism of cancer cells has been applied for the theranostic applications.14-19 For example, cellular glycolysis in the primary breast tumor using

18

F-fluoro-2-deoxy-D-glucose positron emission tomography/computed

tomography has been demonstrated the association with final histopathologic status after neoadjuvant chemotherapy, with greater standard uptake value max values for good responsible compared to the less responsible lesions.20 Glucose-conjugated chitosan nanoparticles have been developed for specific recognition and interaction with glucose transporters (Gluts) overexpressed by 4T1 murine mammary carcinoma cells.21 Glucose-coated iron oxide nanoparticles have electively internalized by the overexpressed Gluts pancreatic adenocarcinoma BxPC3 cells.22 Many previous studies have demonstrated the high uptake of glucose-based nanomaterials with the overexpression of Gluts in a wide variety of malignancies. Furthermore, the overexpression of Gluts has been verified to be associated with tumor progression in various malignant tumors.23-27 Therefore, Gluts could be used as a promising pathway to deliver nanomaterials inside cancer cells. Although many glucose-based nanomaterials have been applied to image cancer cells by the pathway of Gluts, there is still lack of the design of glucose-based nanomaterials to detect the tumor progression. More importantly, in the high grade cancer, the aerobic glycolytic metabolism has correlated with tumor aggressiveness.28-31 The development of glucose-based nanomaterials is urgently required for the target-specific imaging and evaluation of aerobic glycolytic metabolism in the cancer cells with Gluts



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overexpression. The advancements of fluorescent inorganic nanomaterials for the cellular targeting and imaging are important for cancer diagnosis in current clinical applications.32-38 Various types of fluorescent inorganic nanomaterials included metals, metal oxide and semiconductor have been developed as contrast agents for fluorescence imaging during the last decade.39-42 Furthermore, by the surface modifications with antibody, peptide, aptamer or small molecule, the inorganic nanomaterials have been utilized as fluorescent molecular imaging agents for specific cell targeting.43-45 For example, BRCAA1 monoclonal antibody-conjugated Fe3O4-CdTe nanomaterials have been used to image their distribution in gastric cancer tissues by targeted fluorescence imaging and magnetic resonance imaging.46 Europium-doped gadolinium oxide nanoparticles conjugated with nucleolin-targeted AS1411 aptamer have been used as a trimodal contrast agent for cancer-targeting molecular imaging with fluorescence, computed tomography and magnetic resonance.47 ZnS-capped CdSe core-shell quantum dots conjugated with triblock copolymer and monoclonal antibody (J591) have shown sensitive and multicolor fluorescence for prostate cancer targeting and imaging in living animals.48 Compared with traditional organic fluorophores, fluorescent inorganic nanomaterials could provide sharp images, long-term imaging times, high photostability and multiple imaging modalities. With the surface modification of glucose, inorganic nanomaterials can be designed as a fluorescent molecular contrast agent for the overexpressed Gluts cancer cells.

With

tunable

fluorescence,

such

fluorescent

inorganic-based

nanomaterials are very promising candidates for applications spanning from primary tumor detection to further evaluation of tumor aggressiveness by aerobic glycolytic metabolism in research and clinics. The inorganic nanomaterials of gold nanoclusters (AuNCs) composed by tens of


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gold atoms have attracted intensive attention as fluorescent probes because of their ultrafine size, excellent photostability, low toxicity and high water solubility.49-53 Great efforts have been made in the applications of fluorescent AuNCs for detection and cell imaging.54-57 For the application of protease detection, the decrease in fluorescence intensity of bovine serum albumin-AuNCs caused trypsin has been applied for the detection of trypsin in the range of 0.01-100 g/mL.58 Molecular contrast agent of AuNCs with orange-red fluorescence has been demonstrated as a fluorescent probe to reveal the tumor location in CL1-5 tumor-bearing mice.59 Target-specific probe of AuNCs conjugated with cyclic arginine-glycine-aspartic acid peptide has been exploited to contrast the integrin αvβ3 overexpressed melanoma A375 cells using fluorescence imaging.60 The increasing developments of AuNCs as fluorescent molecular contrast agents have brought significant impacts on the improvement of diagnostic accuracy for cancer detection. Up to now, fluorescent AuNCs can be easily synthesized with ligands included phosphine, thiol, DNA, dendrimer, peptide and protein.61-64 Among the gold clusters, thiolate-protected AuNCs have been intensively investigated because of their well-defined structure, highly stable optical property and facile surface modification.65-69 Moreover, the fluorescence intensity and fluorescence wavelength of AuNCs can also be tuned by manipulating the number of gold atoms in the core and ligand on the surface. Therefore, surface modification of glucose onto thiolate-based AuNCs could be a potential candidate as a target-specific fluorescent probe to image cancer cells with Gluts overexpression. Additionally, the fluorescence change of AuNCs caused by the metabolism of glucose on the AuNCs surface could also be used to evaluate the aggressiveness of Gluts overexpressed cancer cells. However, to the best of our knowledge, there is lack of the development of glucose-conjugated AuNCs for target-specific fluorescence imaging and even to assist the evaluation of the



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aggressiveness for Gluts overexpressed cancer cells. In this work, glucose conjugated gold nanoclusters (glucose-AuNCs) were synthesized

and

well characterized.

The

water-soluble

and

biocompatible

glucose-AuNCs were applied for cancer-targeting molecular fluorescence imaging in Gluts overexpressed U-87 MG cancer cell line by confocal microscopy. Furthermore, the potential application of glucose-AuNCs in quantitative analysis of glucose metabolism was investigated by the change of fluorescence intensity caused by glycolytic enzymes form U-87 MG cancer cells. The kinetics behavior of glucose metabolic cleavage for glucose-AuNCs was also measured via curve fitting from the plot of the fluorescence change versus reaction time.

Experimental Section Preparation of Glucose-Conjugated Gold Nanoclusters The glucose-conjugated gold nanoclusters (glucose-AuNCs) were prepared by a two-step approach. First, AuNCs were synthesized with organic ligand of glutathione by previous one-pot green method with some modifications.70 Briefly, 30 mL of HAuCl4 aqueous solution (1% w/w) was added to 30 mL of 25 mM L-glutathione aqueous solution in water bath at 40 °C under vigorous stirring. In the beginning, the color of HAuCl4 and L-glutathione solution changed from yellow to dark brown. The HAuCl4 and L-glutathione solution was then reacted under vigorous stirring in water bath at 40°C in the dark environment for 6 days. After 6 days, the stable fluorescence intensity of AuNCs indicated that the preparation of AuNCs was completed. The fluorescence spectra of AuNCs with different reaction time were shown in the supporting information as Figure S1. The pale yellow solution contained AuNCs was obtained. For further purification, AuNCs solution was subsequently centrifuged at 15000 rpm for 5 min. The supernatant contained AuNCs was precipitated by adding


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ethanol (two volumes of supernatant). Afterward, the yellow cloudy mixture was centrifuged at 18000 rpm for 10 min. After centrifugation, AuNCs were precipitated at the bottom of the centrifuge tube. The precipitate of AuNCs was redispersed in deionized water. Secondly, to prepare glucose-AuNCs, 30 mL of AuNCs solution (0.22 mM) was added to 30 mL of 1-Thio-β-D-glucose sodium salt solution (0.64 mM). After 24 h, the yellow solution of glucose-AuNCs was added to ethanol (two volumes of glucose-AuNCs solution) and then centrifuged at 20000 rpm for 45 min. The precipitate of glucose-AuNCs was redispersed in deionized water. Finally, the solution of glucose-AuNCs was stored at 4°C for following experiments.

Cell Culture Protocols of U-87 MG and SVG p12 Cell Lines The U-87 MG and SVG p12 cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM) medium (Gibco; Thermo Fisher Scientific Inc., Waltham, MA) supplemented with 10% fetal bovine serum (FBS) (HyClone; GE Healthcare Life Sciences, Logan, UT), penicillin (100 unit/mL), and streptomycin (100 g/mL).

Cell Viability Assays of AuNCs and Glucose-AuNCs by U-87 MG Cells Cell

viability

was

determined

by

the

3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT) dye reduction assay. U-87 MG cells at 1×104 cells/well were cultured in 96-well plates (Falcon, Becton Dickinson, NJ) for 24 h and then washed twice with PBS. Afterward, U-87 MG cells were incubated in α-DMEM medium and treated with AuNCs and glucose-AuNCs (final concentrations at 100, 50, 25, 12.5 and 6.25 µg/mL) for 24 h. For the cell viability assay, serum-free α-DMEM medium containing MTT (0.5 mg/mL) was added to each well and then incubated for 4 h in the absence of light. After 4 h, the medium was removed and precipitates were dissolved in DMSO (100 µL). Absorbance at 565 nm was measured using a plate



reader. Experiments were performed in quadruplicate.

Confocal Fluorescence Microscopy for AuNCs and Glucose-AuNCs U-87 MG and SVG p12 cells were seeded onto coverslips and cultured in 6-well culture plates (2×105 cells per well) for 18 h, then washed twice with PBS, incubated in DMEM medium and treated with the AuNCs and glucose-AuNCs (100 µg/mL) for 24 h. The cells were washed twice with PBS to remove free AuNCs and glucose-AuNCs and fixed by paraformaldehyde (4%). Permeabilization of the cell membrane was induced by Triton X-100 (0.25%). Nuclei and actin were stained with DAPI (Sigma, St. Louis, MO) and Alexa Fluor 488 phalloidin (Thermo Fisher Scientific Inc., Waltham, MA), respectively. The fluorescence of AuNCs, glucose-AuNCs, actin, and nuclei were observed by confocal fluorescence microscopy (TCS-SP5, Leica Microsystems, Solms, Germany).

Glycolytic Enzymes Extraction from U-87 MG cell line Glycolytic enzymes are essential for Glioblastoma Multiforme (GBM) proliferation. U-87 MG, a GBM cell line, was cultured in 10 cm dish (1×106 cells) for 48 h to 80% confluence, and then washed twice with phosphate buffered saline (PBS). Cells were suspended by trypsin and collected by centrifugation at 1000 g for 5 min. U-87 MG cancer cells were washed twice with PBS to remove residual trypsin and FBS. Cells were lysed by adding 500 µL distilled and deionized water and repeating the procedure of thawing and freezing for three times. The glucosidase containing cell lysate was obtained from supernatant by centrifuged at 12000 g for 5 min.

Results and Discussion Structural Characterizations of Glucose-AuNCs


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The syntheses of fluorescent glucose-AuNCs involved two major steps. First, AuNCs were prepared with the ligand of glutathione via one-pot green method. The TEM image of AuNCs was shown in the supporting information (Figure S2). Secondly, AuNCs were conjugated with 1-Thio-β-D-glucose to form glucose-AuNCs. The average size, shape and chemical composition of glucose-AuNCs were examined by TEM. The glucose-AuNCs exhibited an approximately spherical shape as shown in the TEM image of Figure 1a. The average sizes of AuNCs and glucose-AuNCs were respectively calculated to be 2.8 and 2.6 nm based on 100 nanoclusters in the TEM images. The average size of glucose-AuNCs was smaller than that of AuNCs. The reason can be ascribed to core etching of AuNCs by 1-Thio-β-D-glucose as demonstrated in previous studies.71,72 The histograms of nanocluster size distributions of AuNCs and glucose-AuNCs and their Gaussian fitting curves were provided in the supporting information as Figure S3 and Figure S4, respectively. In Figure 1b, the EDX analysis of glucose-AuNCs showed that the weight percentage of gold was 73.41%

in

the

glucose-AuNCs.

The

weight

ration

of

glutathione

to

1-Thio-β-D-glucose was calculated to be 0.45 (see supporting information). The TEM image and EDX analysis demonstrated that AuNCs were obtained by the two-step approach. The ESI-MS spectra of AuNCs and glucose-AuNCs were applied to calculate their molecular weights as shown in Figure S5 and Figure S6. To further confirm the conjugation of glucose with AuNCs, both AuNCs and glucose-AuNCs were characterized by FTIR. In the FTIR spectra of Figure 2, AuNCs exhibited the characteristic IR bands of amine (NH stretch; 3270 cm-1) and carbonyl (CO stretch; 1762 cm-1). These IR bands were provided by the glutathione. Also, the disappearance of the IR band of thiol (SH stretch; ~2526 cm-1) confirmed that the glutathione was attached covalently on the gold surface via the SH group. After the conjugation of glucose onto AuNCs surface, the glucose-AuNCs showed the



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characteristic IR bands of alcohol (OH stretch; 3268 cm-1), aldehyde (CO stretch; 1740 cm-1) and ether (CO stretch; 1000~1300 cm-1). The jagged peaks of CO stretching band (1740 cm-1) of glucose-AuNCs can be ascribed to that there were two CO

stretching

bands

from

carbonyl

of

glutathione

and

aldehyde

of

1-Thio-β-D-glucose. The jagged peaks of CO stretching band also indicated that glutathione and 1-Thio-β-D-glucose were both conjugated onto the surface of glucose-AuNCs. Overall, the IR data confirmed that, after adding 1-Thio-β-D-glucose into AuNCs, the glucose was successfully conjugated onto the surface of AuNCs to form glucose-AuNCs based on the Au-S bond.

Optical Properties of Glucose-AuNCs The UV-Vis absorption and fluorescence spectra were measured before and after AuNCs conjugated with glucose. In Figure 3a, the disappearance of the surface plasmon absorption of AuNCs and glucose-AuNCs was attributed to the high oxidation states of AuNCs. Therefore, AuNCs and glucose-AuNCs lack free electrons to generate the coherent oscillations.73,74 The XPS spectra showed that AuNCs and glucose-AuNCs contain lots of Au(I) (see supporting information, Figure S7). In Figure 3b, the fluorescence spectra of AuNCs (0.22 mM) and glucose-AuNCs (0.22 mM) showed the maximum fluorescence intensities at 609 nm and 610 nm, respectively. The details for calculations of concentrations of AuNCs and glucose-AuNCs were shown in the supporting information. The fluorescence quantum yields of AuNCs and glucose-AuNCs were separately calculated to be 0.68% and 1.2% in comparison with rhodamine 6G. The inset in Figure 3b showed the orange-red fluorescence of AuNCs and glucose-AuNCs with the irradiation by hand-held UV lamp. Many studies have proposed that the fluorescence of AuNCs could be ascribed to ligand-metal charge transfer between thiolate and AuNCs.75-77 During the


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ligand-metal charge transfer, the electrons were transferred from sulfur atom of surface glutathione to the core of AuNCs. Furthermore, in comparison with AuNCs, the increase of fluorescence quantum yield of glucose-AuNCs could be attributed to the increase of Au(I) on their surface after conjugation of 1-Thio-β-D-glucose because the binding energy of Au(4f5/2) of glucose-AuNCs was increased from 84.1 eV to 84.2 eV.78 Therefore, we supposed that the fluorescence intensity of glucose-AuNCs could be changed following the metabolism of glucose on the surface of glucose-AuNCs. This fluorescence property makes glucose-AuNCs as a potential indicator for quantitative analysis of the aggressiveness of Gluts overexpressed cancer cells.

Cell Viability Assay The cell viability assays of AuNCs and glucose-AuNCs were respectively evaluated in a U-87 MG cancer cell line by MTT assay. The water-soluble AuNCs and glucose-AuNCs with different concentrations (100, 50, 25, 12.5 and 6.25 µg/mL) were examined to investigate their cytotoxicities. In Figure 4, the MTT assay revealed high cell viabilities (> 90%) for AuNCs and glucose-AuNCs. The results indicated that AuNCs and glucose-AuNCs were both biocompatible for the U-87 MG cancer cells. We therefore studied the applications of AuNCs and glucose-AuNCs as molecular contrast agents for Gluts overexpressed U-87 MG cancer cells.

Targeting and Imaging of Gluts Overexpressed Cancer Cell The Gluts overexpressed human malignant glioma cell line (U-87 MG) and normal human fetal glial cell line (SVG p12) were used to investigate the target-specific fluorescence imaging by using AuNCs and glucose-AuNCs as fluorescent contrast agents. As shown in Figure 5a, the confocal microscope images showed no detectable fluorescence of AuNCs and glucose-AuNCs in the normal SVG



p12 cells. The results indicated that no efficient uptake of AuNCs and glucose-AuNCs happened by the normal SVG p12 cells. In Figure 5b, there is no fluorescence of AuNCs in the U-87 MG cancer cells with Gluts overexpression. In contrast, under the same imaging condition, the fluorescence of glucose-AuNCs (red pseudocolor) was clearly observed with high signal-to-background ratio in the Gluts overexpressed U-87 MG cancer cells. The result showed that only the fluorescent glucose-AuNCs were taken up by U-87 MG cells. Overall, it was suggested that glucose-AuNCs entered into the Gluts overexpressed U-87 MG cancer cells via the pathway of Gluts due to the glucose on the surface. Therefore, the results of fluorescence imaging clearly demonstrated that glucose-AuNCs exhibited the capability as a fluorescent contrast agent for the Gluts overexpressed cancer cells targeting.

Glucose Metabolism of Glucose-AuNCs by Glycolytic Enzymes from U-87 MG Cancer Cell To investigate the glucose metabolic cleavage of glucose-AuNCs, the glycolytic enzymes from U-87 MG cancer total cell lysate were added to the solution of glucose-AuNCs and then the fluorescence changes of glucose-AuNCs were measured with different reaction time. In the control experiments, there are no significant changes of fluorescence intensities of glucose-AuNCs with 25 mM EDTA and AuNCs with glycolytic enzymes from U-87 MG cancer cell as shown in the supporting information (Figure S8 and Figure S9). In contrast, as shown in Figure 6a, the fluorescence intensity of glucose-AuNCs decreased with the increase of reaction time. After 4 h, the steady state of the fluorescence intensity of glucose-AuNCs indicated that the glucose metabolic cleavage of glucose-AuNCs was accomplished by the glycolytic enzymes. The results showed that the fluorescence change of glucose-AuNCs caused by the glycolytic enzymes could be a promising fluorescent


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indicator to analyze the cancer aggressiveness. To further study the glucose metabolic cleavage rate of glucose-AuNCs by glycolytic enzymes from U-87 MG cancer cell, the integrated area under fluorescence spectra of glucose-AuNCs at different reaction time (At) was calculated as a reference of fluorescence intensity. The obtained At was plotted as a function of different reaction time as shown in Figure 6b. The curve of At versus t in Figure 6b was plotted based on the first-order kinetics: At = A∞+A*e-kt

The curve of At versus t showed an exponential decrease and reached the minimum A∞ of 242808 with the rate constant k of 0.84. The value of A∞ indicated that with the glucose metabolic cleavage by glycolytic enzymes, glucose on the surface of glucose-AuNCs was metabolized and then glucose-AuNCs were become AuNCs. The remained AuNCs also exhibited fluorescence to result in the value of A∞. Compared A0 with A∞, the change of the area under fluorescence spectra of glucose-AuNCs was 98082 as definition of A in the equation. The value of A showed that in our experiments, the maximal change of area under fluorescence spectra of glucose-AuNCs from t=0 to t=∞ was 98082. Overall, the fitted line of first-order kinetics was the most appropriate one to describe the mechanism of glucose metabolic cleavage of glucose-AuNCs by glycolytic enzymes from U-87 MG cancer cell. More importantly, in the equation, the rate constant k can be a promising parameter to reveal the glucose metabolic cleavage rate of glucose-AuNCs for quantitative analysis of the cancer aggressiveness.

Conclusions In summary, the fluorescent glucose-AuNCs were successfully developed with good



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water solubility and high biocompatibility. The glucose-AuNCs were also demonstrated as a target-specific fluorescent probe for Gluts overexpressed U-87 MG cancer cell line. The glucose metabolic cleavage of glucose-AuNCs by glycolytic enzymes from U-87 MG cancer cell was measured by the change of fluorescence intensity of glucose-AuNCs. The integrated area under fluorescence spectra of glucose-AuNCs was plotted as a function of reaction time. The fitted curve of At versus t based on the first-order kinetics was the most appropriate one to describe the mechanism of glucose metabolic cleavage of glucose-AuNCs by glycolytic enzymes. Moreover, in the equation of the first-order kinetics, the rate constant k could be a promising parameter to reveal the glucose metabolism rate of glucose-AuNCs for quantitative analysis of the cancer aggressiveness. Overall, our studies showed a new route for the analysis of glucose metabolism based on the target-specific fluorescent probe of glucose-AuNCs for Gluts overexpressed cancer cells.

Acknowledgements This

work

was

supported

by

MOST

105-2119-M-038-002-MY2,

MOST

106-2622-E-038-001-CC2, MOST 105-2314-B-038-016, Taipei Medical University and National Taiwan Normal University. The authors would like to acknowledge Ms. Yuan-Chin Hsiung for her excellent technical support at TMU Core Facility.

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Figures and Captions

Figure 1. (a) TEM image of glucose-AuNCs. The nanoclusters exhibit nearly spherical shape with the average size of 2.6 nm. (b) EDX analysis of glucose-AuNCs.


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Figure 2. The differences in between FTIR spectra of AuNCs (black) and glucose-AuNCs (red) confirm the glucose conjugation with AuNCs.

Figure 3. (a) UV-Vis absorption spectra of AuNCs (black) and glucose-AuNCs (red). (b) Fluorescence spectra of AuNCs (black) and glucose-AuNCs (red). The fluorescence spectra of AuNCs (0.22 mM) and glucose-AuNCs (0.22 mM) were measured at excitation wavelengths of 410 nm. The quantum yields of fluorescence from AuNCs and glucose-AuNCs were respectively calculated to be 0.68% and 1.2% using R6G as the standard. The inset in Figure 3b showed the orange-red fluorescence of AuNCs (left) and glucose-AuNCs (right) with the irradiation by hand-held UV lamp.



Figure 4. Evaluation of cell viability by MTT assay with the range of 100-6.25 µg/mL of AuNCs or glucose-AuNCs in U-87 MG cancer cells.


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Figure 5. Confocal microscopic images of (a) SVG p12 normal cells and (b) U-87 MG cancer cells. The blue, green and red pseudocolors represent the fluorescent signals of nuclei (stained with DAPI), actin (stained with Alexa Fluor 488 phalloidin) and glucose-AuNCs, respectively. In the control experiments, the cells were only cultured with DMEM medium. The scale bars are 50 µm.



Figure 6. (a) Fluorescence spectra of glucose-AuNCs with glycolytic enzymes from U-87 MG cancer cell at different reaction time. The fluorescence spectra of glucose-AuNCs were obtained with the excitation of 410 nm wavelength. (b) Plot of the integrated area under fluorescence spectra at different reaction time (At) of glucose-AuNCs as a function of different reaction time (t). The fitted curve of At versus t was plotted based on first-order kinetics. The value of R2 for the fitted curve is 0.99.


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TOC


Quantitative Analysis of Glucose Metabolic Cleavage in Glucose

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