Simple and Real-Time Colorimetric Assay for Glycosidases Activity

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Simple and real-time colorimetric assay for glycosidases activity using functionalized gold nanoparticles and its application for inhibitor screening Zhanghua Zeng, Shin Mizukami, and Kazuya Kikuchi Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 25 Sep 2012 Downloaded from http://pubs.acs.org on October 2, 2012

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Analytical Chemistry

Simple and real-time colorimetric assay for glycosidases activity using functionalized gold nanoparticles and its application for inhibitor screening Zhanghua Zeng, Shin Mizukami, and Kazuya Kikuchi* Division of Advanced Science and Biotechnology, Graduate School of Engineering, Osaka University 2-1 Yamadaoka, Osaka 565-0871, Japan [email protected] RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required according to the journal that you are submitting your paper to)

ACS Paragon Plus Environment

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ABSTRACT The development of real-time assays for enzymes has been receiving a great deal of attention in biomedical research recently. Self-immolative elimination is the spontaneous and irreversible disassembly of a multi-component construct into its constituent fragments through a cascade of elimination processes in response to external stimuli. Here, we reported a simple and real-time colorimetric assay for glycosidases (β-galactosidase and β-glucosidase). Self-immolative elimination was utilized to release amines to give rise to aggregation and color change by electrostatic attraction after cleavage of the trigger by enzymes displayed on functionalized gold nanoparticles (Gal-Lip-AuNPs and Glc-Lip-AuNPs). The detection limits for β-galactosidase and β-glucosidase were as low as 9.2 and 22.3 nM at 20 min, and they improved slightly over time. Thus, glycosidase activity was detected successfully in real time, and this technique could be used for glycosidase inhibitor screening based on real-time colorimetric variation.

KEYWORDS: Gold nanoparticles, Surface plasmon resonance, Glycosidase detection, Self-immolative, Colorimetric assay


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INTRODUCTION The development of highly sensitive, simple, rapid, and real-time methods for detecting enzyme activity is of critical importance in biomedical studies and early diagnosis of diseases. Glycosidases that exist in living systems catalyze the hydrolysis of glycosidic bonds in carbohydrates and glycoconjugates into low-molecular weight monosaccharides and oligosaccharides, and they are involved in various important biological processes.1-3 For instance, β-glucosidase is involved in metabolic diseases such as diabetes, viral or bacterial infections, and cancer, and β-galactosidase is involved in regulating the expression of the lac operon in E. coli.4-6 Probes to detect the activity of such glycosidases could be useful in evaluating their biological activity. Recently, several elegant probes that utilize fluorescence or MRI to detect glycosidase activity have been reported.7-20 However, the experimental procedures requiring instrument measurements hamper the simple utility.21 Therefore, the development of simpler methods to detect enzymatic activity is important. In 1997, Mirkin and co-workers developed gold nanoparticles (AuNPs) to probe polynucleotides by exploiting their distance-dependent optical change properties.22 AuNPs as colorimetric sensors have been gaining much attention in biomedical application because of their simplicity, rapidity, flexible synthesis, lack of toxicity to living system, and useful optical and electronic properties.23-27 The surface plasmon resonance (SPR) of AuNPs is affected by size alterations, the medium, and inter-particle distance. Such variation can be identified through changes in color, and SPR changes in the visible region depend on the concentration of the biomolecular analytes. This property of AuNPs was used to assay enzymatic activity.28-32 In this method, the enzymes hydrolyze their substrates to give free thiol ligands, which have a high affinity for gold atoms through the formation of Au−S bonds, which are stronger than non-covalent bonds. The thiol ligands bind to non-covalent ligand-capped AuNPs and induce their aggregation to result in a change in color. The disadvantage of this method is interference by intrinsic thiol-containing molecules such as glutathione (GSH), and the two-steps in the process hamper the real-time determination of enzyme activity. In contrast, enzyme substrate ligand-capped


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AuNPs are used in a one-step process, and they enable direct and practical determination of enzyme activity in real time.24,33,34 Self-immolative elimination is the spontaneous and irreversible disassembly of a multi-component construct into its constituent fragments through a cascade of electronic elimination processes in response to external stimuli. This process has been extensively used to design prodrugs, because the active molecule can be released by cleavage of the protecting group.35,36 The release of active molecules by self-immolative elimination prompted us to consider the feasibility of applying this process to the surface of AuNPs to assay real-time enzyme activity. Here, we designed and synthesized two lipoic acid derivatives (Scheme 1) in which disulfide moieties could be anchored onto gold atoms, and moieties with water-soluble sugar protecting groups could be cleaved by glycosidases (β-galactosidase and β-glucosidase) to induce self-immolative elimination through the p-hydroxybenzyl alcohol linker to produce primary amines. Under physiological conditions at pH 7.4, the amine group should be positively charged, and the positively charged ammonium can interact with negatively charged lipoate groups on neighboring nanoparticles. This interaction results in aggregation when the lipoate and amino group-modified lipoic acid derivative are co-coated on AuNPs. As a result, the aggregation induces a colorimetric change from wine red to blue.37

EXPERIMENTAL SECTION Materials and instruments. General chemicals were purchased from Tokyo Chemical Industries, Wako Pure Chemical, or Aldrich Chemical Company and were used without further purification. β-Galactosidase

from

E.

coli,

(1S,6S,7R,8R,8aR)-1,6,7,8-tetrahydroxyindolizidine

β-glucosidase (castanospermine)

from were

almonds, purchased

and from

Sigma-Aldrich. 1H NMR and 13C NMR spectra were recorded on a JEOL JNM-LA400 instrument (300 MHz for 1H NMR and 75 MHz for 13C NMR). Electrospray ionization mass spectra were obtained on a Waters LCT-Premier XE. UV-Vis spectra were measured on a Shimadzu UV-2450. Dynamic light


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scattering (DLS) for size distribution, and ζ-potential were measured on a Horiba nano Partica SZ-100 analyzer. Preparation of citrate-capped gold nanoparticles. Citrate-capped gold nanoparticles were synthesized following previously described methods.38 First, 30 mL of a 2.0 mM aqueous solution of hydrogen tetrachloroaurate hydrate was refluxed for 30 min, and then, 3.5 mL of 75 mM of sodium citrate was quickly added. The color of the solution changed from yellow to wine red. The mixture was boiled for another 30 min and then cooled to room temperature. Preparation of Gal-Lip-AuNPs and Glc-Lip-AuNPs. Ten milliliters of the prepared citrate-capped gold nanoparticles was centrifuged at 10,000 rpm. The pellet was collected and re-dispersed in Millipore water; this process was repeated 3 times. The pellet was collected and re-suspended in 5 mL of PBS (pH 7.4, 50 mM, 10 mM KCl, 10 mM MgCl2). Five milliliters of 0.1 mM Gal-Lip (Glc-Lip) in PBS and 5 mL of 0.1 mM lipoic acid in PBS were mixed and quickly added to the citrate-capped gold nanoparticle solution. The mixture was stirred at room temperature for 4 h. The insoluble residue was filtered out, and the filtrate was centrifuged at 10,000 rpm. The pellet was collected and re-dispersed in PBS. The obtained pellet was re-dispersed in 10 mL PBS after 3 rounds of centrifugation and dispersion. The final pellet was collected and re-dispersed in PBS. Gal-Lip-AuNPs and Glc-Lip-AuNPs were very stable in PBS and could be stored at 4°C for more than 3 months. Enzymatic assay using the absorbance of Gal-Lip-AuNPs and Glc-Lip-AuNPs. Different volumes of enzyme stock solution were added to 2.0 nM Gal-Lip-AuNPs and Glc-Lip-AuNPs in PBS (pH 7.4, 50 mM, 10 mM KCl, 10 mM MgCl2) at 37°C. The concentrations of β-galactosidase and β-glucosidase were 0, 0.01, 0.02, 0.08, 0.3, 1.0, and 2.5 µM. The enzymatic reaction was conducted in a UV-Vis spectrometer by incubating Gal-Lip-AuNPs (Glc-Lip-AuNPs) together with the different concentrations of the enzymes at 37 °C. Dynamic light scattering (DLS) for size distribution and ζ-potential. Gal-Lip-AuNPs and Glc-Lip-AuNPs (2 nM) in PBS (pH 7.4, 50 mM, 10 mM KCl, 10 mM MgCl2) and 1.0 µM


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β-galactosidase and β-glucosidase were incubated at 37°C. The time course of the size distribution and ζ-potential of the nanoparticles interacting with the enzymes were measured. Detection of galactose (glucose) using Amplex® Red Galactose (Glucose) and galactose (glucose) oxidase assay. Each reaction contained 50 µM Amplex® Red reagent, 1.0 U/mL horseradish peroxidase (HRP), 2.0 U/mL galactose (glucose) oxidase, and the indicated amount of galactose (glucose) in reaction buffer. Reaction solutions were incubated at 37°C. After 60 min, fluorescence was measured using excitation at 530 nm and fluorescence detection at 582 nm. The fluorescence change at 582 nm demonstrated sensitivity and linearity of the assay at low levels (0–15 µM) of galactose (glucose). The concentration of an analyte containing galactose (glucose) could be obtained within 15 µM using this method (Figure S11). Determination of enzymatic reaction yield on the surface of AuNPs. Ten milliliters of the prepared citrate-capped gold nanoparticle was centrifuged at 10,000 rpm. The pellet was collected and re-suspended in 5 mL of PBS (pH 7.4, 50 mM, 10 mM KCl, 10 mM MgCl2). Five milliliters of 0.1 mM Gal-Lip (Glc-Lip) in PBS and 5 mL of 0.1 mM lipoic acid in PBS were mixed and quickly added to the citrate-capped gold nanoparticle solution. The mixture was stirred at room temperature for 4 h. The insoluble residue was filtered out, and the filtrate was centrifuged for 20 min at 13,000 rpm. The pellet and supernatant were collected separately after 3 rounds of centrifugation and re-dispersion. The obtained supernatants were combined, and the concentration of Gal-Lip (Glc-Lip) was determined using Amplex red reagent. The concentration of Gal-Lip (Glc-Lip) anchoring AuNPs was quantified according to the amount of galactose (glucose) in the original and surplus Gal-Lip (Glc-Lip) (Scheme S3 and Scheme S4). The pellet (Gal-Lip-AuNPs and Glc-Lip-AuNPs) was re-dispersed in PBS for enzymatic reaction. Gal-Lip-AuNPs (Glc-Lip-AuNPs) were re-dispersed with various concentrations of β-galactosidase (β-glucosidase) in PBS (pH 7.4, 50 mM, 10 mM KCl, 10 mM MgCl2) at 37°C. The AuNPs solution was centrifuged at different reaction times. The pellet and supernatant were collected separately after 3


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rounds of centrifugation and re-dispersion. The combined supernatants were concentrated by lyophilization and re-dissolved in an appropriate volume of PBS. The supernatant containing galactose (glucose) was subjected to the Amplex red reagent method to quantify the concentration. The enzymatic reaction yield (conversion) at different times was determined by the amount of galactose (glucose) in the absence and presence of the enzyme (Scheme S4). Inhibition assay for enzymatic activity using Gal-Lip-AuNPs and Glc-Lip-AuNPs. In the inhibition assay for the enzymes, the mixed solution containing 1.0 µM enzyme (β-galactosidase and β-glucosidase), different concentrations of inhibitors (0.001, 0.01, 0.1, 1.0, 10, 50, 400, 1000, and 2000 µM D-galactal or castanospermine) and 2.0 nM Gal-Lip-AuNPs or Glc-Lip-AuNPs was incubated for 20 min at 37°C. Pictures were taken, and the absorbance of each sample was recorded using a UV-Vis spectrophotometer. The IC50 value was determined based on the titration curve of the absorbance at 700 nm for each sample versus the logarithmic inhibitor concentration.

RESULTS AND DISCUSSION The synthetic routes to obtain the glycosidase substrate ligands (Gal-Lip) are summarized in Scheme S1. Glc-Lip was obtained following the same route. Details regarding the synthesis, as well as the characterization of intermediates and substrate ligands, are provided in the Supporting Information. Real-time colorimetric assay for glycosidases. To verify the self-immolative elimination induced by the enzymes on the surface of the AuNPs, the SPR change of functionalized AuNPs at different time points was measured. The functionalized gold nanoparticles (Gal-Lip-AuNPs or Glc-Lip-AuNPs, 2.0 nM) capped with the enzyme substrate ligand Gal-Lip (Glc-Lip) and lipoic acid at a ratio of 1:1 showed a typical SPR peak at 521 nm, i.e., a red shift of 2 nm with respect to the citrate-capped gold nanoparticles, and good stability in PBS (Figure 1b). The addition of β-galactosidase caused a time-dependent decrease of the peak absorbance at 521 nm, along with a gradual increase at approximately 700 nm that was induced by the aggregation (Figure 1a). Similar results were observed


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for Glc-Lip-AuNPs with β-glucosidase (Figure S2). The aggregation of Gal-Lip-AuNPs and Glc-Lip-AuNPs could be clearly observed by the naked eye as a shift from wine red to blue color over time (Figure 1e and Figure S9). This colorimetric assay can also be used in a McFarland standard No. 0.5 barium sulfate turbid solution (Figure S13). Driving force of aggregation. We hypothesized that the driving force underlying AuNPs aggregation was the electrostatic attraction between the protonated amine produced after enzymatic hydrolysis by self-immolative elimination and the anionic lipoate at pH 7.4, as illustrated in Scheme S2. To test this hypothesis, the neutral ligand Lip-PEG400 (Figure S1) was chosen. When the negatively charged lipoate ligand on AuNPs co-capped with the enzyme substrate ligand was replaced by Lip-PEG400, SPR variations were not detected in the presence of the enzyme, and the original wine red color remained constant. These results confirm that a negatively charged ligand is required for enzyme detection in this system, and the driving force for nanoparticle aggregation is the mutual electrostatic attraction between enzymatically generated positively charged amines and negatively charged carboxylates. Limit of detection. An enzyme assay using Gal-Lip-AuNPs or Glc-Lip-AuNPs (2.0 nM) was further performed by incubating an ensemble solution containing different concentrations of β-galactosidase or β-glucosidase (0, 0.02, 0.2, 1.0, and 2.5 µM). Figure 1d and Figure S2d show the time-dependent absorbance change at 700 nm with various concentrations of the enzyme. As expected, a high enzyme concentration caused a remarkable increase in absorbance change because of the increased speed of enzymatic hydrolysis (Figure S3). The limit of detection for β-galactosidase and β-glucosidase using Gal-Lip-AuNPs and Glc-Lip-AuNPs within 20 min at S/N = 3 was determined as 9.2 and 22.3 nM, respectively, which improved to 2.9 and 9.8 nM at 90 min, respectively (Figure S4). Interference of thiol-containing molecules. Glutathione (GSH), which exists at high concentrations in cells, was chosen as a sample molecule to study the potential interfering effect of thiol-containing molecules on this enzyme assay system. In contrast to the previous two-steps assay method, the Gal-Lip-AuNPs and Glc-Lip-AuNPs exhibited good stability in a high concentration of GSH (5 mM),


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and the effect of GSH on the enzyme assay was almost negligible (Figure S5). Variation of ζ-potential and size. The enzyme-triggered elimination was confirmed by measuring the ζ-potential variation. The ζ-potentials of Gal-Lip-AuNPs and Glc-Lip-AuNPs were −24.3 mV and −24.1 mV without the enzymes, respectively, and increased gradually over time in the presence of enzymes (Figure S6). The ζ-potentials of Gal-Lip-AuNPs and Glc-Lip-AuNPs became −2.7 mV and −2.6 mV at 8 min after the addition of the enzymes. The increase in the ζ-potential was likely the result of the increased production of positive charges through self-immolative elimination. Then, the variation of nanoparticles in the enzymatic reaction was confirmed by transmission electron microscopy (TEM). In the absence of the enzyme, Gal-Lip-AuNPs and Glc-Lip-AuNPs were mono-dispersed (Figure 2a, c). However, upon addition of the enzyme, most of the nanoparticles were aggregated (Figure 2b, d). Dynamic light scattering (DLS) is also useful for determining the diameter and size distribution of AuNPs with sufficient temporal resolution. The DLS data at different time points clearly show that the hydrodynamic size of AuNPs gradually increased upon addition of the target enzyme for both Gal-Lip-AuNPs and Glc-Lip-AuNPs. The original size of Gal-Lip-AuNPs and Glc-Lip-AuNPs was 15.1 nm, and these particles grew to 47 and 44 nm at 1 min, 105 and 115 nm at 2 min, 170, and 176 nm at 4 min, 230 and 221 nm at 6 min, and 396 and 372 nm at 8 min after the addition of the enzyme (Figure S7). It is likely that many amines were generated on the surface of AuNPs and interacted with carboxylate by electrostatic attraction. These DLS data are consistent with TEM results. The hydrodynamic size variation at different pH values was examined 20 min after the addition of the enzyme. Under highly acidic and basic conditions (pH 3 and 11), the hydrodynamic size of Gal-Lip-AuNPs and Glc-Lip-AuNPs nearly returned to the original size without the enzyme (Figure S8). The sample color also recovered from blue to the original wine red. These results confirm that the driving force underlying nanoparticle aggregation was the mutual electrostatic attraction among AuNPs. Enzymatic reaction yield on the surface of AuNPs. To determine the enzymatic reaction yield on the


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surface of the AuNPs, the Amplex Red assay was performed to determine the concentration of galactose or glucose produced after hydrolysis. The principle of this assay and procedure are illustrated in Scheme S3 and Figure S11. The enzymatic reactions of Gal-Lip and Glc-Lip (100 µM) with 1.0 µM enzyme were completed at 100% yield within 5 min in PBS (Figure S10). However, on the surface of the AuNPs, the yield was much less than that in the solution phase with 1.0 µM enzyme and a lower amount of substrate, i.e., 5.0 µM Gal-Lip and Glc-Lip on 2.0 nm Gal-Lip-AuNPs and Glc-Lip-AuNPs (Figures 3a and 3b). Although the enzymatic reactions were accelerated in the presence of a higher enzyme concentration, the maximum yield was 82%. This maximum yield was also obtained with a low enzyme concentration condition when the reaction time was prolonged. The reaction yield at detection limit was determined to be 4% ca 200 nM amine on the surface of AuNPs. It appears that the decrease in the enzymatic reaction rate on the surface of AuNPs caused by the bulky conjugation of enzyme substrates on AuNPs and the extensive aggregation of the AuNPs fully blocks further enzymatic hydrolysis.39 The correlation of enzymatic reaction yields and spectral changes was studied by measuring the ratio of the absorbance value at 700 and 521 nm versus the reaction yield. On 2.0 nm Gal-Lip-AuNPs and Glc-Lip-AuNPs incubated with 1.0 µM β-galactosidase and β-glucosidase, the spectral changes (A700/A521) versus the reaction yield are shown in Figures 3c and 3d. A700/A521 corresponds to the yield, i.e., the quantity of ammonium generated, regardless of enzyme type. Colorimetric enzyme inhibitor screening using Gal-Lip-AuNPs and Glc-Lip-AuNPs. This fast, simple, convenient, and real-time colorimetric assay for β-galactosidase and β-glucosidase activity motivated us to further extend the application for enzyme inhibitor screening.

D-Galactal

and

castanospermine (Figure S12) are well-known inhibitors of β-galactosidase and β-glucosidase,40,41 respectively, and were chosen to demonstrate the application of our system to the screening of enzymatic inhibitors. As shown in Figure 4, D-galactal and castanospermine exhibited potent inhibition to β-galactosidase and β-glucosidase activities, respectively. The color change of the AuNPs became slow in the presence of the inhibitors. As the concentration of inhibitors increased, the color approached


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the original wine red (Figure 4a). The color remained nearly constant when the concentration of D-galactal

reached 1.0 mM, which indicated complete inhibition of β-galactosidase activity, and when

the concentration of castanospermine reached 0.1 mM, which indicated complete inhibition of β-glucosidase activity. The inhibitors decreased the enzymatic activity, and fewer amines were produced on the surface of AuNPs, which resulted in less aggregation of AuNPs, which was visualized as reduced color change. Gal-Lip-AuNPs and Glc-Lip-AuNPs can be utilized to screen β-galactosidase and β-glucosidase inhibitors using the naked eye. This simple, efficient, real-time colorimetric screening for enzyme inhibitors may provide a promising option for clinical assays. Inhibition efficiency. To quantify the inhibition efficiency of

D-galactal

and castanospermine for

β-galactosidase and β-glucosidase, the absorbance at 700 nm of Gal-Lip-AuNPs and Glc-Lip-AuNPs (2.0 nM), together with different concentrations of D-galactal and castanospermine (from 0.001 to 2000 µM), was recorded after a 20-min incubation. The inhibition efficiency (IC50) values for D-galactal and castanospermine toward β-galactosidase and β-glucosidase were determined to be 482 µM and 46.4 µM, respectively, according to the plots of the absorbance change at 700 nm versus the concentration of the inhibitors (Figure 4b,c).

CONCLUSION We designed and developed a new, simple, one-step, real-time colorimetric assay for β-galactosidase and β-glucosidase on the basis of primary amine generation by self-immolative elimination on the surface of Gal-Lip-AuNPs and Glc-Lip-AuNPs. Complex instruments are

not required in this assay. In

principle, β-galactosidase and β-glucosidase hydrolyze their specific substrate groups to induce self-immolative elimination on the surface of AuNPs and afford positively charged ammonium through the p-hydroxybenzyl alcohol linker, resulting in electrostatic aggregation with anionic lipoate on the neighboring nanoparticle surfaces. The aggregation of AuNPs induced a gradual color change that could be observed by the naked eye. The detection limits for β-galactosidase and β-glucosidase were as low as


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9.2 and 22.3 nM, respectively. This property of Gal-Lip-AuNPs and Glc-Lip-AuNPs should be applicable for the detection of gene expression through use of glycosidase for a reporter gene assay. Extended application of Gal-Lip-AuNPs and Glc-Lip-AuNPs to enzyme inhibitor screening was demonstrated. Moreover, the present detection strategy utilizing self-immolative elimination may become a general method to detect enzyme activity through the introduction of specific substrates on the surface of AuNPs.

FIGURE CAPTIONS Figure 1. (a,b) Time-dependent absorbance spectral changes of Gal-Lip-AuNPs (2.0 nM) in the presence (a) and absence (b) of β-galactosidase (1.0 µM). (c) Time-dependent absorbance spectral change of AuNPs capped with Lip and Lip-PEG400 (1:1) (2.0 nM) in the presence of β-galactosidase (1.0 µM). (d) Plot of absorbance of Gal-Lip-AuNPs at 700 nm versus reaction time in various concentrations of β-galactosidase (0, 0.02, 0.2, 1.0, and 2.5 µM). (e) Colorimetric visualization assay using Gal-Lip-AuNPs (3.0 nM) for β-galactosidase (2.5 µM) at various time points (0, 2, 5, and 10 min). All samples were suspended in 50 mM sodium phosphate buffer (pH 7.4, 10 mM KCl, 10 mM MgCl2) at 37°C. Figure 2. (a–d) TEM images of Gal-Lip-AuNPs (2.0 nM) (a,b) and Glc-Lip-AuNPs (2.0 nM) (c,d) without (a, c) and with β-galactosidase (1.0 µM) (b) or β-glucosidase (1.0 µM) (d) after 20 min incubation at 37°C. All samples were suspended in PBS (pH 7.4, 50 mM phosphate buffer, 10 mM KCl, 10 mM MgCl2) at 37°C. Scale bar: 100 nm. Figure 3. Time-dependent variation of the enzymatic reaction yield of 2.0 nM Gal-Lip-AuNPs with β-galactosidase (1.0 µM) (a) and Glc-Lip-AuNPs with β-galactosidase (1.0 µM) (b). Plot of the spectral changes (A700/A521) versus reaction yield (c and d). Figure 4. (a) Visualization of β-galactosidase and β-glucosidase inhibition using Gal-Lip-AuNPs and ACS Paragon Plus Environment

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Glc-Lip-AuNPs (2.0 nM) in different concentrations of D-galactal (down) and castanospermine (up) (0.01, 0.1, 1.0, 10, 50, 400, 1000, and 2000 µM). The control lane does not contain enzyme or inhibitor. (b,c) Plot of absorbance at 700 nm of Gal-Lip-AuNPs versus logarithmic concentration of D-galactal (b) and Glc-Lip-AuNPs versus logarithmic concentration of castanospermine (c).


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SCHEME TITLES Scheme 1. Proposed illustration for the detection of glycosidase activity by Gal-Lip- or Glc-Lip-modified AuNPs (Gal-Lip-AuNPs, Glc-Lip-AuNPs).


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(16) Miranda, O. R.; Li, X.; Garcia-Gonzalez, L.; Zhu, Z.-J.; Yan, B.; Bunz, U. H. F.; Rotello, V. M. J. Am. Chem. Soc. 2011, 133, 650–653. (17) Que, L.; Domaillend, D.; Chang, C. Chem. Rev. 2008, 108, 1517–1549. (18) Mizukami, S.; Matsushita, H.; Takikawa, R.; Sugihara, F.; Shirakawa, M.; Kikuchi, K. Chem. Sci. 2011, 2, 1151–1155. (19) Li, L.; Zemp, R. P.; Lungu, G.; Stoica G.; Wang, L. V. J. Biomed. Optics 2007, 12, 020504. (20) Liu, L.; Mason R. P. PLoS ONE 2001, 5, e12024 (21) Weissleder, R; Ross, B. D.; Rehemtulla, A.; Gambhir, S. S. Molecular Imaging: Principles and Practice, Hamilton, Ontario, Canada: BC Decker Inc. 2010; pp542–573. (22) Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science 1997, 277, 1078–1081. (23) El-Sayed, I. H.; Huang, X.; El-Sayed, M. A. Nano Lett. 2005, 5, 829–834. (24) Xu, X.; Han, S. M.; Mirkin, C. A. Angew. Chem., Int. Ed. 2007, 46, 3468–3470. (25) Lee, J. S.; Ulmann, P. A.; Han, M. S.; Mirkin, C. A. Nano Lett. 2008, 8, 529–533. (26) Lee, S.; Cha, E. J.; Park, K.; Lee, S. Y.; Hong, J. K.; Sun, I. C.; Kim, S. Y.; Choi, K.; Kwon, I. C.; Kim, K.; Ahn, C. H. Angew. Chem., Int. Ed. 2008, 47, 2804–2807. (27) Cheng, Y.; Samia, A. C.; Meyers, J. D.; Panagopoulos, I.; Fei, B.; Burda, C. J. Am. Chem. Soc. 2008, 130, 10643–10647. (28) Guarise, C.; Pasquato, L.; De Fillippis, V.; Scrimm, P. Proc. Natl. Acad. Sci. USA. 2006, 103, 3978–3982. (29) Liu, R.; Liew, R.; Zhou, J.; Xing, B. Angew. Chem. Int. Ed. 2007, 46, 8799–8803. (30) Choi, Y.; Ho, N.; Tung, C. Angew. Chem. Int. Ed. 2006, 46, 707–709. (31) Jiang, T.; Liu, R.; Huang, X.; Feng, H.; Teo, W.; Xing, B. Chem. Commun. 2006, 1972–1974. (32) Wang, M.; Gu, X.; Zhang, G.; Zhang, D.; Zhu, D. Langmuir 2006, 25, 2504–2507. (33) Wang, Z.; Levy, R.; Fering, D. G.; Burst, M. J. Am. Chem. Soc. 2006, 128, 2214–2215.


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17


a

b 20 min

0 min 20 min

0.6

0.6 0

0.4

0.4 0.2

0.2 0.0 400

0.8

Absorbance

Absorbance

0.8

600

0.0 400

800

600

Wavelength (nm)

800

Wavelength (nm)

c

d 0.6

0.8 0 min 20 min

0.6

Absorbance

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 21

0.4 0.2 0.0 400

600

0.4 0.3 0.2 0.1 0.0

800

2.5 µM 1.0 µM 0.5 µM 0.2 µM 0 µM

0.5

0

Wavelength (nm)

0

2

5

5

10

15

Time (min)

20

25

10 min

e

Figure 1. a

b

c

d

Figure 2.


18

Page 19 of 21

a

b

1.0

Reaction yields

Reaction yields

0.8 0.8 0.6 0.4 0.2 0.0 0

20

40

60

80

0.6 0.4 0.2 0.0 0

100

20

40

60

80

100

Time (min)

Time (min)

d 1.2

1.2

1.0

1.0

0.8

0.8

A700/A521

A700/A521

c

0.6 0.4 0.2

0.6 0.4 0.2 0.0

0.0 0.0

0.2

0.4

0.6

0.0

0.8

0.2

0.4

0.6

0.8

Reaction yields

Reaction yields

Figure 3.

a Control

0.01

0.1

1.0

10

50

400

1000

2000 µM

c 0.4 0.3 IC50:482 µM 0.2 0.1 -4

-3

-2

-1

0

1

2

3

4

Absorbance at 700 nm

b Absorbance at 700 nm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.5 0.4 0.3

IC50:46.4 µM

0.2 0.1

log[D-galactal/(µM)]

-4

-3

-2

-1

0

1

2

3

4

log[castanospermine/(µM)]

0.5

Figure 4.


19


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

HO HO

Page 20 of 21

Au

OHOH O O

HO HO O HO O OH

HO

OH

O O

Au

OH OH

Au

HO HO

O O

O O OH

O

OH

OH

glycosidase

OH OH OH

OH

O OH OH

OH

Au Au

S

Au SS

S S S OHOH O + CO2 O +

HO HO

O O

O

O

Au S S SS SS

OH

O

O

O

NH O

O O

O OH

R= HN O

HO

O

H3N S

O

HO OH Gal-Lip OH HO

R

NH O

O

O

O

HO

O

O

OH Glc-Lip

S

OH O Lipoic acid (Lip)

Scheme 1.


20

Page 21 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


For TOC only


21

Simple and Real-Time Colorimetric Assay for Glycosidases Activity

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