Synthesis of Fluorescent Gold Nanodot–Liposome Hybrids for

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Synthesis of Fluorescent Gold Nanodot−Liposome Hybrids for Detection of Phospholipase C and Its Inhibitor Wei-Yu Chen,† Li-Yi Chen,† Chung-Mao Ou,† Chih-Ching Huang,*,▲,‡,§,⊥ Shih-Chung Wei,‡ and Huan-Tsung Chang*,∥,† †

Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan Institute of Bioscience and Biotechnology and §Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung 20224, Taiwan ⊥ School and College of Pharmacy, Kaohsiung Medical University, Kaohsiung 80708, Taiwan ‡

S Supporting Information *

ABSTRACT: We report the synthesis of fluorescent 11-mercaptoundecanoic acid−gold nanodot−liposome (11-MUA−Au ND/Lip) hybrids by incorporation of gold nanoparticles (∼3 nm) and 11-MUA molecules in hydrophobic phospholipid membranes that self-assemble to form small unilamellar vesicles. A simple and homogeneous fluorescence assay for phospholipase C (PLC) was developed on the basis of the fluorescence quenching of 11-MUA−Au ND/Lip hybrids in aqueous solution. The fluorescence of the 11-MUA−Au ND/Lip hybrids is quenched by oxygen (O2) molecules in solution, and quenching is reduced in the presence of PLC. PLC catalyzes the hydrolysis of phosphatidylcholine units from Lip to yield diacylglycerol (DAG) and phosphocholine (PC) products, leading to the decomposition of Lip. The diacylglycerol further interacts with 11-MUA−Au NDs via hydrophobic interactions, leading to inhibition of O2 quenching. The 11-MUA−Au ND/Lip probe provides a limit of detection (at a signal-to-noise ratio of 3) of 0.21 nM for PLC, with high selectivity over other proteins, enzymes, and phospholipases. We have validated the practicality of using this probe for the determination of PLC concentrations in breast cancer cells (MCF-7 and MDA-MB-231 cell lines) and nontumor cells (MCF-10A cell line), revealing that the PLC activity in the first two is at least 1.5-fold higher than that in the third. An inhibitor assay using 11-MUA−Au ND/Lip hybrids demonstrated that tricyclodecan-9-yl potassium xanthate (D609) inhibits PLC (10 nM) with an IC50 value of 3.81 ± 0.22 μM. This simple, sensitive, and selective approach holds great potential for detection of PLC in cancer cells and for the screening of anti-PLC drugs.

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spectroscopy approach is useful for the detection of PLC, it requires tedious labeling and modification of expensive substrates.8 Fluorogenic substrates are alternative probes for the sensitive detection of PLC and for the real-time analysis of enzyme kinetics; however, low catalytic turnover of enzymes acting on the synthetic substrates has limited their application.10 Recently, colorimetric, fluorometric, and mass spectrometric assays using phospholipid-functionalized nanomaterials have been developed for the detection of phospholipase.11 However, they require tedious processes for labeling the substrate or target analytes and the use of expensive fluorophores or coupling reagents. Moreover, they are rarely applied to the selective detection of phospholipases [e.g., phospholipase A2 (PLA2), PLC, and phospholipase D (PLD)] in biological samples. Thus, the development of selective, sensitive, cost-effective, and robust assays for monitoring the

he development of highly sensitive, simple, and robust methods for detecting enzyme activity is of critical importance for point-of-care diagnostics and drug screening in biomedical applications.1 Phospholipases degrade phospholipids to produce free fatty acids, lysolipids, choline, and phosphatidic acid.2 They are involved in a range of important biological processes, including metabolism, digestion, inflammation response, membrane trafficking, and intercellular signaling.2,3 Phospholipase C (PLC), an enzyme of the phospholipase superfamily, catalyzes the hydrolysis of the phosphate ester bond in phospholipids to yield diacylglycerol (DAG) and phosphocholine (PC).4 This hydrolysis reaction has been implicated in aberrant choline metabolism in some cancer cells.5 Fluorometric spectroscopy, acid−base titration, 31P NMR spectroscopy, and chromatography-coupled absorption or fluorescence detection have been employed for the determination of PLC activity or concentration in cancer cells and bacteria.6−9 In general, chromatography and acid−base titration-based assays have limitations, such as low sensitivity, and require large substrate molecules.7,9 Although the 31P NMR © XXXX American Chemical Society

Received: July 4, 2013 Accepted: August 21, 2013

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phospholipase-catalyzed hydrolysis reaction of phospholipids remains a challenge. Fluorescent gold (Au) and silver (Ag) nanodots (NDs)/ nanoclusters (NCs) have received great attention in the detection of analytes of interest (e.g., metal ions and biopolymers) and in cell imaging, mainly owing to advantages such as easy preparation and conjugation, high biocompatibility and stability, and large Stokes shifts.12−15 Au and Ag NDs/NCs possess molecular-like optical properties, depending on their size, oxidation state, and surface properties.15 Polymers, proteins, and DNA have been employed as templates for the preparation of water-soluble, highly stable, and fluorescent Au and Ag NDs/NCs.15−18 Phospholipid-functionalized nanomaterials (e.g., carbon-, metallic-, metal oxide-, and semiconductor quantum-dot-based nanoparticles) have been shown to be useful as sensitive biosensors and selective drugs for cancer cells.11,19−21 In this study, we incorporated 11-mercaptoundecanoic acidcapped-Au NDs (11-MUA−Au NDs) into liposomes, allowing the synthesis of fluorescent 11-MUA−Au ND/Lip hybrids (Scheme 1). On the basis of inhibition of O2-induced

Scheme 2. Schematic Representation of the Operation of the 11-MUA−Au ND/Lip Probe for the Detection of Phospholipase C through Control of O2-Induced Fluorescence Quenching by the 11-MUA−Au ND/Lip Probe

Scheme 1. Schematic Representation of the Synthesis of the Fluorescent 11-MUA−Au NDs/Lip Hybrids

(Geel, Belgium). Tris(hydroxymethyl)aminomethane (Tris) and calcium chloride were purchased from J. T. Baker (Phillipsburg, NJ). Cholesterol and 1,2-dimyristoyl-sn-glycero3-phosphocholine (DMPC) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Tricyclodecan-9-yl potassium xanthate (D609) (98%) was purchased from Enzo Life Sciences (Farmingdale, NY). Sephadex G-100 was purchased from GE Healthcare Bio-Sciences (Piscataway, NJ). Bovine serum albumin (BSA), hemoglobin, myoglobin, cytochrome C, transferrin, trypsin, protease from Aspergillus melleus, phosphatase, ribonuclease A, β-lactoglobuin, β-casein, actin monomer, trypsinogen, lysozyme, carbonic anhydrase, PLA2 (14.5 kDa) from bovine pancreas, PLD (200 kDa) from Arachis hypogaea, and PLC (43 kDa) from Clostridium perfringens (Clostridium welchii) were purchased from Sigma (St. Louis, MO). The Amplex Red/Phosphatidylcholine-specific PLC assay kit (A12218) was purchased from Molecular Probes (Eugene, OR). Milli-Q (Millipore, Billerica, MA) ultrapure water was used for all experiments. Synthesis of Fluorescent 11-MUA−Au ND/Lip Hybrids. For the synthesis of 11-MUA−Au ND/Lip hybrids, DMPC (1.67 mM), cholesterol (1.33 mM), and 11-MUA (16.7 mM) were dissolved in chloroform (3 mL) in a 100 mL roundbottom flask.22 The chloroform was evaporated in a rotary evaporator under vacuum at 35 °C for 30 min, and then the flask was flushed with a nitrogen (N2) stream (60 lb/in.2) for 2−6 h to remove any residual traces of organic solvent. The dried 11-MUA molecules and phospholipid film were hydrated with 5 mL of the THPC−Au NPs, which was followed by sonication for 30 min in a bath sonicator (35 °C) to produce multilamellar vesicles (MLVs) containing Au NPs. The mixtures were then left to react for 48 h in the dark at ambient temperature to form the fluorescent 11-MUA−Au ND/Lip hybrids after the Au NPs were etched by 11MUA.12d,23 Following removal of the precipitates by centrifuging at 8000g for 20 min, the supernatant was mixed with

fluorescence quenching of the 11-MUA−Au NDs, the hybrids were used for the detection of PLC in biological samples. We employed fluorescence spectroscopy, dynamic light scattering (DLS), and liquid chromatography/electrospray ionization mass spectrometry (LC/ESI MS) to investigate the PLCmediated hydrolysis reaction of the hybrids. The hydrolysis products of DAG stabilized the 11-MUA−Au NDs, reducing the O2-induced fluorescence quenching of 11-MUA−Au NDs in solution under vigorous vortexing (Scheme 2). Our inhibition-based assay for PLC shows high sensitivity and selectivity for the determination of PLC concentration in breast cancer cells (MCF-7 and MDA-MB-231 cell lines) and nontumoral cells (MCF-10A cell line). This developed assay was further employed as an inhibitor assay for PLC.

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EXPERIMENTAL SECTION Chemicals. Tetrachloroauric acid trihydrate (HAuCl4· 3H 2 O), 11-mercaptoundecanoic acid (11-MUA), tetra(hydroxymethyl)phosphonium chloride (THPC), acetonitrile (ACN, ≥99.9%, HPLC grade), and aqueous formic acid (FA) solution (8% in water, HPLC grade) were purchased from Sigma-Aldrich (Milwaukee, WI). Chloroform (≥99%), hydrochloric acid (37%, AR grade), and acetic acid (99.8%, biochemical grade) were obtained from Acros Organics B

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Please see the Supporting Information for the details on the synthesis and characteristics of Au NPs, the characterization of fluorescent 11-MUA−Au ND/Lip hybrids, cell culture, and liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometer (LC/ESI-Q-TOF MS) measurements.

phosphate buffer (5 mL, 5.0 mM, pH 7.0). To obtain narrowly distributed small unilamellar vesicles (SUVs), the solution was extruded through a polycarbonate membrane (100 nm pore size) (Whatman, Inc., Phillipsburg, NJ) 11 times.21 The fluorescent 11-MUA−Au ND/Lip solution (5 mL) was further purified on a Sephadex G-100 column to remove free phospholipids, unincorporated fluorescent 11-MUA−Au NDs, and other impurities. The collected fraction (5 mL) of 11MUA−Au ND/Lip hybrids was stable in the dark at 4 °C for 2 weeks. For simplicity, the concentration of the 11-MUA−Au ND/Lip hybrids was designated as 1×. Sensing of PLC and Inhibitory Assay. Aliquots (325 μL) of 5.0 mM Tris-HCl buffer (pH 7.4) containing 1.0 mM CaCl2 and PLC (0−100 nM; ∼0−0.075 unit; one unit will produce 1.0 μM water-soluble organic phosphorus from egg yolk L-αphosphatidylcholine per minute at pH 7.3 at 37 °C) were maintained in an ice bath for 30 min. Solutions of 11-MUA−Au ND/Lip hybrids (0.1×, 50 μL) were added separately to each of the PLC solutions, which were then incubated for another 20 min at ambient temperature. The mixtures were saturated with oxygen (O2) from ambient air by vigorous vortexing (1600 rpm) at ambient temperature for 45 min and were mixed with 125 μL of Tris-acetate solution (200 mM, pH 6.0), followed by equilibration at ambient temperature for 10 min. The mixtures were then transferred separately into 96-well microtiter plates, and their fluorescence spectra were recorded using a monochromatic microplate spectrophotometer (Synergy 4, BioTek, Winooski, VT) upon excitation at a wavelength of 370 nm. For the inhibitor assay of D609, various concentrations of D609 (0−50 μM) were preincubated with PLC (10 nM) in 450 μL of Tris-HCl (5.0 mM, pH 7.4) solution containing 10 μM BSA for 30 min before determination of the activity of PLC using 11-MUA−Au ND/Lip hybrids. The half-maximal inhibitor concentration (IC50) value of D609 for PLC was calculated using the titration curve of the emission wavelength at 530 nm for each sample according to the equation Y = 1/(1 + 10(X − log IC50)), where X = the logarithm of the inhibitor concentration and Y = the measured percent activity at a given inhibitor concentration. All enzyme-activity-inhibitor assays for the determination of IC50 values were performed in triplicate. Analysis of PLC in Cell Extracts. Cell lysis of MCF-7, MDA-MB-231, and MCF-10A (1 × 107 cells/mL) was accomplished by sonication four times in an ice bath for 30 s each.24 The cell lysis solutions were then centrifuged at 3750g for 10 min to collect the lysed protein in the supernatants.24 Aliquots (100 μL) of the extracted proteins of cell line samples (diluted 20-fold) were spiked with standard PLC solutions at concentrations of 0−10 or 0−20 nM. The spiked samples were then diluted to 375 μL with solutions containing the 11-MUA− Au ND/Lip probe (0.01×), 5.0 mM Tris-HCl buffer (pH 7.4), and 1.0 mM CaCl2 and subsequently incubated for 20 min at ambient temperature. The mixtures were saturated with O2 from ambient air by vortexing (1600 rpm) at ambient temperature for 45 min and then mixed with 125 μL of Trisacetate buffer (200 mM, pH 6.0) and equilibrated at ambient temperature for 10 min. The PLC activities in the cell extracts were also determined using the Amplex Red PC−PLC assay kit.5b,6b,24 The mixtures were then transferred separately into 96-well microtiter plates, and their fluorescence spectra were recorded using a monochromatic microplate spectrophotometer upon excitation at a wavelength of 370 nm.

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RESULTS AND DISCUSSION Preparation of Fluorescent 11-MUA−Au ND/Lip Hybrids. First, we prepared Au NPs (≈3.0 nm) by reduction of AuCl4− mediated by THPC, which acted as a reducing and capping agent.25 As indicated in Scheme 1, 11-MUA−Au ND/ Lip hybrids were prepared using 11-MUA, DMPC, cholesterol, and Au NPs. In particular, the 11-MUA−Au NDs could be readily entrapped in the liposome. Figure 1 displays the

Figure 1. UV−vis absorption, excitation, and emission spectra of 11MUA−Au ND/Lip hybrids (0.1×) in Tris-HCl solutions (5.0 mM, pH 7.4). The emission spectrum of the 11-MUA−Au ND/Lip hybrids was obtained upon excitation at a wavelength of 370 nm, while that for the excitation spectrum was obtained at an emission wavelength of 530 nm.

absorption, excitation, and emission spectra of 11-MUA−Au ND/Lip hybrids. Briefly, we inferred that the absorption of the Au NDs at 375 nm (ε = ≈2.3 × 106 M−1 cm−1) originated in the ligand-to-metal ND charge transfer (LMNCT; S → Au) mixed with ligand-to-metal−metal charge transfer (LMMCT; S → Au−Au) on the particle surface (Figure 1).26 The fluorescence of 11-MUA−Au NDs was a combination of that from Au NDs and Au(I)−SR.23,27−29 We suggest that the fluorescence of our 11-MUA−Au NDs mainly originated from the Au ND/polynuclear Au(I)−thiol (core/shell) complexes and that the QY and stability of 11-MUA−Au NDs was highly dependent on ligand coverage.23,27−29 The long (>100 ns) fluorescence lifetime of 11-MUA−Au NDs was assigned to metal-perturbed intraligand phosphorescence, which is a lowlying triplet state populated via intersystem crossing from the lowest singlet state.23,27,28 Although the QY of 11-MUA−Au ND/Lip hybrids (∼3%) in comparison with quinine (QY 53%) is close to that of 11-MUA−Au NDs,23 the 11-MUA−Au ND/ Lip hybrids were more stable at various pH values and were resistant to cysteine-induced fluorescence quenching (Figure S1 in the Supporting Information). As shown in Figure S2 (in the Supporting Information) and Figure 2, the fluorescence spectra and hydrodynamic radius of as-prepared 11-MUA−Au NDs in the absence and presence of 1.0 mM CaCl2 in 5.0 mM TrisHCl (pH 7.4) are almost the same, indicating that 11-MUA− Au NDs are very stable in the buffer solution. C

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function.4 We found almost no fluorescence quenching of 11MUA−Au ND/Lip hybrids in the N2-purging solution in the absence or presence of PLC (Figure S3 in the Supporting Information). Accordingly, we suggest that the fluorescence quenching of 11-MUA−Au ND/Lip hybrids under vigorous vortexing in air is due to the oxidation of Au−S bonds on the surface of 11-MUA−Au NDs by O2 molecules.30 Oxygen (O2) molecules permeate the liposome bilayer membrane easily to contact the surface of 11-MUA−Au NDs because of their small size and lack of charge.31 Consequently, oxidation of the Au−S bonds with the penetrated O2 molecules occurred, inducing fluorescence quenching of 11-MUA−Au NDs (Figure S3 in the Supporting Information). The 11-MUA−Au NDs are mainly distributed in the interior vesicle of the liposome. In our previous study, we revealed that the Au−MUA complexes on the surfaces of Au ND cores play a significant role in determining the fluorescence of 11-MUA−Au NDs.12d We employed dynamic light scattering (DLS) measurement to investigate the hydrodynamic diameter and size distribution of 11-MUA−Au ND/Lip hybrids (0.1×) in the absence and presence of PLC (100 nM) in 5.0 mM Tris-HCl (pH 7.4) containing 10 μM BSA and 1.0 mM CaCl2. Figure 2B shows that the hydrodynamic size of 11-MUA−Au ND/Lip hybrids was ∼110 nm (curve a) in the absence of PLC. After reaction with PLC (100 nM), two sizes of ∼8 and ∼600 nm were observed (curve b). The sharp peak for 8 nm corresponds to the 11-MUA−Au NDs (curve c), revealing that the 11-MUA− Au ND/Lip hybrid nanoassembly underwent degradation by PLC to release 11-MUA−Au NDs. In addition, the aggregation of degraded liposome products resulted in a broadened band at ∼600 nm. We observed almost the same DLS spectra of 11MUA−Au NDs in the presence (curve c in Figure 2B) and absence (curve d in Figure 2B) of 10 μM BSA, revealing that the sharp peak at 8 nm is originally from 11-MUA−Au NDs. Figure S4 (in the Supporting Information) shows microscopic images of the 11-MUA−Au ND/Lip hybrids (0.5×) in the absence and presence of PLC (1.0 μM). The fluorescence images show bright green fluorescence of the 11-MUA−Au ND/Lip hybrids upon excitation at 460 nm. After 11-MUA−Au ND/Lip hybrids reacted with PLC for 30 min, they degraded and 11-MUA−Au NDs were released. Thererfore, no fluorescent spots of 11-MUA−Au ND/Lip hybrids were observed. Because of their low fluorescence quantum yield (∼3%), it is very difficult to observe the fluorescence of 11MUA−Au NDs with an insensitive CCD detector. Next, we employed LC/ESI-Q-TOF MS to analyze the 11MUA−Au ND/Lip hybrids in the absence and presence of PLC. As shown in Figure S5A and S5B (in the Supporting Information), MS spectra of the hybrids display four peaks at m/z 678.51, 700.40, 1355.79, and 1377.99 that correspond to [DMPC + H]+, [DMPC + Na]+, [2DMPC + H]+, and [2DMPC + Na]+, respectively, which were eluted at 25.32 and 25.41 min in the absence and presence of PLC, respectively. In the presence of PLC, we observed the hydrolysis products of DAG at m/z 1047.88 for [2DAG + Na]+, which was eluted at 28.18 min (Figure S5C in the Supporting Information). We also conducted ESI-Q-TOF MS to characterize the purified products of DAG-stabilized 11-MUA−Au NDs that were obtained from the hydrolysis of 11-MUA−Au ND/Lip hybrids (5.0 mL, 1×) with PLC (10 μM). The resultant solution was centrifuged at 18000g for 30 min. After removal of the supernatant solution, the precipitate (DAG-stabilized 11MUA−Au NDs) was resuspended in 0.5 mL of ultrapure

Figure 2. (A) Fluorescence spectra of 11-MUA−Au ND/Lip hybrids (a,b) without and (c,d) with vigorous vortexing in the (a,c) absence and (b,d) presence of 100 nM PLC. (B) DLS spectra of 11-MUA−Au ND/Lip hybrids in the (a) absence and (b) presence of PLC and 11MUA−Au NDs in the (c) absence and (d) presence of BSA. Solutions were prepared in 5.0 mM Tris-HCl (pH 7.4) containing 1.0 mM CaCl2.

Degradation of Fluorescent 11-MUA−Au ND/Lip Hybrids by PLC. PLC catalyzed the hydrolysis of the phosphate ester bond in the phospholipid selectively at the glycerol side to yield the hydrophobic DAG and hydrophilic phosphocholine.4 As a result, the structure of the 11-MUA−Au ND/Lip hybrids was decomposed through PLC-mediated hydrolysis of the phospholipids, and the 11-MUA−Au NDs were released from the nanoassembly (Scheme 2). The O2induced fluorescence quenching of DAG-stabilized 11-MUA− Au NDs that were formed via long-carbon-chain hydrophobic interactions between DAG and 11-MUA was lower than that of 11-MUA−Au NDs. Figure 2A shows that little or no change in fluorescence intensity of 11-MUA−Au ND/Lip hybrids (0.1×; the concentration of the as-prepared 11-MUA−Au ND/Lip hybrids was designated as 1× occurred in 5.0 mM Tris-HCl (pH 7.4) containing 1.0 mM CaCl2 in the absence and presence of 100 nM PLC (curves a and b) without vigorous vortexing, revealing good stability (fluorescence) of the DAGbinding 11-MUA−Au NDs, whereas their fluorescence values were only ∼5 and ∼65% (curves c and d, respectively) of their original values under vigorous vortexing. In addition, we noted that the hydrodynamic diameters of the 11-MUA−Au NDs (curve c in Figure 2B) and the DAG-binding 11-MUA−Au NDs (curve b in Figure 2B) are almost the same. We noted that Ca2+ ions were an essential cofactor for activating the PLC D

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water. As shown in Figure S5 (spectrum D), the MS spectrum of the DAG-stabilized 11-MUA−Au NDs displays a peak at m/ z 535.43 for [DAG + Na]+, strongly supporting the idea that the DAG molecules were bonded to 11-MUA−Au NDs. Selectivity and Sensitivity. To further test the specificity of this assay toward PLC, we incubated aliquots of mixtures of 11-MUA−Au ND/Lip hybrids (0.01×) and BSA (10 μM) in the presence of PLC (10 nM) and other possible interfering proteins including BSA, hemoglobin, myoglobin, cytochrome C, transferrin, trypsin, protease, phosphatase, ribonuclease A, βlactoglobulin, β-casein, actin, trypsinogen, lysozyme, carbonic anhydrase, PLA2, and PLD (each 100 nM). In order to mimic physiological conditions in which background proteins are present, especially serum albumin, we prepared the solutions in the presence of BSA. As shown in Figure 3, none interfered Figure 4. Fluorescence response of a 11-MUA−Au ND/Lip probe (0.01×) for PLC (0−100 nM). Inset: linear range of the plot of (IF − IF0)/IF0 at 530 nm against the PLC concentration (0.50−50 nM). IF0 and IF are the fluorescence intensities of the 11-MUA−Au ND/Lip probe in the absence and presence of PLC, respectively. Error bars represent the standard deviations from three repeated experiments. Other conditions were as described in Figure 2A.

standard addition method (Figure S6 in the Supporting Information), the 11-MUA−Au ND/Lip system provided a linear response to PLC in the first two extracts at 0−10 nM and in the last spiked extract at 0−20 nM. Table 1 lists the concentrations of PLC in the extracted samples from the three cell lines determined by our assays and the commercial Amplex Red PC−PLC assay. According to values from the Student’s ttest, the results from our present approach show no significant differences from those obtained in the Amplex Red PC−PLC assay. The concentrations of PLC from the nontumor (e.g., MCF-10A) and the MCF-7 and MDA-MB-231 cell extracts (1 × 107 cells/mL) were 111.5 ± 9.3, 165.8 ± 13.8, and 376.4 ± 33.9 nM, respectively, in accordance with the previously determined 1.5−6-fold higher activity of PLC in breast cancer cells compared to nontumor cells.5b Inhibitory Assays of PLC. This simple, highly sensitive, and convenient fluorescent assay for PLC activity motivated us to extend its application further to developing an enzymeinhibitor assay. D609 is a well-known inhibitor of phosphatidylcholine-specific PLC.5b,6b We incubated 11-MUA−Au ND/Lip hybrids (0.01×) with 10 nM PLC containing 10 μM BSA in the presence of D609 over the range of 0.05−50 μM (Figure 5). From the plot of quenching efficiency (IF /IF0; IF0 and IF are fluorescence intensities of 11-MUA−Au ND/Lip hybrids in the absence and presence of D609, respectively) against the log concentration of D609, we determined the IC50 value of D609 toward PLC (10 nM) in the presence of 10 μM BSA to be 3.81 ± 0.22 μM. This IC50 value of D609 for PLC agrees with that (4.27 ± 0.39 μM) determined by the Amplex Red PC−PLC assay.5b,6b These results suggest that the 11-MUA−Au ND/Lip probe has a high potential for screening inhibitors of PLC.

Figure 3. Relative fluorescence increase (IF − IF0)/IF0 at 530 nm of 5.0 mM Tris-HCl solutions (pH 7.4) containing 11-MUA−Au ND/Lip hybrids (0.01×) and BSA (10 μM) on addition of 10 nM PLC or 100 nM other proteins or enzymes. IF0 and IF are the fluorescence intensities of 11-MUA−Au ND/Lip hybrids in the absence and presence of interfering proteins or PLC, respectively. Error bars represent the standard deviations from three repeated experiments. Other conditions were as described in Figure 2A.

with detection by the 11-MUA−Au ND/Lip hybrids (0.01×) of PLC in the presence of BSA (10 μM). Although the other phospholipases PLA2 and PLD can catalyze the hydrolysis of phospholipids,2 they did not interfere in the determination of PLC. PLA2 and PLD catalyzed the hydrolysis of the phospholipids, yielding free fatty acid, lysophosphatidylcholine, choline, and phosphatidic acid.2 The low value of (IF − IF0)/IF0 in the presence of PLA2 or PLD suggested relatively weak interactions between these products and 11-MUA−Au NDs. Furthermore, we tested the sensitivity of 11-MUA−Au ND/ Lip hybrids for the detection of PLC. As shown in Figure 4, a linear correlation (r = 0.990) was observed between the value of (IF − IF0)/IF0 of 11-MUA−Au ND/Lip hybrids and PLC concentration over the range of 0.50−50 nM. The limit of detection (LOD; signal-to-noise ratio of 3) for PLC was 0.21 nM in the presence of 10 μM BSA (i.e., a 5 × 105-fold higher concentration). These results suggest that the interaction between PLC and background protein (BSA) is weaker relative to that of PLC with the liposome. The 11-MUA−Au ND/Lip hybrids provided a relatively low LOD and a wider dynamic range (2 orders of magnitude) than those provided by existing optical sensors for PLC analysis.10 Detection of PLC in Cell Extract. To test the practicality of this assay, we performed analyses of PLC in the cell extracts from MCF-7, MDA-MB-231, and MCF-10A cell lines. Using a

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CONCLUSIONS We devised a novel, simple, and convenient platform for the preparation of 11-MUA−Au ND/Lip hybrids that can be used for the detection of PLC. PLC selectively hydrolyzed their specific phosphatidylcholines from liposomes to yield DAG, which further minimized the O2-induced fluorescence quenching of 11-MUA−Au ND/Lip hybrids. Under the optimal conditions, the 11-MUA−Au ND/Lip hybrids provided high E

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Table 1. Determination of the Concentrations of PLC in Three Cell Extracts by the Amplex Red PC−PLC Assay Kit and 11MUA−Au ND/Lip Hybrids

a

cell linea

spiked [PLC] (nM)

11-MUA−Au NDs/Lip hybrids [mean ± SD (nM, n = 5)]

Amplex Red PC−PLC assay kit [mean ± SD (nM, n = 5)]

Student’s t-test values between the two approachesb

MCF-10A MCF-7 MDA-MB-231

0−10 0−10 0−20

111.5 ± 9.3 165.8 ± 13.8 376.4 ± 33.9

120.8 ± 13.7 190.2 ± 20.6 409.1 ± 41.7

1.26 2.20 1.36

Concentration of cell lines is 1 × 107 cells/mL. bThe Student’s t-test value is 2.306 at the 95% confidence level.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the National Science Council of Taiwan under contract NSC 101-2113-M-002-002-MY3.

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Figure 5. Dose-dependent inhibition of PLC using 11-MUA−Au ND/ Lip hybrids (0.01×) in the presence of 10 nM PLC and D609 (0−50 μM). IF0 and IF are the fluorescence intensities of the 11-MUA−Au ND/Lip probe in the absence and presence of D609, respectively. Error bars represent the standard deviations from three repeated experiments. Other conditions were as described in Figure 2A.

sensitivity (LOD: 0.21 nM) and high selectivity for the detection of PLC. Furthermore, we determined PLC concentration in cell extracts, confirming that PLC activity in breast cancer cells is at least 1.5-fold higher than in normal cells. We also demonstrated that our 11-MUA−Au ND/Lip probe could be further applied for the study of the interaction of PLC with its inhibitor, D609. To the best of our knowledge, this is the first report of the use of fluorescent metallic NDs and phospholipids for biosensing applications. We believe that by using varied phospholipids, our 11-MUA−Au ND/Lip hybrids could be further applied for the detection of other phospholipases.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (H.-T.C.). Fax and Tel.: 011886-2-3366-1171. *E-mail: [email protected] (C.-C.H.). Fax: 011-886-22462-2034. Tel.: 011-886-2-2462-2192 (ext. 5517). Present Addresses

∥ Professor Huan-Tsung Chang: Department of Chemistry, National Taiwan University, 1, Section 4, Roosevelt Road, Taipei 10617, Taiwan. ▲ Professor Chih-Ching Huang: Institute of Bioscience and Biotechnology, National Taiwan Ocean University, 2, Beining Road, Keelung 20224, Taiwan.

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Analytical Chemistry

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