Article pubs.acs.org/ac
Fluorogenic Quantum Dot-Gold Nanoparticle Assembly for Beta Secretase Inhibitor Screening in Live Cell Youngseon Choi,†,§ Yoojin Cho,†,⊥ Minjung Kim,† Regis Grailhe,‡ and Rita Song*,† †
Medicinal Chemistry Laboratory, Institut Pasteur Korea (IP-K), 696 Sampyeong-dong, Bundang-gu, Seongnam-Si, Gyeonggi-Do, 464-400, South Korea ‡ Neurodegeneration & Applied Microscopy Laboratory, Institut Pasteur Korea (IP-K), 696 Sampyeong-dong, Bundang-gu, Seongnam-Si, Gyeonggi-Do, 464-400, South Korea S Supporting Information *
ABSTRACT: We have developed a novel fluorogenic nanoprobe prepared from the assembly of CdSe/ZnS quantum dot (QD) and gold (Au) nanoparticles in which QD was conjugated with a specifically designed β-secretase (BACE1) substrate peptide, which was allowed to bind to the Ninitrilotriacetate (Ni-NTA) modified Au nanoparticles. This coordination-mediated binding of the QD with Au nanoparticles via Ni-NTA-histidine (His) interaction resulted in highly efficient quenching of QD fluorescence through a distancedependent fluorescence resonance energy transfer (FRET) phenomenon. The prequenched QD-Au assembly recovered the fluorescence in the presence of the BACE1 enzyme after incubation in vitro. The high quenching efficiency of AuNP and robust QD fluorescence signal recovery upon BACE1 enzymatic digestion enabled us to visualize BACE1 activity in living cells, which further allowed us to generate the half maximal inhibitory concentration (IC50) values for BACE1 inhibitors in the cell-based assay utilizing a high throughput system (HTS). These results suggest the potential application of QD-AuNP assembly toward the HTS drug screening system as a robust and efficient probe to identify active molecules in BACE1-related diseases such as Alzheimer’s disease. robust and efficient fluorogenic probe for BACE1 enzymatic assay. CdSe/ZnS core−shell QDs are novel types of fluorophores with a size-tunable wavelength, great photostability, and narrow emission with broad excitation spectrum.11−14 AuNPs have been widely used in various biological assays due to their surface plasmon resonance and as a universal quencher for diverse fluorophores due to the well-known longrange (up to 20 nm) nanometal surface energy transfer.15−19 The unique characteristics of QDs have been successfully applied to various FRET-based assay systems.13,20−22 Recently, various hybrid structures of QD and gold nanoparticles or gold nanorods have been successfully implemented for detection of small molecule analytes including Pb2+ and trinitrotoluene (TNT)23,24 as well as biomacromolecular analytes such as proteases,25 human IgG,26 or DNA.27 Remarkably, these studies employed small-ligand exchanged QDs with their hydrodynamic diameter of less than 10 nm, under the consideration that high FRET efficiency greatly depends on the distance of donor−acceptor.28,29 However, the inherent stability issues of the small-sized QDs, compared with the polymer-coated QDs possessing a high quantum yield and long-term stability in biological buffers, have limited their practical uses in the fields of biology and medicine. While the polymer-coated QDs with
transmembrane aspartic protease, β-secretase, involved in the processing of amyloid precursor protein (APP) generates toxic amyloid-β peptide (Aβ) aggregates which are known to cause Alzheimer’s disease (AD).1,2 β-Secretase (BACE1) is known to be a key enzyme for the production of Aβ peptide, thus being regarded as an important drug target in the treatment of AD.3,4 Currently, BACE1 inhibitor assays in vitro have been performed by the method of either sandwich enzyme linked immunosorbent assay (ELISA) for detection of Aβ production5 or cell-based assay measuring BACE1 activity using the fluorescence resonance energy transfer (FRET) mechanism.6−8 The FRET-based assay generally utilizes fluorogenic substrates such as dual-dye labeled synthetic peptides or recombinant APP-reporter fusion protein constructs,9,10 in which peptide substrates containing specific BACE1 cleavage sites emit fluorescence upon BACE1 digestion. Despite the known sensitivity and wide applicability of the FRET-based detection system in the cellular BACE assay, there have been inherent problems such as low solubility of peptide substrates due to the presence of organic fluorophore labeling and the low signal-tonoise ratios caused by the overlapping of excitation and emission spectral profiles of donor-acceptor dyes. These issues often require usage of high concentrations of enzymes in order to obtain reliable results.10 Herein, we described CdSe/ZnS quantum dot (QD) and gold nanoparticles (AuNPs) assembly, which can be used as a
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© 2012 American Chemical Society
Received: June 13, 2012 Accepted: September 7, 2012 Published: September 7, 2012 8595
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size of 12−15 nm, which is smaller and monodispersed than other reported polymer-coated QDs (>20 nm). The fluorescence quenching of the polymer-coated QD induced by the Ni-NTA-AuNPs (5 nm in diameter) was recovered in the presence of the BACE1 enzyme in vitro as well as in living cells. The reproducibility and robust signal generation of QDs allowed us to apply this probe to image-based high throughput screening for discovery of BACE1 inhibitors.
the PEG surface exhibit a higher quantum yield (>60%) and better stability,30,31 there have been few studies on the FRET application of the polymer-coated QDs due to their large size and the resulting low FRET efficiency with small molecular acceptor dyes.32 The potential of AuNPs as a universal quencher prompted us to use the polymer-coated QD as a fluorescent energy donor. In this study, a novel BACE1 enzymatic assay was developed using QD-AuNPs self-assembly prepared from the reaction of the polymer-coated QD-peptide conjugate and Ni-nitrilotriacetic acid (Ni-NTA)-modified AuNPs by a well-known Nihistidine (His) interaction (Scheme 1).14,33,34 The substrate
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RESULTS AND DISCUSSION Characterization of QD-Peptide Conjugate. In order to prepare QD-Au assembly via Ni-NTA-His interaction, we first activated amino-PEG-QD 1 (emission peak at 540 nm) with sulfo-succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC) heterobifunctional linker 2 in 1× PBS (pH 7.4) for 30 min to yield SMCC-activated QD 3, which was further reacted with 5 equiv of 6His-containing BACE1 substrate peptide 4 for 16 h at room temperature (see the Supporting Information Scheme S1). The peptide consists of N-terminal cysteine, C-terminal 6xHis, and a BACE1 cleavage site (EVNLDAEF) flanked by L-Aib-2Gs for minimizing steric hindrance (CLAib-2G-EVNLDAEF-2G-AibL-6H; italic for enzyme recognition site; Aib = amino-isobutyric acid). The unreacted peptides were purified by membrane filtration (10 kDa molecular-weight-cutoff). Successful conjugation of peptide 4 to QD 3 was confirmed by agarose gel electrophoresis (see the Supporting Information Figure S1). The reaction of amino-PEG-QD 1 with the SMCC linker resulted in an increase of net negative charge due to the consumption of terminal amino groups. Furthermore, the conjugation of BACE1 substrate peptide 4, which possesses a
Scheme 1. Schematic Representation of BACE1 Enzyme Assay Using Pre-Quenched Polymer-Coated QD and 5 nm Ni-NTA-Modified Au Nanoparticle Assemblya
a
Thick, black curved line is the BACE1 substrate.
peptide was designed to contain 6xHis residues to induce selfassembly with the Ni-NTA modified AuNPs. The size of the polymer-coated QD used in this study was strictly controlled by gel permeability chromatography (GPC) to attain an average
Figure 1. (a) Quenching profile of QD-peptide conjugate 5 with 5 nm Ni-NTA-AuNP. (b) Control experiments with 5 nm AuNP without Ni-NTA moiety (mPEG5k-modified AuNP) and a smaller size Ni-NTA-AuNP (1.8 nm), where the stoichiometric quenching phenomenon was not observed at the molar ratio (AuNP/QD) up to 10. (c) Time-course monitoring of PL quenching of QD-peptide conjugate in the presence of 3 equiv of 5 nm Ni-NTA-AuNP. Over 85% quenching was observed after 2 min of incubation at room temperature, as compared with a nonsignificant decrease of QD-peptide fluorescence. Excitation wavelength = 430 nm, slit = 3 nm, and integration time = 0.3 s. The concentration of QD was 10 nM in 10 mM phosphate-100 mM NaCl buffer (pH 7.4). (d) Dynamic light scattering analysis of the starting QD and AuNP (amino-PEG-QD, 5 nm Ni-NTAAuNP), the QD-peptide conjugate, and the resulting QD-Au assemblies formed at different molar ratios (1:1, 1:3, and 1:5). 8596
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Figure 2. Agarose gel electrophoresis characterization. (a) Various AuNPs used in this study; 1% agarose gel was run in 0.5× Tris-borate buffer at 100 V for 10 min. The loading amount was 50 pmol for 1.8 nm Ni-NTA-AuNP and 1 pmol each for 5 nm citrate-AuNP, mPEG5k-AuNP, and NiNTA-modified 5 nm AuNP. (b) Visualization of QD-peptide interaction with 5 nm Ni-NTA-AuNP at varying molar ratios from 1 to 5. The loading amount of QD and 5 nm Ni-NTA-AuNP was 2 pmol and 1 pmol, respectively. 1% agarose gel was run at 100 V for 10 min in 0.5× Tris-borate buffer (pH 8). The fluorescence image was captured by a LAS3000 image analyzer (430 nm excitation) using a long-pass filter (530 nm). The color image was taken by a Canon DSLR camera at ambient light.
between QD and AuNP, rather than diffusion-driven nonspecific quenching by AuNPs. In fact, it was found that the nonspecific quenching of QD PL occurred in the presence of over 200 nM Ni-NTA-AuNP, which is reportedly known as the collisional quenching effect of AuNPs.35 To elucidate the kinetics of the fluorescence quenching effect, we carried out time-course monitoring of PL intensity of the QD-peptide conjugate in the presence of 5 nm Ni-NTAAuNP (Figure 2c). Upon addition of 3 equiv of 5 nm Ni-NTAAuNP to the 10 nM QD-peptide solution, over 85% quenching was observed only after 2 min of incubation at room temperature. In comparison with the nonsignificant decrease of QD-peptide PL itself, this rapid quenching phenomenon indicates the high potential of AuNPs as efficient energy acceptors of QD fluorescence. The hydrodynamic diameter measured by DLS analysis showed that the size of the QD-peptide conjugate was slightly increased (∼15 nm) after the peptide conjugation to aminoPEG-QD (∼11 nm). Also, QD-Au assembly prepared at different molar ratios (1:1, 1:3, and 1:5) showed a gradual increase up to ∼21 nm with increasing molar ratio up to 5. The saturating profile of the overall size of the QD-Au assemblies indicates that there is a limitation on the number of AuNPs attached to QD, which is found to be saturated with 3 AuNPs per QD. Although the number of peptide molecules conjugated to QD was calculated as an average of 10 as described above, only three AuNPs were bound to QD probably due to the steric hindrance induced by the bulky 5 nm AuNPs. In addition, the specific association of QD-peptide with 5 nm Ni-NTA-AuNP via Ni-His interaction was further confirmed by gel electrophoresis (Figure 2). The retardation of QD-peptide mobility was observed only when QD-peptide was incubated with Ni-NTA-AuNP, not mPEG5k-AuNP or amino-PEG-QD with Ni-NTA-AuNP. We could clearly observe the gradual quenching of QD-peptide PL with decreasing mobility as the molar ratio of AuNP to QD increased; the QD fluorescence was not noticeable on the gel at the molar ratio of 3, which reflects the results of the quenching experiment (∼85% quenching efficiency) in Figure 2a. These results strongly suggest the successful binding of Ni-NTA-AuNP with the QDpeptide conjugate via Ni-His interaction, leading to a specific quenching of the highly luminescent polymer-coated QDs with
net negative charge at pH 8, led to an increase in the electrophoretic mobility of QD-peptide conjugate 5. The net charge of peptide at pH 7 was calculated to be −2.5 according to the peptide property calculator (www.innovagen.se). These results were also corroborated by the zeta potential measurement performed in pH 7.4, in which the QD-peptide conjugate showed higher negative charges (−22 ± 2.5 mV) than the starting amino-PEG-QD 1 (−9 ± 3.1 mV). The fluorescence emission spectra of the final QD-peptide conjugate was also compared with that of amino-PEG-QD at the concentration of 10 nM each, which did not exhibit significant shift (λem = 525 nm and λex = 430 nm) with a narrow emission bandwidth of ∼16 nm at half-maximum. However, a slight decrease of QD fluorescence (∼18%) was observed after conjugation. Furthermore, the number of peptide molecules per QD was determined through the bicinchoninic acid (BCA) assay by determining the unreacted peptide concentration collected after membrane filtration, which yielded an average of 10 ± 2 peptide molecules per QDs (see the Supporting Information Figure S2). Specific Interaction of QD-Peptide with 5 nm Ni-NTAAuNPs. To test the fluorescence quenching profile of QD by AuNP, purified QD-peptide conjugate 5 was first mixed with various molar ratios (1−5) of 5 nm Ni-NTA-AuNP 6 in PBS buffer for 1 h to yield the final QD-Au assembly 7 via Ni-His interaction. Binding of 5 nm Ni-NTA-AuNP with the QDpeptide conjugate resulted in a gradual decrease of QD photoluminescence (PL) intensity with increasing molar ratio of AuNP to QD, which resulted in the maximum quenching efficiency (QE) of 93% (Figure 1a). With only 3-fold excess of Ni-NTA-AuNP, the QE of QD-peptide already reached 88%. The saturation profile of the quenching efficiency implies that the binding of QD and AuNP occurs in a stoichiometric manner. To confirm that this interaction is specifically mediated by the Ni-His interaction, two additional control experiments were performed. As shown in Figure 1b, in the presence of either 5 nm AuNPs without the Ni-NTA moiety (mPEG5kAuNP), the QD-peptide conjugates did not show significant quenching. Also, a small AuNP (1.8 nm Ni-NTA-AuNP) did not induce significant quenching over the molar ratio (AuNP/ QD) up to 10. These results suggested that the quenching phenomena occurred via a specific Ni-NTA-His interaction 8597
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Figure 3. (a) Fluorogenic property of QD-Au assembly (1:3) after incubation with different concentrations of BACE1 enzyme at pH 7.0 at room temperature for 1 h. The QD fluorescence at 525 nm was measured in a spectrofluorimeter with 430 nm excitation. (b) Comparison of PL recovery of QD-Au prepared at different molar ratios of Ni-NTA-AuNP to QD-peptide at a fixed concentration of BACE1 enzyme (0.25 μM). The final QD concentration of all the QD-Au assemblies was fixed at 10 nM. The error bars represent the standard deviations from the means from triplicate measurements using a GraphPad Prism 5 software.
These results suggest the sensitivity and specificity of the QDAu assembly probe for BACE1 enzymatic activity. It is remarkable that the QD-Au assembly probe showed improved sensitivity compared with the reported value from the company, in which at least 2 μg per 100 μL of assay buffer is required to achieve the maximum fold-increase of the fluorogenic peptide substrate. Although not directly comparable due to the different assay format, the lower dynamic range of the fluorogenic signal of the current QD-Au system (∼maximum 3.5-fold) compared to the conventional fluorogenic assay (∼5-fold) may be attributable to (1) the hindered accessibility of the BACE1 enzyme to the substrate peptides between the bulky QD and 5 nm AuNPs and (2) the neutral buffer condition (pH 7.0) we used because of some instability of QD-Au at pH 4.5, a condition in which conventional fluorogenic assay is mostly performed.37 Despite the potential decrease of the optimum activity of BACE1 at the neutral pH, the high photoluminescence and stability of polymer-coated QDs and the highly efficient and specific quenching by AuNP in the current QD-Au assay format allowed us to obtain the fluorogenic assay results with a reproducible signal-to-ratio of over 3, which are considered important criteria for assessing the applicability toward high throughput screening in a 96-well assay format. Live Cell Imaging of the QD-Au Assembly in HEK293 Cells Overexpressing BACE1. In order to test the feasibility of the QD-Au probe for the cell-based Alzheimer drug screening in a high-throughput screening (HTS) format, we performed live cell imaging of the QD-Au assembly in HEK293 cells overexpressing BACE1, which were generated by transfecting the BACE1 plasmid according to the standard procedures mentioned in the experimental section (see the Supporting Information for detail). First, we confirmed the overexpression of BACE1 using the Turbofectin-based transfection method via immunofluorescence analysis including fluorescence activated cell sorter (FACS) and Western blotting. The cells showed increased BACE1 expression in the cytoplasmic region after 24 h of transfection with 1 μg of BACE1 cDNA plasmid in the presence of 3 μL of Turbofectin; transfection efficiency measured by FACS was nearly 20% (see the Supporting Information Figure S3). After 1 h of incubation at 37 °C, the probe showed a recovery of fluorescence signal mostly in the perinuclear regions of the cells, as noted by the
the BACE1 substrate peptide with high efficiency in a stoichiometric manner. In Vitro Evaluation of QD-Au Assembly as Fluorogenic Probe for BACE1. A highly efficient, specific, and stoichiometric quenching profile of the QD-Au assembly ensures the applicability of the probe as a robust sensing probe for the BACE1 enzyme. Given the saturation of quenching efficiency over the molar ratio of 3, QD-Au assembly at 1:3 molar ratio (QD/Au) was prepared for further in vitro assay experiments and live cell imaging experiments. This probe, QD-Au (1:3), shows ∼90% quenching efficiency and a hydrodynamic diameter of ∼20 nm in 1× PBS buffer (pH 7.4). In vitro evaluation of the BACE1 enzymatic activity was performed by incubating 25 μL of 20 nM QD-Au assembly 7 with varying concentrations of the BACE1 enzyme at 37 °C for 1 h in 0.5× PBS buffer (pH 7.0). The concentrations of BACE1 ranged from 0.48 to 4.8 μg per 50 μL of the digestion mixture (0.14−2.4 μM, molecular weight = ∼48 kDa). PL measurement at 525 nm showed that the fluorescence of QD-Au increased up to 3.5-fold at the minimal BACE1 concentration of 0.25 μM (Figure 3a). The saturation profile of the fold-increase of QD PL with regard to varying enzyme concentrations indicates that the prequenched QD-Au assembly probe can respond to BACE1 with a detection limit of 0.5 μg (0.15 μM) in 50 μL of the assay buffer solution. We also tested varying QD-Au assemblies prepared at different molar ratios (1−5) for their fluorogenic property (Figure 3b). QD PL showed a maximum fold-increase at the QD-Au assembly prepared at 1:3 molar ratio in the presence of 0.25 μM of BACE1. This result indicates that there exists an optimal number of AuNPs to ensure the maximum foldincrease of QD PL after enzymatic reactions. An excess amount of AuNPs (i.e., QD-Au assembly prepared at 1:5 molar ratio) rather decreased the recovery of QD PL, potentially due to the nonspecific quenching of AuNPs remaining after enzymatic digestion. Furthermore, as a control experiment, when the QD-Au probe was incubated with other aspartic proteases such as HIV1 protease, which cleaves Tyr-Pro residue in SQNYPIVQ sequences,36 the PL recovery of the QD-Au assembly probe did not occur (data not shown); the BACE1 enzyme cleaves the Leu-Asp residues in the recognition sequence (EVNLDAEF). 8598
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Figure 4. (a) Representative live cell confocal microscope image of HEK293 cells transfected with BACE1 plasmid after 1 h of incubation with 20 nM of QD-Au (1:3) assembly. (b) Colocalization of recovered fluorescence of the QD-Au assembly mainly with the Golgi apparatus and endosomal compartments as stained with the NBD-ceramide-BSA complex reagent. For easy identification, cell nucleus was noted with “N”, and the periphery was marked with a dotted line. Scale bars = 10 μm.
Figure 5. (a) High through-put image analysis algorithm from Operetta Harmony 2.0, which automatically detects the number of cells based on the nucleus stained by Hoechst 33342 (white) and finds the cytoplasmic region per cell, followed by calculation of QD fluorescence signal generated in ̈ or transfected with BACE1 plasmid) obtained from the cytoplasmic region only. (b) Representative fluorescence images of HEK cells (naive Operetta (multiple fields combined, upper) and the magnified views (lower) of the regions which show the recovery of QD fluorescence in the cytoplasm (arrows). The nucleus was stained by Hoechst 33342 (blue). Scale bars = 50 μm.
punctated green dots (Figure 4a). In addition, costaining with the NBD-ceramide-BSA complex, which preferably visualizes the Golgi apparatus and endosomal compartments, also showed that the QD signal was colocalized with the Golgi apparatus ̈ HEK cells or mock control (Figure 4b).37,38 Untreated naive (transfected with empty vector) as controls exhibited no fluorescence in the cells (see the Supporting Information Figure S4). Automated Cell-Based Assay Using QD-Au Assembly. With the proven specificity and efficiency of the fluorescence
recovery of the QD-Au assembly in vitro, we were able to apply the QD-Au-based BACE1 probe to the HTS system using an automated fluorescence microscope for potential drug discovery applications. Image acquisition and subsequent quantification of QD PL recovery in the cytoplasmic region in the 96-well format was automatically performed using Operetta (Harmony 2.0, Perkin-Elmer). An image analysis algorithm from Harmony 2.0 software (Perkin-Elmer) allowed us to automatically detect the number of cells based on the stained nucleus by Hoechst 33342 and find the cytoplasmic region per each cell, followed 8599
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Figure 6. (a) Quantification result of specific fluorescence recovery of QD-Au assembly in BACE1-transfected cell. (b) Dose response curves generated from image-based high throughput screening assay of BACE1 inhibitors using QD-Au assembly in the Operetta HTS system; 0% inhibition was defined as the normalized PL recovery ratio of QD-peptide after BACE1 treatment in the absence of inhibitors, while 100% inhibition was in the presence of the highest concentration of inhibitors. The data fitting was performed using a nonlinear log[inhibitor] vs response mode in GraphPad Prism 5 software.
by calculation of the QD fluorescence signal generated in the cytoplasmic region only (Figure 5a). The representative HTS ̈ and cell images clearly showed a difference between the naive the BACE1-transfected cells with regard to the QD signals generated in the cytoplasm, as marked with arrows in Figure 5b. In addition, to test the feasibility of BACE1 inhibitor screening application, we tested several commercially available BACE1 inhibitors (I, II, IV) and compared the QD fluorescence signal per cells. BACE1 inhibitor I (BSI I) is a statin-based peptidomimetic inhibitor with IC50 values of 30 or 5.6 nM reported in the literature.1 BACE1 inhibitor II (BSI II) is a cell-permeable and reversible inhibitor N-benzyloxycarbonyl-Val-Leu-leucinal (Z-VLL-CHO) with the reported IC50 values of 700 nM or 2.5 μM in CHO cells.39 BACE1 inhibitor IV (BSI IV) is another cell-permeable isophthalamide compound containing a hydroxyethylamine moiety with the IC50 values of 15 nM for human BACE1 and 29 nM for BACE1-transfected HEK293 cells.10 Figure 6a shows the quantification results of QD spot ̈ or the BACE1-transfected HEK number in either the naive cells, which were incubated with 20 nM of QD-Au (1:3) assembly for 1 h in the presence or absence of the inhibitors. QD spot numbers reached up to approximately 90 per cells in ̈ cells or mock the absence of the inhibitor, while the naive vector-transfected cells did not show any fluorescence recovery at the same concentration of the QD-Au probe. To exclude the potential nonspecific signal generation, we also treated the ̈ HEK cells with a high concentration of the probe (i.e., naive QD-Au 40 nM), which exhibited a negligible effect on the generation of the fluorescent spots per cell. However, in the presence of 10 nM each of BSI I, II, and IV, the QD spot numbers significantly decreased up to 50−60 per cell compared to the BSI control peptide (the same as the substrate peptide sequence). Figure 6b summarized the dose−response curves generated from the image analysis after converting the QD spot numbers per cell to the relative QD PL recovery ratio. When compared with the BSI control peptide, the relative QD fluorescence recovery in the presence of the inhibitors decreased in a concentration-dependent manner. Table 1 summarizes the resulting inhibitory concentration at the 50%
Table 1. Comparison of IC50 Values of Known BACE1 Inhibitors (BSIs) by Automated Cell-Based Assay Using Fluorogenic QD-Au Probe
a
Peptide sequences for peptidomimetic inhibitors (I, II) and control peptide (the same with the SCP); molecular structure for nonpeptidomimetic cell-permeable inhibitor (IV). bThe known IC50 values are from the supplier (Anaspec or Calbiochem Inc.) and the literature.39
(IC50) values of each inhibitor. Overall, the IC50 values of the tested inhibitors were found to be in the range of the reported values.1,39,40
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CONCLUSIONS Novel fluorogenic quantum dot (QD)-gold nanoparticle (AuNP) assembly was prepared via Ni-His interaction and utilized for the detection of beta-secretase 1 (BACE1) activity in vitro and in living cells. Specific binding of 5 nm Ni-NTAAuNP with the QD-peptide conjugate via Ni-His interaction resulted in an quenching of the highly luminescent polymercoated QDs bearing the BACE1 substrate peptide with high efficiency in a stoichiometric manner. In vitro evaluation of QD-Au assembly as a fluorogenic probe for BACE1 showed that fluorescence of the prequenched QD PL increased up to 3.5-fold at the minimal BACE1 concentration of 0.25 μM. The QD-Au assembly probe can respond to BACE1 with a detection limit of 0.5 μg (0.15 μM) in 50 μL of the assay buffer solution. Fluorescence recovery of QD with high efficiency and sensitivity, and the robust QD signal further enabled visualization of BACE1 activity in live cells and led to reproducible IC50 values for the BACE1 inhibitors by cell-based visual screening in the high-throughput screening format. The 8600
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present assay system using polymer-coated QDs and 5 nm NiNTA-AuNP may become an alternative tool for solution-based conventional HTS screening applications of other proteaserelated diseases such as Alzheimer’s diseases, AIDS, and cancer.
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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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AUTHOR INFORMATION
Corresponding Author
*Tel: +82-31-8018-8230. Fax: +82-31-8018-8014. E-mail:
[email protected]. Present Addresses §
Drug Metabolism and Pharmacokinetics Team, IP-K, South Korea. ⊥ Drug Biology Group, IP-K, South Korea. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MEST), Gyeonggido (No. (K204EA000001-09E0100-00110), KISTI, National R&D Program (No. 2011-0019144), and General Scientist Program (No. 2011-0027197). We are grateful to Dr. Jiho Kim and Joohyun Lee in the Neurodegeneration & Applied Microscrocopy group in Institut Pasteur Korea for providing BACE1 inhibitors and a fruitful discussion.
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dx.doi.org/10.1021/ac301574b | Anal. Chem. 2012, 84, 8595−8601
