Enzymatic Immuno-Assembly of Gold Nanoparticles for Visualized

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Enzymatic Immuno-Assembly of Gold Nanoparticles for Visualized Activity Screening of Histone-Modifying Enzymes Zhen Zhen, Li-Juan Tang, Haoxu Long, and Jian-Hui Jiang* State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, P. R. China S Supporting Information *

ABSTRACT: Activity screening of histone-modifying enzymes is of paramount importance for epigenetic research as well as clinical diagnostics and therapeutics. A novel biosensing strategy has been developed for sensitive and selective detection of histone-modifying enzymes as well as their inhibitors. This strategy relies on the antibody-mediated assembly of gold nanoparticles (AuNPs) decorated with substrate peptides that are subjected to enzymatic modifications by the histone-modifying enzymes. This design allows a visual and homogeneous assay of the enzyme activity using antibodies without any labels, which circumvents the requirements to prefunctionalize the antibody and affords improved assay simplicity and throughput. Additionally, the use of antibody-based recognition of modified peptides could offer improved specificity as compared with existing techniques based on the enzyme coupled assay. We have demonstrated this strategy using a histone methyltransferase acting on histone H3 (Lys 4) and a histone acetyltransferase acting on histone H3 (Lys 14). The results reveal that the absorption peak characteristic for AuNPs decreases dynamically with increasing activity of the enzymes with concomitant visualizable color attenuation, and subnanomolar detection limits are readily achieved for both enzymes. The developed strategy can thus offer a robust and convenient visualized platform for screening the enzyme activities and their inhibitors with high sensitivity and selectivity.

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epigenetic research as well as clincal diagnostics and therapeutics. There are several methods currently available for activity screening of histone-modifying enzymes. Many of these involve the use of some coupled reactions to detect the byproduct (nonpeptide products) of coenzymes from the enzymecatalyzed reactions.6 For example, homogeneous assay approaches for histone acetyltransferases (HAT) have been described via directly detecting the product of coenzyme A from acetylation reactions using some thiol-specific chromogenic or fluorogenic reagents.7 Techniques for homogeneous assay of histone methyltransferases (HMT) have also been developed on the basis of enzymatic detection of S-adonosylhomocysteine (SAH) using SAH hydrolase or nucleosidase.8 These coupled assays, however, are susceptible to endogenous compounds having similar reactivity as the coenzyme products and homologous enzymes acting on different sites of the substrate peptide.9 As an alternative to the coupled assays, direct detection of the modified peptide products represents an attractive option to circumvent the aforementioned drawbacks. In this situation, the modified peptides can be detected in a straightforward way using mass spectrometry.10 More com-

istone proteins and their assemblies with DNA and nucleosomes are the essential building blocks of eukaryotic chromatin. A diverse array of post-transcriptional modifications often occur on the amino-terminal tail domains of the proteins. These modifications can generate synergistic or antagonistic interaction affinities for chromatin-associated proteins, which controls the structural organization of chromatin and its transcriptional status.1 The combinatorial nature of histone amino-terminal modification thus reveals an epigenetic marking system or a “histone code” that greatly expands the information potential of the genetic code and constitutes a critical regulatory mechanism in many fundamental biological processes.2 Post-translational modifications of amino-terminal tail domains of histones include acetylation, methylation, phosphorylation, ubiquitination, and ADP-ribosylation.3 These modifications take place through enzymecatalyzed addition or removal of certain molecules, such as acetyl or methyl groups, phosphate, ubiquitin, and ADPribose.4 Dysregulation of histone-modifying enzymes is expected to increase susceptibility to various diseases such as cancer, diabetes, inflammatory diseases, neurodegenerative diseases, and cardiovascular diseases.5 Therefore, activities of histone-modifying enzymes have become significant biomarkers for these diseases, and screening for inhibitors of the enzymes represents an effective and valuable approach to chemotherapy as well as chemoprevention. In the context, activity screening of histone-modifying enzymes is of paramount importance for © 2012 American Chemical Society

Received: December 22, 2011 Accepted: March 29, 2012 Published: March 29, 2012 3614

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experiment was a histone 3 tail with its sequence as ART KQT ARK STG GKA PRK QLA TKE EEE EEE EEE EEE NNL AC, where K denotes the lysine site (Lys 4) that can be methylated by SET 7/9 HMT and K denotes the lysine site (Lys 14) that can be acetylated by PCAF HAT. The control peptide was E EEE EEE EEE EEE NNL AC, which was deigned to be significantly shorter than the substrate to mitigate the steric hindrance. Both peptides were synthesized and purified (>95% purity) by A’Peptide Co. Ltd. (Shanghai, China). Preparation of Peptide-Modified AuNPs. AuNPs were synthesized by citrate reduction of HAuCl4 according to documented protocols (more details in Supporting Information).35,36 The peptide-modified AuNPs, which serve as the artificial substrates for HMT and HAT, were prepared according to the reported method37 with some modifications. Briefly, the peptide-modified AuNPs were prepared by adding a 500 μL aliquot of peptide mixture (aqueous solution of 50 μM substrate peptide plus 50 μM control peptide) to 500 μL of AuNPs solution under vigorous stirring for 24 h. The unmodified peptides were removed via centrifugation at 12 000g for 15 min followed by resuspension of the sediment in 500 μL of 10 mM phosphate buffer (PB, pH 7.2). This step was repeated three times to sufficiently remove all excess peptides. Subsequently, the peptide-modified AuNPs were redispersed in 500 μL of PB (10 mM, pH 7.2) and stored at 4 °C for downstream assays. The final concentration of peptidemodified AuNPs was ∼13 nM, assuming that there was no significant loss of AuNPs during the preparation process. Activity Analysis of Histone Metyltransferase (HMT). The HMT reaction was started by adding a 30 μL aliquot of the peptide-modified AuNPs (∼3.2 nM in AuNP concentration) in 30 μL of reaction mixture composed of 50 mM Tris-HCl (pH 8.5), 100 mM NaCl, 1 mM EDTA, 0.01% Tween-20, 80 μM SAM, and a given concentration of the SET 7/9 HMT (5 μL). After a 1 h incubation at 37 °C, 1 μL of methyl-histone H3 (Lys 4) monoantibody (6.7 μM) was added to the reaction mixture and incubated for 10 min at the room temperature on a shaker (Eppendorf, Hamburg, Germany). We also performed control experiments with an equal volume of BSA solution in place of the enzyme and an equal volume of water in place of SAM, respectively. In the inhibitor assay, the SET 7/9 HMT (2 μL, 12 μM) was mixed with 3 μL of sinefungin dilution of varying concentration followed by a pre-equilibration for 10 min at room temperature. Then, the enzyme mixture (5 μL) was included in the reaction mixture (the final volume was 60 μL). Then, the assay of HMT activity in the presence of the inhibitors was performed according to the protocol described above. Activity Analysis of Histone Acetyltransferase (HAT). The HAT reactions were performed by adding a 30 μL aliquot of the peptide-modified AuNPs (∼3.2 nM in AuNP concentration) in a 30 μL reaction mixture containing 100 mM HEPES (pH 7.5), 0.01% Tween-20, 200 μM Acetyl CoA, and a given concentration of the PCAF HAT (5 μL) followed by incubation at 25 °C for 1 h. Then, 1 μL of acetyl-histone H3 (Lys 14) antibody (6.7 μM) was added to the reaction mixture and incubated for 10 min at the room temperature on a shaker (Eppendorf, Hamburg, Germany). We also performed control experiments with 5 μL of BSA solution in place of the enzyme, an equal volume of water in place of Acetyl CoA, and 5 μL of pre-equilibrated mixture of the PCAF HAT (2 μL, 9 μM) with anacardic acid (3 μL, 4 mM), respectively.

monly, the modified peptides can be analyzed using certain antibodies or other binding proteins that specifically recognize the histone modifications, and the enzymatic activity can be detected on the basis of immunosorbent assay11 or fluorescence resonant energy transfer.12 Despite of their improvement in specificity and stability, methods of this kind typically involve the use of antibodies conjugated with enzyme or fluorescence labels. This may complicate the assay development, since each step of the preparation of antibody conjugates should be carefully optimized in order to guarantee the desired stability. Herein, we develop a novel biosensing strategy for visual screening of the activities of histone-modifying enzymes without the need of labeled antibodies. This strategy relied on the antibody-mediated assembly of gold nanoparticles (AuNPs) decorated with substrate peptides that are subjected to enzymatic modifications by the histone-modifying enzymes. AuNP-based assembly has become a useful platform in analytical chemistry because of its high sensitivity comparable to fluorescence assay and exquisite capability of visual detection using “naked” eyes.13,14 It has been implemented directly for a variety of targets such as DNA,15−17 metal ions,18,19 small molecules,20−22 proteins,23−26 and even cancer cells.27 With the combination of enzymatic reactions, it has also been demonstrated for screening of DNA binders,28−30 genetic mutations, and enzyme activities.31−34 To our knowledge, there is no report concerning the use of AuNP-based assembly for detecting histone-modifying enzymes. Herein, we have developed for the first time a novel biosensing strategy for visual and homogeneous screening of the activities of histonemodifying enzymes using antibodies without any labels. The use of label-free antibody offers high specificity intrinsic in the immuno-recognition, while circumvents the requirements to prefunctionalize the antibody and thus affords improved assay simplicity and throughput. This strategy is demonstrated using two model systems, a histone methyltransferase acting on histone H3 (Lys 4) and a histone acetyltransferase acting on histone H3 (Lys 14). The developed strategy may create a robust, convenient, and visualized platform for screening the enzyme activities and their inhibitors with high sensitivity and selectivity.

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EXPERIMENTAL SECTION Chemicals and Materials. Human recombinant SET 7/9 histone methyltransferase (HMT) and pCAF histone acetyltransferase (HAT) were purchased from Cayman Chemical (Ann Arbor, MI, USA). Acetyl coenzyme A sodium salt (Acetyl CoA) was obtained from Sigma Aldrich Chemical Co. Sadenosyl-methionine (SAM) was from New England BioLabs (Ipswich, MA, USA). Sinefungin and anacardic acid were purchased from Enzo Life Science Inc. (Farmingdale, NY, USA). Acetyl-Histone H3 (Lys 14) and methyl-Histone H3 (Lys 4) monoclonal antibodies were obtained from Cell Signaling Techonology Inc. (Danvers, MA, USA). Bovine serum albumin (BSA) was provided by National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). All other chemicals were of analytical grade and obtained from Sinopharm Chemical Reagent Co. Ltd. (Beijing, China). Stock solution of anacardic Acid (2 mM) was prepared with dimethysulfoxide (DMSO) and stored at −20 °C in the dark. All other solutions were prepared using ultrapure water, which was obtained through a Millipore Milli-Q water purification system (Billerica, MA, USA) and had an electric resistance of >18.3 MΩ. The substrate peptide used in the 3615

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Scheme 1. Illustration of Biosensing Strategy for Histone-Modifying Enzyme (HME) Based on Antibody-Mediated Assembly of AuNPs Decorated with Substrate Peptides Subjected to Enzymatic Modifications

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RESULTS AND DISCUSSION Design of Immunoassembly-Based Biosensing for Histone-Modifying Enzymes. This developed biosensing strategy relies on the antibody-mediated assembly of AuNPs decorated with substrate peptides that are subjected to enzymatic modifications by the histone-modifying enzymes. Scheme 1 illustrates the general principle of the immunoassembly-based biosensing strategy for visual screening of histone-modifying enzyme activity. The AuNPs are decorated via mixed self-assembly with a substrate peptide and a control peptide. The substrate peptide is designed to have a substrate sequence at the N′ terminal and a thiolated, negatively charged spacer sequence at the C′ terminal. This spacer peptide can minimize the steric hindrance in enzymatic reactions and enhance the stability of the peptide-decorated AuNPs. The control peptide is designed to only have the spacer sequence, which can control the surface density of the substrate peptide on AuNPs. In the presence of an active histone modifying enzyme such as HMT or HAT, the substrate peptide is modified at a specified site with a certain group such as methyl for HMT or acetyl for HAT. After the addition of an immunoglobin G (IgG) antibody specific to this modifed peptide (a methylated peptide sequence for HMT or an acetylated peptide sequence for HAT), the peptides subjected to enzymatic modifications can be bound by the divalent IgG antibody at its two binding sites. This triggers a network-like assembly of the peptidemodified AuNP and thus induces a significant variation in the plasmon resonance absorption peak with a visualized color change. Because the antibody-mediated assembly of AuNPs is highly selective to the modified peptides obtained in the enzymatic reaction, the resulting absorption spectral response

can then give an indicator for the activity of the histone modifying enzyme. On the basis of the aforementioned principle, the developed immunoassembly-based biosensing strategy has several advantages over traditional techniques because of its ability to allow sensitive visual detection of enzymatic activities, perform homogeneous single-phase assays, combine the specificity of antibody to enzymatic product, and circumvent prefunctionalization of the antibody. It may hold great potential as a very generic approach for activity screening of histone modifying enzymes and other enzymes for protein post-translational modifications. To demonstrate the potential, we chose the SET 7/9 HMT that specifically targets histone 3 at lysine 4 (H3 K4) and the pCAF HAT that selectively acetylates lysine 9 on histone 3 (H3 K9) as the model system. Both modifications on histone 3 can promote a transcriptionally active conformation of the chromatin. Immunoassembly-Based Colorimetric Biosensing Strategy for SET 7/9 HMT Assay. Figure 1 depicts typical absorption spectral responses of the biosensing strategy in the assay of SET 7/9 HMT. The peptide-decorated AuNPs were well dispersed in aqueous solution, displaying a red color with a single surface plasmon absorption peak centered at 522 nm (curve a in Figure 1A). After incubation of the peptidedecorated AuNPs (final concentration of ∼1.6 nM) with 400 nM HMT in the presence of 80 μM SAM, no appreciable color change appeared in the solution and the absorption peak remained unchanged (curve b in Figure 1A), implying that the enzymatic modification reaction had little effect on the stability of the peptide-decorated AuNPs. Followed by the addition of antibody (final concentration of 112 nM) against methylated H3 K4, the solution showed a rapid color fading and became 3616

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addition of antibody also showed no remarkable color change in this system, which revealed that the presence of specific substrate peptide was indispensable for the active enzymatic methylation of H3 K4. Additionally, we performed a control experiment in which the peptide-decorated AuNPs were incubated with HMT in the presence of its inhibitor, sinefungin (100 μM), as well as its cofactor SAM. As anticipated, no significant color change was observed after the addition of antibody, though the absorption spectrum displayed a slight decrease, which might be attributed to incomplete inhibition of the HMT activity in 100 μM sinefungin inhibitor. Taken together, the color change and spectral responses in our assays were highly specific to the methylation of H3 K4 catalyzed by active HMT, implying our immunoassembly-based colorimetric biosensing strategy provided a selective and visualized platform for activity screening of HMT. Dynamic Light Scattering Characterization of the Biosensing Strategy for SET 7/9 HMT Assay. To further verify the mechanism of our biosensing strategy, dynamic light scattering (DLS) analysis was performed to inspect the assembly of AuNPs in the assays. Because the network-like assembly of AuNPs produced AuNP aggregates with a large hydrodynamic diameter, DLS analysis could provide straightforward evidence for the assembly of AuNPs. As shown in Figure 2, the peptide-decorated AuNPs gave an average hydrodynamic diameter of ∼33.6 nm, which was much larger than the core size (∼12 nm) of AuNPs because of the additional peptide and hydration layers. After the peptidedecorated AuNPs reacted with 400 nM HMT in the presence of SAM, their average hydrodynamic diameter almost remained

Figure 1. Typical absorption spectra obtained in assays of SET 7/9 HMT: (A) substrate peptide-decorated AuNPs (1.6 nM) (a); substrate peptide-decorated AuNPs reacting with HMT (400 nM) plus SAM (80 μM) (b); substrate peptide-decorated AuNPs reacting with HMT plus SAM followed by adding methylated H3 K4 antibody (112 nM) (c). (B) substrate peptide-decorated AuNPs reacting with HMT but without SAM followed by adding antibody (a); substrate peptide-decorated AuNPs reacting with SAM but without HMT followed by adding antibody (b); control peptide-decorated AuNPs reacting with HMT and SAM followed by adding antibody (c); substrate peptide-decorated AuNPs reacting with HMT plus inhibitor sinefungin (100 μM) and SAM followed by adding antibody (d); substrate peptide-decorated AuNPs reacting with HMT plus SAM followed by adding antibody (e). The insets are the photographs for the corresponding systems.

almost colorless after 10 min with the absorption peak decreased by ∼65% (curve c in Figure 1A), a typical behavior of the network-like assembly of AuNPs. Because of the high specificity of the antibody to the peptide modification, the methylation of H3 K4 was anticipated, suggesting an active enzymatic reaction in incubation of the peptide-decorated AuNPs with HMT. Further control experiments were performed to verify that the antibody-triggered assembly of the peptide-decorated AuNPs was mediate by active enzymatic methylation of H3 K4, as shown in Figure 1B. In the control experiments when either HMT or SAM was not included in the peptide-decorated AuNPs solution during the incubation step, no color change was obtained after the addition of antibody. This suggested active enzymatic reactions could only occur in the presence of both HMT and SAM. Alternatively, we prepared AuNPs decorated with the control peptide but without the substrate peptide and incubated the control peptide decorated AuNPs with HMT in the presence of SAM. It was observed that the

Figure 2. Hydrodynamic sizes of AuNPs determined by DLS analysis: (a) peptide-decorated AuNPs; (b) peptide-decorated AuNPs reacting with HMT plus SAM; (c) peptide-decorated AuNPs reacting with HMT plus SAM followed by addition of methylated H3 K4 antibody; (d) peptide-decorated AuNPs reacting with HMT but without SAM followed by addition of methylated H3 K4 antibody; (e) peptidedecorated AuNPs reacting with SAM but without HMT followed by addition of methylated H3 K4 antibody. 3617

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unchanged (∼33.8 nm). In contrast, after adding the antibody against methylated H3 K4 in the enzymatic reaction system, a substantial increase in the average hydrodynamic diameter (∼362.4 nm) was observed for the AuNPs, which gave an immediate evidence for the assembly of the peptide-decorated AuNPs into large aggregates. In addition, in the control experiments in which either HMT or SAM was absent in the enzymatic reaction mixture, there was also no remarkable variation in the average hydrodynamic diameters of AuNPs even after the addition of antibody to the methylated peptide. These results were in good consistence with those obtained with absorption spectral measurements, which further confirmed that methylation of the substrate peptide allowed the antibody-mediated assembly of AuNPs in large aggregates, and such an assembly was highly specific for the active HMT reaction. Effect of Mixed-Assembly of Substrate Peptide and Control Peptide. The antibody-mediated assembly of AuNPs requires effective enzymatic reaction with the substrate peptides as well as viable antibody-mediated interparticle cross-linking of the peptide-decorated AuNPs. Both reactions were highly dependent upon surface coverage of the substrate peptides on AuNPs. Because of the theoretical difficulties in predicting the optimal surface coverage for the substrate peptides, we chose the mixed assembly to prepare the peptide-decorated AuNPs in order to control the surface coverage of the substrate peptides. In this case, the peptide-decorated AuNPs were synthesized via self-assembly using a mixture of the substrate peptide and the control peptide in varying molar ratio (Figure S1 in Supporting Information). When the molar ratio of the substrate peptide to the control peptide was larger than 1:1, the absorbance responses became less sensitive to HMT with increasing percentage of the substrate peptide, suggesting in this case the steric hindrance at the peptide-decorated AuNPs had substantial effect on the enzymatic reaction or the antibodymediated interparticle cross-linking. With the molar ratio of the substrate peptide to the control peptide less than 1:1, we also obtained smaller absorbance responses, the decrease of absorbance at 522 nm, to HMT with decreasing percentage of the substrate peptide. This indicated that less surface coverage of substrate peptide might induce lower efficiency in enzymatic reaction and antibody-mediated interparticle crosslinking. So, the maximized absorbance response was achieved in the case when the molar ratio of the substrate peptide to the control peptide was 1:1. This optimal surface density of the substrate peptide on AuNPs was estimated to be ∼203, which was obtained using a fluorescein-labeled substrate peptide for the decoration and determining its concentration via direct fluorescence measurements after dissolving the AuNPs with 80 mM KCN and 0.8 mM K3Fe(CN)6.38 Quantitative Activity Screening of SET 7/9 HMT Using the Biosensing Strategy. The ability of the developed biosensor for quantitative activity screening of the SET 7/9 HMT was further investigated. A series of samples containing the SET 7/9 HMT of different concentrations were incubated with the peptide-decorated AuNPs in the presence of 80 μM SAM, and then antimethylated H3 K4 antibody was added. Figure 3 displays typical absorption spectral responses of the developed strategy in the assays. It was observed in Figure 3A that the red color of the peptide-decorated AuNPs solutions were gradually attenuated with increasing HMT concentration, suggesting the peptide-decorated AuNPs were assembled into larger aggregates in the presence of higher HMT concentration.

Figure 3. (A) Photographs of the biosensing responses to HMT of varying concentrations. (B) Typical absorption spectral responses of the biosensing strategy to of varying concentrations. (C) Corresponding peak absorbance readings versus HMT concentrations.

These visual observations were consistent with the absorption spectral measurements. As shown in Figure 3B, the absorption peaks were found to show a gradual decrease with increasing HMT concentration with a slight concomitant red shift from 522 to 527 nm. A plot of the absorbance readings at 522 nm versus the SET 7/9 HMT concentrations, as depicted in Figure 3C, revealed a dynamic correlation between the peak absorbances and the HMT concentrations in the range from 1 to 400 nM. A quasilinear correlation was obtained to the logarithmic concentration ranging from 1 to 200 nM with a detection limit of 0.2 nM in terms of the rule of 3 times standard deviation over the blank response (Figure S2 in Supporting Information). This detection limit was over 10 times better than existing methods for HMT assays (The detection limits were ∼3.9 μM and 5 nM, respectively).8b,39 Furthermore, the colorimetric biosensor was found to show very desirable reproducibility due to its homogeneous assay format. The relative standard deviations (RSDs) of peak absorbance readings were 1.3%, 1.5%, 1.4%, 1.7%, and 2.4% in four repetitive assays of 4 nM, 20 nM, 60 nM, 150 nM, and 200 3618

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Immunoassembly-Based Biosensing Strategy for PCAF HAT Assay. To demonstrate the generality of the developed strategy for quantitative activity screening of histone modifying enzymes, we further adapted this strategy to the assay of a HAT, the PCAF HAT (Figure S4 in Supporting Information). After the peptide-decorated AuNPs were incubated with 200 μM acetyl CoA in the absence of HAT followed by the addition of antibody (final concentration of 112 nM) against acetylated H3 K9, there was no appreciable change in the red color for the AuNPs solution. In contrast, when the peptide-decorated AuNPs were incubated with 300 nM HAT in the presence of acetyl CoA followed by the addition of the antibody, an obvious attenuation of the red color was observed for the solution of the peptide-decorated AuNPs. This indicated that the peptide-decorated AuNPs were subjected to an antibody-mediated assembly into large aggregates, thus manifesting an active HAT modification of the substrate peptide. Furthermore, when the peptide-decorated AuNPs were incubated with 200 μM acetyl CoA, HAT, and a HAT inhibitor, anacardic acid (200 μM), we also did not observe significant color change in the peptide-decorated AuNPs solution. The absorption spectral measurements were also in good agreement with these observations. These results implied that our biosensor strategy could be used for the assay of the PCAF HAT activity and its inhibitor. The absorption peaks were also observed to decrease in dynamic correlation with increasing HAT concentration in the range from 2 to 300 nM with a slight concomitant red shift from 522 to 526 nm (Figure S5 in Supporting Information). A plot of the absorbance readings at 522 nm versus the logarithmic HAT concentrations revealed a quasilinear correlation between the peak absorbances and the HAT concentrations in the range from 2 to 200 nM with the detection limit estimated to be 0.5 nM. This detection limit was among the best in existing methods for HAT assays (The detection limits were ∼10 nM and 0.5 nM, resectively).7,12 The RSDs of peak absorbance readings were 1.1%, 1.5%, 1.8%, 1.7%, and 1.2% in four repetitive assays of 2 nM, 20 nM, 40 nM, 100 nM, and 160 nM PCAF HAT. These findings implied that the developed strategy provides a sensitive and robust platform for activity assay of HAT.

nM HMT. Therefore, we might conclude that the developed colorimetric biosensor held potential for quantitative activity assay of the SET 7/9 HMT with desirable sensitivity and reproducibility. HMT has been recognized as a significant therapeutic target, and the screening of its inhibitors can provide potential drugs for the treatment of related diseases. Hence, the colorimetric biosensor was further investigated for quantitatively screening of the inhibitor for the SET 7/9 HMT using a model inhibitor, sinefungin. In the assay, the HMT was preincubated with sinefungin of different concentrations for 10 min. The resulting mixture was introduced in the reaction solution to initiate the enyzmatic reaction with the peptide-decorated AuNPs followed by the addition of antibody against methylated H3 K4. Figure 4

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CONCLUSIONS We developed a novel biosensing strategy for sensitive and specific activity screening of the histone-modifying enzymes based on assembly of the peptide-decorated AuNPs into network structures mediated by an antibody specifically recognizing the modified peptide. To our knowledge, this strategy represented the first example of the use of the AuNPbased assembly for detecting histone-modifying enzymes. This design allowed a visual and homogeneous assay of the enzyme activity using antibodies without any labels, which circumvented the requirements to prefunctionalize the antibody and thus afforded improved assay simplicity and throughput. The results revealed that this strategy could be implemented conveniently through two homogeneous reactions and enabled the screening of the enzyme activity and its inhibitors with high sensitivity and selectivity. Compared with existing techniques for histone-modifying enzyme detection based on coupled assay or mass spectrometry, this strategy offered improved specificity because of the incorporation of antibody-based recognition of the modified peptides, which could eliminate most interferences in complex biological matrixes. This method might hold

Figure 4. (A) Typical absorption spectral responses of the biosensing strategy to sinefungin, a HMT inhibitor, of varying concentrations. (B) Corresponding peak absorbance readings versus sinefungin concentrations.

depicts the absorption spectral responses in the assays of sinefungin of varying concentrations. The absorption peaks were observed to increase dynamically with increasing sinefungin concentration in the range from 10 nM to 100 μM with a concomitant blue shift from 527 to 522 nm. The absorbance readings at 522 nm displayed a quasilinear correlation to the logarithmic concentrations of sinefungin in the range from 50 nM to 20 μM (Figure S3 in Supporting Information), and the detection limit was estimated to be 10 nM. The RSDs of absorbance readings were 1.3%, 1.8%, 1.5%, 1.2%, and 1.1% in four repetitive assays of 50 nM, 100 nM, 500 nM, 1 μM, and 10 μM sinefungin. These findings demonstrated that our strategy could provide a convenient, sensitive method for quantitatively screening of the inhibitors of HMT. 3619

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great potential for activity assay of the enzymes in practical samples such as cell extracts. Additionally, it could be further extended for screening the specificity of the enzymes and the study of reaction mechanism by mutating the amino acid sequences. Using substrate peptides for other proteinmodifying enzymes and the corresponding antibodies, this method could create a very generic approach for activity screening of various enzymes for protein post-translational modifications.

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ASSOCIATED CONTENT

S Supporting Information *

Description of other experimental procedures and additional figures. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: 86-731-88821961. Fax: 86-731-88821916. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by NSFC (21025521, 21035001, 21190041), National Key Basic Research Program (2011CB911000), CSIRT Program, and NSF of Hunan Province (10JJ7002).

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dx.doi.org/10.1021/ac203385v | Anal. Chem. 2012, 84, 3614−3620