Hyperbranched Polyester-Based Fluorescent Probe for Histone

Nov 18, 2013 - College of Materials Science & Engineering, State Key Laboratory of Luminescent Materials & Devices, South China University of Technolo...
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Hyperbranched Polyester-Based Fluorescent Probe for Histone Deacetylase via Aggregation-Induced Emission Changmin Yu,† Yinglong Wu,† Fang Zeng,*,† Xizhen Li,† Jianbin Shi,‡ and Shuizhu Wu*,† †

College of Materials Science & Engineering, State Key Laboratory of Luminescent Materials & Devices, South China University of Technology, Guangzhou 510640, China ‡ College of Materials Science & Engineering, College of Science, Beijing Institute of Technology, Beijing 100081, China S Supporting Information *

ABSTRACT: Aberrant expression of histone deacetylases (HDACs) is related to various types of cancer and is associated with increased proliferation of tumor cells. Hence, the detection of HDAC activities is of great significance for medical sciences as well as biological diagnostics. Herein, we report a hyperbranched polyester-based one-step fluorescent assay for HDAC. This assay system consists of two water-soluble components: the hyperbranched polyester coupled with the acetylated lysine groups (H40-Lys(Ac)) and the negatively charged TPE derivative bearing two sulfonic acid groups (TPE-2SO3−). HDAC triggers the deacetylation of H40-Lys(Ac), thereby turning the electroneutral polymer into the positively charged one. Consequently, complexation occurs between the positively charged polymer and the negatively charged TPE-2SO3−, thereby leading to the formation of nanoaggregates due to electrostatic interaction. Eventually, the fluorescence enhancement as a result of AIE effect is achieved. This assay system is operable in aqueous media with very low detection limit of 25 ng/mL. The system is capable of detecting HDAC in such biological fluid as serum, and this strategy may provide a new and effective approach for enzyme assay. disease genes.7,8 Therefore, the detection of HDAC activities is of great significance for medical sciences as well as biological diagnostics. To better detect and understand HDACs’ activity, intensive efforts have been made to develop chemical probes that determine their levels or interrogate their mechanisms and function.9−12 A variety of approaches have been developed for investigating HDACs’ activities and function, such as HPLC,9,10 mass spectrometry,11 inhibitor competition,12,13 and immunohistochemical,14 electrochemical,8 and fluorescent techniques.15,16 Among these methods, fluorescence technique offers distinct advantages including high sensitivity, convenience and real-time detection.17−37 Several fluorescent HDACs probes have been reported to date.15,16,38−42 For instance, Jung’s group developed the first fluorescence-based HDAC assay which relied on the use of a coumarin-labeled ε-acetyl-lysine;38,39 later the same group

1. INTRODUCTION The reversible acetylation of histones influences the structure and transcriptional activity of chromatin. Histone acetylation plays crucial roles in cellular functions such as DNA replication, transcription, differentiation, and apoptosis.1,2 The level of histone acetylation regulates the interaction of nuclear proteins such as RNA polymerase, hormone receptors, and other activator or repressor proteins with DNA. Whereas the acetylation of histones is catalyzed by histone acetyltransferases, their deacetylation reaction is accomplished by histone deacetylases (HDACs). Histone deacetylases (HDACs) are hydrolytic enzymes that catalyze the removal of acetyl groups from ε-N-acetylated lysine residues of histones and other cellular proteins.3,4 It has been demonstrated that, the aberrant expression of histone deacetylases (HDACs) is related to various types of cancer and is associated with increased proliferation of tumor cells.5−7 Hence, HDACs have become the major targets of drug development. In particular, in the case of abnormal level or activity of HDAC, there would be an aberrant acetylation level of histone which could result in reactivating specific silenced © 2013 American Chemical Society

Received: October 18, 2013 Revised: November 11, 2013 Published: November 18, 2013 4507

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Figure 1. Schematic illustration for the assay system, and its fluorescent response to histone deacetylase.

developed a more elegant fluorescent HDAC assay based on a boradiazaindacene derivative.40 Kikuchi et al. ingeniously designed a one-step fluorescent probe for HDAC, which is based on a coumarin-conjugated peptide; in their probe, the deacetylation reaction by HDAC triggered the removal of the carbonate ester on coumarin, thus switching on the fluorescence response.41 Recently they also reported a small-molecular fluorescent probe for HDAC; for their approach, both N-α-tertbutoxycarbonyl-N-ε-acetyl-L-lysine and sulfonate were coupled onto tetraphenylethylene (TPE); upon deacetylation, the ε-amine of lysine complexed with the sulfonate group owing to electrostatic interaction; as a result, the TPE’s free rotor motions was restricted and became highly fluorescent.42 Despite of recent progress in the fluorescent HDAC probes and sensors, there is still a high demand to improve the available probes for utilization in biological samples such as body fluids with respect to their water solubility, low detection limit, ease of use and biocompatibility. Toward this end, we sought to prepare a hyperbranched polyester-based one-step fluorescent assay for HDAC. The hyperbranched polyester H40 was employed as the scaffold of this assay system, due to its water-solubility, high molecular weight nature, globular architecture and high number of chain end functionalities.43−50 This assay system consists of two components: the first one is

the hyperbranched polyester coupled with the acetylated lysine groups (H40-Lys(Ac)), which is electroneutral; and the second is the negatively charged TPE derivative containing two sulfonic acid groups (TPE-2SO3−). In the absence of HDAC, the two-component assay system is nonfluorescent. While in the presence of HDAC, the acetyl moieties are removed from H40-Lys(Ac), leaving the polymer with a large number of ε-amine groups which become protonated under physiological conditions. Consequently, the electroneutral polymer globules become positively charged and complexation occurs between the globules and the negatively charged TPE-2SO3− molecules; and the complexation further leads to the formation of nanoaggregates as well as the fluorescence enhancement of the TPE moieties as a result of aggregation-induced emission (AIE effect); the AIE effect was first reported by Tang et al, and has been applied in fluorescent probes and sensors.51−61 The schematic illustration for the fluorescent turn-on detection is shown in Figure 1. The main advantage of using the H40 polymer as the scaffold for the system is that, H40 molecules turn into polyelectrolytes in the presence of HDAC, and the polyelectrolyte-involved Columbic interaction between TPE2SO3− molecules and the positively charged H40 molecules can facilitate the stabilized complexation as well as the aggregates’ formation, this may reduce the dissociation of the 4508

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H40/TPE-2SO3− complexes and lead to lower detection limit compared to that of the small-molecule-based AIE systems.

product was precipitated out from the solution. The product was collected by filtration and washed with ethanol and acetone twice. The white solid of TPE-2SO3− was obtained in 60% yield. 1H NMR (400 MHz, DMSO-d6, δ ppm): 7.30−7.39 (m, 2H), 6.84−7.10 (m, 12H), 6.64−6.71 (m, 4H), 3.87−3.99 (m, 4H), 2.53−2.57 (t, 4H), 1.91−1.98 (m, 4H). MS (ESI): m/z 629.2 [M + Na]+. 2.6. Synthesis of TPE Derivative Containing One Sulfonic Acid Group (TPE-SO3−). TPE-OH (0.174 g, 0.5 mmol) was added into 10 mL of anhydrous ethanol under nitrogen. A mixture of NaOEt (0.048 g, 0.7 mmol) in 10 mL of ethanol was added dropwise and stirred for 1 h. And then 1,3-propane sultone (0.06 g, 0.482 mmol) in 5 mL of ethanol was added. The mixture was vigorously stirred for 12 h until a white product was precipitated out from the solution. The product was collected by filtration and washed with ethanol and acetone twice. A white solid of TPE-SO3− was obtained in 40% yield. 1 H NMR (400 MHz, DMSO-d6, δ ppm): 7.08−7.18 (m, 9H), 6.94− 6.99 (m, 6H), 6.83−6.85 (d, 2H), 6.66−6.68 (d, 2H), 3.93−3.97 (t, 2H), 2.52−2.55 (t, 2H), 1.93−1.97 (t, 2H). MS (ESI): m/z 468.8 [M − H]−. 2.7. Fluorometric Analysis. Fluorescence spectra were recorded with the excitation at 345 nm. The assay was prepared by dissolving H40-Lys(Ac) and TPE-2SO3− in HEPES buffer. The measurement was conducted every 10 min for samples containing the assay (final concentration: H40-Lys(Ac) 1.5 μM and TPE-2SO3− 20 μM) and NAD+ (300 μM) with or without Sirt1 in 20 mM HEPES buffer (pH 8.0) containing 0.2 mM Tris, pH 8.0, 150 mM NaCl, 3 mM KCl, 1 mM MgCl2 and 1% DMSO (pH 8.0) at 37 °C. The fluorescence spectrum change of the sensing system upon the addition of varied amounts of Sirt1 was recorded after 2 h of mixing. For the inhibition assay, tenovin-6 was added to the reaction mixture at a final concentration of 1 mM. 2.8. Sirt1 Detection in Biological Fluid Samples. The serum was obtained from a local hospital: first, the blood was centrifuged for 20 min at 3000 rpm. Then the supernatant was collected as soon as possible and storied at 2−8 °C for use. For Sirt1 detection in serum samples, the fluorescence measurements were conducted for samples containing the assay (final concentration: H40-Lys(Ac) 1.5 μM and TPE-2SO3− 20 μM) and NAD+ (300 μM) with or without Sirt1 in 20 mM HEPES buffer (pH 8.0) at 37 °C (for the samples with added Sirt1, the measurements were conducted after 2 h of mixing). The final concentration of the serum in the test solution is 200-fold diluted. 2.9. Measurements. 1H NMR spectra were recorded on a Bruker Avance 400 MHz NMR spectrometer. Mass spectra were obtained through a Bruker Esquire HCT Plus mass spectrometer. UV−vis spectra were recorded on a Hitachi U-3010 UV−vis spectrophotometer. Fluorescence spectra were recorded on a Hitachi F-4600 fluorescence spectrophotometer. FT-IR spectra were measured using a MAGNA 760 (USA, Nicolet Instrument). The particle size and distribution was determined by dynamic light scattering (DLS) on a Malvern Nano-ZS90 particle size analyzer at a fixed angle of 90° at 25 °C. Transmission electronic microscopy (TEM) observation was carried out on a JEM2010 HR transmission electron microscopy (Japan).

2. EXPERIMENTAL SECTION 2.1. Materials. Hyperbranched bis-MPA polyester-64-hydroxyl, generation 4 (H40), sirtuin 1 (Sirt1), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 4-hydroxylbenzophenone and benzophenone were purchased from Sigma-Aldrich and used as received. N-α-tert-Butoxycarbonyl-N-ε-acetyl-L-lysine was obtained from J&K Chemical Ltd.. Tenovin-6 was obtained from Cayman Chemical. 4-Dimethylaminopyridine (DMAP), sodium ethoxide (NaOEt), and N,N′-diisopropylcarbodiimide (DIC) was purchased from Alfa Aesar. Sirt1 ELISA Kit was purchased from Global Biotech Inc.. Ethanol and other solvents were analytically pure reagents and distilled before use. The water used throughout the experiments was the triple-distilled water which was further treated by ion exchange columns and then by a Milli-Q water purification system. 2.2. Synthesis of the H40-Lys(Ac). Briefly, H40 (0.03 g, 0.26 mmol hydroxyl groups) was dissolved in 15 mL of anhydrous THF in a 50-mL flask with moderate stirring for 30 min at room temperature under N2 atmosphere. After the polymer was completely dissolved, N-α-tert-butoxycarbonyl-N-ε-acetyl-L -lysine (0.756 g, 2.62 mmol), DIC (0.396 g, 3.15 mmol), and DMAP (0.016 g, 0.131 mmol) were added into the solution. After the mixture being stirred for 48 h at room temperature, the solvent was removed under vacuum. Afterward, the crude product was dissolved in water, and then dialyzed against ethanol for 48 h and against water for 72 h (MW cutoff: 3500). Finally the product was dried under vacuum. 2.3. Synthesis of TPE Derivative Containing Two Hydroxyl Groups (TPE-2OH). Zinc dust (2.9 g, 44 mmol) and 4-hydroxybenzophenone (2.0 g, 10 mmol) were placed into a 250 mL, two-necked, round-bottom flask equipped with a condenser. The flask was evacuated under vacuum and flushed with dry nitrogen for three times. After addition of 100 mL anhydrous THF, the mixture was cooled to 0 °C and TiCl4 (2.5 mL, 22 mmol) was slowly injected. The mixture was slowly warmed to room temperature, stirred for 0.5 h, and then refluxed overnight. The reaction was quenched by 10% aqueous K2CO3 solution. The mixture was extracted with diethyl ether three times and the combined organic layer was washed with brine twice and dried over anhydrous sodium sulfate. After solvent evaporation, the crude product was purified on a silica-gel column using petroleum ether/ethyl acetate (v/v 1:1, Rf = 0.50) as eluent. A yellow solid of TPE-2OH was obtained in 88% yield. 1H NMR (400 MHz, DMSO-d6, δ ppm): 9.29−9.31 (d, 2H), 7.04−7.17 (m, 6H), 6.92−6.97 (m, 4H), 6.70−6.75 (m, 4H), 6.47−6.53 (m, 4H). MS (ESI): m/z 362.8 [M − H]−. 2.4. Synthesis of TPE Derivative Containing One Hydroxyl Group (TPE-OH). Briefly, into the round-bottom flask were added zinc dust (2.9 g, 44 mmol), 4-hydroxy diphenyl ketone (2.2 g, 12 mmol), and benzophenone (2.0 g, 10 mmol), and then the flask was evacuated of air under vacuum and flushed with dry nitrogen three times. After addition of 100 mL of anhydrous THF, the mixture was cooled to 0 °C and TiCl4 (2.5 mL, 22 mmol) was slowly injected. The mixture was stirred for 0.5 h at room temperature, and then refluxed overnight. The reaction was quenched by 10% aqueous K2CO3 solution and extracted with diethyl ether three times and the combined organic layer was washed with brine twice. The mixture was dried over anhydrous sodium sulfate. The crude product was purified on a silica-gel column using DCM/petroleum ether (v/v 4:1, Rf = 0.50) as eluent. A yellow solid of TPE-OH was obtained in 50% yield. 1H NMR (400 MHz, DMSO-d6, δ ppm): 9.34 (s, 1H), 7.06− 7.14 (m, 9H), 6.93−6.98 (m, 6H), 6.73−6.75 (d, 2H), 6.47−6.52 (d, 2H). MS (ESI): m/z 346.8 [M − H]−. 2.5. Synthesis of TPE Derivative Containing Two Sulfonic Acid Groups (TPE-2SO3−). TPE-2OH (0.44 g, 1.2 mmol) was dissolved in 20 mL of absolute ethanol under nitrogen. A mixture of NaOEt (0.23 g, 3.4 mmol) in 20 mL of ethanol was added dropwise and stirred for 1 h. After the colorless solution turned into orange-red, 1,3-propane sultone (0.28 g, 2.3 mmol) in 20 mL of ethanol was added slowly. The mixture was vigorously stirred for 12 h, and a white

3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of the Assay System. To prepare the assay system, its two components, TPE-2SO3− and H40-Lys(Ac), were first synthesized respectively (Scheme S1, Supporting Information), and they were then dissolved in HEPES buffer. The synthesized compounds were characterized by 1H nuclear magnetic resonance (1H NMR), mass spectrometry (MS) and Fourier transform infrared spectroscopy (FTIR) (Figures S1−S6). Taken together, these results confirm the successful synthesis of H40-Lys(Ac) and TPE-2SO3−. In addition, the graft ratio (mole percentage of N-α-tert-butoxycarbonyl-N-ε-acetyl-L-lysine groups relative to the total hydroxyl groups in H40 before the esterification reaction) for the resulting polymer H40-Lys(Ac) has been determined to be 47.5% according to the 1H NMR spectrum of H40-Lys(Ac). 4509

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3.2. Fluorescent Response of the Sensing System toward HDAC. Sirtuin 1 (Sirt1) belongs to the family of NAD+-dependent histone deacetylases (HDAC), which are enzymes that cleave acetyl groups through the involvement of the NAD+ cofactor. To investigate the assay system’s fluorescent response toward Sirt1, we measured the fluorescent spectra of the system (1.5 μM H40-Lys(Ac) and 20 μM TPE2SO3−; namely, the Lys(Ac) concentration is 45 μM based on the graft ratio of 47.5%, and the SO3− concentration is 40 μM) in the absence or presence of Sirt1. The fluorescence spectra of the system were periodically recorded during incubation of the assay system with Sirt1 (1.0 μg/mL) at 37 °C in HEPES buffer (pH 8.0) containing 300 μM NAD+, as shown in Figure 2. Figure 3. Plot of the fluorescence intensity of the assay system (1.5 μM H40-Lys(Ac) and 20 μM TPE-2SO3−) at 465 nm versus reaction time in the presence of different amounts of Sirt1 in 20 mM HEPES buffer (pH 8.0) containing 300 μM NAD+. (λex = 345 nm).

Figure 2. Time course of fluorescence spectra of the assay system (1.5 μM H40-Lys(Ac) and 20 μM TPE-2SO3−) in the presence of Sirt1. The measurement was conducted with Sirt1 (1.0 μg/mL) in 20 mM HEPES buffer (pH 8.0) containing 300 μM NAD+. Spectra measured every 10 min are displayed. (λex = 345 nm).

In the absence of Sirt1, the fluorescence intensity of the system is very weak due to the lack of aggregation of the AIE-active fluorophore TPE-2SO3− in HEPES buffer. Upon addition of Sirt1, the deacetylation of H40-Lys(Ac) generates H40-Lys, which possesses protonated aliphatic ε-amine groups. The electrostatic interactions between the positively charged H40-Lys and the negatively charged TPE-2SO3− results in the formation of nanoaggregates, and TPE-2SO3− serves as the cross-linking agent for the globular polyelectrolyte. As a result, the fluorescence intensity of the system increases significantly due to the AIE effect of TPE-2SO3−. Besides, this time-course experiments show that the enzymatic reaction is almost completed within 2 h. The kinetic activity of the enzymatic reaction was determined by measuring the time-course experiments of the system at three different concentrations of Sirt1, as shown in Figure 3. And the kinetic activity is similar to literature report.41 After considering the effects of the globular polymer concentration (Figure S7), pH value (Figure S8), and the enzymatic reaction time (Figure 2), we determined the optimized conditions. And under the optimized conditions (reaction at 37 °C for 2 h in HEPES buffer in the presence of 300 μM NAD+), the fluorescence spectral change of the assay system toward Sirt1 at varied concentrations is shown in Figure 4A. We also established a working curve by plotting the emission intensities at 465 nm (I465) versus Sirt1 concentration, as shown in Figure 4B. The fluorescence of the solution changes from dark to bright blue upon addition of Sirt1 (Figure S9). It can be seen that, the fluorescence intensity increases steadily with the increasing Sirt1 concentration. And the detection limit is determined as 0.025 μg/mL (Figure S10).

Figure 4. (A) Fluorescence spectra of the assay system (1.5 μM H40Lys(Ac) and 20 μM TPE-2SO3−) in the presence of different amounts of Sirt1 in 20 mM HEPES buffer (pH 8.0) containing 300 μM NAD+. (B) Fluorescence intensity at 465 nm for the assay system (1.5 μM H40-Lys(Ac) and 20 μM TPE-2SO3−) in pH 8.0 HEPES buffer as a function of Sirt1 concentration (λex = 345 nm).

Furthermore, the enzymatic reaction was also examined by using inactivated Sirt1 which was achieved by heating to 100 °C for 10 min. Under this condition, the enhancement in the fluorescence intensity of the assay system is restrained and almost coincides with that in the absence of Sirt1, as shown in Figure 5. These results indicate that nonspecific interaction between the assay system and the enzyme is not observed and that the fluorescence intensity increase in the enzymatic reaction results from the electrostatic interactions between the deacetylated productthe positively charged polymer and the negatively charged TPE-2SO3−. In addition, the effects of NAD+ on the fluorescence intensity of the assay system were 4510

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derivate containing only one SO3− and compared the fluorescent change after Sirt1 was added into the H40-Lys(Ac)/TPE2SO3− and the H40-Lys(Ac)/TPE-SO3− solutions separately (Figures S13 and S14). It is obvious that, TPE-SO3− with only one SO3− group can not induce the formation of nanoaggregate and no fluorescence enhancement can be observed. To examine whether H40-Lys(Ac) is deacetylated by Sirt1 and turns into the positively charged polymer, 1H NMR measurements were conducted. H40-Lys(Ac) was mixed with Sirt1 and NAD+ in HEPES buffer for 2 h, and the resultant polymer product was precipitated out and purified by redissolution and precipitation for three times, afterward the resultant polymer product was further purified by dialyzing against water for 72 h and then dried under vacuum, finally 1H NMR measurements were carried out for the dried polymer product, and the result is shown in Figure 6. For the polymer

Figure 5. Fluorescent intensity at 465 nm for the assay system (1.5 μM H40-Lys(Ac) and 20 μM TPE-2SO3−) plotted against the incubation time in the presence and absence of various additives. (λex = 345 nm).

also determined. The fluorescence intensity is only slightly increased after addition of 300 μM of NAD+ alone into the assay system. However, the fluorescence intensity amplified by the enzyme reaction is significantly higher than that by only NAD+. Moreover, the enzymatic reaction in the presence of a potent Sirt1 inhibitor, tenovin-6, was investigated.62 The fluorescent intensity of the assay system with tenovin-6 (1 mM) in the presence of Sirt1 (1 μg/mL) was measured (Figure 5). The results show that, with the addition of tenovin-6, there is no significant difference in the fluorescence intensity of the system compared with that in the absence of Sirt1. The enzyme inhibition by tenovin-6 further demonstrates that the deacetylation is indeed induced by Sirt1. In addition, upon enzyme inhibition by tenovin-6, the polymer was purified, and 1 H NMR and IR measurements were conducted to confirm the intactness of the polymer structure, as shown in Figures S11 and S12. The results again prove that the deacetylation is induced by Sirt1. For Sirt 1, the location of acetylated lysine generally does not significantly affect the enzyme’s activity. While for other HDAC subtypes, these enzymes may selectively recognize substrates and the nature of the acyl leaving group.63 In addition, it has been demonstrated that, substrates with steady-state acetylation levels (for lysine) are effective substrates for HDAC.64 Furthermore, several researchers also found that, as for Lys(Ac) (acetylated lysine) conjugated to organic compounds such as coumarin, BODIPY and tetraphenylethene, Sirt1 can successfully catalyze the deacetylation of Lys(Ac) moieties.40−42 As for the substrate H40-Lys(Ac) in our system, the substrate activity is comparable to some reported researches;40−42 hence, we suppose it can act as an effective substrate for Sirt1. 3.3. Proposed Detection Mechanism. On the basis of the fluorescent response of the assay system toward Sirt1, the detection mechanism is proposed as follows: upon addition of Sirt1 into the assay system, the deacetylation of H40-Lys(Ac) generates primary aliphatic ε-amine groups which are protonated under physiological conditions (pKa ∼ 10.5),41,42 thus the polymer becomes positively charged. Subsequently, the polymer electrostatically interacts with the negatively charged TPE-2SO3− molecules to form nanoaggregates, leading to the AIE-based fluorescence enhancement (Figure 1). The TPE-2SO3− molecules are supposed to act as the cross-linker between H40 polymers in the nanoaggregates. To prove the cross-linking role of TPE-2SO3−, we also synthesized the TPE

Figure 6. 1H NMR spectra of the polymer before (upper) and after (lower) addition of Sirt1.

upon being treated by Sirt1, a new peak at 2.84 ppm (b′) appears, which corresponds to the signals of methylene protons upon deacetylation of H40-Lys(Ac). And the acetyl proton signal of H40-Lys(Ac) is reduced accordingly. On the basis of the 1H NMR measurements, it can be estimated that, the deacetylation degree is around 50%. To further prove the detection mechanism, we carried out transmission electron microscopy (TEM) and dynamic light scattering (DLS) measurements for the assay systems in the absence and presence of Sirt1, as shown in Figure 7 and Figure S15. TEM and DLS analyses show that no nanoaggregates form in the solution in the absence of Sirt1. While in the presence of Sirt1, nanoaggregates form, and the size of the aggregates increases with the increasing amount of Sirt1. And the diameter of the nanoaggregates ranges from 25−250 nm, depending on the added amount of Sirt1. This detection approach is based on the Columbic interaction of the oppositely charged electrolytes and the subsequent complex formation. Generally, the complexes will reach a complexation-dissociation equilibrium in water; and given the mechanism of the current approach, the dissociation is unfavorable to sensitive detection. To reduce the dissociation, in this study a globular polymer H40 was employed, which turns into polyelectrolyte in the presence of Sirt1. The polyelectrolyte dissociates into a macroion and counterions in polar solvent like water. One typical feature of polyelectrolytes is that they can make their counterions exhibit low activity 4511

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Figure 7. HR-TEM images of the assay systems with the addition of Sirt1 (0.025, 0.25, 1.0, and 5.0 μg/mL respectively) placed on copper grids. Scale bar: 100 nm.

Table 1. Determination of Sirt1 in Serum (200-Fold Diluted) determined endogenous Sirt1 (μg/mL) by Elisa Kit

determined endogenous Sirt1 (μg/mL) by this probe

added Sirt1 (μg/mL)

combined Sirt1 (μg/mL)

0.041 ± 0.005a

0.044 ± 0.004b

− 0.08 0.06 0.04 0.03

− 0.124 0.104 0.084 0.074

measured (μg/mL) by this probec − 0.113 0.112 0.092 0.079

± ± ± ±

0.011 0.005 0.007 0.008

recovery (%) 107.3 91.1 107.7 109.5 106.8

a The Sirt1 level in the 200-fold diluted serum sample is 0.041 μg/mL, which means the endogenous Sirt1 concentration in undiluted serum is ca. 8.2 μg/mL determined by Elisa Kit. bThe Sirt1 level in the 200-fold diluted serum sample is 0.044 μg/mL, which means the endogenous Sirt1 concentration in undiluted serum is ca. 8.8 μg/mL determined by the assay system herein. cThe data are summarized as mean ± standard deviation (SD).

coefficient.65−67 In this fluorescent assay system, TPE-2SO3− acts as one of the counterions for the polyelectrolyte, and a fraction of counterions (known as the condensed fraction of counterions) is at the surface of the globular macroion. Moreover, as the fluorescent indicator of the assay system, TPE-2SO3− exhibits weaker solvation in water than other counterions, hence it may form much stable complex with the charged H40. Thus, we suppose, the TPE-2SO3−/charged H40 complex can exhibit low extent of dissolution and ensure higher detection sensitivity as compared with that of similar AIE sensing system using small-molecule components. 3.4. HDAC Detection in Biological Fluid Samples. To evaluate the efficacy of this assay system in real samples, the system was applied to measure Sirt1 in serum. Since there is relatively high concentration of Sirt1 existing in serum (8−10 μg/mL),68 the serum samples were diluted 200-fold for the measurements. In this study, the determined Sirt1 levels are listed in Table 1. The endogenous (originally existing) Sirt1 in diluted serum sample was determined by the assay system herein using the calibration curve (Figure 4B) as standard, and the endogenous Sirt1 levels for the undiluted serum sample is then calculated as 8.8 μg/mL determined by the assay system. Furthermore, the assay system was also compared with the commonly used commercial assay kit for Sirt1, an Elisa Kit.

And the endogenous Sirt1 level in serum determined by the Elisa Kit is 8.2 μg/mL, which is close to that determined by our system. For Elisa Kit, the antibody-based colorimetric assay, the detection process requires complex multiple procedures; for the assay system herein, the detection process is quite convenient and could serve as a one-step straightforward assay. Also, the recovery of added known amounts of Sirt1 into the serum samples is in general more than 91% by the assay system, which suggests the accuracy and reliability of the present method for Sirt1 determination. In addition, the precision of the assay system was also compared with Elisa Kit (Table 1). Precision was determined using the relative standard deviation. It is clear that, the assay system herein displays at least comparable or slightly better precision than Elisa Kit, since fluorescence technique usually exhibits higher precision than colorimetric method. Compared with other detection techniques including HPLC, mass spectrometry and colorimetric method (e.g., Elisa Kit, the antibody-based colorimetric assay), the probe herein is quite convenient and could function as a straightforward one-step assay for HDAC.

4. CONCLUSION In summary, we have developed a novel hyperbranched polyester-based fluorogenic assay system for HDAC activity 4512

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detection. The deacetylation of the polymer H40-Lys(Ac) generates positive charges on the polymer, which results in the complexation through electrostatic interaction between the negatively charged TPE-2SO3− and the positively charged polymer, leading to the formation of nanoaggregates and the corresponding fluorescence enhancement via the AIE effect. The use of the globular polymer as the scaffold for the assay system can ensure low detection limit. In addition, the system can be used for Sirt1 detection in biological fluid like serum. This system could serve as a one-step straightforward fluorescent assay for histone deacetylases. This strategy may offer a technically simple approach for designing sensing systems with low detection limit for detecting other analytes in biological samples.

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ASSOCIATED CONTENT

* Supporting Information S

Synthetic routes, 1H NMR spectra, determination of graft ratio, mass spectra, FTIR spectra, photographs, determination of the detection limit, time course of the fluorescence spectra, plot of the fluorescence intensity of the assay system and the control system at 465 nm versus reaction time, size distribution of the nanoaggregates determined by dynamic light scattering (DLS), and absorption spectrum. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*(F.Z.) Telephone: +86-20-22236262. Fax: +86-20-22236363. E-mail: [email protected]. *(S.W.) E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge the financial support by the National Key Basic Research Program of China (Project No. 2013CB834702), the NSFC (21025415 and 21174040), and “the Fundamental Research Funds for the Central Universities, SCUT”.

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