SCIENCE CHINA Chemistry • ARTICLES • · SPECIAL TOPIC · Aggregated-Induced Emission
September 2013
Vol.56 No.9: 1234–1238
doi: 10.1007/s11426-013-4917-6
A highly sensitive “turn-on” fluorescent probe for bovine serum albumin protein detection and quantification based on AIE-active distyrylanthracene derivative WANG ZiLong, MA Ke, XU Bin*, LI Xing & TIAN WenJing* State Key Laboratory of Supramolecular Structure and Materials; Jilin University, Changchun 130012, China Received April 13, 2013; accepted May 2, 2013; published online July 10, 2013
A sulfonated 9,10-distyrylanthracene derivative with aggregation-induced emission (AIE) property is designed and synthesized. It shows a highly sensitive and selective fluorescence enhancement property for bovine serum albumin (BSA) protein detection and quantification. Analysis on the interaction between the probe molecule and BSA reveals the essential role of the hydrophobic cavities of the protein folding structure. aggregation-induced emission, fluorescent probe, distyrylanthracene derivative
1 Introduction Protein analysis is of fundamental importance for the proteome research that aims to decipher biological processes at protein level [1]. The fluorescence luminescent (FL) techniques are extensively used methods for chemical analyses and bioassay, because they offer the advantages of high sensitivity, low background noises and wide dynamic ranges. Many fluorescent dyes for protein assays have been developed by utilizing the changes in their photophysical properties caused by their chemical reactions or physical interactions with proteins [2–5]. However, most of the FL bioprobes show lengthy procedure, small Stokes shifts and nonlinear calibration curves. More importantly, an undesirable problem of the conventional FL probes is the aggregation of dyes. FL dyes usually exhibit strong emission and high quantum yield in their dilute solution, but remarkably decreased intensity of emission and quantum yield when they are in aqueous media or bound to proteins in high concentration, due to “aggregation caused quenching”(ACQ) [6, *Corresponding authors (email:
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7], which makes it particularly difficult to assay lowabundance biomacromolecules. There is thus an urgent need and challenge for the development of novel FL probes, which can keep strong emission and high fluorescence quantum yield in aggregation or solid-state. Since the intriguing phenomenon, aggregation -induced emission (AIE) was reported by Tang’s group [8], various AIE active dyes have been developed by many research groups. For example, silole [9], tetraphenylethene [10] and their derivatives [11–13] show intense emission in the aggregation state, which make them ideal candidates for high-tech applications in the practically useful solid state, such as organic light emitting diodes (OLED), organic solid state laser and chemical sensors. Recently, bioprobes using AIE molecules have attracted more and more attention because of their advantages of label free and “turn-on” properties for biological detection and imaging [14–18]. Anthracene and its derivatives constitute a very famous class of fluorophores that have been widely used in the development of FL sensors [19, 20] because of their excellent photoluminescence properties and chemical stability. We have recently developed a series of 9,10-distyrylanthracene (DSA) derivatives, which possess a typical AIE properties, chem.scichina.com
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i.e. nonluminescent DSA molecules are induced to strong emission by aggregate formation [21–24]. Our study indicates that the restricted intramolecular rotations between the 9,10- -anthylene core and the vinylene segment are the origin of the AIE property [25]. In this study, we report a label-free FL probe for bovine serum albumin (BSA) protein based on a DSA sulphonate derivative compound 2, and screening by taking advantage of the abnormal fluorescent behavior of AIE. The compound shows an excellent fluorescent turn-on behavior for protein detection and it is also selective, which works for native BSA but not its denatured forms. Through the analysis of binding constants, binding sites, interaction type and the thermodynamic properties of the interaction between bioprobe and protein, it is found that hydrophobic interactions play a main role between the probe molecule and BSA and the interaction process is spontaneous.
2 Experimental 2.1
Materials and instrumentation
All reagents and starting materials are commercially available and were used as received. 9,10-Dibromoanthracene was purchased from Acros and used without further purification. Dimethyl acetamide (DMAc) and tetrahydrofuran (THF) were purified by fractional distillation before use as solvents. As shown in Scheme 1, 9,10-bis(4-hydroxystyryl) anthracen (compound 1) was prepared according to literature procedures [25]. Bovine serum albumin (BSA; >98%, fraction V) were purchased as lyophilized crystalline powders from Sigma. The 1H NMR spectra were recorded on Varian Mercury-300 NMR at 298K by utilizing deuterated CDCl3 as solvent and tetramethylsilane (TMS) as standard. MALDI-TOF mass spectrometry experiments were performed on a Kratos MALDI-TOF mass system, and the spectrum was recorded in the linear or reflect mode with anthracene-1,8,9-triol as the matrix. The compound was characterized by Perkin Elmer 2400LS II. UV-vis absorption spectra were recorded on a UV-3100 spectrophotometer. Fluorescence measurements were carried out with RF-5301PC. The relative fluorescence quantum yield (ΦF) was determined by the standard method using quinine sulfate in 0.1 N H2SO4 solution as a reference. 2.2
UV and FL spectra
BSA was dissolved in a pH 7.0 phosphate buffer solution (0.1 and 1.0 mg/mL). The stock solution of compound 2 was 1×103 M in water. FL titration was carried out by adding aliquots of BSA solution to 0.1 mL of a stock solution of compound 2, followed by adding an aqueous phosphate buffer (10 mM, pH 7.0) to acquire a 10.0 mL solution. The mixture was stirred for half an hour prior to recording its spectrum.
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2.3 Synthesis of sodium 9,10-bis[4-(3-sulfonatopropoxyl)styryl] anthracene (compound 2) To a stirred suspension of NaH (200 mg, 60% in mineral oil, 5 mmol) in dry DMF (5 mL) was added dropwise a solution of compound 1 (828 mg, 2 mmol) in dry DMF (80 mL). After stirring for 45 min at room temperature 1,3-propanesultone (733 mg, 6 mmol) was added, and the resultant mixture was heated at 80 ºC for 10 h. After cooling, ethanol (100 mL) was poured into the reaction mixture, the mixture was filtered and washed with ethanol and acetone twice respectively to give a yellow solid. Yield: 69%. Fluorescence quantum yield (F): 9%; 1H NMR (300 MHz, DMSO-d6): 8.39 (q, 4 H), 7.96 (d, 2 H), 7.74 (d, 4 H), 7.54 (q, 4 H), 7.02 (d, 4 H), 6.85 (d, 2H), 4.13 (t, 4 H), 2.58 (t, 4H), 2.03 (m, 4H); MS (TOF) m/e: 653.4 [(M+2H)+-Na, Calcd 653.1]; Anal. Calcd (%) for C36H32Na2O8S2: C 61.53; H 4.59; S 9.13; Found (%): C 61.31; H 4.89; S 9.21.
3 3.1
Results and discussion Synthesis and characterization
The DSA derivatives were prepared by the synthesis route shown in Scheme 1. The sulphonation of compound 1 by 1,3-propanesultone gave compound 2. The structure of the product molecule was confirmed by 1H NMR, MALDITOF-MS, and elemental analysis. Compound 1 was soluble in common organic solvents such as acetone and chloroform but insoluble in water. Compound 2 was soluble in water. 3.2
AIE effect
Compound 2 was completely soluble and showed weak emission in water，but the increasing viscosity of the solution of compound 2 can increase its FL intensity (Figure 1). At room temperature, the FL intensity of a dilute solution of compound 2 in a viscous glycerol/water mixture (7/3 V/V) was much higher than that in pure water because high viscosity can hamper the intramolecular rotation, leading to the closure of the nonradiative decay channel and thus enhanced FL emission [26]. Figure 2(a) showed the changes of FL spectra before and after the addition of BSA to the compound 2 in the solution (PBS buffer). Water-soluble compound 2 was nonluminescent in a pH 7.0 phosphate buffer. However, the FL intensity
Scheme 1 Synthesis of functionalized compound 2: 9,10-bis[4-(3sulfonat-opropoxyl)styryl] anthracene.
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Figure 1 mixture.
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Emission spectra of compound 2 (10 M) in a glycerol/water
of the buffer solution of compound 2 increased gradually when BSA was added. For instance, the FL intensity of compound 2 (5 M) at 525 nm was enhanced by 40-fold after the concentration of BSA reached 50 μg/mL. The FL intensity increased linearly (R2 = 0.975) with the BSA concentration from 0 to 10 g/mL (Figure 2(a)). Similarly, we can see from Figures 2(c) and (d) that when the concentration of compound 2 was 10 M, the FL intensity of compound 2 at 525 nm was enhanced by 600-fold after the concentration of BSA reached 70 μg/mL. The FL intensity increased linearly (R2 = 0.996) rapidly with the BSA concentration from 0 to 10 μg/mL and increased linearly (R2 = 0.987) gently with the concentration from 10 to 60 g/mL (Figure 2(d)), which indicated that the linear response range of BSA could be broaden by increasing the concentration of compound 2. As indicated in Figure 3, the FL of the ensemble of compound 2 (10 M) and BSA (100 g/mL) was rather strong. However, it was dramatically weakened when cetyltrimethy- -lammonium bromide (CTAB) was added to the BSA solution of compound 2. CTAB has a property of biological degradability, which means it can unfold the BSA chains to be extended chains. The probe molecule was thus folding-structure-sensitive, implying that the probe is only sensitive to the folding structure of BSA, because the surfactant molecules of CTAB unfolded the BSA chains and destroy the native hydrophobic regions of the protein, which quenched the fluorescence. Figure 4 shows the selectivity of the biosensor for BSA analysis. The fluorescence spectrum of compound 2 (10 M) at 520nm was measured in the presence of control proteins, such as cyt-c, papain, pepsin, trypsin, DNA-ct, lysozyme and transferrin under the same conditions using the same concentration of 100 g/mL. It is indicated that none of control proteins can perform rather obvious fluorescence intensity than BSA. It confirms that compound 2 is a very useful probe for the specific detection of BSA.
Figure 2 Change in the FL spectrum of compound 2. (a) Compound 2 (5 M) with the addition of BSA in an aqueous phosphate buffer (pH 7.0); (b) plot of FL intensity vs BSA concentration for (a) (R2 = 0.975, BSA: 0–10 g/mL); (c) compound 2 (10 M) with the addition of BSA in an aqueous phosphate buffer (pH 7.0); (d) plot of FL intensity vs BSA concentration for (c) (R2 = 0.996, BSA: 0–10 g/mL; R2 = 0.987, BSA: 10–60 g/mL).
3.3 Origin of the interaction between compound 2 and BSA According to Figure 2, the FL intensity of the buffer solu-
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(BnQ). Figure 6 shows the lg(I0I)/I versus lg[Q]. With the equation above, we can get n (the binding sites of the binding of compound 2 to BSA). It equals to the slope of the line in Figure 6. Figure 5 performed the similar experiment in the different temperature, 298 K and 303 K. Then we put the date into Van’t Hoff equation: G 0 RT ln KT d ln K / d (1 / T ) H 0 / T
Figure 3 Effect of BSA (100 g/mL) and/or CTAB (0.6 mg/mL) on the FL spectrum of a buffer solution of compound 2 (10 M).
(4)
S (G H ) / T The binding constant, binding sites, Gibbs free energy, entropy, enthalpy of compound 2 to BSA were shown in Table 1. H0 values don’t change with the temperature and S0 values can be achieved by Eq. (4). The negative value of G0 revealed that the interaction process between the compound 2 and BSA was spontaneous. And the positive H0 and S0 values indicated that hydrophobic interaction played a main role in the binding of compound 2 to BSA [28, 29]. With the temperature increasing, the binding sites and binding constant increased gradually meaning the binding interaction strengthened gradually. To briefly conclude, the interaction of compound 2 to BSA was proved to be hydrophobic interaction. 0
0
0
Figure 4 Fluorescence intensity (I520 nm) of compound 2 (10 M) in the presence of different proteins (100 g/mL) in PBS buffer. ex = 425 nm.
tion of BSA at 525 nm increased gradually when compound 2 was added. But, the FL intensity of the solution at 340 nm decreased gradually with the addition of compound 2 due to the quenching of the BSA FL when compound 2 added. By using the following binding Eq. (1) [27], the interaction between compound 2 and BSA can be explained. B + nQ→BnQ (1) Here, B represents BSA, Q represents compound 2, n represents the binding sites of the binding of compound 2 to BSA. And the binding constant expression can be expressed like this [27]: [B nQ] KT (2) [B] [Q]n It can be transformed into this form [27]: lg(I0I)/I = lg KT + nlg[Q] (3) where I is the FL intensity of the solution at 340 nm (the summit of the BSA fluorescence emission peak) in Figure 5. I is in direct proportion to the concentration of BSA. I0 is the FL intensity of the solution with no compound 2 at 340 nm. So (I0I) is in direct proportion to the concentration of
Figure 5 Change in the FL spectrum of BSA with the addition of compound 2 to an aqueous phosphate buffer (293 K, pH 7.0).
Figure 6 Plots of lg(I0I)/I vs lg[Q]. I was the FL intensity of the solution at 340 nm in Figure 4. Q represents compound 2.
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Table 1 The binding constant, binding sites, Gibbs free energy, entropy, enthalpy of the binding of compound 2 to BSA T (K)
KT (106 M1)
n
293 298 303
5.0276 8.7541 12.337
1.244 1.288 1.331
G0 (kJ/mol) 37.59 39.60 41.13
H0 (kJ/mol) 65.89 65.89 65.89
S0 (J/(mol K)) 353.17 353.99 353.20
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4 Conclusions 14
In summary, a water-soluble sulphonate DSA derivative with aggregation-induced emission characteristics was developed, which can be applied as a FL biological probe to detect BSA in quantitation. The nonluminescent sulphonate DSA derivative becomes emissive in the presence of BSA. The AIE bioprobe shows a linear calibration curve at BSA (0–60 g/mL), enabling the protein quantitation over a wide concentration range. Studies on the interaction between AIE bioprobe and BSA reveal the essential role of the hydrophobic cavities of the protein folding structure.
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18 This work was financially supported by the National Basic Research Program of China (973 Program, 2013CB834702, 2009CB623605), the Natural Science Foundation of China (21074045, 21204027, 21221063), the Research Fund for the Doctoral Program of Higher Education of China (20120061120016) and the Project of Jilin Province (20100704).
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