A Water-Soluble Conjugated Polymer with Pendant Disulfide

We investigated a water-soluble conjugated polymer (WSCP) with pendant disulfide linkages to poly(ethylene glycol) (PEG) chains, which is a highly eff...
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A Water-Soluble Conjugated Polymer with Pendant Disulfide Linkages to PEG Chains: A Highly Efficient Ratiometric Probe with Solubility-Induced Fluorescence Conversion for Thiol Detection Jie Li,†,‡ Congcong Tian,†,‡ Yan Yuan,†,‡ Zhen Yang,†,‡ Chao Yin,†,‡ Rongcui Jiang,†,‡ Wenli Song,†,‡ Xiang Li,†,‡ Xiaomei Lu,†,‡ Lei Zhang,†,‡ Quli Fan,*,†,‡ and Wei Huang*,†,‡ †

Key Laboratory for Organic Electronics & Information Displays (KLOEID) and Institute of Advanced Materials, Nanjing University of Posts & Telecommunications, Nanjing 210046, China ‡ Jiangsu-Singapore Joint Research Center for Organic/Bio- Electronics & Information Displays and Institute of Advanced Material, Nanjing Tech University, Nanjing 211816, China S Supporting Information *

ABSTRACT: We investigated a water-soluble conjugated polymer (WSCP) with pendant disulfide linkages to poly(ethylene glycol) (PEG) chains, which is a highly efficient ratiometric probe with solubility-induced fluorescence conversion for thiol detection. This WSCP was doped with a low-bandgap fluorophore, 1,4-dithienyl benzothiadiazole (DBT), and was modified with PEGs by disulfide linkages to increase its water solubility. The free probe exhibited good solubility in aqueous solution (28 mg/mL) and showed purple fluorescence because of the low doping ratio of DBT. The separation of water-soluble PEG chains from the conjugated backbone induced by the cleavage of the disulfide linkages would lead to a significant decrease of the water solubility of the probe. The combined utilization of scanning electron microscopy, dynamic light scattering, and fluorescence spectrophotometer further confirmed that decreased solubility produced an aggregation of the hydrophobic conjugated backbone and subsequently increased fluorescence resonance energy transfer efficiency from the conjugated backbone to DBT which manifested as fluorescence conversion from purple to red. The fluorescence ratiometry (I628/I420) of the probe varied from the lowest value of 0.095 to 1.15 (12-fold maximum enhancement). The detection limit was 2.56 μg/mL (0.021 mM). The WSCP probe was confirmed to be a good sensing material with high selectivity for thiols by examining various biological molecules. We also successfully achieved the imaging of intracellular thiols in HeLa cell. Considering that the disulfide could be replaced by other cleavable linkages, such a fluorescence ratiometry induced by decreased solubility could be utilized for detecting other chaincleavable biomolecules, which would contribute to the development of new probes based on conjugated polymers.

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the conjugated backbone (donor)16−18 to design an effective ratiometric WSCP. Using the WSCPs modified with pendantcharged groups (ionic WSCPs), the method of binding the fluorophore to the backbone by electrostatic interaction could lead to the enhancement of FRET efficiency and meristic fluorescent changes. However, the electrostatic interaction lacks specificity. Thus, ionic WSCPs usually combine nontarget molecules with heterogeneous charges. The intrinsic hydrophobic backbones are still prone to aggregation and interaction with hydrophobic substrates, thereby resulting in decreased quantum yield (QY) and unexpected nonspecific binding.19 To overcome these drawbacks, poly(ethylene glycol) (PEG), a neutral water-soluble polymer, was introduced into WSCPs to improve water solubility and QY, reduce undesirable aggregation, and eliminate nonspecific interactions with

INTRODUCTION Over the past decade, fluorescent conjugated polymers (CPs), especially water-soluble conjugated polymers (WSCPs), have shown significant potential for biological applications, such as biosensing, bioimaging, and drug delivery.1−5 Considering their amplification of the collective response and unique lightharvesting ability, WSCPs used for bioimaging or biosensors generally include two categories of detection mechanisms, namely, fluorescence intensity6,7 (one-channel signal) and fluorescence ratiometry8−11 (generally based on fluorescence resonance energy transfer (FRET), dual-channel signal). Compared with fluorescence intensity, which is significantly influenced by variable factors, such as photobleaching, probe concentration, intracellular microenvironment, or light source stability, ratiometry is highly preferred because of its built-in correction property (ratiometric dual-channel signal), which could efficiently reduce the influence of the external environment.12,13 A second-signal fluorophore, as an acceptor, is generally bonded to the target biomolecule14,15 or doped into © XXXX American Chemical Society

Received: October 30, 2014 Revised: January 28, 2015

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Macromolecules Scheme 1. Synthesis of the Thiol Probe PF-DBT-PEGa

a Reagents and conditions: (a) Bispinacolatodiboronmin, Pd(dppf)2Cl2 and KOAc, dimethylformamide (DMF), 90 °C, 12 h; (b) tetrakis(triphenylphosphine)Pd(0), K2CO3 (2 M), 85 °C, 72 h; (c) trifluoroacetic acid (TFA), room temperature (RT), 24 h; (d) 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (EDCi), N-hydroxysuccinimide (NHS), RT, 24 h; (e) glacial acetic acid, methanol, RT, 48 h.

and utilized. To theoretically prove the hypothesis, we selected a thiol-cleavable group, i.e., disulfide bond, to connect PEG chains and to sense biological thiol-containing molecules, which extensively exist in organisms and play vital roles in the measurement of physical health as one of the most important antioxygens.27−30 In our responding system, the low molar ratio of DBT and pendant hydrophilic PEG chains could reduce the original energy transfer efficiency (0.095) of the CP in aqueous solution because the molecules are free in this state with a large spatial distance between the donor and the acceptor. When the disulfide linkages were cleaved by thiols in the biological environment, we anticipated the reduction of the water solubility of the WSCPs and the subsequent aggregation of the conjugated backbone. In this study, we observed that shortening of intramolecular and intermolecular distances enhanced the energy transfer efficiency between conjugated backbone and low-bandgap groups and resulted in a significant ratiometric change of fluorescence intensity (1.15) as well as an intuitionistic conversion in the color of fluorescence. Thus, the detection of linkage-cleavable biomolecules and intracellular distribution can be achieved by using a single WSCP probe. Notably, this solubility-induced sensing system can further be universally applied in other biological chain-cleavable molecules by simply changing the cleavage-specific linkages.

biomacromolecules or cells. However, further functionalization of the PEG chains with folate or other target groups was required to generate WSCPs that are specific to the target biomacromolecules or cells.20−22 In addition, these WSCPs only showed one-signal fluorescence in detection. Moreover, these WSCPs are inapplicable in the detection of other types of target substances, especially those with specific chain-cleavable ability. Thus, developing neutral WSCPs with ratiometric signals would achieve superiority in the detection of chaincleavable molecules. In this study, we investigated a WSCP with pendant disulfide linkages to PEG chains, which was a highly efficient ratiometric probe with solubility-induced fluorescence conversion for thiol detection. Low-bandgap fluorophores, 1,4-dithienyl benzothiadiazole (DBT), were introduced into polyfluorene at 5 mol % to generate a pair of ratiometric signals. PEG side chains were used to increase the water solubility of the CP, reduce the aggregation of the conjugated backbone, and eliminate nonspecific interactions. The key points of specific detection are the cleavage-specific linkages between the conjugated backbone and the pendant hydrophilic PEG chains. At present, several types of cleavage-specific linkages, such as disulfide bonds,23 reactive oxygen species linkages,24 pH response linkages,25 and enzyme-cleaved linkages,26 have been reported B

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appeared, thereby further confirming that the final product, PFDBT-PEG, was obtained. Absorption and Emission Spectra of PF-DBT-PEG. The optical behaviors of pure PF and PF-DBT-PEG were compared with each other to demonstrate the possibility of FRET between PF and DBT in the polymer structure (shown in Figure 2). As shown in the absorption spectra of PF-DBT-PEG (Figure 2A), a strong absorption peak at 380 nm from PF groups and a broad absorption band ranging from 450 to 600 nm from DBT groups were clearly exhibited. For efficient FRET, the donor and acceptor should fulfill two principles, i.e., the emission band of the donor overlaps with the absorption band of the acceptor, and a suitable distance (less than 10 nm) exists between the donor and acceptor.32 In Figure 2B, when excited with 380 nm irradiation, PF-DBT-PEG exhibited a typical emission peak from the PF groups at 400−550 nm, which well overlapped with the absorption spectra of DBT groups. Therefore, theoretical feasibility of energy transfer from PF to DBT existed. In the fluorescence spectra of PF-DBTPEG, a low fluorescence emission peak within 560−700 nm from DBT can be observed, thereby indicating the presence of intramolecular energy transfer from PF to DBT. This low FRET efficiency can be attributed to the small doping ratio of DBT and the good water solubility of PF-DBT-PEG, which dramatically reduced the intrachain and interchain aggregations. As reported in a previous study,33 a high doping ratio of DBT resulted in a high FRET efficiency of the CP. In the literature, when the doping ratio of DBT reached 10%, FRET efficiency could nearly reach the maximum, and when the doping ratio of DBT was 1%, FRET efficiency was much lower. In this study, we should control the doping ratio of DBT to detect thiolcontaining molecules through aggregation-enhanced FRET. If a higher doping ratio was selected, then efficient FRET would already exist before the addition of thiol-containing molecules. Thus, ratiometric fluorescence change would not be evident after adding thiol-containing molecules. When a lower doping ratio was used, FRET was indeed lower before adding the thiolcontaining molecules. However, aggregation-induced FRET might not be efficient after adding thiol-containing molecules because of the extreme low doping ratio. In this study, 5% doping ratio of DBT was selected because the FRET efficiency of WSCP before adding thiol-containing molecules was still lower and could provide evidently enhanced FRET during the cleavage of the soluble group of WSCP in the presence of thiolcontaining molecules at this doping ratio. In addition, the polymer has bright photoluminescence in water with QY of 0.31, which was measured using quinine sulfate in 0.1 M H2SO4 (QY = 0.577) as the standard. Stability of Ratiometry. The emission spectra and the corresponding emission ratios of I628/I420 are shown in Figure 3 to illustrate the ratiometric stability of the CP under different concentrations. Figures 3A and 3B show the fluorescence spectra of PF-DBT-PEG in a series of concentrations (from 1 to 0.1 μg/mL) in pH 7.4 phosphate-buffered saline (PBS) buffer and the corresponding emission ratios of I628/I420, respectively. Results revealed a drastic reduction of emission intensity when the concentration decreased. Meanwhile, no evident change in the emission ratio of I628/I420 was observed, thereby exhibiting the independence of the emission ratio of I628/I420 from the concentration. Furthermore, considering that diverse intracellular pH values may influence the fluorescence of the CP, the potential pH effects on the emission of PF-DBT-PEG at RT were

RESULTS AND DISCUSSION Synthesis of the Ratiometric Polymer Probe, PF-DBTPEG. The synthesis process is shown in Scheme 1. Monomers A and B were synthesized step by step in advance and then copolymerized with the low-bandgap acceptor (DBT) (with a molar ratio of 45:50:5) to obtain pristine CP with protected carboxyl groups, i.e., polyfluorene-co-1,4-dithienylbenzothiadiazole. Subsequently, deprotected carboxyl groups were modified with 2-(pyridyldithio)ethylamine (PDA) by amidation to introduce the disulfide bonds into the side chain of the conjugated backbone. Finally, the pyridine unit was easily replaced by PEG-SH, which endowed the polymer with good water solubility (28 mg/mL). Basic characterization methods, such as nuclear magnetic resonance spectroscopy (NMR), gel permeation chromatography (GPC), and elemental analysis (EA), were used to confirm the structure of the material. The chemical structure of each products was also confirmed by the 1 H NMR and 13C NMR spectra, shown in Figures S5−S7 (1H NMR) and Figures S8, S11−S14 (13C NMR) in the Supporting Information. Time-of-flight mass spectrometry (TOF-MS) of monomers A and B was performed on a Micromass GC-TOF in DCM (Figures S9 and S10). The number-average molecular weight (Wn) and polydispersity values were 5206 and 1.84 for PF-DBT-COOR (calculated to have 12 repetitive units on each polymer), 3858 and 1.62 for PF-DBT-COOH, 6939 and 1.36 for PF-DBT-PDA, and 18 860 and 1.29 for PF-DBT-PEG (with 11 PEG chains on each polymer), as determined by GPC with tetrahydrofuran (THF) as the solvent and polystyrene as the standard. The content of disulfide groups in PF-DBT-PEG was measured by EA (Table S1) and calculated to be 0.758 mmol/g and 11 per macromolecule. For PF-DBT-PDA and PF-DBTPEG, we confirmed that the pyridine units of PF-DBT-PDA were replaced by PEGs to generate the final product, PF-DBTPEG, by comparing the peaks at 8.12 and 6.95 ppm for pyridine units (present in PF-DBT-PDA but absent in PF-DBT-PEG) and 3.30 ppm for PEG units (present in PF-DBT-PEG but absent in PF-DBT-PDA) in the 1H NMR spectra shown in Figure 1. Fourier transform infrared spectroscopy (FT-IR, Figure S1) showed that the two peaks at 1414 and 751 cm−1 (belong to pyridyl31 in PF-DBT-PDA) disappeared in PF-DBTPEG. However, a new peak at 1111 cm−1 (belong to PEG)

Figure 1. 1H NMR spectra of PF-DNT-PEG and PF-DBT-PDA. C

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Figure 2. (A) Normalized absorption spectra of polyfluorene (blue circle) and PF-DBT-PEG (black square) in dichloromethane. (B) Normalized emission spectra of polyfluorene (blue circle) and PF-DBT-PEG (black square) in dichloromethane. λex = 380 nm.

Figure 3. (A) Fluorescence spectra of PF-DBT-PEG in a series of concentrations in pH 7.4 PBS buffer (λex = 380 nm). (B) Changes of the emission ratio of I628/I420 with the corresponding concentration.

investigated. As shown in Figure 4, almost no change in the emission intensity and ratio was observed in the free probe over

Figure 5. Visualized pictures of PF-DBT-PEG (10 μg/mL) in (a) bright field (daylight) and (b) dark field (λex = 380 nm) in the presence (1) and absence (2) of Cys (5 mg/mL, 41 mM) in 24 h.

in the dark field (λex = 380 nm). Meanwhile, in the absence of Cys, PF-DBT-PEG still exhibited excellent water solubility and a mixed purple fluorescence. These visible phenomena intuitively showed that the decreased water solubility of the probe, which might be caused by cleavage of the disulfide bond, could actually enhance the FRET efficiency for applying the ratiometric method. The aggregation-induced red-shifted bands in the absorption and emission spectra normally existed in CPs. In this study, we observed normalized absorption and emission spectra of PF-DBT-PEG (1 μg/mL) before and after the addition of Cys (500 μg/mL, 4.1 mM) (Figure S2). Notably, approximately 10 nm red-shift in the absorption spectra and 5 nm red-shift in the emission spectra were observed. Such redshifts in the absorption and emission spectra indicated the successful formation of π-aggregation of the conjugated backbone in the presence of cysteine via the cleavage of the soluble group. Scanning electron microscopy (SEM) and dynamic light scattering (DLS) were used to determine the influence of solubility on the aggregation status of the probe (1 μg/mL) in

Figure 4. Fluorescence intensity changes of PF-DBT-PEG (1 μg/mL) in different pH values at RT.

an extensive pH ranging from 4 to 9, indicating that such stable photophysical properties of PF-DBT-PEG in various pH environments were valuable in intracellular detection. Given that the ratiometry of the polymer was not susceptible to the concentration and pH values, our probe would have good application potential in cell imaging and detection. Response to Cys. To investigate if thiols could lead to the initial prospection of fluorescence conversion, intuitive pictures were taken in bright (daylight) and dark (λex = 380 nm) fields, as shown in Figure 5, for the direct visualization of the influence on the aggregation status and fluorescence of the CP with the addition of Cys, a common thiol-containing biological molecule. The CP with a relatively high concentration (10 μg/mL) in PBS buffer (pH 7.4) was pretreated with Cys (5 mg/mL, 41 mM) for 4 h. Evident precipitation of PF-DBTPEG occurred in PBS buffer in the presence of Cys, and the emission color changed from pristine purple to red fluorescence D

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Macromolecules the presence of Cys (500 μg/mL, 4.1 mM). The aggregation status of PF-DBT-PEG (1 μg/mL) in PBS buffer (pH 7.4) was measured by DLS with the addition of Cys in a successive time period (Figure 6). The particle size increased drastically from

PEG chains from the conjugated polymer, we added Cys (0.1 g/mL, 0.83 M) to PF-DBT-PEG (0.1 g/mL) until no fluorescence was observed in the aqueous solution, thereby ensuring that the conjugated backbone aggregated and precipitated completely. After filtration, the aqueous solution was collected, and its components were subsequently analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The typical molecular weight signal of the PEG group was observed in aqueous solution (Figure 8), thereby confirming that PEG chains had

Figure 6. Diameter of PF-DBT-PEG (1 μg/mL) nanoparticles in pH 7.4 PBS buffer changes with time with the addition of Cys (500 μg/ mL, 4.1 mM).

24 to 228 nm within 0 to 40 min, and visible precipitates could be observed after 35 min. The corresponding SEM and DLS images at specific periods, such as 0, 6, 27, and 45 min, are shown in Figure 7. Small particles were formed during the initial time period. As time elapsed, the molecules continuously gathered together to generate larger particles, and their morphology became irregular, indicating that the addition of Cys could actually lead to effective aggregation of the CP probe. DLS data showed the same variation tendency of particle size (Figure 7B). Such increased particle size may be due to the decreased water solubility of CP caused by the cleavage of the PEG chains through breakage of the disulfide bond. The thiol/ disulfide cleavage reactions played a critical role in maintaining the cellular redox balance via the glutathione (GSH)/disulfide redox couple34 and were further utilized to construct fluorescent thiol probes.35,36 To directly prove the cleavage of

Figure 8. MALDI-TOF-MS picture of the components in aqueous solution after aggregation occurred.

been successively cleaved from CPs and had led to the reduction in solubility of CPs and enhancement of aggregation of hydrophobic conjugated backbones. The optical response of the probe to thiols was further observed. The normalized fluorescence spectra of the ratiometric probe PF-DBT-PEG (1 μg/mL) in pH 7.4 PBS buffer in the presence of Cys (0−1000 μg/mL, 0−8.25 mM) for 40 min are shown in Figure 9A. After adding Cys, a significant decrease of the fluorescence intensity at 420 nm and a gradual enhancement at 628 nm were observed. Furthermore, a 12-fold maximum enhancement in the emission ratio of I628/ I420 (Figure 9B) from 0.095 (without Cys) to 1.15 (with Cys)

Figure 7. (A) SEM of PF-DBT-PEG (1 μg/mL) nanoparticles in pH 7.4 PBS buffer at different times when Cys (500 μg/mL, 4.1 mM) was added: (a) 0, (b) 6, (c) 27, and (d) 45 min. (B) DLS analysis of PF-DBT-PEG (1 μg/mL) nanoparticles in pH 7.4 PBS buffer at different times upon the addition of Cys (500 μg/mL, 4.1 mM): (e) 0, (f) 6, (g) 27, and (h) 45 min. E

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Figure 9. (A) Normalized fluorescence spectra (excitation at 380 nm) of PF-DBT-PEG (1 μg/mL) in pH 7.4 PBS buffer in the presence of Cys (0− 1000 μg/mL and 0−8.25 mM) for 40 min. (B) Tendency chart of the fluorescence ratio (628−420 nm) of PF-DBT-PEG (1 μg/mL) in the presence of Cys with various concentrations from 0 to 1000 μg/mL (0−8.25 mM) in pH 7.4 PBS buffer for 40 min.

was observed after Cys at 500 μg/mL was added (4.1 mM that reached the saturated ratiometric effect of the probe). All the experimental results revealed the evident enhancement of FRET efficiency in the CP after the addition of Cys. We also observed that our probe had a similar detection mechanism for GSH (Figure S3). In our system, the cleaved disulfide bond by Cys resulted in the separation of the water-soluble PEG chain from the hydrophobic conjugated backbone and, consequently, the drastically decreased water solubility of the CPs. Decreased solubility further led to the enhanced aggregation of the hydrophobic conjugated backbone and the shortened distance between the donor and acceptor in the polymer, thereby significantly improving the FRET efficiency and changing the fluorescence (from purple to red). Detailed studies on the quantitative relationship between FRET efficiency and Cys were conducted. The fluorescence ratio of I628/I420 showed a good linear relationship with the concentration of Cys from 1 to 200 μg/mL (Figure S4). The detection limit (S/N = 3) of the polymer probe was calculated to be 2.56 μg/mL (0.021 mM). The good stability and large ratiometric fluorescence range indicated the high sensitivity of the CP-based probe to Cys. As reported previously, GSH is one of the thiols with the highest relevant levels in human cells; the highest GSH level is approximately 1 mM in normal cells and 10 mM in cancer cells.37 The detection limit of our polymer probe is much smaller than the aforementioned GSH (thiols) concentration in normal and cancer cells. Thus, our probe is suitable for biological monitoring applications in cells. The emission ratio of PF-DBT-PEG (1 μg/mL) in pH 7.4 PBS buffer with Cys (500 μg/mL, 4.1 mM) over time at RT is shown in Figure 10 to further illustrate FRET efficiency

changes over time. Upon the addition of Cys, a significant enhancement in the emission ratio of I628/I420 (from 0.095 to 1.15) was observed, an evident red fluorescence was detected at 7 min, and a plateau of the fluorescence ratio was reached at 40 min. This result coincided with the changes of particle size with time, as previously mentioned, which further confirmed that the aggregation of the CP probe improved the intermolecular and intramolecular FRET efficiencies. Selectivity. Non-thiol-containing molecules (Ala, Gln, Gly, Leu, Pro, Val, Thr, Tyr, Glu, and His) and thiol-containing molecules (GSH or Cys) were examined in pH 7.4 PBS buffer (Figure 11) to evaluate the selectivity of PF-DBT-PEG. In Figure 11A, compared with the free probe, PF-DBT-PEG (1 μg/mL) in pH 7.4 PBS buffer, thiol-containing molecules (GSH or Cys, 4.1 mM in pH 7.4 PBS) induced a significant response in the emission ratio (from 0.095 to 1.15). By contrast, non-thiol-containing molecules (4.1 mM in pH 7.4 PBS) and their mixture induced no visible ratiometric changes (approximately 0.095). Cys and the mixture of Cys with nonthiol-containing molecules (Ala, Gln, Gly, Leu, Pro, Val, Thr, Tyr, Glu, and His) were examined in the PF-DBT-PEG solution to further investigate the potential interference of the two types of molecules to the fluorescence of PF-DBT-PEG. As shown in Figure 11B, the mixtures produced the same results as the fluorescence ratio of PF-DBT-PEG (1.15) with Cys. These facts showed that PF-DBT-PEG had a good selectivity for thiols and that non-thiol-containing molecules would not interfere during detection. Then, images of the fluorescence of PF-DBT-PEG (1 μg/ mL) in pH 7.4 PBS buffer were obtained to further reveal the change of the emission ratio (Figure 12). In the presence of several typical non-thiol-containing molecules (4.1 mM), such as Tyr, Ala, Gly, and their mixtures, the fluorescence was purple and showed no difference from the free probe. However, evident red fluorescence appeared in the presence of thiolcontaining molecules (Cys or GSH, 4.1 mM). All of the aforementioned data confirmed the good selectivity of PFDBT-PEG in sensing thiols. Cell Imaging. A CLSM image was used to visualize the fluorescent changes in HeLa cells and to sense intracellular thiols using the WSCP probe (Figure 13). For comparison purposes, two groups of HeLa cells were tested. One group was pretreated with N-ethylmaleimide (1 mM) to inhibit the activity of free thiols in living cells (Figures 13F−J,L), and the other was a normal group without any pretreatment (Figures 13A−E,K). Three fluorescence channels, namely, blue, red, and

Figure 10. Fluorescence intensity ratio of I628/I420 of PF-DBT-PEG (1 μg/mL) changes with time in the presence of Cys (500 μg/mL, 4.1 mM) in pH 7.4 PBS buffer at RT. F

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Figure 11. (A) Fluorescence intensity ratio of I628/I420 of PF-DBT-PEG (1 μg/mL) changes in the absence and presence of various amino acids (4.1 mM) in pH 7.4 PBS buffer: (1) no analytes; (2) Ala; (3) Gln; (4) Gly; (5) Leu; (6) Pro; (7) Val; (8) Thr; (9) Tyr; (10) Glu; (11) His; (12) Cys; (13) GSH; and (14) the mixture of various amino acids (Ala, Gln, Gly, Leu, Pro, Val, Thr, Tyr, Glu, and His) without Cys and GSH. (B) Fluorescence intensity ratio of I628/I420 of PF-DBT-PEG (1 μg/mL) changes with Cys and the mixture of Cys and various amino acids (4.1 mM) in pH 7.4 PBS buffer: (1) Cys; (2) Ala + Cys; (3) Gln + Cys; (4) Gly + Cys; (5) Leu + Cys; (6) Pro + Cys; (7) Val + Cys; (8) Thr + Cys; (9) Tyr + Cys; (10) Glu + Cys; (11) His + Cys.

consistent with the results of the extracellular test. Thus, we could visually distinguish thiol content from its distribution in living cells through fluorescence change. Figure 13K (normal HeLa cells) and Figure 13L (thiols-controlled HeLa cells) were the ratio images of the HeLa cells incubated with PF-DBT-PEG constructed from two collection windows, thereby resulting in average emission ratios of 1.12 and 0.23, respectively. Consequently, the contents of thiols in cells were calculated to be approximately 1.95 mM (normal HeLa cells) and 0.3 mM (thiol-controlled HeLa cells). All these data showed that the probe (PF-DBT-PEG) is functional in thiol detection and imaging in vitro.

Figure 12. Fluorescence of PF-DBT-PEG (1 μg/mL) in the absence and presence of various amino acids (4.1 mM) in pH 7.4 PBS buffer with λex = 380 nm: (1) absence; (2) Cys; (3) Tyr; (4) Ala; (5) Gly; (6) GSH; (7) Cys + Tyr + Ala + Gly; (8) GSH + Tyr + Ala + Gly; (9) Tyr + Ala + Gly.

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CONCLUSIONS We designed and synthesized a WSCP with pendant disulfide linkages to PEG chains, which was a highly efficient ratiometric probe with solubility-induced fluorescence conversion for thiol detection. Once the specific cleavable linkages (disulfide) in the response structure were cleaved by thiol-containing molecules, the solubility of the probe was reduced immediately and the conjugated backbone aggregated together. Consequently, the intermolecular FRET would be enhanced by the shortened intramolecular and intermolecular distances. As a seldom reported ratiometric WSCP for thiol detection, this probe showed good selectivity and sensitivity. The dynamic fluorescence ratio range and good ratiometric stability of

the overlay, were examined after HeLa cells were incubated with the ratiometric probe PF-DBT-PEG (0.05 mg/mL) for 30 min at 37 °C. As shown in Figures 13A−E, almost no fluorescence signal from the blue channel, a fine fluorescence signal from the red channel, and an evident red fluorescence in the overlay channel were observed in the normal group without any pretreatment. By contrast, when free intracellular thiols were controlled in HeLa cells by N-ethylmaleimide (1 mM) for 30 min (Figures 13F−J), the fluorescence signal in the blue channel was stronger than the previous signal but weaker than the signal in the red channel. In addition, the overlay channel showed a prime mixed purple fluorescence, which was

Figure 13. Confocal laser scanning microscopy (CLSM) images of living HeLa cell (λex = 380 nm): (A) CLSM image of the cells incubated with PFDBT-PEG (0.05 mg/mL) for 30 min at 37 °C; (B) fluorescence image of (A) from the blue channel; (C) fluorescence image of (A) from the red channel; (D) overlay of the fluorescence images; (E) overlays of the fluorescence images with a nonconfocal phase contrast image; (K) ratio image of (A); (F) differential interference contrast image of the cells pretreated with N-ethylmaleimide (1 mM) for 30 min and then loaded with PF-DBTPEG (0.05 mg/mL) for 30 min; (G) fluorescence image of (F) from the blue channel; (H) fluorescence image of (F) from the red channel; (I) overlay of the fluorescence images; (J) overlays of the fluorescence images with a nonconfocal phase contrast image; and (L) ratio image of (F). G

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Macromolecules

with a yield of 95%. 1H NMR (400 MHz, CDCl3, ppm): δ = 7.80 (t, J = 8.54 Hz, 4 H), 7.71 (d, J = 7.68 Hz, 2 H), 2.38 (t, J = 8.37 Hz, 4 H), 1.44 (d, J = 8.37 Hz, 4 H), 1.37 (s, 24 H), 1.29 (s, 18 H) (Figure S5). 13 C NMR (100 MHz, CDCl3, ppm): 172.8, 147.9, 143.8, 134.4, 129.0, 119.7, 83.8, 79.9, 54.5, 34.4, 30.0, 28.0, 25.0 (Figure S8). Element analysis calculated (%) for C39H56B2O8, C 69.54, H 8.37, O 18.98. TOF-MS (m/z): [M + H]+ calculated, 674.48; found, 674.4 (Figure S10). Synthesis of PF-DBT-COOR. Monomer A (1.0 g, 1.72 mmol), monomer B (1.223 g, 18.1 mmol), and DBT (0.042 g, 0.09 mmol) were mixed in an aluminum foil-packed flask with a molar ratio of 45:50:5. Tetrakis(triphenylphosphine)Pd(0) was dissolved in a degassed mixture of toluene (25 mL) and K2CO3 (2 mol/L, 5 mL). The mixture was stirred at 85−90 °C for 72 h under a nitrogen atmosphere. After cooling down to RT, the mixture was extracted by chloroform and concentrated. Then, the desired polymer was precipitated in acetone three times. PF-DBT-COOR was precipitated in petroleum ether and filtered to yield 1.02 g (red powder, 65.7% yield) of the pure polymer. 1H NMR (400 MHz, CDCl3, ppm): δ = 7.87 to 7.41 (br, m, 6 H), 8.19 (s, 0.15 H), 2.5 (s, 8 H), 1.3 (s, 18 H) (Figure S6). 13C NMR (100 MHz, CDCl3, ppm): 173.2, 149.6, 141.0, 127.4, 122, 120.2, 80.5, 54.4, 35.1, 30.5, 28.4 (Figure S11). Synthesis of PF-DBT-COOH. PF-DBT-COOR (1 g) was dissolved in 15 mL of 10% TFA/dichloromethane and stirred at RT under a nitrogen atmosphere for 24 h. After PF-DBT-COOR was evaporated, the crude product was precipitated in petroleum ether and filtered to remove impurities. Then, the desired solid product was obtained as red powder (0.75 g, 99% yield). 1H NMR (400 MHz, dimethyl sulfoxide [DMSO]-d6, ppm): δ = 7.99 to 7.39 (br, m, 6 H), 8.27 (s, 0.15 H), 2.5 (s, 8 H) (Figure S6). 13C NMR (100 MHz, DMSO-d6, ppm): 174.5, 158.9, 149.8, 140.2, 127.1, 121.8, 60.2, 54.3, 29.5 (Figure S12). Synthesis of PF-DBT-PDA. PF-DBT-COOH (0.308 g, 1 mmol of monomers), EDCi (3.83 g, 20 mmol), and NHS (5.75 g, 50 mmol) were dissolved in 10 mL of anhydrous DMSO and stirred for 30 min at RT. Then, 0.744 g of PDA (4 mmol, synthesized following the steps in the study of Zugates38) was added dropwise into the solution. After 24 h, the solvent was removed by freeze-drying, and the residue was dissolved in 10 mL of chloroform. The organic phase was washed with water (20 mL) three times and then concentrated and precipitated with anhydrous ether twice. The final solid was desiccated in a vacuum oven. A red powder was obtained with a yield of 60% (0.375 g). Mn = 6939, PDI = 1.36. 1H NMR (400 MHz, CDCl3, ppm) is shown in Figure S6. 13C NMR (100 MHz, CDCl3, ppm): 172.7, 159.1, 149.6, 137.0, 121.2, 68.1, 54.6, 38.51, and 29.7 (Figure S13). Synthesis of PF-DBT-PEG. PF-DBT-PDA (0.2 g, 0.3 mmol of the monomers) and PEG-SH (Mw = 1000 Da, 1.2 g, 6 mmol) were dissolved in 20 mL of methanol. After stirring at RT for 48 h, the crude product, PF-DBT-PEG, was evaporated and then dialyzed in water for 72 h. The final product was obtained by freeze-drying (red powder, 0.6 g, 90% yield). Mn = 18 860, PDI = 1.29. The content of the disulfide groups in PF-DBT-PEG was calculated to be 0.758 mmol/g, as shown in Scheme S1 (the content of S−S is approximately half the content of sulfur atoms). The average PEG chains on each polymer were calculated to be 11 (the content of PEG in the polymer can approximately be attributed to the difference of the average molecular weight between PF-DBT-PEG and PF-DBT-PDA). 1H NMR (400 MHz, CDCl3, ppm) is shown in Figure S7. 13C NMR (100 MHz, CDCl3, ppm): 170.3, 70.6, 59.0, 37.3, and 29.5 (Figure S14).

WSCP indicated its excellent property in imaging thiol in living cells. Furthermore, this type of design can be extended as a good detection platform through which this solubility-induced sensing system has further universality of application in other biological chain-cleavable molecules by simply changing the cleavage-specific linkages.

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EXPERIMENTAL SECTION

Materials and Instruments. All amino acids were purchased from Sigma-Aldrich Chemical Co. and were used as received. Other chemicals were purchased from Sigma-Aldrich, Acros, and Alfa and were used as received. THF was purified by distillation from sodium in the presence of benzophenone. Other organic solvents were used without any further purification. All reactions were conducted under a nitrogen atmosphere. NMR spectra were recorded on a Bruker Ultra Shield Plus 400 MHz NMR. CDCl3 was used as the solvent. TOF-MS of monomers A and B was conducted on a Micromass GC-TOF in DCM. FT-IR spectra were recorded on a Shimadzu IR Affinity-1 spectrometer by dispersing samples in KBr. MALDI-TOF-MS was conducted on a Bruker Autoflex without the matrix under the reflector mode for data acquisition. The UV−vis absorption spectra were recorded on a Shimadzu UV-3600 UV−vis−NIR spectrophotometer. Photoluminescent spectra were recorded at RT using a fluorescence spectrophotometer with excitation and emission slit widths of 5.0 nm. 1 H NMR spectra were recorded on NMR (400 MHz) spectrometers, using tetramethylsilane as an internal standard. The particle diameters were measured by DLS using a 90 Plus particle size analyzer (Brookhaven Instruments). SEM images were recorded using a scanning electron microscope. Cell imaging was conducted by CLSM. Element content was detected by an elemental analyzer (EA, VARIO EL III). GPC analysis of the polymers was conducted on a Shim-pack GPC-80X column with THF as the eluent at a flow rate of 1.0 mL/min at 35 °C and polystyrene as the standard. The data were analyzed by using the software package provided by Shimadzu Instruments. Cell Culture. The human cervical cancer HeLa cell line was obtained from the American Type Culture Collection and cultured in Dulbecco’s modified Eagle’s medium (Gibco BRL, Gaithersburg, MD) supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco BRL, Gaithersburg, MD). Cells were cultured at 37 °C in a humidified chamber containing 5% CO2. Synthesis of Monomer A. 2,7-Dibromofluorene (3.3 g, 10.2 mmol), tert-butyl acrylate (5.25 g, 41 mmol), and tetrabutylammonium bromide (0.25 g, 0.78 mmol) were mixed together and protected by nitrogen. Then, toluene (50 mL) and KOH (2 M, 5 mL) were added using injectors. The mixture was stirred for 12 h at RT. Afterward, the mixture was washed successively by water and HCl aqueous solution, extracted by dichloromethane, and then dried by Na2SO4. The mixture was filtered, and the filtrate was rotary evaporated to obtain a yellow solid. The resultant solid was purified by SiO2 chromatography, and monomer A (white solid) was obtained with a yield of 70%. 1H NMR (400 MHz, CDCl3, ppm): δ = 7.53 (d, J = 8.73 Hz, 2 H), 7.48 (d, J = 6.27 Hz, 4 H), 2.30 (t, J = 8.18 Hz, 4 H), 1.46 (t, J = 8.36 Hz, 4 H), 1.33 (s, 18 H) (Figure S5). 13C NMR (100 MHz, CDCl3, ppm): 172.2, 150.0, 139.1, 131.1, 126.5, 122.1, 80.4, 54.1, 34.4, 29.8, 28.0 (Figure S8). Element analysis calculated (%) for C27H32Br2O4, C 55.88, H 5.56, O 11.03. TOF-MS (m/z): [M + H]+ calculated, 580.35; found, 580.2 (Figure S9). Synthesis of Monomer B. Monomer A (2.13 g, 3.7 mmol), bispinacolatodiboronmin (2.4 g, 9.5 mmol), and Pd(dppf)2Cl2 and KOAc (1.7 g, 17 mmol) were mixed in a round-bottom flask. The flask was covered with aluminum foil to prevent the absorption of light. DMF was added to dissolve the reactant. The mixture was stirred and refluxed at 90 °C under N2 protection. After 12 h, the mixture was dissolved in DCM and filtered to eliminate the catalyst. Afterward, the mixture was washed with water seven times to remove DMF and HCl aqueous solution. Na2SO4 was used to dry the organic solution. Then, the mixture was evaporated to obtain a solid and purified by SiO2 chromatography. The final product was obtained as a white powder

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ASSOCIATED CONTENT

S Supporting Information *

Figures showing 1H NMR, 13C NMR, TOF-MS, UV spectra, PL spectra, FT-IR spectra, and elemental analysis. This material is available free of charge via the Internet at http://pubs.acs.org. H

DOI: 10.1021/ma5021775 Macromolecules XXXX, XXX, XXX−XXX

Article

Macromolecules

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(30) Harwood, D. T.; Kettle, A. J.; Brennan, S.; et al. J. Chromatogr., B 2009, 877, 3393. (31) Robert, R. W.; Takushi, K. Bioconjugate Chem. 1990, 7, 96. (32) Morawetz, H. Science 1988, 240, 172. (33) Hu, Q.; Zhou, Q. M.; Zhang, Y.; et al. Macromolecules 2004, 37, 6299. (34) Giles, N. M.; Giles, G. I.; Jacob, C. Biochem. Biophys. Res. Commun. 2003, 300, 1. (35) Pires, M. M.; Chmielewski. J. Org. Lett. 2008, 10, 837. (36) Pullela, P. K.; Chiku, T.; Carvan, M. J.; Sem, D. S. Anal. Biochem. 2006, 352, 265. (37) Hong, R.; Han, G.; Fernandez, J. M.; Kim, B. J.; et al. J. Am. Chem. Soc. 2006, 128, 1078. (38) Gregory, T. Z.; Daniel, G. A.; Steven, R.; Ingrid, E. B. L.; Robert, L. J. Am. Chem. Soc. 2006, 128, 12726.

AUTHOR INFORMATION

Corresponding Authors

*E-mail [email protected], Tel +86 (25) 8586 6360, Fax +86 (25) 8586 6369 (Q.F.). *E-mail [email protected], Tel +86 (25) 5813 9001, Fax +86 (25) 5813 9988 (W.H.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Basic Research Program of China (No. 2012CB933301 and 2012CB723402), the Ministry of Education of China (No. IRT1148), and the National Natural Science Foundation of China (No. 21222404, 61378081, 51173080, and 21104033) as well as the Natural Science Foundation of Jiangsu Province of China (No. BZ2010043, NY211003, and BM2012010).

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REFERENCES

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DOI: 10.1021/ma5021775 Macromolecules XXXX, XXX, XXX−XXX