Fluorescence Ratiometric Assay Strategy for Chemical Transmitter of

Oct 9, 2015 - A new water-soluble conjugated poly(fluorene-co-phenylene) derivative (PFP-FB) modified with boronate-protected fluorescein (peroxyfluor...
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Fluorescence Ratiometric Assay Strategy for Chemical Transmitter of Living Cells Using H2O2‑Sensitive Conjugated Polymers Yunxia Wang, Shengliang Li, Liheng Feng, Chenyao Nie, Libing Liu,* Fengting Lv, and Shu Wang* Beijing National Laboratory for Molecular Science, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, People’s Republic of China S Supporting Information *

ABSTRACT: A new water-soluble conjugated poly(fluorene-co-phenylene) derivative (PFP-FB) modified with boronate-protected fluorescein (peroxyfluor-1) via PEG linker has been designed and synthesized. In the presence of H2O2, the peroxyfluor-1 group can transform into green fluorescent fluorescein by deprotecting the boronate protecting groups. In this case, upon selective excitation of PFP-FB backbone at 380 nm, efficient fluorescence resonance energy transfer (FRET) from PFP-FB backbone to fluorescein occurs, and accordingly, the fluorescence color of PFP-FB changes from blue to green. Furthermore, the emission color of PFP-FB and the FRET ratio change in a concentration-dependent manner. By taking advantage of PFP-FB, ratiometric detection of choline and acetylcholine (ACh) through cascade enzymatic reactions and further dynamic monitoring of the choline consumption process of cancer cells have been successfully realized. Thus, this new polymer probe promotes the development of enzymatic biosensors and provides a simpler and more effective way for detecting the chemical transmitter of living cells. KEYWORDS: water-soluble conjugated polymers, FRET, detection, chemical transmitter, living cells Compared with fluorescent intensity-based assays, the technique that relies on fluorescence resonance energy transfer (FRET) presents a large Stokes shift and results in a ratiometric fluorescence signal,15 which measures two different emission intensities and thereby eliminates the environmental influence.16 Recently, water-soluble conjugated polymers (CPs) have attracted more interest as optical platforms for biochemical sensors.17−20 Compared with small molecule counterparts, the CPs possess signal-amplifying effects through coordinating the action of absorbing units in the skeleton of CPs. Meanwhile the transfer of the excitation energy along the backbone of CPs to an acceptor also amplifies the detection signal.21−23 Combining fluorescent ratiometric technique offered by FRET with the signal-amplification property of CPs, CPs have been utilized for sensitive and selective detection of disease-related biomarkers.24 For most reported methods, intermolecular FRET from CPs to fluorephores rationally incorporated to analytes dominated. Previously, we have reported a water-soluble cationic polyfluorene (PF-FB) as a fluorescence probe to optically detect hydrogen peroxide (H2O2) and glucose in serum.25 However, the positively charged CPs can nonspecifically bind with cells, which is adverse to cell detection in situ. Herein, we present a new water-soluble conjugated poly(fluorene-co-phenylene) derivative (PFP-FB) with boronate-protected fluorescein

1. INTRODUCTION Choline, known as a kind of vitamin B, is an essential component of membrane phospholipids,1 and it is the precursor of acetylcholine (ACh), which is one of the important brain chemicals involved in memory.2 Numerous studies have suggested most tumor tissues contain a large amount of phosphorylcholine, while the choline metabolite of corresponding normal tissues is present at low or undetectable levels.3−5 ACh acts as a classical neurotransmitter that appears in the peripheral nervous system and central nervous system.6 Both choline and ACh play an important role in many physiological activities, including learning-memory, emotion, myokinesis, and so on. Furthermore, abnormal levels of choline and ACh can trigger a range of illnesses such as Alzheimer’s and Parkinson’s disease, and other nerve disorders. Therefore, the quantitative determination of choline and ACh is of great significance in biological sciences and clinical analysis. Due to the complexity of samples analysis, enzyme-based sensors are very competitive because of the high sensitivity, specificity, and fast response. Numerous methods involving the enzyme cascade have emerged in recent years for monitoring choline and ACh,7−9 for instance, colorimetric, electrochemical, chemiluminescent, and fluorescent detections.10−13 Among these methods, fluorescent technique has obvious advantages over others, such as rapidity, sensitivity, and real-time monitoring.14 Thus, development of a novel fluorescent probe will provide a simpler and more effective way for choline or ACh detection. © XXXX American Chemical Society

Received: August 4, 2015 Accepted: October 9, 2015

A

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Scheme 1. (a) Schematic Representation of the Sensing Strategy Using PFP-FB To Monitor the Choline Consumption Process of Living Cells. (b) Reaction of PFP-FB with H2O2

new polymer probe itself shows good ratiometric fluorescence response to H2O2. Based on the ratiometric fluorescent response of PFP-FB to H2O2 that was produced from choline and ACh under AChE/ChOx enzyme-coupled reaction, choline and ACh are detected with high sensitivity and specificity. More

covalently linked to the PEG side chain. The neutral PEG chains enhance the water solubility of PFP-FB, which allows for interrogating biological substrates. The remaining carboxyl groups of side chains not only reduce nonspecific interaction with cells but also allow for the further modification of CPs. This B

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ACS Applied Materials & Interfaces Scheme 2. Synthetic Route of PFP-FB

intensities, it is possible to assay H2O2. For the case without H2O2, the peroxyfluor-1 covalently links to the side chain of PFPFB, existing as a colorless and nonfluorescent lactone form.26−28 Since the concentration of H2O2 is directly proportional to the choline concentration, the change of fluorescence ratio can be utilized to detect the choline concentration. Because living cancer cells consume the choline in a proliferation process, PFPFB could successfully monitor the choline concentration change with cancer cells proliferation. Synthesis and Characterization of PFP-FB. Scheme 2 outlines the procedures for the synthesis of PFP-FB. Reaction of compound 1 with N-hydroxysuccinimide (NHS) in the presence of N,N′-dicyclohexylcarbodiimide (DCC) gave compound 2 in 78% yield. Then compound 2 reacted with HOOC-PEG-NH2 to afforded compound 3 in 80% yield. Compound 4 was prepared by palladium-catalyzed Suzuki cross-coupling reaction29 of compound 3 with benzen-1,4-bis(boronic acid)-propane-1,3diol diester in the presence of 2.0 M K2CO3 aqueous solution and

importantly, the choline consumption process of living cancer cells has been successfully monitored by the H2O2-sensitive PFPFB system.

2. RESULTS AND DISCUSSION Sensing Strategy of PFP-FB for Choline. The ratiometric fluorescence assay for choline and monitoring the choline consumption process of cells using PFP-FB and enzyme-coupled reaction is illustrated in Scheme 1a. The cell culture medium usually contains a specific amount of choline to meet the requirement of cell growth. In the presence of ChOx, choline is oxidized by ChOx to produce H2O2. With reaction of H2O2, the boronate-protected fluorescein (peroxyfluor-1) group can generate green fluorescent fluorescein (Fl) by deprotecting the boronate protecting groups (Scheme 1b). Then Fl acts as the acceptor of the efficient intramolecular FRET from the fluorene unit, which results in changes of blue to green fluorescence ratios. By triggering the ratio change of blue to green emission C

DOI: 10.1021/acsami.5b07172 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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spectrum of the Fl, which indicates that the fluorene unit and Fl are good energy transfer donor and acceptor pairs for FRET.31 However, the green emission band centered at 522 nm of Fl is not observed in the emission spectrum of PFP-FB since the Fl is boronate-protected and no FRET occurs. The fluorescence quantum yield (QY) of PFP-FB is calculated to be 21% using quinine sulfate in 0.1 M H2SO4 (η = 55%) as the standard. Optical Response of PFP-FB to H2O2. In order to verify the sensing strategy, intramolecular FRET of PFP-FB in the presence of H2O2 was investigated. As shown in Figure 2a, with 380 nm excitation, a maximum peak around 424 nm is shown in the emission spectrum of PFP-FB in phosphate buffer solution and no obvious emission peak is observed for the peroxyfluor-1 unit. Upon treatment with H2O2, the peroxyfluor-1 unit emission at 522 nm appears and the fluorene unit emission at 424 nm decreases significantly. All of the results indicate that treatment with H2O2 generates a fluorescent Fl as energy acceptor and an efficient intramolecular FRET occurs from fluorene to Fl. The FRET ratio of Fl to fluorene emission intensities (I522 nm/I424 nm) increases from 0.24 without H2O2 to 1.20 after H2O2 treatment; a 5-fold FRET ratio is observed. Meanwhile, the apparent emission color changes from blue to green under UV light (Figure 2b), indicating the realization of visual detection. The emission spectrum changes of PFP-FB treated with different concentrations of H2O2 are shown in Figure 2c. With the increasing concentration of H2O2, the emission intensity at 424 nm decreases gradually and that centered at 522 nm increases obviously. The fluorescence changes of PFP-FB in a concentration-dependent manner demonstrate that PFP-FB is a H2O2sensitive probe. The value of I522 nm/I424 nm increases gradually and reaches a plateau at 2000 μM with the increasing concentration of H2O2 (Figure S1). Figure 2d shows I522 nm/ I424 nm) as a function of H2O2 incubation time. A nearly linear FRET ratio increase is observed with the H2O2 incubation time from 0 to 30 min, which indicates that PFP-FB can be employed

Pd(dppf)Cl2 in DMF (25% yield). Reaction of compound 5 with NHS in the presence of 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide hydrochloride (EDCI) gave compound 6 in 87% yield. Reaction of compound 6 with tert-butyl(6-aminohexyl)carbamate gave compound 7 in 90% yield. Compound 8 was obtained by reaction of compound 7 with bis(pinacolato)diboron in the presence of potassium acetate and Pd(dppf)Cl2 in 1,4-dioxane (41% yield). Compound 8 was treated with trifluoroacetic acid (TFA) and triethylamine to give compound 9 containing free primary amino groups. Compound 9 was linked covalently to the side chain of compound 4 in the presence of NHS and EDCI to afford target co-polymer PFP-FB. The photophysical properties of PFP-FB were evaluated in aqueous solution. As shown in Figure 1, PFP-FB displays a

Figure 1. Normalized absorption and emission spectra of PFP-FB and fluorescein in water. The excitation wavelength is 380 nm for PFP-FB and 480 nm for fluorescein.

maximum peak at 375 nm in the UV−vis absorption spectrum. The emission spectrum exhibits a typical characteristic of polyfluorenes30 that presents a maximum peak at 424 nm and a shoulder peak at 446 nm upon excitation at 380 nm. The emission spectrum of PFP-FB overlaps well with the absorption

Figure 2. (a) Emission spectra of PFP-FB in the absence and presence of H2O2. (b) Photograph of PFP-FB in the absence and presence of H2O2 under UV light (λmax = 365 nm). (c) Emission spectra of PFP-FB treated with different concentrations of H2O2 from 0 to 800 μM. The incubation time is 30 min. (d) FRET ratio (I522 nm/I424 nm) as a function of the H2O2 (3.0 mM) incubation time. [PFP-FB] = 4.6 μM (in repeat units). The experiments were performed in phosphate buffer (pH 7.4). The excitation wavelength is 380 nm. D

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Figure 3. (a) Emission spectra of PFP-FB/ChOx in the absence and presence of choline. (b) Emission spectra of PFP-FB/ChOx as a function of the choline concentration. (c) FRET ratio (I522 nm/I424 nm) as a function of the choline concentration. (d) FRET ratio (I522 nm/I424 nm) of PFP-FB to different analytes. [PFP-FB] = 4.6 μM (in repeat units), [ChOx] = 0.84 U·mL−1, [Ch] = 100 μM, [ACh] = 100 μM, and the concentration of the other analytes is 1 mM. The incubation time is 5 h, and the excitation wavelength is 380 nm.

Figure 4. (a) Emission spectra of PFP-FB/AChE/ChOx in the absence and presence of ACh. (b) Emission spectra of PFP-FB/AChE/ChOx with adding different concentrations of ACh. (c) FRET ratio (I522 nm/I424 nm) as a function of the acetylcholine concentration. (d) Linear relationship between I522 nm/I424 nm and acetylcholine concentration. [PFP-FB] = 4.6 μM (in repeat units), [AChE] = 0.26 U·mL−1, and [ChOx] = 0.84 U·mL−1. The incubation time is 30 min, and the excitation wavelength is 380 nm.

as an efficient probe to quantitatively detect and real-time monitor H2O2 concentration. Fluorescence Detection of Choline Based on PFP-FB. Enzyme-coupled assays have attracted much attention owing to their high sensitivity and selectivity,32,33 and many substrates can be catalyzed by their respective oxidases to generate H2O2.34

Based on this strategy, using H2O2-sensitive PFP-FB combined with ChOx, choline detection was investigated. Physiological temperature 37 °C was chosen as the optimal condition in favor of enzyme reaction (Figure S2). As shown in Figure 3a, the addition of choline to the solution of PFP-FB and ChOx in phosphate buffer could trigger efficient FRET from fluorene E

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coefficient of 0.999 (Figure 4d). Thus, PFP-FB presents a good potential to assay ACh combined with the AChE/ChOx cascade reaction. Monitoring Choline Consumptions of Living Cells by PFP-FB. To further validate the practicality of the proposed assay strategy, we used the PFP-FB probe combined with enzymatic reaction for dynamically monitoring the choline consumption process of cancer cells. As shown in Figure 5, I522 nm/I424 nm of PFP-FB decreases gradually with the increasing incubation time of the Jurkat T cell and NCI-H82 cell. For comparison, the FRET ratio of PFP-FB in the medium without cells almost remains the same within 72 h. A549 and HeLa cells were also monitored, and similar results were obtained (SI Figure S4). This is consistent with the fact that cell growth requires nutrients, and it reflects the consumption of choline by living cells over time. These results indicate that the PFP-FB probe could act as the choline indicator for dynamically monitoring the choline’s consumption process of cells.

units to Fl. It can be seen that the addition of ChOx has little effect upon the emission of PFP-FB. These observations demonstrate that the ChOx catalyzed the oxidation of choline to generate H2O2, which leads to an efficient FRET response of PFP-FB. The concentration-dependent emission spectral changes of PFP-FB with the choline concentration ranging from 0 to 100 μM were further investigated. The intensity of the emission band centered at 424 nm decreases gradually and that centered at 522 nm increases distinctly with the increasing concentration of choline (Figure 3b). And I522 nm/I424 nm of PFPFB as a function of choline concentration from 0 to 1 mM are summarized in Figure 3c. With the increasing concentration of choline from 0 to 400 μM, the value of I522 nm/I424 nm increases gradually and reaches a plateau at 400 μM. More importantly, a good linear relationship between I522 nm/I424 nm and the choline concentration is observed over the range from 0 to 100 μM. Because the average concentration of choline in the serum is 10.8 μM,35 PFP-FB presents a good potential to assay choline in blood samples. In order to testify the selectivity of this assay for the detection of choline, the possible interferences, including dopamine (DA), glucose (Glu), and aspartic acid (AA), were investigated. Because of the high substrate specificity of the enzyme ChOx, there is no significant FRET ratio change of PFPFB in response to the preceding analytes (Figure 3d). These results suggest that the sensing system of PFP-FB possesses high selectivity toward choline. Detection of Acetylcholine Based on Enzyme-Coupled Reaction and PFP-FB. To demonstrate the possibility that PFP-FB coupling with various enzymes combination could realize diverse substrates detection via enzyme-coupled reactions, AChE and ChOx were chosen as a proof of concept to detect ACh. Figure 4a depicts the emission spectra without and with the addition of ACh to the solution of PFP-FB, AChE, and ChOx in the phosphate buffer. Upon addition of ACh, efficient FRET from fluorene units to Fl is observed. Meanwhile, the emission spectrum of PFP-FB is not changed in the presence of any enzyme (AChE and ChOx) or ACh. And different concentrations of enzymes have little effect on I522 nm/I424 nm of PFP-FB (Supporting Information (SI) Figure S3). These results indicate that AChE catalyze hydrolysis of ACh to form choline, which in turn is oxidized by ChOx to generate H2O2 that leads to the efficient FRET response of PFP-FB (Scheme 3). Figure 4b

3. CONCLUSION In summary, we have designed and synthesized a new poly(fluorene-co-phenylene) (PFP-FB) containing a boronateprotected fluoran pendent. By combining the superior optical properties of PFP-FB with the fluorescent ratiometric technique offered by FRET, this new polymer probe shows good potential to detect H2O2. Based on the efficient FRET response of PFP-FB to H2O2 that was generated from choline and ACh under cascade enzymatic reactions of AChE/ChOx coupling, a new optical assay for choline and ACh has been developed. The proposed method realizes choline and ACh detection in a simple and selective manner and presents high specificity to choline and ACh, respectively. We further utilized the present method to dynamically monitor the choline consumption process of living cancer cells. Thus, this new polymer probe promotes the development of enzymatic biosensors and provides a simpler and more effective way for detecting chemical transmitter of living cells. 4. EXPERIMENTAL SECTION Materials and Measurements. All chemicals were purchased from Acros, Aldrich Chemical Co., or Alfa-Aesar and used as received. All organic solvents were purchased from Beijing Chemical Works and used as received. NH2−PEG10−CH2CH2COOH with the purity of 95% was purchased from Yarebio Co., Ltd. (Shanghai, China). HeLa cells, A549 cells, and Jurkat T cells were obtained from the cell culture center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). NCI-H82 cells were purchased from Biopike Technology (Beijing, China). Dulbecco’s modified Eagle medium (DMEM), RPMI 1640 medium, and phosphate buffer saline (PBS) were purchased from Hyclone (Beijing, China). 1H NMR and 13C NMR were obtained from a Bruker Avance 400 MHz spectrometer. High resolution mass spectra were obtained using Solarix 9.4T. UV−vis absorption spectra were taken on a JASCO V-550 spectrophotometer. Fluorescence spectra were measured using a Hitachi F-4500 fluorometer equipped with a xenon lamp excitation source. Photographs of the polymer solutions were taken using a Canon EOS 550D digital camera under a hand-held UV lamp (ZF-7A, Shanghai Gucun Electron Optic Instrument Factory) with λmax = 365 nm. Gel permeation chromatography (GPC) measurement was performed on a Waters-410 system against polystyrene standards with DMF as eluent. Synthesis of Compound 3. Compounds 1 and 2 were synthesized as previously reported.36 To a solution of compound 2 (116.4 mg, 0.16 mmol) in CH2Cl2 (10 mL) was added NH2−PEG10−CH2CH2COOH (169.5 mg, 0.32 mmol). The resulting solution was stirred for 48 h at room temperature. After being washed with water, the solvent was

Scheme 3. AChE/ChOx Enzyme Cascade Reaction

shows the concentration-dependent emission spectral changes of PFP-FB with the ACh concentration ranging from 0 to 100 μM. The intensity of the emission band centered at 424 nm decreases gradually and that centered at 522 nm increases obviously with the increasing concentration of ACh. And the FRET ratio changes of PFP-FB as a function of ACh concentration ranging from 0 to 400 μM are given in Figure 4c. The value of I522 nm/ I424 nm increases gradually with increasing ACh concentration initially and gradually reaches a plateau. A good linearity was obtained in the range from 5 to 100 μM with a correlation F

DOI: 10.1021/acsami.5b07172 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 5. I522 nm/I424 nm changes of PFP-FB as an indicator for the choline consumption process of Jurkat T cells (a) and NCI-H82 cells (b). removed under vacuum to afford compound 3. 1H NMR(400 MHz, CDCl3, ppm): δ 7.51 (d, 2H), 7.44 (s, 2H), 7.41 (d, 2H), 6.27 (br, 2H), 3.74 (t, 4H), 3.61 (m, 72H), 3.50 (t, 4H), 3.37 (m, 4H), 2.57 (t, 4H), 1.99 (t, 4H), 1.89 (m, 4H), 1.38 (m, 4H), 1.07 (m, 4H), 0.58 (m, 4H). 13 C NMR (100 MHz, CDCl3, ppm): δ 173.3, 152.2, 139.0, 130.3, 126.1, 121.5, 121.2, 70.5, 70.2, 69.9, 66.8, 55.6, 40.0, 39.1, 36.4, 33.8, 29.5, 25.3, 23.4. HRMS (MALDI-TOF; m/z, [M + Na]+): calcd, 1595.6231; found, 1595.6228. Synthesis of Polymer 4. A mixture of monomer 3 (157.6 mg, 0.1 mmol) and benzen-1,4-bis(boronic acid)-propane-1,3-diol diester (24.6 mg, 0.1 mmol) in 10 mL of DMF and 4 mL of 2.0 M K2CO3 aqueous solution was degassed, and then Pd(dppf)Cl2 was added. The resulting mixture was stirred at 80 °C for 2 days under nitrogen. After cooling to room temperature, 2.0 M HCl was added to neutralize the base. Then the mixture was extracted with dichloromethane (3 × 60 mL). The combined organic layer was washed with water (20 mL) twice and with brine once and then dried over anhydrous MgSO4 for 30 min. After removing the solvent, the crude product was re-dissolved in about 1 mL of CH2Cl2 and then added into 100 mL of acetone. The precipitate was collected by centrifugation. The procedure was repeated twice to obtain a brown solid (46 mg, 25%). 1H NMR (MeOD, 300 MHz, ppm): δ 7.87−7.58 (br, 10H), 3.7 (br, 4H), 3.59 (br, 72H), 3.45 (br, 4H), 3.30 (br, 4H), 2.53 (t, 4H), 1.98 (br, 8H), 1.39 (br, 4H), 1.20 (br, 4H), 0.75− 0.66 (br, 4H). Synthesis of Compound 6. To a solution of compound 525 (169 mg, 0.25 mmol) in DMF (10 mL) was added NHS (51.8 mg, 0.45 mmol) and EDCI (95.8 mg, 0.5 mmol). The resulting solution was stirred for 48 h at room temperature. The solvent was removed under vacuum, and the residue was purified by silica gel chromatography with petroleum ether/ethyl acetate (v/v = 2:1) as eluent to afford a white solid (151 mg, 87%). 1H NMR (400 MHz, CDCl3, ppm): δ 8.37 (d, 1H), 8.16 (d, 1H), 7.83 (s, 1H), 7.69 (s, 2H), 7.41 (d, 2H), 6.5 (d, 2H), 2.88 (s, 4H). 13C NMR (75 MHz, CDCl3, ppm): δ 168.7, 160.4, 150.7, 133.6, 132.3, 131.7, 130.5, 128.9, 126.6, 126.1, 125.9, 117.4, 96.2, 25.6. HRMS (MALDI-TOF; m/z, [M + H]+): calcd, 693.8854; found, 693.8854. Synthesis of Compound 7. To a solution of compound 6 (310 mg, 0.45 mmol) in DMF (10 mL) was added tert-butyl(6-aminohexyl)carbamate (145 mg, 0.67 mmol). The resulting solution was stirred for 24 h at room temperature. The solvent was removed under vacuum, and the residue was purified by silica gel chromatography with petroleum ether/ethyl acetate (v/v = 3:2) as eluent to afford a white solid (323 mg, 90%).1H NMR (400 MHz, CDCl3, ppm): δ 8.11 (d, 1H), 8.01 (d, 1H), 7.64 (m, 3H,), 7.36 (d, 2H), 6.50 (d, 2H), 3.30 (m, 2H), 3.02 (br, 2H), 1.49 (m, 2H), 1.38 (s, 11H), 1.26 (m, 4H). 13C NMR (75 MHz, CDCl3, ppm): δ 168.3, 165.3, 156.3, 153.2, 150.7, 141.7, 133.3, 129.1, 127.4, 126.4,125.5, 122.7, 118.0, 95.8, 81.4, 79.2, 39.6, 30.1, 28.9, 28.4, 25.7, 25.4. HRMS (ESI; m/z, [M + H]+): calcd, 795.0423, found, 795.0423. Synthesis of Compound 8. Compound 7 (158.8 mg, 0.2 mmol), bis(pinacolato)diboron (203 mg, 0.8 mmol), potassium acetate (117.6 mg, 1.22 mmol), and Pd(dppf)Cl2 (163 mg, 0.2 mmol) were dried under vacuum overnight in a 25 mL flask before use. Then 8 mL of fresh dioxane were added, and the resulting solution was heated at 50 °C for 2 h under a nitrogen atmosphere. After cooling to room temperature,

water (20 mL) was added and the mixture was extracted with dichloromethane (3 × 60 mL). The combined organic layer was dried over anhydrous MgSO4 for 30 min. The solvent was removed under vacuum, and the residue was purified by silica gel chromatography with CH2Cl2 as eluent to afford yellow solid (70 mg, 41%). 1H NMR (400 MHz, CDCl3, ppm): δ 8.07 (s, 2H), 7.69 (s, 2H), 7.55 (s, 1H), 7.38 (d, 2H), 6.52 (d, 2H), 3.37 (m, 2H), 3.08 (m, 2H), 1.58 (m, 2H), 1.44 (m, 2H), 1.39 (s, 9H), 1.32 (m, 4H), 1.26 (s, 24H). 13C NMR (100 MHz, CDCl3, ppm): δ 168.4, 165.5, 156.4, 153.2, 150.8, 141.8, 133.5, 129.3, 129.2, 127.9, 127.6, 126.5, 125.6, 122.8, 118.2, 95.9, 83.0, 81.6, 39.8, 30.1, 29.1, 28.5, 25.8, 25.5, 24.6. HRMS (MALDI-TOF; m/z, [M + H]+): calcd, 795.4121; found, 795.4190. Synthesis of Compound 9. To a solution of compound 8 in 5 mL of methanol was added 5 mL of trifluoroacetic acid (TFA) and then stirred at room temperature overnight. The solvent was removed under reduced pressure. The solid was re-dissolved in 5 mL of methanol, and 1 mL of triethylamine was added. The resulting solution was stirred overnight. After being washed with water, the solvent was removed to afford a yellow liquid. The liquid was washed with water. 1H NMR (400 MHz, MeOD, ppm): δ 8.1 (t, 2H), 7.69 (s, 2H), 7.55 (s, 1H), 7.41 (d, 2H), 6.84 (d, 2H), 3.25 (t, 2H), 2.85 (t, 2H), 1.58 (br, 2H), 1.51 (br, 2H), 1.33 (s, 24H), 1.25 (br, 4H). 13C NMR (100 MHz, MeOD, ppm): δ 170.1, 167.8, 155.5, 151.7, 142.8, 132.3, 130.7, 129.0, 128.3, 126.4 124.4, 123.7, 122.1, 118.4, 85.6, 83.4, 75.9, 40.9, 40.5, 29.9, 28.3, 27.3, 26.9, 25.2. HRMS (MALDI-TOF; m/z, [M + H]+): calcd, 695.3670; found, 695.3671. Preparation of Polymer PFP-FB. To a solution of compound 9 (9.3 mg, 13.4 nmol) and polymer 4 (10 mg, 6.7 nmol) in CH2Cl2 (4 mL) was added NHS (1.5 mg, 13.4 nmol) and EDCI (2.6 mg, 13.4 nmol). The resulting solution was stirred for 24 h at room temperature. Then the crude product was added into 8 mL of acetone. The precipitate was collected by centrifugation to obtain a brown solid (3.5 mg, 20%). 1H NMR (300 MHz, MeOD, ppm): δ 7.87−7.58 (br, 10H), 3.7 (br, 4H), 3.59 (br, 72H), 3.45 (br, 4H), 3.30 (br, 4H), 2.53 (t, 4H), 1.98 (br, 8H), 1.39 (br, 4H), 1.20 (br, 4H), 0.75−0.66 (br, 4H). GPC: Mw = 14470; Mn = 11920, PDI = 1.21. Assays for H2O2. H2O2 was added to a solution of PFP-FB in phosphate buffer (pH = 7.4) at room temperature ([H2O2] = 3 mM). After incubating for 30 min, the fluorescence spectra were measured with an excitation wavelength of 380 nm. Assays for Choline. To a solution of choline in phosphate buffer (pH = 7.4), PFP-FB and choline oxidase (ChOx) were added ([PFPFB] = 4.6 μM in RUs and [ChOx] = 0.84 U·mL−1). After incubation for 5 h at 37 °C, the fluorescence spectra were measured with an excitation wavelength of 380 nm. PFP-FB and ChOx were separately added to a solution of dopamine (DA), glucose (Glu), and aspartic acid (AA) in phosphate buffer (pH = 7.4) ([PFP-FB] = 4.6 μM in RUs and [ChOx] = 0.84 U·mL−1). After incubation for 5 h at 37 °C, the fluorescence spectra were measured with an excitation wavelength of 380 nm. Assays for Acetylcholine. To a solution of acetylcholine in phosphate buffer (pH = 7.4), PFP-FB, AChE, and ChOx were added ([PFP-FB] = 4.6 μM in RUs, [AChE] = 0.26 U·mL−1, and [ChOx] = 0.84 U·mL−1). After incubation for 5 h at 37 °C, the fluorescence spectra were measured with an excitation wavelength of 380 nm. G

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ACS Applied Materials & Interfaces Cell Culture. HeLa and A549 cells were cultured in DMEM supplemented with 10% fetal bovine serum at 37 °C in a humidified incubator containing 5% CO2. NCI-H82 and Jurkat T cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum at 37 °C in a humidified environment with 5% CO2. Assay for Choline Consumption of Cells. Jurkat T and NCI-82 cells were seeded in 48-well plates at a density of 4 × 105 cells/mL. After incubation for 1, 4, 8, 12, 24, 48, and 72 h, PFP-FB and ChOx were added to the medium ([PFP-FB] = 4.6 μM in RUs and [ChOx] = 0.84 U·mL−1). After incubation for 5 h at 37 °C, the corresponding fluorescence spectra were measured with an excitation wavelength of 380 nm. HeLa and A549 were seeded in 48-well plates at a density of 2 × 105 cells/mL. After incubation for 0.5, 2, 8, 11, 24, and 48 h, PFP-FB and ChOx were added to the medium ([PFP-FB] = 4.6 μM in RUs and [ChOx] = 0.84 U·mL−1). After incubation for 4 h at 30 °C and 180 rpm, the corresponding fluorescence spectra were measured with an excitation wavelength of 380 nm.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.5b07172. Figures S1−S9 showing FRET ratios and NMR and HRMS spectra (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (S.W.). *E-mail: [email protected] (L.L.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work described herein was supported by the National Natural Science Foundation of China (Grant Nos. 21003140 and 21373243) and the Major Research Plan of China (Grant Nos. 2011CB932302 and 2011CB808400).

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REFERENCES

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DOI: 10.1021/acsami.5b07172 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

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DOI: 10.1021/acsami.5b07172 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX