Aggregation-Induced Energy Transfer of Conjugated Polymer

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Aggregation-Induced Energy Transfer of Conjugated Polymer Materials for ATP Sensing Qianling Cui, Yu Yang, Chuang Yao, Ronghua Liu, and Lidong Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b12525 • Publication Date (Web): 01 Dec 2016 Downloaded from http://pubs.acs.org on December 4, 2016

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ACS Applied Materials & Interfaces

Aggregation-Induced Energy Transfer of Conjugated Polymer Materials for ATP Sensing Qianling Cui,#, † Yu Yang, #, † Chuang Yao,‡ Ronghua Liu,† and Lidong Li*,† † State Key Lab for Advanced Metals and Materials, School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing 100083, China ‡ College of Mechanical and Electrical Engineering, Yangtze Normal University, Chongqing 408100, China

Keywords: fluorescence, water-soluble conjugated polymer, FRET, fluorescence enhancement, ATP sensing Abstract Water-soluble conjugated polymers are attractive fluorescent materials for applications in chemical and biological sensing. The molecular wire effect of such polymers amplifies changes in the fluorescence signal, which can be used for detecting various analytes with high sensitivity. In this work, we report an efficient ratiometric fluorescent probe based on a water-soluble conjugated polymer, which showed high sensitivity and selectivity towards adenosine 5′-triphosphate (ATP). The macromolecular probe consisted of a polyfluorene backbone doped with 5 mol% 1,4-dithienylbenzothiadiazole

(DBT),

modified

by

bis-imidazolium

and

oligo(ethylene glycol) moieties. Solutions of the polymer emitted purple fluorescence, which changed to red upon addition of ATP molecules. The addition of ATP caused the polymer to aggregate, which enhanced fluorescence resonance energy transfer 1

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efficiency from the fluorene segments to DBT units, leading to an increase in red emission. The ratio of the fluorescence at these different wavelengths (I655/I423) showed a strong dependence on the ATP concentration. PF-DBT-BIMEG also exhibited high selectivity for ATP sensing over other representative anions and discriminated

it

from

adenosine

5′-diphosphate

(ADP)

and

adenosine

5′-monophosphate (AMP). This can be explained by the much stronger electrostatic interactions between the polymer and ATP than the interactions between the polymer and ADP or AMP, as confirmed through molecular dynamics simulations.

Introduction In recent years, water-soluble conjugated polymers (CPs) have drawn attention because of their potential applications in chemical and biological sensing, bioimaging, drug delivery, diagnosis, and therapeutics.1-8 CPs possess many attractive photophysical properties, such as strong and broad light absorption, good photostability, and tunable absorption-emission spectra. Electrons delocalize along the π-conjugated backbones of CPs and can amplify changes in fluorescent signals, which is especially valuable for constructing sensitive chemo- or biosensors. Detection mechanisms can be classified into two categories: measuring changes in fluorescence intensity at a single wavelength9-12; and measuring the change of a ratio of fluorescence at two wavelengths (ratiometric fluorescence).13-22 Ratiometric fluorescence detection is based on fluorescence resonance energy transfer (FRET) between an energy donor and an acceptor. This interaction is strongly dependent on 2


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the distance between the energy donor and acceptor, and can be altered by analyte interactions leading to variation in the FRET efficiency and corresponding signal output. Compared with measuring fluorescence intensity at a single wavelength, ratiometric methods have the potential for colorimetric responses that can be seen by the naked eye and signals that are affected less by environmental interference. Adenosine-5′-triphosphate (ATP) is an important biomolecule, which acts as an energy source for many cellular events.23-24 Detection of ATP is an important target for investigations of biological processes. To this end, some fluorescent chemosensors based on zinc complexes have been designed and used for ATP detection under biological pH conditions.25-28 However, the single recognition site in these fluorescent probes limits their selectivity to ATP compared with that of other phosphate anions. Taking advantage of the specific recognition, some aptamer-based fluorescence sensing assays have been developed for the selective detection of ATP.29-32 Nevertheless, to design and synthesize fluorescent probe for ATP sensing with high sensitivity and selectivity is still urgently required. Because of the unique π-delocalized backbone and molecular wire amplifying effect, CPs is an attractive candidate for constructing the ATP sensing platform. A few examples of CPs as ATP sensors have been reported, based on electrostatic interactions between positively charged quaternary ammonium and phosphatase.33 These water-soluble CPs have shown promise for applications in ATP detection, especially in biological ATP sensing and imaging. Nevertheless, the electrostatic attraction of the quaternary ammonium was non-specific and undesirable 3



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self-aggregation occurred. This was caused by the CP’s limited water solubility and such issues have limited the practical application of CPs. Recently, the imidazolium group has been reported to be an efficient recognition site for ATP, which can also discriminate between tri-, di-, and monophosphate groups.34-35 This group has been identified as a candidate recognition structure for ATP sensing and imaging. Combining the advantages of CPs and this ATP recognition site might allow more efficient ATP sensing. In this work, we designed a water-soluble conjugated polymer containing imidazolium side chains, which acts as a highly efficient ratiometric probe for ATP detection based on an aggregation-enhanced FRET mechanism. Its chemical structure is shown in Scheme 1. A polyfluorene (PF) backbone was doped with 5 mol% of 1,4-dithienylbenzothiadiazole (DBT), a low-bandgap fluorophore, to provide a pair of fluorescent signals. Two positively charged imidazolium units were introduced onto the side chains, and acted as ATP binding sites. Nonspecific interactions typically occur between positive polyelectrolytes and negative species. This causes the intrinsically hydrophobic backbone to aggregate upon interaction with hydrophobic substrates, reducing fluorescent quantum yields. To resolve this problem, oligo(ethylene glycol) (EG) moieties were used to enhance the water solubility, and reduce nonspecific interactions and undesirable aggregation. Because of the strong hydrophilicity of the di(1H-imidazol-1-yl)methane dication (BIM) and EG moieties, the CP PF-DBT-BIMEG dissolved well in aqueous solution. The low doping ratio of DBT and inefficient intra-molecular FRET from the fluorene segments to DBT caused 4


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solutions of PF-DBT-BIMEG to emit purple fluorescence upon excitation, derived mainly from emission of the PF backbone. When PF-DBT-BIMEG interacted with ATP molecules, PF-DBT-BIMEG/ATP complex were formed via electrostatic attractions induced by polymer aggregation. This effect reduced the spatial distance between the donor and acceptor components, which in turn enhanced the intra- or/and inter-chain FRET efficiency. The fluorescence color of the mixture changed from purple to red, and the fluorescence ratio (I655/I423) could be used to calculate the ATP concentration. The interaction between PF-DBT-BIMEG and ATP was also explored by molecular dynamics simulations. Experimental Section Materials. PF-DBT-BIMEG was synthesized as reported in our previous work.36 The sodium salt of ATP, the adenosine 5′-diphosphate (ADP) and adenosine 5′-monophosphate

(AMP)

sodium

salts,

and

2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) were bought from Sigma-Aldrich. Ultrapure Millipore water (18.6 MΩ·cm) was used throughout the experiments. Preparation of PF-DBT-BIMEG Solution and ATP Sensing. An aqueous stock solution of PF-DBT-BIMEG (1 mg/mL) was prepared by dissolving the solid in water. This solution was stored in the dark at 4 °C before use. Aqueous solutions of the ATP sodium salt were prepared freshly and used immediately. A 2-µL portion of the PF-DBT-BIMEG stock solution (1 mg/mL) was added to 10 mM HEPES buffer (pH = 7.4) in a 1 cm × 1 cm cuvette. Aliquots of ATP solution were then added to reach the 5



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final target concentration, while maintaining the total volume of the sample at 1 mL. After mixing, the fluorescence spectra of the aqueous solutions were measured. The excitation wavelength was set at 380 nm, and the emission spectra were monitored from 400 to 750 nm. The intensity ratio at 655 nm and 423 nm (I655/I423) was used to calculate the ATP concentration. Preparation of PF-DBT-BIMEG/ATP Nanoparticles. For preparation of the fluorescent nanoparticles 1 µL of fresh ATP solution (1 mM or 0.01 M) was added into 1 mL of an aqueous solution of PF-DBT-BIMEG (2 µg/mL) and mixed thoroughly. The mixture was dropped onto a cleaned silicon/glass wafer and then freeze-dried for use in subsequent measurements. Molecular Dynamics Simulation. To investigate the interaction between PF-DBT-BIMEG and the nucleoside phosphates in water, we simulated a system comprising a simplified PF-DBT-BIMEG molecule P1 (structure is illustrated in Scheme 2) and five molecules of ATP, ADP or AMP in water. We compared this system with a reference system without ATP, ADP or AMP. Ions of Na+ or Cl− were inserted randomly into the solvent to preserve electrical neutrality. The resulting ATP system had one P1, five ATP, 1655 water molecules, eight Cl− and 20 Na+ ions; the ADP system had one P1, five ADP, 1661 water molecules, eight Cl− and 15 Na+ ions the AMP system had one P1, five AMP, 1687 water molecules, eight Cl− and 10 Na+ ions; the reference system had one P1, 1759 water molecules and eight Cl− ions. Simulations were performed with the Gromacs 5.0.4 package.37 GROMOS 54A7 force field with the united atom convention for CH and CH2 groups was used and 6


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water was simulated by the single-point charged intermolecular potential model.38 The systems were evolved under NPT ensemble dynamics for 10 ns at 300 K. The gmx energy program was used to calculate the short-range Lennard–Jones interaction energies E(LJ-SR) and Coulomb interaction energies E(Coul-SR) between P1 and ATP, P1 and ADP, P1 and AMP, or between P1 and water. Characterization. The hydrodynamic diameters of the particles were determined by a Nano ZS90 Zeta-sizer (Malvern Instruments Ltd., UK). Ultraviolet-visible (UV-vis) absorption spectra were measured on a Hitachi U3900 spectrophotometer. Fluorescence spectra of the samples were recorded by a Hitachi F-7000 spectrometer equipped with a Xe lamp as the light source. The slit widths for excitation and emission, and photomultiplier tube voltage were 5 nm and 700 V, respectively. The emission spectra were recorded in the range 400–750 nm with excitation at 380 nm. The morphology of the nanoparticles was observed with a high-resolution field emission scanning electron microscope (HR-SEM, JEOL JSM-7401F) operating at an accelerating voltage of 3.0 kV. Samples for SEM observation were prepared by adding a drop of a freshly prepared mixture of PF-DBT-BIMEG and ATP onto a clean silicon wafer and then freeze drying. Photographs of the solutions were captured by a Nikon D-7000 camera under a hand-held UV lamp with 365 nm excitation. Fluorescence microscopy images of the CP nanoparticles were recorded using an Olympus FV1000-IX81 confocal laser scanning microscope through a 100× objective, with 330–380 nm excitation produced by a 100 W mercury lamp light source.

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Results and Discussion The synthesis and characterization of the water-soluble CP used in this work, PF-DBT-BIMEG, has been described previously.32 The chemical structure of the polymer is shown in Scheme 1. The PF backbone of the polymer is a commonly used donor owing to its large energy gap, which gives a bright blue fluorescence.39 The low-energy gap fluorophore DBT was selected as the energy acceptor and doped into the main chain at a 5% molar ratio.40 The polymerization degree of the PF-DBT backbone was calculated to be around 32 based on the molecular weight of the polymer precursor, and the DBT ratio was calculated to be near 5% as reported in previous work.36 The PF-DBT backbone has been shown to behave as a main chain that displays intra-chain FRET.40-42 The BIM group, which acts as the ATP binding site, was covalently linked to the fluorene side chains and EG moieties were added to further increase the water solubility and reduce nonspecific interactions. The nearly complete substituation of BIMEG groups on the side chains was proved by the N/C ratio of the PF-DBT-BIMEG.36 The obtained PF-DBT-BIMEG was soluble in water and polar organic solvents such as dimethyl sulfoxide (DMSO).

Scheme 1. Chemical structure of fluorescent probe PF-DBT-BIMEG and schematic 8


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illustration of the ATP detection mechanism based on aggregation-enhanced intra- and inter-molecular FRET. Efficient FRET only takes place at short distances (less than 10 nm) between the acceptor-donor pair. The emission band of the donor should also overlap well with the absorption band of the acceptor.43 It has also been reported that inter-chain FRET is much more efficient than intra-chain FRET because of the increased transfer dimensionality and stronger electronic coupling.36 To study the optical properties of PF-DBT-BIMEG we measured its absorption and emission spectra in water and DMSO. Figure 1a shows that the shapes of the absorption spectra in the different solvents were similar, exhibiting an absorption maximum around 400 nm and a broad absorption band from 450 to 600 nm, which are assigned to the PF backbone and doped DBT units, respectively. The corresponding fluorescence spectra of PF-DBT-BIMEG were also measured in these solvents under excitation at 380 nm (Figure 1b). Both spectra were dominated by emission from the donor PF segments centered at 423 nm. The overlap between the emission band of PF and the absorption band of DBT indicates the possibility of FRET from PF to DBT. Weak emission from DBT in the range 600–700 nm was observed, indicating low FRET efficiency. This was attributed to the relatively low doping ratio of the DBT and the good dissolution of the polymer chains. Excitation spectra measuring the emission at 655 nm showed an excitation peak around 380 nm. This peak overlapped with a similar peak in the excitation spectra measuring the emission at 420 nm, indicating efficient FRET from PF to DBT (Figure 1c). Furthermore, DMSO is known to be a good solvent for 9



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charged conjugated polymers. The emission spectrum of PF-DBT-BIMEG in H2O showed only a slight increase in DBT emission compared with that in DMSO indicating that the polymer was well dispersed in both H2O and DMSO. We used dynamic light scattering (DLS) to further study the molecular state of PF-DBT-BIMEG in H2O. The DLS results in Figure 1d revealed a single narrow distribution with an average size less than 1.0 nm, corresponding to the hydrodynamic diameter of individual polymer chain. This implies that PF-DBT-BIMEG was dispersed as individual chains in water, owing to the strong hydrophilicity of the BIMEG side chains and inter-chain charge repulsion. The well-dispersed nature of PF-DBT-BIMEG in water extended the distance between the donor fluorene segments and acceptor DBT units leading to inefficient FRET. Thus, under UV light irradiation, the fluorescence of the PF-DBT-BIMEG solution appeared purple from the combination of blue emission from PF and weak red emission from DBT.

Figure 1. Normalized absorption (a) and fluorescence spectra (b) of PF-DBT-BIMEG 10


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in H2O and DMSO excited at 380 nm. (c) Fluorescence excitation spectra of PF-DBT-BIMEG in H2O with emission at 420 nm (black curve) and 655 nm (red curve), respectively. (d) Hydrodynamic diameter of PF-DBT-BIMEG in H2O determined by DLS. For chemical and biological sensing applications a good fluorescent probe requires a fluorescence ratio that is stable to environmental variations and changes in the overall fluorescence intensity. Emission spectra of PF-DBT-BIMEG were recorded at different concentrations and pH values to evaluate the ratiometric stability of the fluorescence signals. Figure 2a shows the fluorescence spectra of different concentrations of PF-DBT-BIMEG (1–15 µg/mL) measured in 10 mM HEPES buffer (pH=7.4). Figure 2b shows the corresponding variation in the I655/I423 emission ratio. As the polymer concentration increased, the emission intensity increased drastically; however, only a slight increase was observed for the I655/I423 emission ratio. This indicates that the PF-DBT-BIMEG I655/I423 emission ratio was not strongly affected by the polymer concentration over the tested range. The effects of solution pH on the fluorescence spectra and the I655/I423 emission ratio of PF-DBT-BIMEG are shown in Figure 2c. It showed no obvious changes in the fluorescence intensity as the solution pH was increased from 4 to 8, and the I655/I423 emission ratio also remained stable. Furthermore, the effect of temperature on the emission spectra of the polymer was also determined in the temperature range from 5 °C to 60 °C, as displayed in Figure 2d. The rise in temperature from 5 °C to 60 °C only led to 10% reduction in fluorescence intensity of PF emission at 423 nm, while the I655/I423 ratio still kept 11



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constant during the temperature range. Thus, the photophysical properties of the PF-DBT-BIMEG remained stable over a relatively wide concentration, pH or temperature range, which might be a useful feature for detection of ATP, especially for potential application in complicated intracellular environment.

Figure 2. (a) Fluorescence spectra of PF-DBT-BIMEG at different concentrations in 10 mM HEPES buffer (pH = 7.4). (b) Variation of I655/I423 emission ratio with polymer concentration. (c) Fluorescence spectra of PF-DBT-BIMEG (2 µg/mL) at different pH values in 10 mM HEPES buffer. Inset is the variation of the I655/I423 emission ratio with solution pH. (d) Fluorescence spectra of PF-DBT-BIMEG (2 µg/mL) at temperature of 5 °C, 35 °C, and 60 °C. Inset is the variation of the I655/I423 emission ratio in the temperature range of 5–60 °C. The chemical structure of PF-DBT-BIMEG features nearly four positive charges on each repeating unit and this high charge density allows the polymer to combine with species with opposing charges. ATP is composed of adenine, pentose sugar and three 12


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negatively charged phosphate groups. In aqueous solution, a strong electrostatic attraction is expected between the bis-imidazolium groups of the polymer and phosphate groups of ATP, accompanied by a hydrophobic interaction between the adenine base and the hydrophobic polymer backbone. The formation of this complex reduces the distance between the donor PF and acceptor DBT units leading to enhanced inter-chain FRET efficiency and changes in the fluorescence signals. To test if addition of ATP could affect the fluorescence of the PF-DBT-BIMEG solution we photographed the fluorescing solutions under irradiation by a hand-held UV lamp before and after addition of ATP. In Figure 3a, in the absence of ATP, the PF-DBT-BIMEG solution appeared purple. After addition of ATP to a final concentration of 1 mM, the fluorescence of the solution clearly changed to red. Notably the fluorescence changed immediately after ATP addition and no incubation time was required. Furthermore, no obvious precipitation occurred during the test indicating the formation of fully soluble complexes in the mixture. We investigated the changes in the photophysical properties by measuring emission spectra. The corresponding emission spectra are shown in Figure 3b. A large difference was observed in the fluorescence spectra after the addition of ATP. The intensity of the emission peak around 423 nm decreased considerably, while that of the band centered at 655 nm increased. Accordingly, the I655/I423 emission ratio increased from 0.06 to 5.40, representing a 90-fold increase, demonstrating an enormous enhancement in the FRET efficiency. We then determined the fluorescence response of PF-DBT-BIMEG in the presence 13



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of ATP at different concentrations, as shown in Figure 3c. These results clearly show that the PF emission band around 423 nm was reduced as the ATP concentration increased, while the band at 655 nm increased slightly. The overall fluorescence intensity diminished gradually, which is attributed to aggregation-induced self-quenching, resulting from π-stacking between the main chains of the conjugated polymer in the tight aggregates.44 The reduced emission intensity was obvious even at ATP concentrations as low as 1×10−10 M, which can be attributed to amplified quenching phenomenon and the molecular wire effect of the conjugated polymer.45 However, the I655/I423 emission ratio increased rapidly as the ATP concentration was increased. Figure 3d showed that the I655/I423 emission ratio displayed a good linear relationship within the ATP concentration range of 30–70 µM. This relationship was fitted by a linear function of the form I655/I423 = 3.195 + 0.015 (C/µM) with a correlation coefficient of R = 0.9909. Thus, the ATP concentration in samples could be quantitatively determined from the fluorescence intensity ratio. Notably, the limits of detection were at the level of 0.1 nM, which is much lower than previously reported values for ATP detection based on zinc complexes.26-28 This observation confirms the high sensitivity of PF-DBT-BIMEG as a sensing agent for ATP through fluorescence ratiometric analysis.

14


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Figure 3. (a) Photograph of PF-DBT-BIMEG solutions in the absence and presence of ATP (1×10−4 M) under irradiation by a hand-held UV lamp. (b) Fluorescence spectra of PF-DBT-BIMEG solutions in the absence and presence of ATP (1×10−6 M) excited at 380 nm. (c) Fluorescence spectra of PF-DBT-BIMEG solutions with different ATP concentrations. (d) Linear relationship between the fluorescence intensity ratio (I655/I423) and ATP concentration in the range 30–70 µM. We assumed that stable complexes formed as a result of the interactions between PF-DBT-BIMEG and ATP; however, no macroscopic precipitation was observed. We used a scanning electron microscope (SEM) and dynamic light scattering (DLS) to study the morphologies of aggregates. Here, we choose two samples as the representative

examples;

aggregates

formed

from

a

dilute

solution

of

PF-DBT-BIMEG (2 µg/mL) with ATP concentrations of 1×10−5 and 1×10−3 M. When the ATP concentration was low (1×10−5 M), the SEM image (Figure 4a) revealed quasi-spherical nanoparticles with uniform size around 100 nm. As the ATP 15



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concentration increased, larger particles formed, as shown in Figure 4c. The aggregate size distributions were shown to be monodisperse by DLS results, which indicated average diameters of 130 and 300 nm, for the 1×10−5 and 1×10−3 M aggregates, respectively. These measurements agreed with results of the SEM images.

Figure 4. SEM image (a) and DLS results (b) for aggregates formed from PF-DBT-BIMEG solution (2 µg/mL) with an ATP concentration of 1×10−5 M. SEM image (c) and DLS results (d) of aggregates formed from PF-DBT-BIMEG solution (2 µg/mL) with an ATP concentration of 1×10−3 M. The aggregates formed by PF-DBT-BIMEG and ATP were imaged by fluorescence microscopy under irradiation with UV and green light (Figure 5b and 5d, respectively) from a mercury lamp. Under UV light excitation, the nanoparticles emitted bright purple fluorescence (Figure 5a and 5c), consistent with the results above. When a green light source was used a pure red signal was observed, which was assigned to 16


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emission of aggregated DBT segments. Although the fluorescent dots in the microscope images do not reflect the actual size of the nanoparticles, the particles formed at a higher ATP concentration appeared to have larger average sizes. Notably, the nanoparticles with higher ATP concentration (Figure 5c, d) show obviously a higher brightness than that of nanoparticles in Figure 5a and b. This might be explained by the compact structures of the polymer/ATP complex with higher ATP contents. These results indicate the possibility of using these polymer/ATP complexes as fluorescent nanoparticles with bright fluorescence and dual-channel emission. The photophysical properties and stability of these nanoparticles might have potential applications as fluorescent nanoparticles in chemical or biological fields.46

Figure 5. Fluorescence microscopy images of aggregates formed by PF-DBT-BIMEG solution (2 µg/mL) at ATP concentrations of 1×10−5 M (a) (b) and 1×10−3 M (c) (d), irradiated by UV (a) (c) and green light (b) (d), respectively. The aggregation-induced purple-to-red fluorescence change of PF-DBT-BIMEG 17



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shows potential for use as a ratiometric fluorescent probe for ATP sensing. ADP and AMP have similar structures to ATP, but contain one and two phosphate groups, respectively. These compounds can interact with PF-DBT-BIMEG in a similar way to ATP and potentially interfere with ATP sensing. To evaluate the selectivity of this polymeric probe, fluorescence sensing experiments were also performed with ADP and AMP under the same experimental conditions used to study ATP. Figure 6a displays the emission spectra of PF-DBT-BIMEG solutions in the presence of 100 µM of ATP, ADP, or AMP. These spectra were normalized to the emission value at 655 nm to allow a comparison of the relative emission intensity of the 423-nm band. The emission at 423 nm noticeably reduced with the number of phosphate groups on the analyte molecules (i.e., ATP > ADP > AMP). This implies that the higher charge density of the analyte increased inter-chain FRET efficiency. The I655/I423 emission ratio was plotted against the logarithmic concentrations of the three analytes over a wide concentration range from 1 µM to 5000 µM, as shown in Figure 6b. The logarithmic concentration was used as the X axis for convenient comparation in such a wide concentration range. In the range 1-100 µM, the ATP curve showed a rapid increase as the concentration increased, while the emission ratio for both ADP and AMP almost displayed no obvious change, indicating a high selectively for ATP sensing in this concentration range. This can be explained by the fact that the lower charge density of ADP and AMP resulted in weaker interactions with the positively charged polymer probe. When the anlyate concentration increased over 100 µM, the ADP and AMP curve began to rise up slowly. However, the emission ratio of ATP was 18


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still 3-fold higher than that of ADP or AMP, which is a large enough difference to be able to discriminate these analytes from ATP.

Figure 6. (a) Fluorescence spectra of PF-DBT-BIMEG solution (2 µg/mL) in the presence of ATP, ADP or AMP (100 µM). (b) Plots of fluorescence intensity ratio (I655/I423) against ATP, ADP, and AMP logarithmic concentrations in the range 1–5000 µM. We studied the interaction between P1 and ATP, ADP or AMP in water using molecular dynamics simulations as a model to give insight into the selectivity of PF-DBT-BIMEG for ATP sensing over ADP and AMP. The 2-ns structure was selected for analysis because the interaction energy E(Coul-SR) stabilized from this 19



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time onwards. The interaction energies E(LJ-SR) and E(Coul–SR) for the systems are shown in Table 1. In the systems with P1 and ATP/ADP/AMP the average values of E(LJ-SR) and E(Coul–SR) over the last 2 ns were, respectively, 393 and −1927 kJ/mol for ATP, −376 and −1439 kJ/mol for ADP, and −278 and −1479 kJ/mol for AMP. We noticed that the Coulomb interaction energy was much lower than the Lennard-Jones energy in these systems, which indicated that the interactions of P1 and the different phosphates were dominated by electrostatic interactions. The total interaction energy (E(Coul-SR)+E(LJ-SR)) in the ATP system (−2320 kJ/mol) was much lower than those in the ADP and AMP systems (−1815

and −1757 kJ/mol,

respectively). This suggests that the interaction between P1 and ATP was much stronger than those between P1 and ADP or P1 and AMP. This model is consistent with the higher selectivity of P1 for ATP sensing than ADP and AMP. Compared with the reference system (P1/solvent), the total interaction energy (E(Coul–SR)+E(LJ-SR)) of −1719 kJ/mol was much higher than those of the systems with ATP/ADP/AMP (−613, −916, and −657 kJ/mol, respectively). This suggests that the P1-solvent interactions in the systems with ATP/ADP/AMP were much weaker than those in the reference system and indicates reduced water solubility of P1 upon binding to ATP, ADP, and AMP.47 These calculations show that the higher selectivity of P1 for ATP than for ADP and AMP can be attributed to stronger interactions in the P1 and ATP system compared with the P1 and ADP/AMP systems. These findings support our experimental results which showed stable aggregate formation from electrostatic interactions between PF-DBT-BIMEG and ATP. 20


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Scheme 2. Simplified chemical structures of PF-DBT-BIMEG (P1, a), ATP (b), ADP (c) and AMP (d), as used in molecular dynamics simulations.

Table 1. Average interaction energies between P1 and ATP/ADP/AMP, and between P1 and solvent in the reference system (kJ/mol). P1-ATP/ADP/AMP E(Coul

Control system-ATP Control system-ADP Control system-AMP Reference system

E(LJ-S

P1-SOL

Total

E(Coul -SR)

E(LJ-S

Total

-SR)

R)

R)

-1927

-393

-2320

-59

-554

-613

-1439

-376

-1815

-346

-570

-916

-1479

-278

-1757

-103

-554

-657

0

0

0

-1055

-664

-1719

We confirmed the high selectivity of PF-DBT-BIMEG for ATP sensing compared with ADP and AMP; however, other anions could also complex with the probe through electrostatic attractions. To investigate potential interference effects, we selected eleven commonly used anions and studied their influence on the fluorescence responses of PF-DBT-BIMEG in the presence and absence of ATP. It is reported that the average concentration of ATP in mammalian cells is 1-4 mM.24 Thus, here the concentrations of all the analytes were set as 1 mM to illustrate their potential

21



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interference to ATP sensing in real application. Figure 7 shows that these anions only caused slight fluctuations in the fluorescence ratio of PF-DBT-BIMEG in the absence of ATP. This indicates that these anions cannot induce aggregation of PF-DBT-BIMEG even at a high concentration (1 mM). After addition of ATP, a substantial increase of the ratiometric value was observed, which was comparable to the response of solutions with only ATP added. These results indicate that interference effects of these anions are negligible and we conclude that ATP sensing with PF-DBT-BIMEG in aqueous solution is unaffected by common anions.

Figure 7. Fluorescence response of PF-DBT-BIMEG (2 µΜ) to various ions at 1 mM in the absence and presence of ATP (1 mM) at pH 7.4 (10 mM HEPES). 1) H2O 2) Cl−, 3) Br−, 4) I−, 5) CO32−, 6) HCO3−, 7) NO3−, 8) SO42−, 9) HSO3−, 10) PO43−, 11) HPO42−, 12) H2PO4−.

Conclusions In summary, a water-soluble conjugated polymer modified with imidazolium and oligo(ethylene glycol) side chains, shows excellent potential as a ratiometric probe for 22


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ATP detection based on aggregation enhanced FRET. The PF-DBT-BIMEG probe dissolved well in aqueous solutions and emitted purple fluorescence under excitation at 380 nm. This suggested that intra-chain FRET from PF segments to DBT was inefficient in the free polymer. When ATP, containing three phosphate groups, was introduced, strong electrostatic attractions between the PF-DBT-BIMEG and the ATP induced formation of complexes. This brought the donor and acceptor into close proximity and enhanced the FRET efficiency, resulting in a change of the fluorescence color from purple to red. The I655/I423 emission ratio was successfully used to evaluate ATP concentration with limits of detection at the level of 0.1 nM. In addition, this polymeric probe also showed a high selectivity towards ATP over other common anions and species with similar structures including ADP and AMP. Molecular dynamics simulations further confirmed that the interactions of the polymer and nucleoside phosphates were dominated by electrostatic interactions. Thus, we show that PF-DBT-BIMEG is an excellent ratiometric probe for ATP sensing with high sensitivity and selectivity.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (L.L.) Author Contributions †Q.C. and Y.Y. contributed equally to this study. Notes The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (51503015, 51373022), the State Key Laboratory for Advanced Metals and Materials (2016Z-08) and the Fundamental Research Funds for the Central Universities (FRF-TP-15-003A1).

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Aggregation-Induced Energy Transfer of Conjugated Polymer

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