Ion-Induced Aggregation of Conjugated Polyelectrolytes Studied by

Article pubs.acs.org/JPCB

Ion-Induced Aggregation of Conjugated Polyelectrolytes Studied by Fluorescence Correlation Spectroscopy Jie Yang,§ Danlu Wu,§ Dongping Xie, Fude Feng, and Kirk S. Schanze* Department of Chemistry and Center for Macromolecular Science and Engineering, University of Florida, P.O. Box 117200, Gainesville, Florida 32611-7200, United States S Supporting Information *

ABSTRACT: Fluorescence correlation spectroscopy (FCS) is applied to investigate aggregation behavior of an anionic conjugated polyelectrolyte (CPE) featuring branched carboxylate groups (PPE-dCO2) upon the addition of different metal ions. FCS results reveal that monovalent metal ions (Na+, K+) bind to the CPE chain but do not induce aggregation, and divalent metal ions (Ca2+, Cu2+) bind and induce the formation of small aggregates. Iron cations (Fe2+ and Fe3+) bind strongly and induce the formation of large CPE aggregates. This is believed to be because of the propensity of these metals to bind to six ligands and thus bridge two of the triacid units on adjacent PPE-dCO2 chains. A comparison between the Stern−Volmer (SV) fluorescence quenching and the relative diffusion time change from FCS demonstrates that the most efficient SV quenching is observed for the ions that give rise to large increases in FCS diffusion time, underscoring the importance of ion-induced aggregation in the amplified quenching effect.

■

in the early 1970s by Madge, Elson, and Webb,18 which monitors the spontaneous fluctuations of fluorescent intensity of diffusing molecules within a small excitation volume (∼femtoliter). Various processes including Brownian diffusion, chemical reaction, or flow contribute to the fluorescence intensity fluctuation. By applying an autocorrelation function G(τ), the raw fluctuation data is converted to a decay curve, which can be analyzed using an appropriate fitting model. The important dynamic and kinetic information for a particular molecule or particle can be obtained and related to the chemical reaction, physical interaction, or change in chemical environment. FCS has been primarily employed for analysis of biological systems,19−21 but its application to polymers, in particular CPEs, has gained increasing interest throughout the past decade due to the high sensitivity and the capability of single molecule analysis.22,23 Because of their inherent fluorescence, CPEs can be observed directly, avoiding the tedious dyelabeling process and simplifying studies of conformational or diffusional changes. For example, Jayakannan and co-workers used FCS for a systematic study of the influence of chain length and molecular weight distribution on the diffusion dynamics of a CPE at the single molecular or chain level.24 Cotlet and coworkers investigated the solvent polarity effect on chain conformation using FCS measurements,25 and by coupling

INTRODUCTION Conjugated polyelectrolytes (CPEs) are polymers composed of a conjugated backbone with numerous ionic side groups, making them both highly emissive and water-soluble. These attractive characteristics make CPEs a popular platform for a variety of applications,1,2 including light emitting diodes (LEDs),3−5 solar cells,6−8 and chemo- and biosensors.9,10 The addition of oppositely charged small ions into a CPE solution leads to significant changes in the photophysical properties.11,12 Early studies found that some metal ions, such as Pd2+ and Ca2+, exhibit a superquenching ability toward carboxylated poly(p-phenylene ethynylene)s (PPE) in aqueous solution.13−16 The sensitive photophysical response of conjugated polyelectrolytes has been ascribed to either the energy transport properties of the conjugated backbone, the so-called “molecular wire effect,” or a multivalency effect. In the latter case, the cations may be simultaneously bound by more than one carboxylate, belonging either to a single polymer chain or to adjacent polymer chains to form aggregates. Several ion sensors have been developed based on cation-induced CPE fluorescence change.13,17 Although fluorescence spectroscopic studies have fully elucidated CPE aggregation in the literature,13−16 direct evidence of polymer aggregation upon addition of various types and concentration of ions is still lacking. To better interpret the spectral changes of CPEs and to study aggregation, the research described herein makes use of a powerful techniquefluorescence correlation spectroscopy (FCS)a statistics-based analytical technique, first introduced © 2013 American Chemical Society

Received: August 21, 2013 Revised: November 17, 2013 Published: November 22, 2013 16314

dx.doi.org/10.1021/jp408370e | J. Phys. Chem. B 2013, 117, 16314−16324

The Journal of Physical Chemistry B

Article

Scheme 1. Structure of PPE-dCO2, PPE-dNH3 and PPi

amplified quenching mechanism in terms of aggregation/ physical interactions of CPE. The results demonstrate that FCS with 405 nm excitation has great potential for studies of the molecular conformational and diffusional behavior of CPEs.

FCS with a time-correlated single-photon counting (TCSPC), Masuo and co-workers determined the relationship between the probability of single photon emission and the spatial size of the CPE chains.26 Most of these applications utilized laser excitation with wavelength ≥460 nm. However, most of the commonly used CPEs have λmax at shorter wavelength. Consequently, an FCS system equipped with a 405 nm blue diode laser designed specifically for CPE detection was constructed as part of the work reported here. As far as we know, only a few papers focus on the use of FCS with a short wavelength laser, specifically, 405 nm.27,28 Even fewer technical data or details can be found in the literature for the construction of such an FCS system. In the current paper, we provide the details for construction and optimization of an FCS system coupled with a 405 nm diode laser (see Supporting Information). This FCS system was calibrated and applied to investigate the aggregation behavior of two CPEs, induced by oppositely charged ions, successfully extending the application of FCS to shorter wavelengths. The two CPEs (PPE-dCO2 and PPE-dNH3, Scheme 1) studied here have PPE backbones substituted with bulky, highly charged ionic side chains.29,30 Anionic PPE-dCO2 contains carboxylate groups as the side chains, while PPE-dNH3 is cationic with a branched polyamine as the functional group. The introduction of the branched ionic side chains to these polymers reduces the hydrophobic and π−π stacking interactions by increasing the electrostatic repulsion between adjacent polymers.31 In addition, the bulky charged functional groups on these polymers are also able to increase the solubility of the CPE in aqueous solutions.32 Therefore, each of the polymers exhibits similar optical properties in methanol and water, since they exist as free single polymer chains in these solvents. In this paper, the interactions of the CPEs with six metal cations (PPE- d CO 2 ) or pyrophosphate anions (PPE-dNH3, PPi, Scheme 1) were investigated by FCS to observe the relationship between the chain aggregation and fluorescence quenching. The FCS results indicate that addition of Fe2+, Fe3+, and Cu2+, all transition metal ions with high charge density, can readily induce aggregation of PPE-dCO2 chains. Similarly, addition of negatively charged PPi to the cationic PPE-dNH3 system also induces aggregate formation. These findings agree well with the results obtained by fluorescence titration experiments. We explain the fluorescence change by different quenching mechanisms and also explore the

■

EXPERIMENTAL PROCEDURES AND METHODS Theory of FCS. This section follows closely the classical paper reported by Haustein and Schwille.19 FCS is accomplished by focusing an excitation laser beam onto the sample through an objective lens to form an ellipsoid-like femtoliter volume and collecting the fluctuating emission signal within the confocal volume. The correlation function G(τ) defined as G (τ ) =

⟨F(t )F(t + τ )⟩ − ⟨F(t )⟩2 ⟨δF(t )δF(t + τ )⟩ = 2 ⟨F(t )⟩ ⟨F(t )⟩2 (1)

is used to characterize the temporal fluctuations. For eq 1, δF(t) = F(t) − ⟨F⟩ represents the fluctuation of the fluorescence signal F(t) as deviations from the temporal average of the signal ⟨F⟩ at time t. The fluorescence fluctuation is a “fingerprint” for a typical species. A three-dimensional (3D) fitting model, representing a single-component system, is written as G3D(τ ) =

1 1 N1+

1 τ τD

1+

τ ω2τD

(2)

In eq 2, ω, the structure parameter, equates to ωz/ωr, where ωz is the longitudinal radius, while ωr is the transversal or waist radius of the confocal volume; N is the average number of fluorescent molecules in the detection volume; τD is the average time of fluorescent molecules diffusing in the detection volume, which is characteristic for specific molecules or particles. When ωz ≫ ωr, ω → ∞, (1/(1 + (τ/ω2 × τD))1/2) → 1, a twodimensional (2D) model is obtained:

G2D(τ ) =

1 1 N1+

τ τD

(3)

As τ → 0, both G3D(0) and G2D(0) approach 1/N, indicating that the inverse of the intercept of G(τ) on the y axis is the number of fluorescent molecules within the confocal volume. The relationship of τD to the molecular diffusion coefficient D (m2 s−1) is given by 16315



ωr 2 4D

τD =

Article

(4)

The waist radius is obtained from its conversion equation 4Dfreedye ·τD

ωr =

(5)

where Dfreedye is the diffusion coefficient of the standard calibration dye. The translational diffusion coefficient D of a molecule is related to its size by the Stokes−Einstein equation D=

kT 6πηR

(6)

where k is Boltzmann’s constant; T is the temperature; η is the viscosity of the solvent; and R is the hydrodynamic radius. This equation can be used to estimate the sizes of diffusing particles by assuming that the particles have a spherical shape with radius R, which is related to the molecular weight MW of the molecule with a specific gravity v ̅ by 4 V = (MW)ν ̅ = πR3 (7) 3

Figure 1. Schematic diagram of the FCS setup described in the text. L, laser; M1, M2, mirrors; SF, spatial filter; SMF, single mode filter; FP, fiberport; DM, dichroic mirror; MO, microscope objective lens; S, sample; EF, emission filter; PH, pinhole; MMF, multimode fiber; APD, Avalanche Photo Diode; C, correlator; PC, personal computer. Black dashed line represents the outline of the fluorescence microscope.

where V is the volume of molecule. Thus we have ⎛ 3(MW) v ̅ ⎞1/3 ⎟ R=⎜ ⎝ 4π ⎠

(8)

These equations show that the radius R and diffusion coefficient D are weakly dependent on the molecular weight. By combining eqs 6 and 8, we have ⎡ kT ⎛ 4π ⎞1/3⎤ ⎜ ⎟ ⎥(MW)−1/3 D=⎢ ⎢⎣ 6πη ⎝ 3v ̅ ⎠ ⎥⎦

from the excitation light by a dichroic mirror (Chroma, 405 nm), then split by a 50/50 cube splitter. After passing through a 500 ± 20 nm or 590 ± 20 nm bandpass filter, the fluorescence beam was directed into an avalanche photodiode (APD, Perkin−Elmer, SPCM-AQR-14-FC) through a 50 μm inner diameter optical fiber; chambered coverglass (Thermo Scientific, Nunc, Lab-Tek) was used as the container for samples in FCS measurement. In each FCS experiment, the fluorescence fluctuations were measured for 2−10 min. Free fluorescein (D = 3.00 × 10−10 m2 s−1)34 and tetramethylrhodamine (TMR, D = 2.88 × 10−10 m2 s−1)35 were used for calibration. Autocorrelation was processed by a hardware correlator (correlator.com, Flex02−12D). Materials. PPE-dCO2,29 PPE-dNH3,29,30 and oligomer PECO236 were synthesized and prepared according to the reported procedures. The synthesis procedure and characterization of PPE-PEG-dCO2 are provided in the Supporting Information. PPE-PEG-dCO2 was conjugated with ssDNA with sequence 5′-A10CCCAATCACTAA-3′ (Invitrogen), using the synthesis method in the literature.37 The loading ratio was ∼5 ssDNAs per polymer chain. Molecular weights (Mn) of the PPE-dCO2, PPE-dNH3, oligomer PE-CO2, PPE-PEG-dCO2, and PPE-PEG-dCO−DNA were ca. 27 300, 16 600, 1790, 11 000 and 49 000 Da, respectively. (The Mn values for the CPEs were calculated from the Mn of their precursor polymers. The latter were determined by gel permeation chromatography (GPC) analysis.) TMR was purchased from AnaSpec, Inc. Fluorescein was purchased from Fisher. Biotin−TMR (5-(and 6-)tetramethylrhodamine biocytin) was purchased from Invitrogen. Avidin was purchased from Sigma. DNA−TMR was prepared according to the literature.38 All sample solutions were prepared using water distilled and purified by a Millipore purification system (Millipore Simplicity Ultrapure Water System). Buffer solutions were prepared with reagent-grade materials (Fisher). All polymer concentrations are reported as the polymer repeat unit (PRU) concentration. Concentrated

(9)

This relationship is useful for estimation of the molecular weight of a spherical particle from its diffusion coefficient. The effective femtoliter detection volume Veff is obtained from the concentration of solution C and the number of diffusing particles inside the detection volume N:

Veff =

N NAC

(10)

where NA is Avogadro’s constant. The longitudinal radius ωz can be simply obtained from33 Veff = π 3/2ωr 2ωz → ωz =

Veff 3/2 2 π ωr

(11)

As long as the experimental conditions (excitation wavelength, excitation power, coverslip thickness, solvent and immersion medium, emission filter and the position of optic elements) are the same during the measurement, the detection volume does not change. FCS Measurement. The measurements were performed in a setup constructed in house (Figure 1), using an Olympus IX70 epi-fluorescence microscope platform. A 405 nm diode laser (Coherent, CUBE) was employed as the excitation light source. After passing through the spatial filter (Thorlabs, KT110), 405 nm single mode fiber, and fiberport collimator, the laser beam was optimized, expanded, and collimated to a 4.4 mm diameter. The beam was then focused onto the sample through an objective lens (Olympus, 60×, numerical aperture 1.2, water immersion), forming a femtoliter confocal volume. The fluorescence was collected by the same objective, separated 16316



Article

Scheme 2. Structure of TMR, Fluorescein, Biotin−TMR, PE-CO2, and PPE−PEG-dCO2

stock solutions of biotin−TMR and avidin were prepared in 10 mM phosphate buffer (pH 7) to obtain the desired concentrations. Fluorescein was prepared in 10 mM phosphate buffer (pH 8). MES buffer solution (10 mM, pH 6.5) was prepared from 2-(N-morpholino) ethanesulfonic acid and sodium hydroxide. All metal ions were purchased from Sigma-Aldrich Company and used as received. Sodium pyrophosphate was obtained from J. T. Baker Chemical Co. Metal ion titrations were carried out by first mixing the polymer and metal ion solutions and then stirring them for several minutes. The solutions were then allowed to stand overnight in the dark prior to FCS analysis. Fluorescence Spectroscopy. Fluorescence spectra were recorded on a Photon Technology International (PTI) fluorometer and corrected by using correction factors generated with a primary standard lamp. Atomic Force Microscopy. Atomic Force Microscopy (AFM) images were acquired with a Veeco Innova scanning probe microscope in tapping mode using MikroMasch NSC-15 AFM tips with resonant frequencies ∼300 kHz.

Figure 2. (a) Correlation curves for TMR in water. From top to bottom the TMR concentrations were 5, 10, 25, 50, and 100 nM. The inset shows the measured average number of molecules. (b) Effect of molecular weight (MW) on the correlation curves of molecules. Left panel, using 590 nm emission filter. Right panel, using 500 nm emission filter. The black solid lines are the single species fitting curves.

■

RESULTS AND DISCUSSION Characterization and Calibration of the FCS System. Various test experiments were conducted to characterize the performance of the new instrument. The chemical structures of the compounds and polymers used in the work are shown in Scheme 2, along with the acronyms that are used in the text. TMR and fluorescein in aqueous solution were used for calibration of the system with emission filters centered at 590 and 500 nm, respectively. According to the system calibration, the confocal volume is ca. 0.5−1 fL, with ωz in micrometers and ωr in submicrometers. On the basis of the calculation results obtained from eqs 5, 10, and 11, the structure parameter was found to be 11 and 45 for detection at 590 and 500 nm, respectively. A series of TMR samples with varying concentrations was tested with emission detection at 590 nm, and the results are shown in Figure 2a. As expected, because of the inverse

relationship between G(0) and the number of molecules in the confocal volume, a clear decrease of the correlation curve amplitude is observed as sample concentration is increased. The inset plot indicates that the number of molecules N, equal to 1/ G(0), is linear with the increase in concentration. A second test of the FCS setup was conducted by measuring the diffusion behavior of samples with different molecular weight. Using FCS with 590 nm detection (Figure 2b left), TMR (386 Da) with a known diffusion coefficient of 2.88 × 10−10 m2 s−1 was used as the standard to determine the effective volume and the structure parameter. The experimentally observed diffusion time for TMR was 45.0 ± 3.5 μs. Further FCS measurements were carried out on three samples, biotin− 16317



Article

FCS system was to utilize it to probe the interactions of CPE chains with ions in aqueous solution. We also have the goal to correlate the metal ion-induced changes in CPE solutions with the changes in fluorescence. Six different metal cations (Na+, K+, Ca2+, Cu2+, Fe2+, and Fe3+) with a concentration range of 0−25 μM were added to 780 nM aqueous PPE-dCO2 (pH = 6), and the fluorescence spectra were recorded. Figure 3a illustrates

TMR (869 Da), TMR-labeled DNA chain (∼19 300 Da), and biotin−TMR/avidin ([TMR]/[avidin] = 1:10, ∼67 000 Da). The resulting diffusion times were 75.4 ± 0.6, 165 ± 2, and 294 ± 12 μs, respectively. On the basis of eq 4, the measured diffusion coefficient for each species is (1.72 ± 0.17) × 10−10, (0.79 ± 0.09) × 10−10, and (0.44 ± 0.05) × 10−10 m2 s−1, respectively. The diffusion coefficient of biotin−TMR (1.72 ± 0.17) × 10−10 m2 s−1 is close to the value of 1.67 × 10−10 m2 s−1, as previously reported by our group using a different FCS system.39 The diffusion coefficient of avidin/biotin−TMR (0.44 ± 0.05) × 10−10 m2 s−1 compares well with the value of 0.4 × 10−10 m2 s−1, reported for streptavidin40 (another biotinbinding protein that shares almost identical secondary, tertiary, and quaternary structures with avidin41), and with the value of 0.39 × 10−10 m2 s−1, reported for biotin−TMR/avidin ([avidin]/[biotin] = 1:4, 69 000 Da).39 For these molecules/ complex, the diffusion time increases with increasing molecular weight. Assuming that the structures are approximately spherical and that the conditions for the highly diluted aqueous solvents (viscosity and temperature) are the same, on the basis of eq 6, the hydrodynamic radius RH, which can be interpreted as an effective radius, is estimated to be 1.42 ± 0.01, 3.12 ± 0.04, and 5.55 ± 0.03 nm for biotin−TMR, TMR-labeled DNA, and biotin−TMR/avidin, respectively. The hydrodynamic radius of avidin with biotin−TMR 5.55 ± 0.03 nm is larger than the 4 nm value for avidin−biotin complex reported in the literature, where the complex was modeled as a sphere having the same molecular volume.42,43 Considering the nonspherical nature of avidin and the fact that RH is the apparent size of the dynamic hydrated/solvated particle, the difference is within acceptable limits. With 500 nm emission detection (Figure 2b right), fluorescein (332 Da) with diffusion coefficient of 3.00 × 10−10 m2 s−1 was employed as a standard sample, and its experimental diffusion time was 23.5 ± 3.3 μs. The oligomers PE-CO2 (1790 Da), PPE−PEG-dCO2 (∼11 000 Da), and PPE−PEG-dCO−DNA (∼49 000 Da) were also investigated. Their diffusion times from shortest to longest as shown in Figure 2b are, 39.2 ± 4.0, 54.8 ± 3.3, and 112 ± 3 μs. The corresponding diffusion coefficients are (1.80 ± 0.24) × 10−10, (1.29 ± 0.14) × 10−10, and (0.63 ± 0.09) × 10−10 m2 s−1. Although it is difficult to model accurately the conformation of these rigid-rod polymer chains, we can still estimate their sizes by making the same assumptions described above and applying eq 6. The resulting RH values are 1.37 ± 0.18, 1.91 ± 0.19, and 3.91 ± 0.07 nm for PE-CO2, PPE−PEG-dCO2, and PPE− PEG-dCO−DNA, respectively. A plot of diffusion coefficient versus molecular weight is displayed in the Supporting Information (Figure S4). A simple fitting model (red line) indicates an approximately inverse cubic root relationship between the diffusion coefficient D and the molecular weight MW: D = 17.22 × (MW)−0.297

Figure 3. (a) Stern−Volmer plot for aqueous PPE-dCO2 (780 nM, pH = 6) with different metal ions measured by PTI. I0 and I are the fluorescence intensities of PPE-dCO2 before and after the addition of metal ions, respectively. (b) Diffusion time ratio for PPE-dCO2 (780 nM, pH = 6) in water with different metal ions measured by FCS. τd and τd0 are the diffusion time of PPE-dCO2 before and after the addition of metal ions, respectively, and PRU is polymer repeat unit concentration.

the Stern−Volmer (SV) plots of I0/I as a function of the concentration for different metal ions at the maximum emission wavelength of 436 nm for PPE-dCO2, where I0 and I are the emission intensities of polymer before and after the addition of metal ions, respectively. Little quenching is observed for PPE- d CO 2 for [M n+ ] < 2 μM. However, when the concentration increases above 3 μM, a dramatic increase in the slope of the plots is observed for Fe2+, Fe3+, and Cu2+, signaling the onset of a highly efficient quenching process. The fluorescence intensity for PPE-dCO2 begins to decrease very sharply from this point for Fe2+ and Fe3+, until the SV plot levels off. At the highest concentration level, 25 μM, the fluorescence intensity is quenched 97%, 96%, and 95% relative to fluorescence of the unquenched PPE-dCO2 for Fe2+, Fe3+, and Cu2+, respectively. In comparison, all the other ions, namely, Na+, K+, and Ca2+ were unable to induce any significant quenching. The diffusion behavior of 780 nM aqueous PPE-dCO2 with the same six metal ions was investigated via FCS. The normalized FCS correlation curves for PPE-dCO2 with six metal ions at 15 μM are shown in Figure 4a. Although the sizes of the

(12)

When compared to eq 9, it is suggested that the difference in the power value (−0.297 vs −0.33) is due to the spherical model assumed for the rigid-rod polymer. However, the results here are still consistent with the former conclusiona higher molecular weight and larger particle size lead to longer diffusion time. Interaction of Anionic PPE-dCO2 with Metal Cations. Fluorescence and FCS Measurements of PPE-dCO2 in the Presence of Metal Cations. The objective for developing the

Figure 4. (a) Normalized correlation curves for PPE-dCO2 (780 nM) in water (pH = 6) with different ions (15 μM). (b) Count rates for PPE-dCO2 (780 nM) in water (pH = 6) with different ions (15 μM). In (a) the black solid lines are single species fitting curves. 16318



Article

polymer and the polymer/metal complexes are not completely homogeneous, the single species fitting equation (eq 2) was nonetheless used to fit the FCS curves, so that an average diffusion time for each CPE sample could be obtained. As can be seen, the polymer alone has the shortest diffusion time at 60.2 ± 1.8 μs.44 Addition of Na+, K+, and Ca2+ ions induces very little change in the correlation curves, and the diffusion time increases only slightly, to 69.2 ± 3.1 μs for Na+, to 66.8 ± 2.7 μs for K+, and to 90.9 ± 5.2 μs for Ca2+, indicating that the size of the polymer increases little in the presence of these metal ions. However, when the same concentration of Cu2+ is added to the polymer solution, the curve shifts to a much longer diffusion time of 0.406 ms, which is almost 7 times that of the pure polymer. The diffusion curve shifts even further, with a diffusion time of 1.06 ms, upon the addition of Fe2+, suggesting the formation of large aggregates in the presence of this metal ion. The largest change is observed after addition of Fe3+, which features the longest diffusion time of 4.38 ms. From the comparison, we can conclude that the ability of metal ions to induce aggregation increases in the order of K+ ≈ Na+ < Ca2+ < Cu2+ < Fe2+ < Fe3+, which is consistent with the increase of the positive charge density on the metal ions as well as their binding affinities to the carboxylate groups on the polymer side chains. In addition, Fe2+, Fe3+, and Cu2+ are able to bind to multiple carboxylate groups and thus can bridge two or more −COO− units on adjacent PPE-dCO2 chains. Note there are some unusual peaks that appear in the diffusion curve with Fe3+, and this is attributed to the formation of large aggregates. The count rates (corresponding to the fluorescence intensity) versus observation time for polymer/metal ion mixtures at [metal ion] = 15 μM are shown in Figure 4b. Most of the fluctuation profiles have a narrow distribution of fluorescence events, and therefore the fluctuation profiles have a relatively stable baseline.45 However, in some cases,22,23 a few peaks are observed in the fluctuation profiles of some polymer− metal mixtures (such as PPE-dCO2 with Fe2+). Such peaks have usually been attributed to the existence of relatively large particles passing through the excitation volume,39,46 indicating the formation of aggregates. Note that the average count rate, which reflects the corresponding fluorescence intensity, decreases in the order of Fe3+ < Cu2+ < Fe2+ < Ca2+ < K+ < Na+. According to previous reports47 and the results described above, the formation of aggregates can efficiently quench the CPE fluorescence, because the emission is dominated by excitons, which are trapped in the aggregated state. Thus, the above intensity order is consistent with the observed quenching ability of these metal ions. The effect of metal ion concentration on the size of the PPE aggregates was also investigated. The correlation curves for PPE-dCO2/ Fe2+ mixtures at [Fe2+] = 5−25 μM are specifically selected and presented in Figure 5. In general, the diffusion time increases gradually with increasing [Fe2+], reflecting an increase in aggregate size. In particular, there is a considerable increase in size from 5 μM to 7.5 μM, indicating that the formation of very large aggregates is triggered at this point, with [Fe2+] ≈ 7.5 μM as the onset concentration for formation of large CPE aggregates. A small peak is observed near the end of the curve when [Fe2+] = 25 μM. This is presumably because of the formation of larger particles.23 To gain a complete picture of the effect of metal ions on the diffusion behavior of PPE-dCO2, the diffusion time ratios τd/τd0 for PPE-dCO2 in water before and after the addition of metal ions with varying concentrations from 5 μM to 25 μM were

Figure 5. Normalized correlation curves of PPE-dCO2 (780 nM) in water (pH = 6) with different [Fe2+] (0, 5, 7.5, 10, 12.5, 15, 20, and 25 μM). The black solid lines are single species fitting curves.

calculated and are plotted in Figure 3b. Detailed diffusion time ratio data for each ion is summarized in Table S1 of the Supporting Information. On the basis of eqs 4 and 6, the diffusion time τd is proportional to RH, the hydrodynamic radius of the particle. Therefore, any changes in the hydrodynamic radius will affect the mobility of the molecules; a large particle would usually have a longer diffusion time than small particles in a certain fixed volume.48,49 As a result, each of the plots provides insight as to how the size of the polymer changes in the presence of different metal ions at varying concentrations. As illustrated in Figure 3b, the τd/τd0 is close to 1 for Na+ and K+ at all concentrations, suggesting that monovalent metal ions fail to induce the aggregation of PPE-dCO2. The ratio increases to about 2 for Ca2+ at 25 μM, and for Cu2+ the ratio is about 8 at this concentration, indicating that divalent metal ions tend to bind with polymers and induce the formation of small aggregates. However, for Fe2+ and Fe3+, τd/τd0 increases greatly with the increasing of concentration and reaches 70 when [Fe2+] = 25 μM and [Fe3+] = 15 μM. Very large CPE aggregates are believed to be formed in these systems. By comparing panels a,b in Figure 3, a direct relationship between fluorescence quenching and polymer aggregation is observed. It is evident that metal ions display similar trends in polymer fluorescence quenching and aggregation ability. The most efficient SV quenching is observed for the metal ions that give rise to the longest diffusion times, that is, to the largest polymer aggregates, verifying previous reports that indicate polymer aggregation plays an important role in the amplified quenching effect.13−15,50 Moreover, these results also shed light on the origin of the superlinear SV correlations that are frequently observed for CPE-quencher systems. From the FCS results, it is evident that there is a minimum concentration of metal ions needed to induce aggregation (7.5 μM for Fe2+ in this study). Size Calculation and AFM Studies. Although it is difficult to know the exact shapes of the particles in different systems, we assume that they are approximately spherical particles to calculate the approximate hydrodynamic radius, RH. By using eqs 4 and 6, the RH of PPE-dCO2 is calculated to be ∼1.9 nm. After the addition of 15 μM metal ions, the values of RH are 2.2, 2.1, and 2.9 nm for PPE-dCO2 with Na+, K+, and Ca2+, respectively, while for Cu2+, Fe2+, and Fe3+, the calculated RH values are 13, 34, and 141 nm, respectively. Additional information about the size of the mixture is summarized in Table 1. AFM was also used to study the size change of the PPE-dCO2 in the presence of Fe3+ at different concentrations. The CPE/ metal ion solutions were deposited on the surfaces of mica 16319



Article

Table 1. Hydrodynamic Radiusa (RH, nm) of PPE-dCO2 ([PPE-dCO2] = 780 nM) with Different Metal Ions at Different Concentrations concentration of ions (μM) 5 7.5 10 12.5 15 20 25 a

Na+

K+

Ca2+

Fe2+

Fe3+

Cu2+

2.02

2.08

2.04

2.23

2.15

2.93

2.25

2.35

3.69

2.68 14.6 17.4 23.5 34.3 84.4 150

5.76 23.0 35.3 80.7 141 >194 >194

2.52 4.66 6.79 8.55 13.1 14.0 16.7

Typical limits of error on RH are less than 5%.

Figure 7. Fluorescence spectra of PPE-dNH3 solution (1 μM) titrated with PPi in MES buffer (10 mM, pH 6.5).

substrates and dried overnight before being subjected AFM imaging. As shown in Figure 6, pure PPE-dCO2 without any metal ions is well dispersed on the mica substrate, and no large particles are observed. By contrast, deposition of a mixture of the polymer with 5 μM Fe3+ onto mica affords a surface that is decorated with polymer “clusters” with fairly large sizes. As [Fe3+] increases to 30 μM, much larger aggregates are observed, with a significant height of 200 nm. In the latter sample it was difficult to find aggregates on the substrate during sample scanning, indicating that the aggregates formed in dilute solution are few in number, and we conclude they are very large, containing a large number of polymer chains per particle. The AFM image supports the FCS results that Fe3+ can successfully induce strong interchain aggregation of PPE-dCO2. PPi-Induced Aggregation of Cationic PPE-dNH3. In an earlier study,29,30 we reported that PPi can efficiently quench the fluorescence of cationic PPE-dNH3 (structure in Scheme 1). Because of the fact that PPi cannot directly participate in electron or energy transfer processes with the polymer, the most likely origin of the PPi-induced quenching is aggregation of the polymer induced by electrostatic and/or specific host− guest interactions between the polyammonium side groups and diphosphate. In our previous work, direct evidence for aggregation-induced quenching was insufficient, and in the present report we utilize FCS to prove the hypothesis. Initially, fluorescence spectra of 1 μM PPE-dNH3 in MES buffer in the presence of PPi from 0.5 to 10 μM were recorded (Figure 7). When the PPi concentration increases, the strong blue emission at λ = 430 nm decreases, accompanied by the appearance of a broad and structureless green band at around λ = 520 nm. The large red shift (∼ 90 nm) of the fluorescence spectrum suggests that the photoluminescence emanates from a lower energy

state47 and that strong interchain interactions (e.g., π−π stacking) between phenylene rings in adjacent chains may enhance the conjugation effect and lower the overall energy level. To further investigate the origin of the PPi-induced fluorescence quenching of PPE-dNH3, FCS measurements are carried out on 1 μM PPE-dNH3 solutions with varying PPi concentrations of 1, 10, and 25 μM in MES buffer solution. The normalized correlation functions obtained for each mixture are displayed in Figure 8a. The initial diffusion time of PPE-dNH3 is 58.0 μs; however, after the addition of 1 μM PPi, the correlation curve does not change much (τD = 58.5 μs). When [PPi] increases to 10 μM, a shift in the FCS curve is observed, indicating the formation of large aggregates with an estimated diffusion time of 2.03 ms. After 25 μM PPi is added to the polymer solution, a more pronounced shift is observed, and the diffusion time is around 11.8 ms. The result demonstrates that even larger aggregates are formed with increasing [PPi]. The calculated RH are 1.88 nm for PPE-dNH3 itself and 65.7 and 382 nm when [PPi] = 10 μM and 25 μM, respectively. Figure 8b illustrates the count rate of PPE-dNH3 (1 μM)/PPi mixtures with different [PPi] in MES buffer. In the control experiment of PPE-dNH3 without PPi, the baseline is quite stable, with no obvious peaks, and it has a relatively high fluorescence intensity. A large peak appears after the addition of 10 μM PPi to the polymer solution, indicating that large aggregates are passing through the illumination volume. The fluorescence intensity largely decreases when PPi is added. When the [PPi] increases to 25 μM, a higher peak appears that is attributed to the formation of even larger aggregates in the solution. The fluorescence intensity of this system is the lowest,

Figure 6. AFM images of PPE-dCO2 (1 μM) deposited from water onto mica with different [Fe3+] (0, 5, and 30 μM). (A) Polymer is well dispersed when no Fe3+ is added. (B) Small aggregates formed when [Fe3+] = 5 μM. (C) Large aggregates formed when [Fe3+] = 30 μM. Scale bar is shown in lower right in each image; distance shown in scale bar corresponds to scale in x- and y-dimensions in the image. 16320



Article

Figure 8. (a) Normalized correlation curves for PPE-dNH3 (1 μM) with different [PPi] in MES buffer (10 mM, pH 6.5). The black solid lines are single species fitting curves. (b) Count rates for PPE-dNH3 (1 μM) with different [PPi] in MES buffer (10 mM, pH 6.5).

Scheme 3. Mechanism of PPE-dCO2 Quenching by Metal Ions

forms particularly strong complexes with oxygen donors. Consequently, the amplified quenching is the result of the combined effects of both ion-pairing, photoinduced charge and/or energy transfer, and ion-induced aggregation, which is especially pronounced for the polyvalent metal ions. The mechanism is illustrated in Scheme 3. When a small amount of Cu2+, Fe2+, or Fe3+ ([metal ions] < 3 μM) is added to the PPE-dCO2 aqueous solution, the metal cation will bind with the anionic carboxylate groups on the polymer due to electrostatic and specific metal−ligand interactions. At this point, the negative charge density of the polymer is partially decreased, but the polymer still retains the characteristics of molecularly dissolved chains, and photoinduced electron transfer is not pronounced for the electron-deficient metal ions. Thus, only slight quenching of the polymer is observed, and the small size change is not detectable by FCS. When the concentration of metal ions exceeds 3 μM, interchain crosslinking is induced, and small aggregates of PPE-dCO2 form in the solution. As a result, an exciton can migrate along different polymer chains until it encounters a metal ion, where the exciton is quenched. Since any electron acceptor, that is, a metal ion, attached to a single polymer chain is capable of quenching a number of polymer chains in the aggregate, amplified quenching occurs, resulting in the onset of a large decrease of fluorescence in the SV plot in Figure 3a. However, the molecular weight becomes only 3 or 4 times that of the single chain, as only small aggregates form at this point. On the basis of eqs 4 and 9, the change of the molecular weight is roughly proportional to the third power of the molecule’s diffusion time. Therefore, the size of the polymer changes only

and the intensity trend is consistent with the spectra shown in Figure 7. Mechanisms for Aggregate-Induced Quenching. In the first system with PPE-dCO2, the cations are believed to form ion-pair complexes with the anionic polymer. For the transition metal ions, including Cu2+, Fe2+, and Fe3+, the excited-state quenching can occur by either photoinduced electron transfer (polymer to metal) or via an energy transfer process. The latter is possible since each of the transition metal ions has relatively low-lying metal-centered (d−d) excited states.51−53 The alkali and alkaline earth metal ions (e.g., Na+, K+, and Ca2+) have a closed-shell electronic structure and are incapable of accepting electrons from the polymer chains; these ions must quench the polymer via an aggregation-induced quenching mechanism, in which the aggregate states serve as exciton traps. It has been reported that the quenching efficiency of oppositely charged quenchers with CPEs is more efficient when the polymer chains are aggregated, because of interchain exciton migration.50,51,54,55 More extensive aggregation is induced by those ions (Fe3+, Fe2+, and Cu2+) with higher positive charge density compared to Na+ and K+. Moreover, the reason that Fe2+ and Fe3+ ions have higher quenching efficiencies than Cu2+ is likely due to a higher association constant for binding of the ferric and ferrous ions to the carboxylate groups, in contrast to Cu2+, which primarily forms complexes containing four ligands (tetrahedral or distorted octahedral with water in axial position). Fe2+ and Fe3+ form octahedral complexes with six donor atoms, resulting in a greater tendency to cross-link two sets of tri-CO2 pendants on different polymers and form aggregates. In particular, iron(III) 16321



Article

the fluorescence spectra. Thus ion-induced aggregation significantly contributes to the fluorescence quenching of CPE. In this work, the use of a 405 nm laser has successfully expanded the application of FCS to CPE area and established a platform for in-depth study of conformational changes and the diffusion behavior of CPE. FCS is clearly a promising and powerful tool for investigations of the interactions between CPEs and other ions or molecules..

slightly, and only a small increase in the diffusion time is observed (Figure 3b). After a large excess of metal ions is added, PPE-dCO2 is highly cross-linked by the metal cations to form large aggregates. A significant increase of the diffusion time of polymer is observed, corresponding to the large increase of molecular weight, and a large rise of the diffusion time ratio is observed (Figure 3b). This model explains the response lag between the SV plot and the diffusion time ratio plot in Figure 3a,b. The diffusion time ratio increases remarkably at 10 μM Fe2+ and at 7.5 μM Fe3+, while in the SV plot, the fluorescence decreases significantly after the addition of only 3 μM Fe2+ or Fe3+. A similar mechanism can be applied to explain the observations for PPi quenching of PPE-dNH3. PPE-dNH3 exists as a single chain in absence of PPi in MES buffer solution and emits efficient blue fluorescence. After PPi is attached to the NH3+ groups, the negative charge density on PPi neutralizes the polyamine groups and also induces interchain interaction of PPE-dNH3 to form aggregates. The fluorescence of PPE-dNH3 is strongly quenched and shifts to longer wavelength. The FCS result gives solid evidence for formation of large aggregates, especially after adding 10 μM PPi. By introducing FCS, the amplified quenching mechanism has been investigated from a new aspect. More importantly, the comparison of different responses by two techniques based on different mechanisms (fluorescence quenching and FCS) provides insight into the details of molecular interactions during the quenching process.

■

ASSOCIATED CONTENT

S Supporting Information *

Detailed description of the construction and optimization of the FCS system, synthesis of PPE−PEG-dCOOtBu and PPE− PEG-dCOOH including their 1H NMR spectra and GPC trace of PPE−PEG-dCOOtBu, additional FCS diffusion time measurement data. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]fl.edu. Author Contributions §

Danlu Wu and Jie Yang contributed equally and substantially to the experimental work and writing of the manuscript. Notes

The authors declare no competing financial interest.

■

■

ACKNOWLEDGMENTS We acknowledge Dr. Mingxu You for DNA−TMR preparation, Dr. Seoung Ho Lee for PPE-dCO2 synthesis and Dr. Xiaoyong Zhao for PPE-dNH3 synthesis. We also acknowledge the United States Department of Energy (Grant DE-FG02-03ER15484) for support of this work.

CONCLUSION We have successfully constructed an FCS system that utilizes a blue-violet laser as source and provided the details regarding the alignment, optimization, and calibration of the setup. Two example systems were carefully investigated by using the FCS instrument. In the study of PPE-dCO2 with metal ions, Fe3+, Fe2+, and Cu2+, which have much higher fluorescence quenching efficiencies compared to other ions, were shown to induce significant polymer aggregation, on the basis of the FCS results. The charge density and the binding affinity of the metal ions to the carboxylate groups on the polymer chains play an essential role in aggregate formation. The correlation between the SV and FCS diffusion time ratio plots demonstrates that aggregation of the CPE contributes significantly to the amplified quenching phenomenon. Furthermore, the large increase in diffusion time observed by FCS clearly proves that the quenching of PPE-dNH3 is due to a conformational or aggregation-induced mechanism. Both of the results are consistent with the data from steady-state fluorescence spectroscopy. The changes in photophysical properties of CPEs in the presence of oppositely charged ions results from the combined effects of physical quenching by charge transfer or energy transfer and the aggregation state of the polymer chains. It is difficult to distinguish the effects from the two mechanisms based solely on the fluorescence spectroscopy measurements. FCS is capable of measuring polymer size in solution, providing insight and direct information about the physical state of the polymer. With the help of FCS, we are able to explain the fluorescence change caused by different quenching mechanisms in greater detail and also to prove that the amplified quenching effect is strongly correlated with polymer aggregation. Ions with high charge density can induce formation of larger aggregates, consistent with their higher quenching efficiency, as shown in

■

REFERENCES

(1) Jiang, H.; Taranekar, P.; Reynolds, J. R.; Schanze, K. S. Conjugated Polyelectrolytes: Synthesis, Photophysics, and Applications. Angew. Chem., Int. Ed. 2009, 48, 4300−4316. (2) Liu, Y.; Ogawa, K.; Schanze, K. S. Conjugated Polyelectrolytes as Fluorescent Sensors. J. Photochem. Photobiol., C. 2009, 10, 173−190. (3) Onitsuka, O.; Fou, A. C.; Ferreira, M.; Hsieh, B. R.; Rubner, M. F. Enhancement of Light Emitting Diodes Based on Self-Assembled Heterostructures of Poly(p-phenylene Vinylene). J. Appl. Phys. 1996, 80, 4067−4071. (4) Baur, J. W.; Kim, S.; Balanda, P. B.; Reynolds, J. R.; Rubner, M. F. Thin-Film Light-Emitting Devices Based on Sequentially Adsorbed Multilayers of Water-Soluble Poly(p-phenylene)s. Adv. Mater. 1998, 10, 1452−1455. (5) Ma, W. L.; Iyer, P. K.; Gong, X.; Liu, B.; Moses, D.; Bazan, G. C.; Heeger, A. J. Water/Methanol-Soluble Conjugated Copolymer as an Electron-Transport Layer in Polymer Light-Emitting Diodes. Adv. Mater. 2005, 17, 274−277. (6) Mwaura, J. K.; Pinto, M. R.; Witker, D.; Ananthakrishnan, N.; Schanze, K. S., Jr. R. Photovoltaic Cells Based on Sequentially Adsorbed Multilayers of Conjugated Poly(p-phenylene ethynylene)s and a Water-Soluble Fullerene Derivative. Langmuir 2005, 21, 10119− 10126. (7) Jiang, H.; Zhao, X. Y.; Shelton, A. H.; Lee, S. H.; Reynolds, J. R.; Schanze, K. S. Variable-Band-Gap Poly(arylene ethynylene) Conjugated Polyelectrolytes Adsorbed on Nanocrystalline TiO2: Photocurrent Efficiency as a Function of the Band Gap. ACS Appl. Mater. Interfaces 2009, 1, 381−387. (8) Fang, Z.; Eshbaugh, A. A.; Schanze, K. S. Low-Bandgap Donor Acceptor Conjugated Polymer Sensitizers for Dye-Sensitized Solar Cells. J. Am. Chem. Soc. 2011, 133, 3063−3069. 16322



Article

(9) Liu, Y.; Ogawa, K.; Schanze, K. S. Conjugated Polyelectrolyte Based Real-Time Fluorescence Assay for Phospholipase C. Anal. Chem. 2008, 80, 150−158. (10) Ji, E. K.; Wu, D. L.; Schanze, K. S. Intercalation-FRET Biosensor with a Helical Conjugated Polyelectrolyte. Langmuir 2010, 26, 14427− 14429. (11) Thomas, S. W.; Joly, G. D.; Swager, T. M. Chemical Sensors Based on Amplifying Fluorescent Conjugated Polymers. Chem. Rev. 2007, 107, 1339−1386. (12) Zhou, Q.; Swager, T. M. Fluorescent Chemosensors Based on Energy Migration in Conjugated Polymers: The Molecular Wire Approach to Increased Sensitivity. J. Am. Chem. Soc. 1995, 117, 12593−12602. (13) Kim, I. B.; Dunkhorst, A.; Gilbert, J.; Bunz, U. H. F. Sensing of Lead Ions by a Carboxylate-Substituted PPE: Multivalency Effects. Macromolecules 2005, 38, 4560−4562. (14) Tan, C. Y.; Pinto, M. R.; Schanze, K. S. Photophysics, Aggregation and Amplified Quenching of a Water-Soluble Poly(pphenylene ethynylene). Chem. Commun. 2002, 446−447. (15) Jiang, H.; Zhao, X. Y.; Schanze, K. S. Amplified Fluorescence Quenching of a Conjugated Polyelectrolyte Mediated by Ca2+. Langmuir 2006, 22, 5541−5543. (16) Müller, J. G.; Atas, E.; Tan, C.; Schanze, K. S.; Kleiman, V. D. The Role of Exciton Hopping and Direct Energy Transfer in the Efficient Quenching of Conjugated Polyelectrolytes. J. Am. Chem. Soc. 2006, 128, 4007−4016. (17) Kim, I. B.; Bunz, U. H. F. Modulating the Sensory Response of a Conjugated Polymer by Proteins: An Agglutination Assay for Mercury Ions in Water. J. Am. Chem. Soc. 2006, 128, 2818−2819. (18) Magde, D.; Webb, W. W.; Elson, E. Thermodynamic Fluctuations in a Reacting SystemMeasurement by Fluorescence Correlation Spectroscopy. Phys. Rev. Lett. 1972, 29, 705−708. (19) Haustein, E.; Schwille, P. Fluorescence Correlation Spectroscopy: Novel Variations of an Established Technique. Annu. Rev. Biophys. Biomol. Struct. 2007, 36, 151−169. (20) Krichevsky, O.; Bonnet, G. Fluorescence Correlation Spectroscopy: The Technique and Its Applications. Rep. Prog. Phys. 2002, 65, 251−297. (21) Lakowicz, J. R. Topics in Fluorescence Spectroscopy; Plenum Press: New York, 1991. (22) Waldeck, D. H.; Kaur, P.; Yue, H. J.; Wu, M. Y.; Liu, M.; Treece, J.; Xue, C. H.; Liu, H. Y. Solvation and Aggregation of Polyphenylethynylene Based Anionic Polyelectrolytes in Dilute Solutions. J. Phys. Chem. B 2007, 111, 8589−8596. (23) Waldeck, D. H.; Yue, H. J.; Wu, M. Y.; Xue, C. H.; Velayudham, S.; Liu, H. Y. Evolution in the Supramolecular Complexes between Poly(Phenylene Ethynylene)-Based Polyelectrolytes and Octadecyltrimethylammonium Bromide as Revealed by Fluorescence Correlation Spectroscopy. J. Phys. Chem. B 2008, 112, 8218−8226. (24) Murthy, A. V. R.; Goel, M.; Patil, S.; Jayakannan, M. Probing the Role of Chain Length on the Diffusion Dynamics of Pi-Conjugated Polymers by Fluorescence Correlation Spectroscopy. J. Phys. Chem. B 2011, 115, 10779−10788. (25) Xu, Z. H.; Tsai, H. H.; Wang, H. L.; Cotlet, M. Solvent Polarity Effect on Chain Conformation, Film Morphology, and Optical Properties of a Water-Soluble Conjugated Polymer. J. Phys. Chem. B 2010, 114, 11746−11752. (26) Masuo, S.; Nishi, N.; Hosugi, M.; Machida, S.; Itaya, A. Dependence of Single-Photon Emission from Single Conjugated Polymer Chains on Their Spatial Size as Determined by PhotonCorrelation Measurements in Fluid Solution. Chem. Lett. 2010, 39, 780−782. (27) Andrews, A. B.; Guerra, R. E.; Mullins, O. C.; Sen, P. N. Diffusivity of Asphaltene Molecules by Fluorescence Correlation Spectroscopy. J. Phys. Chem. A 2006, 110, 8093−8097. (28) Ghosh, S.; Mandal, U.; Adhikari, A.; Bhattacharyya, K. Study of Diffusion of Organic Dyes in a Triblock Copolymer Micelle and Gel by Fluorescence Correlation Spectroscopy. Chem.Asian J. 2009, 4, 948−954.

(29) Lee, S. H.; Kömürlü, S.; Zhao, X. Y.; Jiang, H.; Moriena, G.; Kleiman, V. D.; Schanze, K. S. Water-Soluble Conjugated Polyelectrolytes with Branched Polyionic Side Chains. Macromolecules 2011, 44, 4742−4751. (30) Zhao, X. Y.; Schanze, K. S. Fluorescent Ratiometric Sensing of Pyrophosphate Via Induced Aggregation of a Conjugated Polyelectrolyte. Chem. Commun. 2010, 46, 6075−6077. (31) Swager, T. M. Iptycenes in the Design of High Performance Polymers. Acc. Chem. Res. 2008, 41, 1181−1189. (32) Lee, K.; Kim, H.-J.; Kim, J. Design Principle of Conjugated Polyelectrolytes to Make Them Water-Soluble and Highly Emissive. Adv. Funct. Mater. 2012, 22, 1076−1086. (33) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; 3rd ed.; Springer: New York, 2006. (34) Chen, Y. Analysis and Applications of Fluorescence Fluctuation Spectroscopy. Ph.D. Dissertation, University of Illinois at UrbanaChampaign, 1999. (35) Schuler, J.; Frank, J.; Trier, U.; Schafer-Korting, M.; Saenger, W. A. Interaction Kinetics of Tetramethylrhodamine Transferrin with Human Transferrin Receptor Studied by Fluorescence Correlation Spectroscopy. Biochemistry 1999, 38, 8402−8408. (36) Feng, F. D.; Yang, J.; Xie, D. P.; McCarley, T. D.; Schanze, K. S. Remarkable Photophysics and Amplified Quenching of Conjugated Polyelectrolyte Oligomers. J. Phys. Chem. Lett. 2013, 4, 1410−1414. (37) Taira, S.; Yokoyama, K. Self-Assembly DNA-Conjugated Polymer for Detection of Single Nucleotide Polymorphism. Biotechnol. Bioeng. 2004, 88, 35−41. (38) You, M. X.; Wang, R. W.; Zhang, X. B.; Chen, Y.; Wang, K. L.; Peng, L.; Tan, W. H. Photon-Regulated DNAEnzymatic Nanostructures by Molecular Assembly. ACS Nano 2011, 5, 10090−10095. (39) Wu, D. L.; Feng, F. D.; Xie, D. P.; Chen, Y.; Tan, W. H.; Schanze, K. S. Helical Conjugated Polyelectrolyte Aggregation Induced by BiotinAvidin Interaction. J. Phys. Chem. Lett. 2012, 3, 1711−1715. (40) Haussling, L.; Ringsdorf, H.; Schmitt, F. J.; Knoll, W. BiotinFunctionalized Self-Assembled Monolayers on GoldSurface-Plasmon Optical Studies of Specific Recognition Reactions. Langmuir 1991, 7, 1837−1840. (41) Green, N. M. Avidin and Streptavidin. Methods Enzymol. 1990, 184, 51−67. (42) Balgi, G.; Leckband, D. E.; Nitsche, J. M. Transport Effects on the Kinetics of Protein-Surface Binding. Biophys. J. 1995, 68, 2251− 2260. (43) Livnah, O.; Bayer, E. A.; Wilchek, M.; Sussman, J. L. 3Dimensional Structures of Avidin and the AvidinBiotin Complex. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 5076−5080. (44) Note that the diffusion time of PPE-dCO2 (Mn = 27 300 Da) (60.2 μs) is close to that of PPE-PEG-dCO2 (Mn = 11 000 Da) (54.8 μs), although their MW is much different. A possible reason is that GPC tends to overestimate the MW of polymer (PPE- dCO2) with longer side-chains and a more rod-like conformation. (45) Blagbrough, I. S.; Adjimatera, N.; Kral, T.; Hof, M. Lipopolyamine-Mediated Single Nanoparticle Formation of Calf Thymus DNA Analyzed by Fluorescence Correlation Spectroscopy. Pharm. Res. 2006, 23, 1564−1573. (46) Holdcroft, S. Determination of Molecular Weights and MarkHouwink Constants for Soluble Electronically Conducting Polymers. J. Polym. Sci., Part B: Polym. Phys. 1991, 29, 1585−1588. (47) Winnik, F. M. Photophysics of Preassociated Pyrenes in Aqueous Polymer-Solutions and in Other Organized Media. Chem. Rev. 1993, 93, 587−614. (48) Chiantia, S.; Kahya, N.; Schwille, P. Raft Domain Reorganization Driven by Short- and Long-Chain Ceramide: A Combined AFM and FCS Study. Langmuir 2007, 23, 7659−7665. (49) Sasmal, D. K.; Mondal, T.; Sen Mojumdar, S.; Choudhury, A.; Banerjee, R.; Bhattacharyya, K. An Fcs Study of Unfolding and Refolding of Cpm-Labeled Human Serum Albumin: Role of Ionic Liquid. J. Phys. Chem. B 2011, 115, 13075−13083. 16323



Article

(50) Tan, C. Y.; Alas, E.; Müller, J. G.; Pinto, M. R.; Kleiman, V. D.; Schanze, K. S. Amplified Quenching of a Conjugated Polyelectrolyte by Cyanine Dyes. J. Am. Chem. Soc. 2004, 126, 13685−13694. (51) Chen, L. H.; McBranch, D. W.; Wang, H. L.; Helgeson, R.; Wudl, F.; Whitten, D. G. Highly Sensitive Biological and Chemical Sensors Based on Reversible Fluorescence Quenching in a Conjugated Polymer. Proc. Natl. Acad. Sci. 1999, 96, 12287−12292. (52) Zhao, X.; Liu, Y.; Schanze, K. S. A Conjugated PolyelectrolyteBased Fluorescence Sensor for Pyrophosphate. Chem. Commun. 2007, 28, 2914−2916. (53) Smith, T. A.; Ramachandran, G.; Gomez, D.; Ghiggino, K. P. Fluorescence Studies on the Conjugated Polyelectrolyte DPS-PPV [Poly (2,5-Dipropoxy Sulphonato Para Phenylene) Vinylene] in Aqueous Solution. Synth. Met. 2005, 152, 17−20. (54) Wang, D. L.; Wang, J.; Moses, D.; Bazan, G. C.; Heeger, A. J. Photoluminescence Quenching of Conjugated Macromolecules by Bipyridinium Derivatives in Aqueous Media: Charge Dependence. Langmuir 2001, 17, 1262−1266. (55) Wang, J.; Wang, D. L.; Miller, E. K.; Moses, D.; Bazan, G. C.; Heeger, A. J. Photoluminescence of Water-Soluble Conjugated Polymers: Origin of Enhanced Quenching by Charge Transfer. Macromolecules 2000, 33, 5153−5158.

16324


Ion-Induced Aggregation of Conjugated Polyelectrolytes Studied by

Recommend Documents