Article pubs.acs.org/Macromolecules
Conformational Transition of Poly(N‑isopropylacrylamide) Single Chains in Its Cononsolvency Process: A Study by Fluorescence Correlation Spectroscopy and Scaling Analysis Fei Wang, Yi Shi, Shuangjiang Luo, Yongming Chen, and Jiang Zhao* Beijing National Laboratory for Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China S Supporting Information *
ABSTRACT: Fluorescence correlation spectroscopy (FCS) has been adopted to investigate the conformational transition of poly(N-isopropylacrylamide) (PNIPAM) single chains with moderate molecular weights in the cononsolvency process. A practical approach of performing accurate FCS measurements with the presence of the refractive index mismatch was developed. The practical and reliable FCS calibration facilitates the acquisition of the hydrodynamic radius (RH) of PNIPAM single chains with the change of the water−ethanol composition. By using the synthesized PNIPAM samples covering a range of degrees of polymerization (N), the scaling analysis in the relationship of RH ∼ Nν exhibits a progressive, re-entrant change of the scaling index (ν) between good solvent (0.57) and poor solvent (∼1/3) condition, which is a reflection of a re-entrant conformational transition of the polymers. Furthermore, the highly asymmetrical feature of the cononsolvency process of single PNIPAM chains was unveiled, which indicates a much stronger effect or interaction of the ethanol molecules to the PNIPAM chain. Comparisons of the present results with previous reports provided new information to the mechanism model of the PNIPAM cononsolvency.
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ally high molecular weights (on the order of 107 g mol−1) was used in order to guarantee a high enough signal-to-noise ratio in extremely diluted solution and an explicit conformational transition. As for single molecule study of relatively short polymer chains by light scattering, however, the low scattering level at extreme dilution condition brings about a challenge, and thus an increase in concentration is desirable, which in turn may result in the chain aggregation due to the poor solvent condition (e.g., at intermediate solvent compositions of cononsolvency). In addition, the fluctuations in the local composition of mixed solvents may generate additional adverse scattering background.11,13 Therefore, it seems considerably difficult to explore the conformation and the consequent conformational transition of single PNIPAM chains of easily accessible yet relatively short chain lengths during the entire cononsolvency process that includes all of good, theta, and poor solvents. From the polymer physics’ point of view, exploring the physics of polymers at the single molecule level provides unique information on the structures and properties of polymer materials.14,15 Its importance also lies in the fact that it helps to elucidate biological processes at single molecule level such as DNA condensation and protein folding.16 Despite numerous successful applications of conventional scattering techniques in polymer science,17 there still remain a number of challenges.
INTRODUCTION Poly(N-isopropylacrylamide) (PNIPAM), one of the mostly studied stimuli-responsive polymers, shows the depressed chain size, solubility, solution viscosity, and gel swelling at certain intermediate mixture compositions of water and the second miscible solvent (methanol, ethanol, tetrahydrofuran, 1,4dioxane, dimethyl sulfoxide, etc.), although each is a good solvent of PNIPAM at room temperature.1 This type of reentrant phenomena is a typical case of the cononsolvency of polymers.1,2 Similarly in pure aqueous solutions, the chain conformation and phase behavior of PNIPAM in the aqueous mixed solvents are determined by the balance between the hydrogen bonding of two solvents onto the amide group and the hydrophobic effect of the pendant isopropyl group and the chain backbone. Although extensive efforts have been made to study the cononsolvency phenomena, including the phase behavior of PNIPAM solutions and the volume phase transition of PNIPAM microgels and bulk gels, the origin of PNIPAM cononsolvency has not been clearly understood.1−12 From the molecular point of view, the chain conformation and conformational change should convey valuable information on the microscopic mechanism of the polymer cononsolvency. There has been a successful experimental study of PNIPAM single chains during its cononsolvency,5 in which static and dynamic laser light scattering have been used to reveal the coil-toglobule-to-coil conformational transition of linear PNIPAM in the mixture of water and methanol, by measuring the radius of gyration (RG) and the hydrodynamic radius (RH) of PNIPAM single chains. However, in this work, PNIPAM with exception© 2012 American Chemical Society
Received: August 24, 2012 Revised: October 20, 2012 Published: November 8, 2012 9196
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Scheme 1. Synthesis of PNIPAMs by RAFT Polymerization and Their Fluorescence Labeling via the Random Incorporation of a Proportional Fluorescent Comonomer (RB-MA) in Approach 1 (for N = 190, 290, 680, and 1260) or via the Chemical Linkage of a Fluorophore at the End of Carboxyl-Terminated PNIPAM (N = 86) in Approach 2
In this paper, reliable and quantitative FCS measurements have been conducted to study the cononsolvency process of PNIPAM single chains in the mixed solvents of water and ethanol. Narrowly distributed linear PNIPAMs covering a range of molecular weights were synthesized in a controllable polymerization and examined by FCS. The scaling analysis of the hydrodynamic radius of single PNIPAM single chains during the cononsolvency was exploited to identify the conformational transition, providing new information about the cononsolvency mechanism.
For example, it is not an easy task for the scattering methods to extract single chain information from rather complex systems, such as systems with multiple charges (polyelectrolytes)18,19 and multicomponent systems in which the scattering from multiple sources buries the signal from the target (e.g., in concentrated intracellular environment). Fluorescence correlation spectroscopy (FCS) has been proved to be a highly sensitive technique to study the single molecule dynamics.20,21 As both dynamic light scattering (DLS) and FCS are photon correlation spectroscopy techniques, FCS monitors the fluctuation of fluorescence signals, by single photon counting, inside a confocal excitation-detection volume (∼10−15 L) and analyzes the temporal correlation function from which the dynamic parameters of the fluorescent probe can be extracted, such as the translational diffusion coefficient (D). The high sensitivity of FCS offers the experimental feasibility to study single polymer chains in extremely dilute solutions. Experimentally, the target polymer chain is labeled by a fluorescent group at a properly low ratio (optimally one fluorescent group per polymer molecule). Because the working concentration of FCS is low (on the order of 10−9 mol L−1), measurements are allowed under concentration ∼3 orders of magnitude lower than that commonly required by laser light scattering. The advantage of the high sensitivity has been demonstrated in the studies of polyelectrolytes,22−24 whose single chain behavior would otherwise be concealed at usual concentrations due to the long-ranged electrostatic interactions. Additionally, FCS as a sensitive fluorescence technique with proper spectral recognition is suitable for the studies of complex multicomponent systems such as the binary solvent systems, which can normally generate scattering signals due to the presence of local density fluctuations in the solvent mixture.11,13 For example, in the study of poly(acrylic acid) in the mixture solvents of 2,6-lutidine and water, DLS suffered from the background scattering from both the critical opalescence of the mixed solvents and the polymer concentration effect.13 In contrast, FCS has been successfully applied to reveal the chain contraction and re-expansion of poly(acrylic acid) of 10−9 mol L−1 in the vicinity of the critical temperature of the same solvent mixture.25 Therefore, FCS is expected to open up a new approach to study the conformation of single chains in binary liquid mixtures, such as close to the critical point25 and during the cononsolvency process.
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EXPERIMENTAL SECTION
Fluorescence Correlation Spectroscopy (FCS). The descriptions of FCS principles can be found in a number of publications.20,21 Briefly, a FCS setup with the confocal detection geometry monitors the fluorescence fluctuation within a small excitation-detection volume of an ellipsoid-like shape. The radii of the confocal ellipsoid are 300− 500 nm in the direction perpendicular to the optical axis and 1−2 μm along the optical axis. The distribution of the laser intensity inside this space is of a Gaussian profile (at ideal optical condition). FCS measures the temporal autocorrelation function of the fluorescence fluctuation, G(τ) = ⟨δI(t)δI(t + τ)⟩/⟨I(t)⟩2, where I(t) denotes the fluorescence intensity at time t and τ the time lag. The numerical fitting of the function adopting the Gaussian profile and threedimensional diffusion model helps to determine the translational diffusion coefficient and the average concentration, ⟨c⟩, of the fluorescence probe. The expression of the autocorrelation function by the three-dimensional Gaussian model is G(τ) = (π3/2w02z0⟨c⟩)−1(1 + 4Dτ/w02)−1(1 + 4Dτ/z02)−1/2, where w0 and z0 are the lateral radius and the half length of the excitation-detection volume, respectively. The RH value of the probe is therefore determined through the Stokes−Einstein equation, RH = kBT/6πηD, where kB, T, and η are Boltzmann constant, the absolute temperature, and solution viscosity, respectively. The details of the FCS setup used in this study are provided in previous publications.22,23 Briefly, the FCS setup is home-built based on an inverted optical microscope (IX-71, Olympus, Japan). A waterimmersion objective (UPlanApo 60×, number aperture = 1.20, working distance = 0.25 mm) was used with its collar correction ring fixed at 0.16 in order to be compatible with the coverslip’s thickness. The output of a solid-state laser of 532 nm worked as the excitation light source. The power density of the excitation laser was ∼4.6 × 107 J s−1 m−2 (13 μW at the sample stage). The focal point of the objective lens was precisely controlled using a piezoelectric nanopositioner (Physik Instrumente, Germany). A sample cell for liquid samples was fabricated with a glass tube with one end epoxyglued onto a coverslip with the thickness of 0.16 mm (Fisherbrand). 9197
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aberrations and possibly misleading FCS measurements.29,30 However, insufficient attention has been paid to this problem in spite of a rich library of experimental FCS studies, which often use various complex specimens (concentrated aqueous solutions of salts,23,31 organic solutions,25 polymer melts,32 and crowded environments,33,34 etc.) with the considerable refractive index mismatch. Motivated by this problem, the optimal experimental condition was explored prior to FCS examinations of PNIPAM single chains. To examine the influence of the refractive index mismatch on FCS, the aqueous solution of glycerol with varied concentrations was chosen as the model media. The refractive index of aqueous glycerol solution varies from 1.333 to 1.364 at 20 °C with the concentration by weight (cglycerol) from 0 to 30%.35 Furthermore, monodisperse fluorescent nanoparticles are adopted as the standard substance to calibrate the parameters of the excitation-detection volume of the setup. Its diffusion coefficient in the model media of various concentrations was calibrated by well-established DLS measurements at five scattering angles (30°, 60°, 90°, 120°, and 150°) (details provided in the Supporting Information). The usual standard substance, rhodamine 6G,20 was not used here because of the recently discovered discrepancy of its diffusion coefficient in water.36−39 The accurate determination of the diffusion coefficient of the fluorescent nanoparticle by DLS allows the calibration of the dimension of excitation-detection volume of FCS when separate measurements of FCS were conducted with the same probe concentrations as in DLS measurements. Figure 2a displays the representative autocorrelation function curves of the same nanoparticle diffusing in pure water and at cglycerol = 25%. The data can be perfectly fitted by a three-dimensional Gaussian model, from which the apparent dimension of the excitation-detection volume is calibrated (data shown in Figure 2b), taking the known values of D determined by DLS. Because of the mismatch of refractive index between the sample medium and the refractive index matching fluid of the objective lens (water for the current case), considerable changes in the excitationdetection volume are exposed. One of the most important parameter, the apparent lateral radius (w0) is found to decrease with the increase of the mismatch of the refractive index at large focal depths, as the refractive index of aqueous glycerol solution is increasingly larger with cglycerol than that of pure water.35 Another important feature is that the mismatch gets increasingly pronounced as the focal point is adjusted further into the solution. Theoretically, the optimal focus profile of the excitation-detection volume is only reached with zero mismatch of the refractive index, and otherwise the volume would change owing to the distortion of the point spread function away from the Gaussian profile and optical aberrations would readily emerge. This refractive index mismatch should not be neglected because a small deviation in w0 would result in a considerable shift in the apparent D values due to the relationship of w02 = 4Dt. This result agrees with a previous computer simulation study which discovers the distortion of the confocal volume due to the refractive index mismatch on the FCS measurements.29 Because of the distortion of the excitation spot, the apparent numerical values of w0 deducted from the idealized three-dimensional Gaussian distribution model cannot accurately describe the distorted focus profile. However, the decrease of the apparent w0 values clearly indicates the distortion of the excitation-detection volume in the presence of the refractive index mismatch. The most important feature is that smaller deviation in w0 is found with smaller focal depths, i.e., when the focal point is positioned less deep into the solution. The deviation becomes insignificant when the focal depth goes down to ∼25 μm (Figure 2b). Separate experiments with an even smaller focal depth (10 μm) gave identical resultsno noticeable deviation was observed. This provides an important yet simple approach to making accurate measurements inside media of varied refractive index by keeping a small focal depth rather than going through complicated refractive index calibration process. With the accurate determination of the dimension of the excitationdetection volume, it is interesting to further check the value of the diffusion coefficient of rhodamine 6G, the widely used small fluorescent molecule as a standard substance for calibration. The
The sample cell was carefully cleaned and later treated by oxygen plasma (Harrick), which generated hydroxyl-group rich surface to avoid the adsorption of probe molecules. Materials. The narrowly distributed, carboxyl acid-coated, fluorescent polystyrene nanoparticle with a diameter of 0.1 μm (F8803, FluoSpheres, Invitrogen) was used as the standard substance for the calibration of the excitation-detection volume. Rhodamine 6G, a small fluorescent molecule often used as a standard substance with a known diffusion coefficient value in water, was purchased from Fluka. The chain transfer agent (CTA), S-1-ethyl-S′-(α′-dimethyl-α″-acetic acid) trithiocarbonate, was synthesized according to a published protocol.26 Other chemicals are listed in the Supporting Information. Synthesis, Characterization, and Fluorescence Labeling of PNIPAM. The radical addition−fragmentation chain transfer (RAFT) polymerization27 was adopted in order to synthesize narrowly distributed PNIPAM, as shown in Scheme 1. A carboxyl-containing trithiocarbonate was used as a highly efficient chain transfer agent. The molar feed ratio was tuned for the purpose of a range of molecular weights. The degrees of polymerization were determined to be 86, 190, 290, 680, and 1260.28 The molecular weight distributions (Mw/ Mn: 1.07−1.18) were determined by size exclusion chromatography (SEC; Waters) equipped with a differential refractive index recorder. The SEC instrument was calibrated with polystyrene standards, using DMF solution of 0.01 mol L−1 LiBr as the eluent. The chemical structure of PNIPAM was confirmed by proton nuclear magnetic resonance. Two approaches were taken to label the PNIPAM with fluorescence molecules: (1) via the RAFT copolymerization with a fluorescent comonomer; (2) via the chemical conjugation of an active fluorophore at the chain end, as shown in Scheme 1. The former used the random incorporation of a commercially available methacrylate comonomer containing a rhodamine B unit (RB-MA; Polysciences). The latter used the chemical conjugation of the carboxyl-terminated PNIPAM chains by an amino-containing sulforhodamine derivative (SulfoRBNH2; Invitrogen). Careful purification was conducted by repetitious precipitation and centrifugal ultrafiltration. The successful purification was verified by the well-defined autocorrelation curves of the diffusion of the labeled polymer measured by FCS (single diffusing species as shown in Figure 3a,b). The labeling efficiency (the x value in Approach
Figure 1. SEC curves of PNIPAM with five N values. DMF containing 0.01 mol L−1 LiBr was used as the eluent at 50 °C. Signals were obtained by a differential refractive index detector. 1 of Scheme 1) has been characterized to be 0.56, 0.24, and 0.15 per chain for PNIPAM of N = 190, 290, and 680, respectively, as typical examples. More details are provided in the Supporting Information. Experimental Optimization of FCS Measurements in the Presence of the Refractive Index Mismatch. FCS measurements may encounter the artifacts arising from improper determination of instrumental parameters.29 Because the refractive index of the water− ethanol mixture varies with the composition and differs from that of water (the refractive index matching fluid of the objective lens), a mismatch exists between the refractive index of the solution and that of the objective lens which can introduce undesirable optical 9198
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Figure 2. (a) Representative normalized autocorrelation function curves of fluorescent nanoparticles diffusing in aqueous solution of glycerol at a small focal depth (25 μm). The weight concentration of glycerol, cglycerol, is displayed. (b) Values of the apparent lateral radius of the excitationdetection volume of FCS, w0, as a function of the refractive index (n) of the solution due to the change in cglycerol. The distance of the focal point in the sample medium away from the coverslip surface (the focal depth, d) is displayed. All measurements were performed with a water-immersion objective (UPlanApo 60×, N.A. = 1.20) at 25 °C. An illustration of the focal depth is provided in the inset of (b).
Figure 3. Normalized autocorrelation function curves of diffusing single chains of PNIPAM with five degrees of polymerizations (N) in pure ethanol (a) and at xEtOH of 0.25 (b). The solid line with each data set denotes the results of the numerical fitting using three-dimensional diffusion model. Rh6G in (a) denotes the results of free fluorescent rhodamine 6G, and its drastic difference from those of polymers indicates the successful labeling and sample purification. (c) The values of hydrodynamic radius (RH) of PNIPAM single chains as a function of xEtOH. The N values are displayed by the corresponding data set. All measurements were conducted at 20 °C. commercial water-immersion objective was adjusted, one by one, to compensate the varied refractive index mismatch. For example, the correction collar was adjusted separately for each solution with its specific refractive index.33 In other studies, without adjusting the correction collar and keeping relatively large focus depths (e.g., 100 μm34), a separate calibration of w0 had to be conducted for every sample. In the present work, the proposed practice by adopting a small and fixed focal depth (e.g., 25 μm) with the proper fixed collar correction positioning is easy to perform for reliable FCS measurements. Therefore, the focal depth was kept at 25 μm in the following parts.
data showed that the diffusion coefficient of rhodamine 6G in pure water was 420 ± 6 μm2 s−1 at 25 °C, corresponding to a hydrodynamic radius of 0.58 nm. This result agrees well with a number of recent publications which utilized other sophisticated methods including microfluidic technique,36 multicolor dual-focus FCS,39 scanning FCS,38 and a combination of pulsed field gradient nuclear magnetic resonance spectroscopy and FCS.37 The present work as well as recent papers leads us to conclude that the widely cited value of 280 μm2 s−1 for rhodamine 6G (at ∼22 °C) has generally been underestimated.20 The effect of the refractive index mismatch for various confocal microscopes has long been studied.30 FCS as a confocal based spectroscopy has been employed for investigations of various sample media. Nevertheless, only several studies have explicitly taken into account the adverse effect of the refractive index mismatch.29,31,33,34 In computer simulations, the effect of deviations in the thickness of a coverslip and the refractive index of the sample solution has been studied. The results showed that the increase in the refractive index mismatch and the focal depth and the deviation of a coverslip thickness distorted the confocal volume and depressed the apparent diffusion coefficient.29 Experimentally, the correction collar of a
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RESULTS AND DISCUSSION Scaling Analysis of Single PNIPAM Chains in the Cononsolvency. On the basis of the mean-field approximation40 and the scaling concept of polymers,41 the universal scaling exponent ν in the relationship of the chain dimension (R) and the number of monomeric units, R ∼ Nv, depends on solvent quality. If polymers are sufficiently long and flexible, the 9199
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Figure 4. Typical double-logarithmic plot of RH of single PNIPAM chains as a function of N under different solvent compositions: (a) xEtOH = 1.0, (b) xEtOH = 0.28, (c) xEtOH = 0.25. Solid lines are the least-squares linear fitting. The fitted ν values are displayed. (d) The ν values as a function of xEtOH. The two data points denoted by circles are obtained from RH values of four (xEtOH = 0.082) and three (xEtOH = 0.091) molecular weights of PNIPAM. The three dotted lines denote the theoretical values of the static scaling index for a random coil (0.588), an undisturbed coil (0.5), and a compact globule (1/3). More details are provided in the Supporting Information.
scaling index ν assumes one of three values: 0.588 for a coil-like chain in an athermal solvent, 0.5 for an undisturbed chain in a theta solvent, and 1/3 for a tightly collapsed globule in a poor solvent. Therefore, the value of ν can identify the conformational state of polymer single chains. Experimentally, studies on the scaling of the dimensions of linear chains of synthetic polymers,42−44 proteins,45,46 and double-stranded DNA47 can be found in a large body of literatures. However, to the best of our knowledge, no experimental investigation has been reported on the scaling law of neutral polymer single chains in solvents ranging from good to theta to poor solvents yet. It should be intriguing to extract the scaling law of PNIPAM single chains throughout the entire cononsolvency process and explore the cononsolvency at the single molecular level. Here FCS was adopted to explore the scaling law of PNIPAM with moderate chain lengths in the mixture of water and ethanol and reveal the conformational transition. Figure 3a,b shows the normalized autocorrelation function of diffusion of PNIPAM single chains in pure ethanol and in the mixture solution with the molar fraction of ethanol (xEtOH) of 0.25. PNIPAM samples of five degrees of polymerization were measured by FCS, and the data showed good features of single diffusing species, indicating that the measurements were based on separate PNIPAM chains. Additional experimental data by photon count histogram showed well-fitted single species of fluorescence brightness (photon counts of fluorescence emitted by a single fluorescent dye in unit time), indicating no existence of aggregation and that the measured diffusion behavior was at single molecular level (more details provided in the Supporting Information).48 The numerical fitting of the autocorrelation function gave D values and corresponding RH values of PNIPAM single chains. These measurements were conducted at the concentration of ∼10−9 M, much lower than the concentration usually used in the conventional scattering experiments. This is the benefit of the high sensitivity of the single photon counting method, the most sensitive optical detection, which enables the measurements at real single molecular level. Furthermore, because the fluorescence signal comes merely from the fluorescent tag, the
signal level does not change when the molecular weight of the polymer is changed, resulting in the constant signal-to-noise ratio regardless of the molecular weight of the polymer. This brings about another advantage of the FCS method over the scattering methods, whose signal-to-noise ratio relies on the molar mass of the polymerthe higher molar mass, the higher signal level. The values of RH for different molecular weights as a function of the molar fraction of ethanol in the mixture solvent are displayed in Figure 3c. All samples showed the re-entrant featurethe PNIPAM chain shrank at first and then reexpanded with the further increase of xEtOH. In the intermediate xEtOH composition beyond 0.09, no uniform fluorescent signal could be detected in the solution, indicating the formation of suspended aggregates. This situation did not allow reliable FCS measurements, and therefore no data of RH could be obtained. Such a situation remained within a range of xEtOH value until it reached the point of xEtOH = 0.25, beyond which further addition of ethanol made the uniform distribution of the single chains reappear and the RH values increased progressively. The re-entrant variation of RH values was asymmetricalit dropped at a small ethanol content (xEtOH ∼ 0.1) and increased progressively within a broad range of xEtOH with the increase of ethanol content. The scaling behavior in the relationship of RH ∼ Nv was investigated at various xEtOH. Values of ν were obtained using the least-squares linear fitting approach in the doublelogarithmic plot of RH versus N. Typical examples for xEtOH of 1.0, 0.28, and 0.25 are presented in parts a, b, and c of Figure 4, and all results are presented in the Supporting Information.49 The data fitting for all samples are satisfactory, providing reliable ν values for all conditions. For the case of pure ethanol and pure water, the corresponding ν value was 0.574 ± 0.006 and 0.561 ± 0.009, respectively. Both values are close to the predicted value for polymers in athermal solvent (ν = 0.588),50 indicating the random coil conformation of PNIPAM chains, which is well soluble in ethanol and in water at 20 °C. An important concern is the possible effect of the fluorescent tag, and this is considered to be minor because of the relatively 9200
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significant on the basis of the thermal blob model.53 However, the cartoon of a thermal blob as an abrupt cutoff in chain statistics is still a distinct oversimplification, as shown previously.54 Short sequences, especially close to the size of a thermal blob, can deviate up to ∼15% from the predicted asymptotic limit of the static scaling relationship for chains with an infinite length.51 Such an effect is even more remarkable for the dynamic scaling law.51,53 This has been observed in both theoretical analysis and simulations, which take into account the internal correlations of a single chain and show the nonuniform swelling of subchains depending on the location along the chain (i.e., unequal size of blobs).55,56 The improved blob models57,58 have shown that practical polymer chains can stay in variously intermediate conformational states with intermediate effective scaling index between typical extremes of ν values, particularly for usually limited chain lengths.52 Our results clearly observed the progressive change in ν from 0.57 (Figure 4a) to intermediate values (Figure 4b) and then to 0.32 (Figure 4c) by using PNIPAMs with the readily accessible lengths, rather than a crossover in the thermal blob model. Very recently, a similar gradual change in ν from ∼0.58 to 0.5 was also found in both the static and dynamic scaling exponents for linear polystyrene.44 Conformational Transition of Moderately Long Chains Evidenced by the Scaling Analysis. The re-entrant coil−globule conformational transition of PNIPAM chains in the cononsolvency process is evidenced by the variation of the scaling index with the increase of the ethanol content. The coil−globule transition (CGT) is the transition of conformations between expanded coils and collapsed globules of linear polymer chains which remain in the one phase region, if the solvent quality switches between good and poor. CGT is a subject of considerable theoretical and experimental interests in polymer science and holds significant implications in many biological systems such as DNA packing and protein folding/ unfolding.16 Abundant studies have experimentally explored CGT in organic solutions, mainly via laser light scattering techniques.59−61 A specific scaling manner of α3|τ|Mw1/2 versus |τ|Mw1/2 was frequently employed to gain master curves, where α is the reduced expansion factor of chain dimensions and τ is the reduced dimensionless temperature with respect to the theta temperature (Θ), τ = 1 − Θ/T. However, for the present cononsolvency system of PNIPAM, the value of theta temperature is difficult to determine because it changes with the solvent composition, and therefore the variable |τ|Mw1/2 is difficult to acquire. It is challenging to employ this analysis in a quantitative way. It is also due to this reason that CGT of the ultralong (∼107 g mol−1) PNIPAM chain and its derivatives was studied62,63 in aqueous solutions. Alternatively, the specific ratio RG/RH was adopted as a criterion to determine that its conformation was a random coil or a tight globule and the corresponding CGT. The variation of ν value with the change of xEtOH shows the re-entrant behavior of the conformational change of the PNIPAM chainthe coil-to-globule-to-coil transition. The gradual decrease of ν value from ∼0.57 at pure water and ethanol situation shows that the collapse of the chain starts from a relatively large chain length scale. When the xEtOH value is increased from 0.0 (the pure water situation) or when this value is decreased from 1.0 (the pure ethanol situation), the gradual decrease of ν value indicates that the chain length scale at which the collapse of the PNIPAM chain occurs becomes smaller and smaller. At the intermediate xEtOH value of 0.09 and
small size of the tag (RH of 0.58 nm for free rhodamine B) compared with the polymer of high molecular weights, which can be an order of magnitude larger. However, its effect can be less ignorable for shorter polymers: for example, for the PNIPAM sample with N of 86, its RH value is about 2 nm, about 4 times that of the free fluorophore. This fact can demonstrate the advantage of scaling analysis, which covers a large span of molecular weight, compared with the mere analysis of the values of hydrodynamic radius. With the change of ethanol content, the ν value also exhibited a re-entrant feature. When xEtOH increased from 0.0 to 0.09, the ν value dropped sharply from 0.57 to ∼0.33. When xEtOH was higher than 0.25, a gradual recovery of ν value from ∼0.32 to 0.57 was observed. The ν values close to 1/3 indicate the globular conformation of PNIPAM at xEtOH of 0.09 and 0.25, due to the poor solvent condition. For the intermediate composition range (0.09 < xEtOH < 0.25), the experimental observation of the single chain behavior has been difficult, particularly for longer chains, due to the formation of polymer aggregation in the poor solvent even at such a very low polymer concentration (less than 1 × 10−9 mol L−1). This observation agrees with the fact that PNIPAM is macroscopically insoluble in this xEtOH range, and therefore PNIPAM single chains are believed to take the compact globule conformation. Compared with the results of other techniques on the scaling analysis of the collapsed state of chain molecules, such as natively folded proteins and polypeptides,45,46 the FCS results reported here demonstrate a much higher accuracy, exhibiting its merit of high sensitivity for the study of single polymer molecules. The dynamic scaling indexes in pure water and pure ethanol (both good solvents for PNIPAM at room temperature) are close to but slightly lower than the theoretical limit of 0.588. This weak discrepancy can be attributed to the relatively low molecular weight of samples and the feature brought by the approach of dynamic scaling. For single polymer chains in a good solvent, the scaling index was originally estimated to be 0.6 using the mean-field approximation40 and scaling concept.41 More precise evaluations with the renormalized group consideration lead to the value of 0.588 in an athermal environment in the asymptotic limit (N → ∞).50 These theories take into account the significant excluded volume interaction, and the static dimension (e.g., the radius of gyration, RG) is mostly studied. Moreover, the dynamic scaling law in the relationship of RH versus N has also been considered with the incorporation of the hydrodynamic interaction.51 The theoretical asymptotic limit of 0.588 of the static and dynamic scaling index in good solvent can be theoretically approached only for very large molecular weights; otherwise, for moderately long chains in good solvent, the actual scaling index is less than the limit value of 0.588. It is particularly remarkable for the dynamic scaling law, which can be interpreted from the different definitions of RH and RG.51,52 As previously stated by Akcasu and Han,51 the dynamic scaling fitting of RH ∼ Nν would result in a scaling exponent lower than the predicted static value in a good solvent. Previous experimental investigations have observed the dynamic scaling index was slightly smaller than static scaling index.43 The progressive change of ν values between ∼0.57 and ∼0.33 implies the effect of finite chain lengths on the dynamic scaling law of polymers. The profound scaling concept has found great applications to predict rich polymer properties.41 For example, the interpretation of the scaling index of single chain dimensions under various solvent quality has been 9201
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PNIPAM chains in a gradual way due to the relatively weak cooperative adsorption of alcohol molecules.8 Therefore, this hypothesis can explain the asymmetrical feature of the present FCS observation. The effect of the molecular weight on the cononsolvency is also investigated. Figure 5 is the plot of the normalized RH of
0.25, the collapse of the chain occurs at a length scale small enough compared with the chain length, and the ν value of ∼1/ 3 for the globular conformation is recovered. This scaling analysis can be regarded as a new way to recognize experimentally the conformational transition of polymer chains, particularly with moderate but more practically accessible lengths. On the Cononsolvency Mechanism of PNIPAM. As shown in Figures 3c and 4d, the present investigation clearly exposes the highly asymmetrical re-entrant feature of the conformation and scaling index of PNIPAM chains changing with the mixture composition, which is representative of the cononsolvency process at the molecular level. With the increase of xEtOH, a steep fall of ν, indicative of the abrupt chain collapsing, occurred at low ethanol content and within a narrow range of xEtOH (mainly 0.07−0.09), whereas a progressive increase of ν, indicative of the chain re-expansion, occurred within a quite broad range of xEtOH (mainly 0.25−0.37) prior to the recovery of a fully expanded conformation at full ethanol content. The collapse of PNIPAM single chains at the low ethanol content indicates that ethanol molecules cast a stronger effect on the PNIPAM conformation than water molecules.6 The results here help to provide new information on the mechanism of the cononsolvency process. Up to now, there have been three major models for the origin of the PNIPAM cononsolvency: (1) the formation of the complex between two solvents induced by the presence of polymer networks;4 (2) the stoichiometric compounds between two solvents which behave as a poor solvent for polymers;5 (3) the competitive adsorption of solvent molecules onto polymer chains.3,7−9 The comparison of FCS data here with previously published results shows no dependence of cononsolvency on the PNIPAM concentration in water−ethanol mixtures. The present linear PNIPAM chains (Mw = 9.7 × 103−1.42 × 105 g mol−1) with extreme dilution (∼10−5−10−4 mg mL−1) stayed in a collapsed state in the range of xEtOH of 0.09−0.25 at 20 °C. The same cononsolvency system exhibited phase separation within a similar range of xEtOH (0.08−0.31) at the commonly dilute solution of linear PNIPAM (Mw = 1.2 × 105 g mol−1) with a higher concentration (1.0%) at 20 °C.6 In a very recent study, PNIPAM microgels (locally concentrated system) experienced strong deswelling within the same range of xEtOH of 0.07−0.32.10 The absence of the polymer concentration dependence has also been found in another cononsolvency system of PNIPAM in mixture of water and methanol.1,5,12 This fact further excludes the first hypothesis4 discussed above, in which the effect of polymer concentration should be expected.1 The highly asymmetrical re-entrant feature of the collapse of the PNIPAM chain with the increasing ethanol content indicates a much stronger effect of ethanol than that of water. This observation agrees well with the mechanism of competitive adsorptionthe recently proposed model in which the cooperative adsorption, in competition, of water and of alcohol molecules onto PNIPAM chains is considered.7−9 Its prediction agrees well with the conformational transition of linear PNIPAM chains and the volume phase transition of cross-linked PNIPAM microgels in the water− methanol mixture. If the adsorption/desorption cooperativity of water molecules is stronger than that of alcohol molecules, the adsorption of a small amount of alcohol can cause the cooperative desorption of a larger amount of water molecules (PNIPAM dehydration) and hence collapse PNIPAM dramatically, whereas further addition of ethanol would expand
Figure 5. Normalized hydrodynamic radius of each PNIPAM sample as a function of the fraction of ethanol (xEtOH). The data are normalized by the RH value at xEtOH of 0.0 for xEtOH < 0.09, and for xEtOH > 0.25, they are normalized by the RH value at xEtOH of 1.0.
PNIPAM of different molecular weight as a function of xEtOH. The normalization was conducted with respect to the RH value at xEtOH of 0.0 for the region of xEtOH < 0.09, and for xEtOH > 0.25, it was normalized to RH value at xEtOH of 1.0. The data show a change of the sharpness of the conformational changethe higher molecular weight, the sharper the conformation change. This observation is in consistence with the sharp conformation transition of the ultralong linear PNIPAM chains (N ≈ 2.5 × 105).5 Compared with the gradual re-entrant volume phase transition of PNIPAM microgels and gels,4,10 which depends closely on the length of the subchains between adjacent cross-linking points,64 the effect of the chain length on the sharpness of the PNIPAM conformation transition is clearly evidenced. On the basis of this feature, it is envisioned that the sharpness of the responsiveness of smart PNIPAM materials can be tuned by changing the effective chain lengths in varied ethanol content. One subtle point is the validity of self-similarity, the key assumption of scaling analysis, of PNIPAM chains with the cooperative adsorptions of water and alcohol molecules. It has been shown that every repeating unit of PNIPAM in aqueous solution is strongly hydrated by up to 11 water molecules in a cooperative way at room temperature.65 As the minimum unit of the chain self-similarity must be one or several repeating units, the scaling analysis can still be applicable here for the PNIPAM cononsolvency process that is subjected to cooperative adsorptions of abundant solvents.
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CONCLUSIONS The ultrahigh sensitivity of fluorescence correlation spectroscopy (FCS) enables measurements of polymers in extremely dilute solution and of relatively low molecular weight. FCS has showed its advantage in the investigation of polymer single chains inside complex multicomponent systems, which used to 9202
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be difficult for conventional scattering measurements. Although the accuracy of FCS measurements is affected by the mismatch of the refractive index between the sample and the objective lens, this difficulty can be overcome by adopting optimal experimental condition, for example, by taking a relatively small penetration depth of the focus at 25 μm. The hydrodynamic radius of moderately long PNIPAM chains in the cononsolvency process has been measured successfully. For the synthesized PNIPAM samples covering a range of molecular weights (Mw: 9.7 × 103−1.42 × 105 g mol−1; N: 86−1260), the dynamic scaling index in the relationship of RH ∼ Nv varied from 0.56 to ∼0.3 and then to 0.57 with the increase of ethanol content from 0.0 to 1.0 and shows a progressive, re-entrant feature of coil-to-globule-to-coil conformational transition. The scaling analysis with the aid of FCS can be considered as a new experimental method to study conformational transition of polymers, particularly with moderate and even shorter chain lengths. The variation of the conformation and scaling index of PNIPAM single chains is highly asymmetrical with the change in the ethanol content, which indicates a much stronger effect of ethanol at the process of competitive cooperative adsorption onto PNIPAM chains, compared with that of water molecules. The comparison of present data and reported results shows no distinct dependence of the cononsolvency process in the water−ethanol mixture on the polymer concentration, further excluding the previously proposed model of the polymerinduced complex formation between two solvents.1 Moreover, the effect of molecular weights on the cononsolvency process is also exposeda higher sharpness of the conformational transition is with PNIPAM of a higher molecular weight.
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ASSOCIATED CONTENT
S Supporting Information *
Synthesis, fluorescence labeling, and characterization (1H NMR, SEC, MALDI-TOF mass spectroscopy, and the labeling efficiency) of PNIPAM, DLS, FCS, PCH, and the fit of scaling index. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research is partially supported by National Natural Science Foundation of China (NSFC 20925416, 51173197) and Chinese Academy of Sciences (KJCX2-YW-H19, KJCX2-EWW09). Our thanks are given to Prof. Taihyun Chang and Mr. Joongsuk Oh (POSTECH) for SEC characterization, to Prof. Charles Han (ICCAS) and Prof. Guangzhao Zhang (USTC) for the helpful discussions on the cononsolvency, and to Dr. Shih-Chu Liao (ISS Inc.) and Prof. Jörg Enderlein (Universität Göttingen) on FCS.
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