Article pubs.acs.org/Macromolecules
Synthesis and Characterization of Dextran−Tyramine-Based H2O2‑Sensitive Microgels Hua Wei,† Jianda Xie,‡ Xiaomei Jiang,§ Ting Ye,† Aiping Chang,† and Weitai Wu*,† †
State Key Laboratory for Physical Chemistry of Solid Surfaces, The Key Laboratory for Chemical Biology of Fujian Province, and Department of Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, Fujian, China ‡ School of Materials Science and Engineering, Xiamen University of Technology, Xiamen 361024, Fujian, China § Clinical Laboratory, Huli Center for Maternal and Child Health, Xiamen 361009, Fujian, China S Supporting Information *
ABSTRACT: We report a type of polymer microgel that can undergo a rapid and highly sensitive volume change upon adding H2O2. Such a H2O2-sensitive microgel is made of dextran−tyramine and horseradish peroxidase (HRP), which are interpenetrated in chemically cross-linked gel networks of poly(oligo(ethylene glycol) methacrylates). Unlike the H2O2-sensitive microgels reported in previous arts that typically involve degradation processes related to H2O2-induced cleavability of specific bonds, the proposed microgels can shrink upon adding H2O2 owing to the HRP-catalyzed coupling reaction of tyramine residues via decomposition of H2O2. While a fast ( 107 M−1 s−1) of the met enzyme (i.e., in the Fe(III) heme state) by H2O2 to give a green enzymatic intermediate (compound I), with the heme iron oxidized to the oxyferryl state (FeIVO) and a π-cation radical on the porphyrin ring; phenols reduce compound I in a one-electron step to compound II and compound II back to the native enzyme in a sequential one-electron step.37−40 We note that this enzymecatalyzed coupling reaction has been employed in the fast insitu formation of hydrogels for tissue engineering.32−34 Recent works entrapped HRP in poly(acrylamide) microgels, where the polymer is responsible to protect the enzyme environment, for detection of phenolic compounds.41,42 Herein, we explore the HRP-catalyzed coupling reaction in the proposed HSM microgels. Under this rational design, different form the H2O2sensitive microgels reported in previous arts that typically involve the H2O2-induced cleavability of specific bonds, the HSM microgels exploit the reaction induced increase in the
2. EXPERIMENTAL SECTION Materials. All chemicals were purchased from Aldrich. 2-(2Methoxyethoxy)ethyl methacrylate (MEO2MA), oligo(ethylene glycol)methyl ether methacrylate (MEO5MA, Mn 300 g/mol), and a cross-linker poly(ethylene glycol) dimethacrylate (PEGDMA, Mn 550 g/mol) were purified with neutral Al2O3. Dextran (Mn 14 000 g/mol) was dried by azeotropic distillation from dry toluene. N,NDimethylformamide (DMF) was dried over CaH2, distilled under vacuum, and stored over molecular sieves. Lithium chloride (LiCl) was dried at 80 °C under vacuum over phosphorus pentoxide. pNitrophenyl chloroformate (PNC), pyridine, tyramine (TA), hydrogen peroxide (H2O2, 30%), horseradish peroxidase (HRP, type VI, 298 purpurogallin unit/mg solid), N,N,N′,N′-tetramethylenediamine (TEMED), and other chemicals were used as received without further purification. The water used in all experiments was of Millipore MilliQ grade. Dex-TA Synthesis. Dex-TA was synthesized according to slightly modified literatures.32−34 Dextran was reacted with PNC to form pnitrophenyl carbonate derivatives, which were then treated with TA by aminolysis. Typically, dextran (1.0 g) was dissolved in DMF (100.0 mL, containing 2.0 g/L LiCl) at 90 °C under nitrogen. After dextran was dissolved, the mixture was cooled down to 0 °C. PNC (440.0 mg) and pyridine (180.0 mg) were added to the solution while stirring. The reaction was allowed to proceed for 12 h. The dextran activated with pnitrophenyl carbonate groups (denoted as Dex-PNC) was precipitated in cold ethanol, filtered and carefully washed with ethanol, and then dried in a vacuum. 1H NMR (D2O; see Figure S1 in the Supporting Information for the spectrum): δ 3.00−4.00 (m, dextran glucosidic 6068
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protons), 4.91 (s, dextran anomeric proton), 7.58 and 8.32 (m, aromatic protons of PNC). Then, Dex-PNC (2.0 g) was dissolved in DMF (22.4 mL, containing 280.0 mg of TA) under nitrogen. The reaction was allowed to proceed for 12 h. The product was precipitated in cold ethanol, filtered, and carefully washed with ethanol. The obtained DexTA was further purified by ultrafiltration (MWCO 3000) against water and isolated after lyophilization. 1H NMR (D2O; see Figure S2 in the Supporting Information for the spectrum): δ 2.75 and 3.05 (m, −CH2−CH2−), 3.30−4.10 (m, dextran glucosidic protons), 5.00 (s, dextran anomeric proton), 6.86 and 7.17 (m, TA aromatic protons). Microgels Synthesis. The HSM microgels were prepared by free radical precipitation copolymerization of MEO2MA, MEO5MA, and PEGDMA using APS/TEMED as an initiating system in the presence of Dex-TA and HRP. A mixture of MEO2MA (126.0 μL), MEO5MA (138.0 μL), PEGDMA (30.0 μL), Dex-TA (0.3 g), HRP (1.8 mg), SDS (50.0 mg), and water (95.0 mL) was poured into a 250 mL threeneck round-bottom flask equipped with a stirrer, a nitrogen gas inlet, and a condenser. The mixture was heated to 40.0 °C under a N2 purge. After 30 min, APS (5.0 mL, 1.5 M) and TEMED (10.0 mL, 0.2 wt %) were added one by one at 10 min interval to initiate the polymerization. The reaction was allowed to proceed for 5 h under the thermostatic conditions. The product was purified by centrifugation (20 000 rpm, 30 min, 35 °C; Thermo Electron Co. SORVALL RC-6 PLUS superspeed centrifuge), decantation, and washed with water. The HSM microgels were further purified by 2 weeks of dialysis (Spectra/Por molecularporous membrane tubing, cutoff 12 000−14 000 Da) against frequently changed water at room temperature (∼22 °C). Laser Light Scattering (LLS) Studies. A standard laser light scattering spectrometer (BI-200SM) equipped with a BI-9000 AT digital time correlator (Brookhaven Instruments, Inc.) and a Mini-L30 diode laser (30 mW, 637 nm) as the light source was used. The very dilute microgel dispersions (5.0 μg/mL) were passed through Millipore Millex-HV filters with a pore size of 0.80 μm to remove dust before LLS measurements. In dynamic LLS (DLS), the Laplace inversion (here the CONTIN method was used) of each measured intensity−intensity time correlation function in a dilute dispersion can lead to a line-width distribution G(Γ). For a purely diffusive relaxation, Γ is related to the translational diffusion coefficient D by (Γ/q2)C→0,q→0 = D, so that G(Γ) can be converted to a translational diffusion coefficient distribution and ⟨Dh⟩ distribution by using the Stokes− Einstein equation, ⟨Dh⟩ = (kBT/3πη)/D, where kB, T, and η are the Boltzmann constant, the absolute temperature, and the solvent viscosity, respectively.43 Kinetics Measurements. The H2O2-sensitive kinetics measurements were conducted on a Bio-Logic SFM300/S stopped-flow instrument equipped with a MOS-250 spectrometer, a temperature controller, and three 10 mL step-motor-driven syringes which can be operated independently to carry out single- or double-mixing. For light scattering detection at a scattering angle θ = 90° and at 22.0 °C, both the excitation and emission wavelengths were adjusted to 335 nm with 10 nm slits. Using either FC-08 or FC-15 flow cells, typical dead times are 1.1 and 2.6 ms, respectively. Both the microgel dispersions and the H2O2 solutions were passed through Millipore Millex-HV filters with a pore size of 0.80 μm to remove dust before the kinetics measurements. The final concentrations of the microgels (5.0 μg/mL) and H2O2 (50.0 μM−1.0 mM) were controlled by varying mixing ratio of the microgel dispersion to the H 2 O2 solutions. Data collection commenced 3.0 ms after activating the pneumatic drive mechanism of the stopped flow apparatus. Moreover, to study the effect of the microgel particle concentration on the characteristic response time τ, the measurements were also carried out for the HSM microgels of the concentration in the range 5−200 μg/mL, with the mole ratio of H2O2/TA being fixed at 1. Each kinetic curve reported here represents an average of at least five consecutive mixing trials. Optical Detection of Glucose in Blood Samples. A representative sample of 62 adults, 20 years of age or older, participated in this study. After an overnight fast, participants underwent an oral glucose-tolerance test in the clinical laboratory.
Typically, blood samples (7.5−15 mL) were collected from the diabetes patients and from healthy donors, and they were centrifuged and processed within 1 h of collection. The impaired fasting glucose (IFG) level ([Glu]actual) was measured using enzyme-based method, GOx-POx method. Principally, glucose is oxidized by glucose oxidase (GOx) to produce gluconate and hydrogen peroxide; the hydrogen peroxide is then oxidatively coupled with 4-aminoantipyrene (4-AAP) and phenol in the presence of peroxidase (POx) to yield a red quinoeimine dye that is measured at 505 nm.44 In the chemistry laboratory, the microgels were put in a dialysis tube (Spectra/Por molecular porous membrane tubing, cutoff 500−1000 Da) which was then inserted into whole blood or serum; then, GOx was added into whole blood or serum. After 15 min, the microgels was sampled. The ⟨Dh⟩ was measured with DLS following the methods mentioned above and repeated for five times at each sample. The apparent IFG levels ([Glu]apparent) were read out based on the calibration model constructed in PBS of known H2O2 concentrations. Other Characterizations. The pH values were measured on a METTLER TOLEDO SevenEasy pH meter. NMR spectra were recorded on a Bruker AVIII 500 MHz solution-state NMR spectrometer. FTIR spectra were recorded on a Thermo Electron Corporation Nicolet 380 Fourier transform infrared spectrometer. TEM images were taken on a JEOL JEM-2100 transmission electron microscope at an accelerating voltage of 200 kV.
3. RESULTS AND DISCUSSION The proposed HSM microgels were prepared by direct polymerization of the MEO2MA and MEO5MA monomers and the cross-linker PEGDMA initiating by APS/TEMED (a redox initiator system, where the TEMED catalyzes the decomposition of the persulfate ion to give sulfate free radicals which initiate the polymerization of monomers)15 in the DexTA/HRP aqueous solution. The preparation of nearly monodisperse POEGMA microgels, which has a lower critical solution temperature below 40 °C in water when the feeding molar ratio of MEO2MA/MEO5MA is about 1.4,45 has been well-established from the precipitation polymerization.45−47 OEGMA can complex with Dex-TA and HRP through the hydrogen bonding between the ether oxygens of OEGMA and the glycosyl groups of Dex-TA as well as the amino groups of HRP.48 The polymerization and cross-linking of OEGMA monomers that are complexed with Dex-TA/HRP can result in narrowly distributed gel particles with a chemically cross-linked POEGMA network interpenetrated by Dex-TA/HRP. In this respect, conducting the synthesis at high temperatures favors the precipitation polymerization to form microgels; however, the increase of temperature may denaturalize HRP (see Figure S3 in Supporting Information for thermal stability of HRP). Therefore, we synthesized the HSM microgels at a appropriate temperature of 40.0 °C. At the end of polymerization, the opalescent color was observed in the dispersion (Figure 1a; when cooling down to room temperature, the dispersion appeared transparent with very light blue color, since the concentration was relative low and the microgels were swollen as shown below). The TEM image shown in Figure 1b displays a typically spherical shape of the HSM microgels. In addition, a remark has to be made concerning the particle size measured from the TEM image: the particle size measured by this technique is somewhat unreliable as the microgels may have a tendency to shrink, flatten, and spread on TEM grid during the sample preparation. This can also lead to a larger apparent polydispersity; individual microgels are not expected to interact with the substrate in a homogeneous fashion.49 It is for these reasons that the particle 6069
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Dex-TA, and dextran), the characteristic amide I band of HRP appeared at 1659 cm−1, which is consistent with the bands for a α-helice structure of HRP.50 The characteristic bands of (1 → 3)-α-D-glucan at 921, 850, and 815 cm−1, the CCH and HCO bonds at 1038 and 1023 cm−1, and the in-plane vibrations (a broad hump centered at 1399 cm−1) of C−H in various C C−H structures of Dex-TA chains were also recorded. The complicated nature of the bands in the 1600−1500 cm−1 range suggests that the aromatic ring bands overlapped with the OH binding vibration bands (at 1544 cm−1) of phenol moieties in tyramine residues.51 The characteristic −COOR band of POEGMA chains appeared at 1728 cm−1.45 Moreover, the characteristic C−O−C bands of the HRP, Dex-TA, and/or POEGMA chains appeared at 1050−1150 cm−1. It is known that the oxidative reaction of HRP and H2O2 can catalyze the coupling reaction of phenol moieties in tyramine residues of Dex-TA.32−34 The coupling of phenols can take place either via a carbon−carbon bond at the ortho positions or via a carbon−oxygen bond between the carbon atom at the ortho position and the phenoxy oxygen.35−38 Figure 4 compares the FTIR spectra of the HSM microgels in the absence (with the H2O2 concentration [H2O2] = 0.0 mM) and the presence ([H2O2] = 1.0 mM) of H2O2. Upon adding H2O2, one can observe a significant decrease of the intensity of the OH binding vibration bands and the C−H in-plane vibration bands of phenols. Similar phenomena on the spectral change have been found on phenols experiencing the coupling reaction.51 The HRP-catalyzed coupling reaction of phenol moieties in tyramine residues of Dex-TA also manifests as a new band at 1034 cm−1, while the CCH/HCO bonds at 1038 and 1023 cm−1 weaken and even become undetectable. The spectral change in the CCH/HCO bands is due to the variation on the chain flexibility present in dextran around the glycosidic bonds.52 In addition, there is no change in the position of the amide I band of HRP at 1659 cm−1, indicating that the HRP is stable with its secondary structure remaining unchanged during the coupling reaction. UV−vis spectroscopy is a versatile tool for further analyzing the characteristic structure of enzymes. The peak position at 403 nm of the Soret absorption of the heme iron in HRP molecule provides the information on HRP conformation. A neglectable peak shift occurred when HRP (see Figure S5 in Supporting Information for UV−vis spectra) was subjected to the microgels synthesis, storage (after 3 months storage at room temperature, the activity reduced to about 89% of the original value; see Figure S6 in Supporting
Figure 1. (a) Photographs (left: taken at room temperature; right: taken after heating to ∼40 °C) and (b) TEM image of the HSM microgels.
sizing in the whole work was performed via DLS, a less perturbing method. Figure 2a shows DLS size distribution of the HSM microgels (5.0 μg/mL, in 5.0 mM PBS of pH 7.4) measured at 22.0 °C. A narrowly size distribution range from 200.1 to 296.2 nm is observed, and the average hydrodynamic diameter (⟨Dh⟩) is determined to be 241.6 nm. The microgels can be well reproduced from batch to batch, with a high yield of ≥86%. Meanwhile, a microgel (serve as a control) without chemical cross-linkage was also prepared following the procedures for the synthesis of the proposed HSM microgels, except that PEGDMA was not added. From Figure 2a, it can be seen that the ⟨Dh⟩ of un-cross-linked microgels (512.9 nm) is much larger than that of the HSM microgels, suggesting that the cross-linking reaction further solidifies and stabilizes the HSM microgels; this is also supported by the fact that after dialysis against water the opalescent solution was retained for the HSM microgels, while the solution became completely colorless for un-cross-linked microgels. The size of the cross-linked microgels is tunable. An increase in the feeding amount of the cross-linker PEGDMA (Figure 3a), or the surfactant SDS (Figure 3b), in the synthesis can significantly reduce the ⟨Dh⟩ of the microgels. All the chemically cross-linked microgels showed good stability (Figure 2b; also see below), with the size distribution kept nearly the same before and after dialysis, or even before and after 3 months storage at room temperature. The good stability of the chemically cross-linked microgels is a key parameter in the following characterization of the volume change upon adding H2O2. FTIR spectroscopy was used to confirm the structure of the purified HSM microgels. In FTIR spectra (Figure 4a; also see Figure S4 in Supporting Information for FTIR spectra of HRP,
Figure 2. (a) DLS size distribution of the HSM microgels (solid columns) and the un-cross-linked microgels (serve as a control; open columns) before dialysis. (b) DLS size distribution of the HSM microgels before (■) and after 3 days dialysis (●) or 3 months storage (▲) at room temperature. All measurements were made in 5.0 mM PBS of pH 7.4 at 22.0 °C. 6070
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Figure 3. DLS size distribution of the microgels synthesized with different feeding amounts of (a) PEGDMA (the feeding amount of SDS was set to 50.0 mg) or (b) SDS (the feeding amount of PEGDMA was set to 30.0 μL). The feeding amounts of MEO2MA, MEO5MA, Dex-TA, and HRP were set to 126.0 μL, 138.0 μL, 0.3 g, and 1.8 mg, respectively. All measurements were made in 5.0 mM PBS of pH 7.4 at 22.0 °C.
that the shrinking behavior of the microgels upon adding H2O2 can elegantly occur over a broad range of 0−1.5 mM (Figure 5c); at the higher [H2O2] of above approximately 1.5 mM, the change in ⟨Dh⟩ becomes faint. The shrinking ratio, in terms of ⟨Dh⟩0.0 mM/⟨Dh⟩3.0 mM, is determined to be 2.2. If we define the detection limit as the [H2O2] at which a 10% ⟨Dh⟩ change can be measured by employing the HSM microgels (5.0 μg/mL), the H2O2 detection limit was approximately 6.8 μM. After subject to the H2O2 aqueous solution, the HSM microgels were recovered simply by centrifugation and reused for the next cycle. The recycling experiments revealed the robustness of the microgels (see Figure S7 in Supporting Information). In at least five recycling experiments, no significant departure of the measured ⟨Dh⟩ against the suppositional ⟨Dh⟩ calculated from the constructed model (Figure 5c) was observed. This suggests that the microgel is easy to handle and facilitates robust stability for H2O2 detection related applications. Therefore, these results can not only provide additional, through indirect, proofs on the HRP-catalyzed coupling reaction but also foreshadow a novel type of H2O2-sensitive microgel. To estimate the response time of H2O2-sensitive size change that is potentially achievable, we have monitored the kinetics based on the scattered light intensity (I) change of the HSM microgel dispersions (5.0 μg/mL) upon adding H2O2. Typical kinetic traces associated with the response process of the HSM microgels upon adding H2O2 are shown in Figure 6a. Clearly, upon adding H2O2, the relative scattered light intensity (It/I0, where It is the scattered light intensity at a certain time t and I0 at t = 3.0 ms after mixing) of the microgel dispersions increased immediately and then leveled off gradually. According to the light scattering theory43,54 and the principles proposed for macroscopic gels,32−34 we interpret our data as follows: the HRP-catalyzed coupling reaction of phenol moieties on the microgel exterior occurs first, which leads to the formation of a shrunk, relatively dense outer shell and an abrupt increase in scattered light intensity; this initial fast process is followed by the HRP-catalyzed coupling reaction of phenol moieties on the inner cores and the relatively diffusion of water out of the shrinking microgels. The overall kinetics of the response process should be retarded by the presence of the initially formed dense surface layer, which is relatively impermeable to the inner fluid and suppresses the excretion of water from the microgel particle interior. In this respect, the change in the relative scattered light intensity might include at least two main opposing contributions among others: (i) the reaction-induced
Figure 4. FTIR spectra of the HSM microgels in the absence (0.0 mM) and presence (1.0 mM) of H2O2.
Information for storage stability), and H2O2. These results confirm that the HRP encapsulated in the microgels has a secondary structure identical to the native state of HRP in the solution. In other words, the proper orientation of the active site of the enzyme was kept during the adsorption. If the active site of the enzyme, the heme group, was denatured, the absorption band would have shifted or disappeared.53 The neglectable change in enzyme activity of HRP encapsulated in the microgels is another key parameter in the characterization of the volume change upon adding H2O2. The HRP-catalyzed coupling reaction of phenol moieties in tyramine residues of Dex−TA can introduce a second crosslinked network into the preformed first network (i.e., the chemically cross-linked POEGMA network), implying a change in the cross-linking density and thus the size of the involved HSM microgels. We then examined the effect of the concentration of H2O2 on the HSM microgels by using DLS. Figure 5a shows the DLS intensity autocorrelation functions (C(τ)) for the HSM microgels dispersed in solutions with different H2O2 concentrations of [H2O2] = 0.0, 0.1, 0.5, and 1.0 mM. In the absence of H2O2 ([H2O2] = 0.0 mM), the diffusion coefficient (D) was 1.9 × 10−8 cm2/s, corresponding to a ⟨Dh⟩ of 241.6 nm (Figure 5b); interestingly, increasing the [H2O2] to 0.1, 0.5, and 1.0 mM increased D to 3.0 × 10−8, 4.0 × 10−8, and 6.0 × 10−8 cm2/s, respectively, indicating an decrease in the ⟨Dh⟩ to 216.4, 165.8, and 129.9 nm, respectively. The decreased ⟨Dh⟩ at the higher [H2O2] verified the shrinking of the microgels upon adding H2O2. A detailed study further indicates 6071
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characteristic response time τ,31 which thus was determined to be 0.21, 0.19, 0.18, and 0.14 s upon adding with [H2O2] = 50.0 μM, 0.1 mM, 0.5 mM, and 1.0 mM, respectively. These τ values should reflect the essential feature of the isolated (rather than interacting) HSM microgel particles upon adding different concentrations of H2O2 because the concentration of the HSM microgels is below the critical value of ca. 10−2 wt % (the dynamic contact concentration) and the interparticle interactions can be completely ignored, as predicted by the concept of the screening length.55,56 This is further confirmed by the result of additional experiments that the τ value is nearly independent of the concentration of the HSM microgels in the range 5−85 μg/mL (Figure 6b). Therefore, these results demonstrate a fast time response of the HSM microgels upon adding H2O2. Regarding that the HRP-catalyzed coupling reaction of phenols in the HSM microgels via decomposition of H2O2 can be affected by the reaction-induced change in the polymer gel networks, which is solely achieved by varying the diffusion of solutions and mass transport in/out of the porous network of the microgels as proposed above, the response time of H2O2sensitive size change of the microgels should be modulated by other ways that can manipulate the microgels. Considering the temperature-responsive volume phase transition behavior of the POEGMA-based microgels (Figure 7a),45−47 we decided to perform an analysis of the influence of temperature on the response time of H2O2-sensitive size change of the HSM microgels. The characteristic response time τ of the HSM microgels (5.0 μg/mL) upon adding H2O2 (1.0 mM) over 10− 38 °C is shown in Figure 7b. It is noted that the τ does not exhibit the typical dependence on the dispersion temperature. Instead of a simple linear relationship (a conventional Arrhenius-type dependence) between ln(τ−1) and T−1 reported previously for the HRP-catalyzed coupling reaction of phenols via decomposition of H2O2,57,58 the change of ln(τ−1) with T−1 can be divided into three regions: at the dispersion temperatures of ≤18 °C (i.e., T−1 ≥ 3.43 × 10−3 K−1), the ln(1/τ) increases with temperature, whereas in the region of ca. 18−33 °C (i.e., 3.27 × 10−3 K−1 < T−1 < 3.43 × 10−3 K−1) the ln(τ−1) decreases with increasing temperature, and at ≥33 °C (i.e., T−1 ≤ 3.27 × 10−3 K−1) the ln(τ−1) increases again; in particular, the ln(τ−1) shows a local minimum right at the volume phase transition temperature (VPTT) of the HSM microgels. Clearly, the response time of the HSM microgels upon adding H2O2 can be modulated by temperature in a nonmonotonous way over a wide range. As indicated above, the modulating on the response time can be explained on the basis of the temperatureresponsive volume phase transition behavior of the microgels. At low temperatures, the microgels are in the fully swollen state; the reactants can relatively easily diffuse through the pores and start up the reaction, so that the τ decreases with temperature, as expected from Arrhenius’ law.59−61 In the vicinity of the transition region, the ⟨Dh⟩ starts to decrease (the microgels shrink), which leads to the expulsion of water and compression of the porous network. This decrease in porosity affects the diffusion of reactants through the polymer gel networks, which slows down as the temperature is increased, thus leading to an increase in the τ. The results indicate that this decrease in the diffusion coefficients is not compensated by the Arrhenius-like increase of reaction rate with temperature. Once the microgels are shrunk to some extent, the diffusion of reactants is only slightly or even no longer affected by changes in the polymer gel networks. If the dispersion temperature
Figure 5. (a) DLS intensity autocorrelation functions (C(τ)) and (b) size distribution for the HSM microgels dispersed in solutions with H2O2 concentrations [H2O2] = 0.0 (■), 0.1 (●), 0.5 (▲), and 1.0 mM (▼). (c) H2O2-dependent ⟨Dh⟩ values. All measurements were made in 5.0 mM PBS of pH 7.4 at 22.0 °C.
shrinking of the microgels cause an increase in scattered intensity due to their higher refractive index difference relative to water; (ii) the reaction reduces the overall mass of the microgel particles due to the excretion of water, leading to a reduction in scattered intensity. Thus, the initial increase in the relative scattered light intensity during the response process might indicate that reaction-induced microgel shrinking dominates initially, and the subsequent gradual decrease in the relative scattered light intensity suggests that the diffusion of small molecules out of the microgel dominates over longer time scales. Nevertheless, the relative scattered light intensity reached a stable value shortly (
