Exploring Microenvironment Acidity Inside the Solvent-Filled Pores of

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C: Surfaces, Interfaces, Porous Materials, and Catalysis

Exploring Microenvironment Acidity Inside the Solvent-Filled Pores of Mesoporous Silica Thin Films via Single Molecule Spectroscopy Jingyi Xie, Jiayi Xu, Xiaojiao Sun, Huan Wang, Daniel A. Higgins, and Keith L. Hohn J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.9b05111 • Publication Date (Web): 29 Jul 2019 Downloaded from pubs.acs.org on August 11, 2019

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The Journal of Physical Chemistry

Exploring Microenvironment Acidity Inside the Solvent-Filled Pores of Mesoporous Silica Thin Films via Single Molecule Spectroscopy Jingyi Xie,a Jiayi Xu, a Xiaojiao Sun, a Huan Wang, a Daniel A Higgins,b,* and Keith L Hohna,* aDepartment

of Chemical Engineering, Kansas State University, 1701A Platt Street, Manhattan, KS 66506, United States

bDepartment

of Chemistry, Kansas State University, 1212 Mid-Campus Drive North, Manhattan, KS 66506-0401, United States

* Corresponding authors: [email protected] and [email protected]

Abstract Single molecule (SM) spectroscopy was used to study the local acidity inside solutionfilled silica mesopores. The dual emission, pH-sensitive dye C-SNARF-1 was used as the fluorescent probe.

Mesoporous materials were prepared as thin films supported on glass

substrates. A microfluidic device was used to seal the upper surface of the films and to allow for their exposure to flowing aqueous solutions of the dye under different pH and ionic strength conditions. Single molecule data were collected by two-color wide-field fluorescence imaging in the pseudo-total internal reflection fluorescence mode. Pairs of fluorescence images were acquired simultaneously in bands centered around 580 nm and 640 nm, each having a 40 nm bandwidth. The ratio of C-SNARF-1 emission in these two bands (I580/I640) is highly sensitive to pH in bulk solution. The SM data show that C-SNARF-1 remains sensitive to pH in mesoporous materials but that the emission ratios obtained are often larger than in bulk solution. Results obtained as a function of ionic strength and interpreted in conjunction with simulations are consistent with

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perturbation of the dye response by coulombic interactions with charged sites (i.e. deprotonated silanol groups) on the pore surfaces. When the mesoporous films are exposed to low pH solutions or high ionic strength buffers, the SM emission ratios are similar to those obtained in bulk solution, while those recorded at intermediate pH are highly variable, due to the aforementioned coulombic interactions. When considered together with the results of simulations, the SM results reveal that the pH inside the mesopores is largely independent of solution pH under low ionic strength conditions and is instead determined by the ionization of the surface silanols. The pH within the mesopores responds most strongly to changes in solution pH at high ionic strength and at low or high pH. The silanol group pKa is estimated to fall between 7.1 and 8.7 from the SM results. Overall, these studies provide new information on the factors affecting the pH within the confined environments of solution-filled silica mesopores. They also provide a better understanding of the efficacy of pH measurements in porous materials made using organic probe dyes.

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Introduction Mesoporous silica materials have attracted considerable attention recently due to their potential for broad application in catalysis,(1, 2) nanofluidic devices,(3, 4) and gas sensors.(5) The pore surfaces within these materials are commonly covered in weakly acidic silanol groups. These groups provide mesoporous materials with many of their functional attributes. For example, owing to their high surface area and acidity, porous silica structures have been found to exhibit high catalytic activities in various processes, including dehydration,(6) hydrolysis,(7) and cracking reactions.(8) Shape-selectivity can also be achieved with mesoporous catalysts, when large molecules are confined inside the pores.(1, 9, 10) In one such study, SBA-15- and MCM-41supported sulfonic acid catalysts were found to be selective for monoglyceride synthesis from glycerol and fatty acids.(9) The silanol surface sites are also very important to applications of mesoporous silica in nanofluidic technologies.(11) Because at least some of the silanol groups remain deprotonated at all but very low pH, they play a profound role in modulating binding of molecules to silica surfaces, and the entry of charged species into silica mesopores.(12) The ensemble-averaged acidity properties of mesoporous silica materials have been studied previously by methods such as temperature-programmed desorption (TPD), FTIR, and NMR spectroscopy.(13-16) While these measurements provide valuable insights into the average properties of the materials, information on the local properties of individual microenvironments is lost. However, the local distribution of acidic sites is known to influence the catalytic activity of meoporous materials.(17) Single molecule spectroscopy and closely related methods are now being employed to probe their local properties.(18-23) The diffusion of dye molecules in hexagonal and laminar mesostructures has been investigated both via fluorescence correlation spectroscopy (FCS)(24-26) and single-molecule tracking (SMT).(27-32)

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wobbling of molecules within the pores has also been investigated,(33, 34) and polarity-sensitive dyes have been used to probe the dielectric properties of the pore-filling medium.(35) Despite extensive interest in these materials, studies of the acidity of silica-based porous materials by single molecule methods are rare. Recent reports include work by Ristanović et al., who studied reaction kinetics at the single turnover level catalyzed by the Brønsted acidity of zeolite ZSM-5.(36, 37) Earlier studies by our groups probed the acidity in silica materials incorporating disordered micropores(24) and in aluminosilica materials comprising ordered mesopores.(38) In both cases, the materials were studied in a semi-dry state, after having been exposed to different aqueous solutions. Both employed the pH-sensitive dye C-SNARF-1, which is also used in the present studies. C-SNARF-1 has been employed in pH measurements since 1991.(39-41) As a dual emission probe, it emits into two different spectral bands associated with its protonated and deprotonated forms. The ratio of the emission in these two bands provides the means to measure the pH of its local environment. In such studies, the response of the dye is first calibrated by determining its emission ratio in solutions having different pH values. While solution phase measurements of pH using C-SNARF-1 are rather simple, several challenges hinder its application as a single molecule probe of Brønsted acidity in mesoporous materials. Most importantly, the pH-dependent response of the dye can change when it is entrapped inside these materials.(42-44) The origins of these changes are often ambiguous,(42-44) but several possible factors are readily identified. For example, it has been reported that adsorption of the dye onto certain surfaces may alter its response.(45-47) Similar effects have also been observed for other pH sensitive dyes.(48) In our own studies of pH in microporous(42) and mesoporous(38) materials, the C-SNARF-1 emission ratio measured from single molecules was often found to be significantly larger and more

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variable than in solution. The variability of the emission ratio was attributed primarily to materials heterogeneity. Unfortunately, it was not clear that all dye molecules probed were actually incorporated within the silica pores. Furthermore, these previous studies were performed with the materials in a semi-dry state and hence, the large emission ratios could have reflected the lack of solvent, causing the dye to respond very differently than in solution.(24, 27, 32, 38, 42, 52) In the present studies, we have revisited the pH-dependent response of C-SNARF-1, this time in ordered mesoporous silica (SBA-15) materials in which the molecules are known to be confined within solution-filled pores. To ensure the mesoporous materials remained hydrated throughout the experiments, they were covered by a PDMS-based microfluidic device. The microfluidic device incorporated two microchannels that allowed the films to be exposed to different aqueous solutions. These solutions were doped to nanomolar levels with C-SNARF-1 and had different pH values and ionic strengths. The local response of the dye within the mesopores was characterized by single molecule spectroscopic methods that involved recording wide field fluorescence images of the samples simultaneously in two emission bands. The new studies reported here offer several advantages over previous investigations of the pH within confined environments. First, each film was well hydrated prior to the start of each experiment and was maintained in that state throughout the experiments. Second, the pH and ionic strength inside the mesopores was adjusted by changing the solutions flowing through the microchannels of the microfluidic device. Third, the pH, ionic strength, and diffusion profiles of the ions found within the mesopores were simulated.

Taken together, the simulated and

experimental data afforded a better understanding of the factors governing the pH and ionic strength, and silanol charge density within the mesopores. Specifically, they reveal that the pH inside the pores was determined by dissociation of the silanol groups and was largely independent

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of external-solution pH under solutions of intermediate pH and low ionic strength. The pH within the mesopores could be controlled by changing the pH of the external solution when the films were exposed to solutions of low pH or under high ionic strength buffers.

The large emission ratios

recorded in our earlier work were again observed in the present studies, demonstrating that they were not caused by a lack of solvent. Rather, coulombic interactions between the dye molecules and deprotonated silanols on the pore surfaces were identified as the likely cause. The knowledge gained from these studies pave the way towards a better understanding of the factors affecting the pH within silica mesopores and also provide knew knowledge on the conditions under which quantitative measurements of pH within nano/mesostructured materials can be made using pHsensitive probe dyes.

Experimental section Materials C-SNARF-1 was purchased from Invitrogen. Both tetraethyl orthosilicate (TEOS, 99 %) and Pluronic P-123 were obtained from Sigma-Aldrich. Zipcone UA (PP1-ZPUA) was obtained from Gelest Inc. Norland Optical Adhesive 74 was obtained from Norland Products. All were used as received and as directed by the supplier.

Sample preparation Mesoporous silica (SBA-15) thin films were prepared on glass microscope coverslips using a dip-coating procedure. The coverslips were cleaned prior to use by rinsing with acetone and high purity (18 M•cm) water. They were subsequently dried under flowing nitrogen, and finally treated in an air plasma for 5 min. To prepare the silica sol for dip coating, P123 (0.1337 g) and TEOS (0.45 g) were dissolved in ethanol (17.4 g) with vigorous stirring. Immediately thereafter,

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water (0.234 g) and 0.1M HCl (0.045 g) were added and the solution was stirred for an additional 2 h at room temperature. During the dip coating process, the coverslip was immersed into the silica sol and withdrawn vertically at a speed of 0.025 mm/s. Dip coating was conducted at room temperature and 40 % relative humidity. The as-prepared thin films were then aged for 48 h and calcined in air at 350 oC for 6 h. Finally, the calcined thin films were hydrothermally treated in 0.1 M HCl solution at 120 oC for 3 h. The microfluidic device was prepared by a replica molding method (See supporting information, Figure S1). Positive reliefs for PDMS molding were prepared on glass slides by a photolithography method, using a printed photomask. A 40:60 (v/v) mixture of Zipcone UA, an acryloxy-terminated siloxane polymer, and Norland Optical Adhesive 74 (NOA 74), an acrylate based polymer were employed as the photoresist. The photoresist mixture was sandwiched between the printed photomask and the glass slide, and was then exposed to UV light through the photomask for a period of 4 s. The remaining unexposed polymer was subsequently rinsed from the surface using acetone.

In preparation of the PDMS block, prepolymer and hardener were

mixed in a 10:1 ratio and cast on the positive relief. The PDMS coated mold was subsequently cured at 80 oC for 48 h. The PDMS block could be easily peeled from the relief after full curing. Assembly of the microfluidic device used to fill the mesopores with solution involved bonding the PDMS block to the mesoporous silica film. For this purpose, both the PDMS block and the freshly prepared, hydrothermally treated SBA15-coated glass coverslip were plasma treated for 5 min and held in contact with each other for 4 h. The PDMS block was irreversibly bound to the mesoporous silica in this process.

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Instrumentation and methods C-SNARF-1 was loaded into the mesopores by flowing 10 nM dye solutions having different pH values and ionic strengths through the two microfluidic channels. The solution flow was maintained for a period of 48 h prior to each experiment and was continued during the SM experiments to ensure the pores remained hydrated. All SM studies were carried out on a wide-field fluorescence microscope that has been described previously (see supporting information, Figure S2 and(53)). Two color imaging with CSNARF-1 loaded into the mesoporous material was employed to assess the local pH. For this purpose, the single molecule fluorescence was divided into two signal beams, using a 605 nm dichroic beam splitter. The two signal beams were individually directed through two band-pass filters: one centered at 580 nm, the other at 640 nm, both having 40 nm passbands. Images at these two wavelengths were simultaneously recorded on a back-illuminated EM-CCD camera (Andor iXon DU-897). Images were acquired under conventional gain, using an integration time of 0.3 s. The individual, well-separated dye molecules appeared as pairs of spots in the two images. Details on image analysis are given in supporting information. The pH-dependent response of the dye was calibrated by recording ensemble averaged emission ratios from C-SNARF-1 aqueous solutions of different pH values on the same microscope used in the SM studies. Small-angle X-ray scattering was used to verify mesoporous silica films were obtained. The instrument and methods employed are also described in supporting information.

Results Experimental design for SM studies The goals of the present studies were twofold: to better understand i) the response of the pH sensitive dye C-SNARF-1 when loaded into the pores and ii) the factors that determine the pH

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inside the solvent-filled pores of mesoporous silica films. In order to allow the films to be continuously exposed to aqueous solution, and to ensure that most of the C-SNARF-1 molecules detected were present inside the pores, a PDMS block incorporating microfluidic channels was attached to the film during sample fabrication. A diagram and photograph of the PDMS block are shown in Figure 1. Several different aqueous solutions having different pH values and ionic strengths were flowed through the microchannels beginning 48 h prior to the start of each experiment. The solution flow was maintained for the duration of each experiment. Both the solvent and the dye molecules migrated laterally into the pores of the SBA-15 film under the PDMS block during the initial 48 h of solution exposure.

Figure 1. (a) Illustration of the microfluidic device used for SM spectroscopy measurements. (b) Image of microfluidic channels and sealed region between the channels. Single molecule data were taken as functions of position in the direction perpendicular to the microchannels in the sealed region.

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Figure 1b shows that the width of the region sealed by the PDMS block was ~ 175 µm, slightly smaller than the field of view in the microscope eyepiece. It is critical for the present experiments that the fluorescence signals come from dye molecules found in the silica mesopores. This was verified by comparing dye-doped samples to several controls (see Figure S6). From these controls, it can be concluded that most of the C-SNARF-1 dye molecules detected in images of the SBA-15 materials were found within the mesopores of the silica films. More spots were generally observed when ordered mesoporous materials were employed and the number of spots observed with increasing film thickness, both indicating the presence of dye molecules within the films.

SM studies of acidity in silica pores Figures 2 shows images of C-SNARF-1 molecules in the PDMS sealed region of a mesoporous silica film for the 640 nm and 580 nm image channels. These data were acquired after exposure of the film to dye solution for 48 h. In order to determine the emission ratio from each molecule, the individual fluorescent spots they produced were fit to Gaussian functions. The amplitude of each Gaussian was used in the emission ratio calculation. The vast majority of molecules detected were found to be immobile on the experimental time scale. While some molecules moved between video frames, these exhibited a “hopping-like” motion indicating they moved rapidly between adsorption sites. These molecules produced round fluorescent spots in each video frame, rather than the blurred streaks expected for molecules that were continuously mobile. Therefore, virtually all of the molecules analyzed in these studies are concluded to be adsorbed to the surfaces of the silica pores or otherwise entrapped within the pores. Very few, if any, molecules appeared to remain in solution and continuously mobile. It is possible that such

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molecules were present in these samples, but the video data obtained indicate that any such molecules would be moving too rapidly to be detected.

Figure 2. (a,b) Single frame images of C-SNARF-1 dye molecules in mesoporous silica films for the 640 and 580 nm detection channels, respectively. The pH of the HCl solution flowing through the adjacent microchannel was 4.7. The grayscale in both images depicts 0 to 500 counts.

The response of the C-SNARF-1 dye to changes in the pH inside the silica pores was investigated by flowing solutions of different pH through the microchannel adjacent to the region being imaged. Aqueous HCl and NaOH solutions were used to adjust the pH inside the pores. At the same time, deionized water was flowed through the microchannel on the other side of the PDMS block, producing what is nominally a pH and ionic strength gradient through the films. Histograms of the SM emission ratios were used to visualize the response of a large population of C-SNARF-1 dye molecules within the mesopores. In this case, the SM emission ratios for the individual molecules were determined by averaging their values across several video frames. Data acquired from five different locations along a direction perpendicular to the expected pH and ionic strength gradient were compiled and plotted in one histogram to ensure that enough data were collected. Figure 3 shows the histograms obtained from the mesoporous silica films when solutions of several different pH values were flowed through the microfluidic channels. 11



The data shown in Figure 3 reveal a complex dependence of the C-SNARF-1 response on the solution pH. First, the emission ratio distributions appear to be monomodal at low pH but are clearly at least bimodal at intermediate and high pH. To better characterize these distributions, they were fit to single Gaussian functions at pH = 1.8 and 2.8 and to a pair of Gaussian functions at the higher pH values. When low pH HCl solutions (pH 1.8 and 2.8) were flowed through the microchannel, small peak emission ratios (1.33 and 1.34) were obtained from the narrow distributions. At pH = 4, two different distributions were observed, with the lower emission ratio distribution peaked at ~ 1.7 and the higher at ~ 4.5. Interestingly, an increase in emission ratio is expected from the bulk solution data for this range of pH values, as shown in Figure S4 in supporting information, and the distribution centered near 1 - 2 is concluded to reflect this trend. The distribution peaked at ~ 4.5 is concluded to be anomalous (see below), as such high values are never observed in bulk solution. With a further increase in the solution pH, the peak position of the distribution found at smaller emission ratios begins to fall, as expected from the bulk results (Figure S4). At pH 4.7, 6, 6.6, and 8.9 values of 1.4, 1.3, 1.2, and 1.0 are obtained. However, the rate of decrease with increasing pH is dramatically smaller than expected from the bulk solution results. Furthermore, the anomalous distribution at higher emission ratios shows no clear trend, although the population of molecules exhibiting high emission ratios abruptly drops at the highest pH employed (8.9).

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Figure 3. SM emission ratio (I580/I640) histograms for C-SNARF-1 in the pores of SBA-15 films. The pH value of the aqueous solution flowed through the microchannel adjacent to the measured region is shown on each histogram. Deionized water was flowed through the other microchannel. Emission ratios were calculated by averaging the values obtained from each molecule across several video frames. The solid blue lines show fits of the data to Gaussian functions.

Similar trends in single molecule emission ratios have been reported previously.(44) For example, both Fu et al.(42) and Sun et al.(38) reported that the standard deviation of the emission ratio distributions reached a maximum at pH 6 or 7 and decreased at both lower and higher pH values. The bimodal distributions shown in Figure 3 were also observed in our previous studies

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of aluminosilicate thin films.(38) However, the imaging in these previous studies was done with the films in a semi-dry state. Hence, it was possible the larger emission ratios were due to the presence of dye molecules in dry environments. The present studies were performed with the silica materials fully hydrated. Hence, it is concluded that the anomalously large values obtained do not result from variations in the solvent content of the individual nanoscale environments.

Simulations of pore properties In order to better understand the nanoscale environments within the SBA-15 pores, simulations of the pH, ionic strength, and surface charge were performed. The equations given below define the model employed (see support information for details). Eqn. 1 is the NernstPlanck equation and gives the flux, Ji, for each electrolyte, i, (where i = H+, Cl-, etc.). Eqn. 2 describes the chemical equilibrium between silanol groups and H+ ions. Eqn. 3 is the Poisson equation and is used to represent the electrostatics of the system. In Eqns. 1-3, Ci represents the concentration of species i; Di is its diffusion coefficient; and  is the electrical potential. The pKa for the dissociation of the weakly acidic silanol groups is reported to fall between 7.1(54) and 8.5.(55) Here, we assume the pKa of the silanol groups is 7.5.(56) To simplify the calculations, steady state conditions are assumed and Dirichlet boundary conditions are employed. ∂𝐶𝑖(𝑥)

𝐽𝑖(𝑥) = ― 𝐷𝑖

∂𝑥

𝐾𝑠𝑖𝑙𝑎𝑛𝑜𝑙 =

𝑧𝑖𝐹

∂𝛷(𝑥) ∂𝑥

― 𝑅𝑇 𝐷𝑖𝐶𝑖

𝐶𝐻 + 𝐶𝑆𝑖𝑂 ― 𝐶𝑆𝑖𝑂𝐻

∑𝑛𝑖𝑧𝑖𝑒 = ― 𝜀𝜀0

∂2𝛷 ∂𝑥2

Eqn. 1 Eqn. 2 Eqn. 3

Figure 4a shows the simulated hydronium ion concentration as the pH changes inside the SBA-15 pores. The results show that when sufficiently dilute acid (< 10-4 M) or deionized water

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are flowed through the microchannels, pH inside the pores is determined primarily by deprotonation of the silanol groups on the pore surfaces. The pH inside the pores is estimated to be ~ 3.4 under these conditions, for silanol groups having pKa = 7.5 and a density of 1.8×109 silanols/µm3.(57, 58) Deprotonation of the surface silanols leaves negative charges on the pore walls. These surface charges, coupled with the low solution ionic strength in the absence of cations other than H+, effectively prevent the protons from either leaving the pores or being neutralized by the external solution. The value of [H+] in this case will be referred to as the “intrinsic acid concentration” and the density of deprotonated silanol groups is approximately equal to the intrinsic acidity inside the mesopores (Figure 4c). The ionic strength (Figure 4b) in solution is half [H+] because Cl- is excluded from the mesopores due to the overlap of the electrical double layers.

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Figure 4. Simulated spatial variations in (a) pH, (b) ionic strength in solution given as log(µ), and (c) deprotonated silanol concentration, also plotted as the log of its value, across the region sealed by the PDMS block. The channel through which deionized water was flowed is at 0 µm, while the channel through which the acidic solutions were flowed is at 175 µm (see legend in a).

When the concentration of HCl flowing through one of the microchannels is higher than the intrinsic acid concentration, deprotonation of the silanol groups is suppressed (Figure 4c). Under these conditions, the external solution sets the pH inside the pores (Figure 4a). Furthermore, the decrease in concentration of deprotonated silanol groups reduces the surface charge density.

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The increase in solution ionic strength (Figure 4b) causes the electrical double layer thickness within the pores to decrease, allowing additional ions (i.e., Cl-) into the pores.

Impact of ionic strength on the emission ratio of C-SNARF-1 Some of the complexities observed in the pH-dependent C-SNARF-1 emission ratios (Figure 3) likely arise from interactions between the dye and the pore surfaces. For example, as both the silica pore surface and C-SNARF-1 dye are charged at intermediate pH, coulombic interactions are expected to occur between the two, but their effect on the emission ratio has not been previously explored. If the emission ratio is at least partly dependent by these interactions, then manipulation of the solution ionic strength should yield changes in its value. To explore the impact of ionic strength on the emission ratio of C-SNARF-1 inside the silica mesopores, an HCl solution of pH 3 was flowed through one microchannel while pH ~ 7 phosphate buffer saline (PBS) solutions of different concentrations were flowed through the other. With this arrangement, the solution pH at one boundary of the PDMS-sealed SBA-15 film was poised near 3, while at the other, the solution was of nearly neutral pH (i.e., 7.4, 7.3 and 7.0 for 1X PBS, 0.2X PBS and 0.05X PBS, respectively). The base PBS solution (1X PBS) contained 0.138 M NaCl and 0.027 M KCl. This solution was diluted to produce 0.2 X and 0.05X PBS solutions. Figure 5 plots representative histograms of the emission ratio acquired under different ionic strength conditions at similar positions in the mesoporous silica films. These positions were ~ 4070 µm from the microchannel through which the PBS buffer solutions were flowed. Two distinct emission ratio distributions peaked near values of 1 and 4 were again observed at low ionic strengths, with a single distribution peaked near 1 occurring at the highest ionic strength. The population of molecules with emission ratios near 4 appears to be greatly reduced at higher ionic

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strengths, providing clear evidence that its appearance can be linked to coulombic interactions between the dye and pore surface.

Figure 5. SM emission ratio (I580/I640) histograms for C-SNARF-1 in SBA-15 films exposed to PBS buffer solution in one microchannel (0 μm) and pH = 3 HCl solution in the other (175 μm). The SM emission ratio histograms obtained from SM data recorded at distances of 44 μm, 71 µm and 76 µm from the buffer filled channel in the presence of 0.05X, 0.2X and 1X PBS, respectively are plotted in (a), (b) and (c).

Further evidence for the importance of coulombic interactions in producing the anomalous emission ratios was obtained by acquiring data as a function of position across the mesoporous films. Figure 6 plots the mean emission ratios obtained as a function of position for all three ionic strengths. It is noteworthy that at every position studied, the average emission ratio was again found to decrease with increasing ionic strength, as depicted by the individual data points (squares, triangles, and diamonds), consistent with the results shown in Figure 5.

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Interestingly, the position dependence of the emission ratio exhibits the opposite trend with respect to the expected local ionic strength. Estimates of the local ionic strength and proton concentration were obtained from the simulations described above and are also plotted as a function of position in Figure 6a,b (red lines). The results in this case show that the mean emission ratio actually decreases as the ionic strength decreases with position. However, it is noteworthy that the proton concentration also increases along the pore axis at the same time. As shown in Figure S4, for pH < ~5, the emission ratio decreases with decreasing pH. Therefore, as the pH is well below 5 in these experiments and decreases with position (see Figure 6), it is concluded that the pH dependence of the emission ratio dominates the position-dependent response, rather than its ionic strength dependence. It is also noteworthy that the simulations predict a decrease in ionic strength within the pores of only about a factor of 8, when the solution ionic strength deceases by a factor of 20 (1X PBS to 0.05X PBS). This observation demonstrates that the ionized silanol groups within the pores play a significant role in determining the ionic strength within the pores in the presence of dilute buffer solutions.

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Figure 6. SM emission ratio for C-SNARF-1, and simulated [H+] and ionic strength (μ) across the PDMS sealed SBA-15 film with PBS buffer solution flowing through one microchannel (0 μm) and pH = 3 HCl solution flow through the other microchannel (175 μm): (a) average emission ratio across the PDMS sealed SBA-15 film when (□) 0.05X PBS, (∆) 0.2X PBS, (◊) 1X PBS solution is flowed through the buffer channel; simulated ionic strength across the PDMS sealed SBA-15 film for (—) 0.05X PBS, (∙∙∙∙) 0.2X PBS, (----) 1X PBS solutions; (b) simulated [H+] across the PDMS sealed SBA-15 film for (—) 0.05X PBS, (∙∙∙∙) 0.2X PBS, (----) 1X PBS solutions.

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Discussion The primary objectives of this study were to better understand the response of the pHsensitive dye C-SNARF-1 to pH within mesoporous materials and to understand the factors governing the acidity/basicity of solutions confined within silica mesopores. Obtaining a better understanding of the dye response within confined environments is critical to its future applicability in studies of solution acidity/basicity within mesoporous heterogeneous catalysts. Indeed, previous studies have shown that pH-sensitive probe dyes that are normally well-behaved in solution often yield broad emission ratio distributions when employed in confined environments.(38, 44) These results may reflect significant heterogeneity in the acidity of the environments themselves, or they may result from a change in the dye response to its environment. One complicating factor in the interpretation of the data reported in some previous studies(38, 42) was that the solvent content of the films being investigated was not well controlled. The present studies employed silica mesopores that had been carefully hydrated to ensure they remained filled with solvent throughout the experiments. Nevertheless, the experiments once again yielded broad, bimodal emission ratio distributions for C-SNARF-1 under certain conditions. As a result, it is concluded that the broad distributions observed in earlier studies(38, 42) were likely not due to heterogeneity caused by uncontrolled hydration of the films. Rather, based on the present results, the broad distributions and large emission ratios are concluded to reflect coulombic interactions between the dye and the pore surface. Figure 3 shows that the largest emission ratios and the broadest emission ratio distributions were obtained for low ionic strength solutions (diluted HCl solutions), when the pH was between 4 and 6.6. These distributions often appear to be bimodal, with one component peaked near an emission ratio between 1 and 2, and the other near 4. Values between 1 and 2 approach those obtained from C-SNARF-1 in bulk

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solutions of the same pH, while emission ratios near 4 are anomalously large. When the pH of the solution in the microchannel was reduced below 4 or increased above 6.6, the emission ratios became significantly less variable, producing narrow distributions dominated by values between 1 and 2. The emission ratios near 4 are thus attributed to molecules that experience strong coulombic interactions with the pore surface, while values between 1 and 2 are concluded to arise from molecules that only weakly interact with the charged surfaces of the pores and hence yield a response more similar to what is observed in bulk solution. The data shown in Figures 5 and 6 provide supporting evidence for the above conclusions. Specifically, these data show that the mean emission ratio from the dye molecules becomes smaller and less variable as the ionic strength is increased. In contrast, experiments performed in bulk solution showed no clear dependence of the C-SNARF-1 emission ratio on solution ionic strength (see Figure S5, supporting information), indicating that the C-SNARF-1 response is not perturbed by the presence of the ions alone. These observations again provide support for the conclusion that strong coulombic interactions between the dye molecules and the pore surfaces are the primary cause of changes in the C-SNARF-1 response to its local environment. The decrease in the dye emission ratio and narrowing of the emission ratio distribution observed at low pH (1.8 and 2.8), as shown in Figure 3, provide supporting evidence for the role of coulombic interactions in altering the dye’s response. The pI of silica is known to be around 2.6.(3) Thus, the surface will carry only slight positive and negative charges, respectively, at pH 1.8 and 2.8, respectively. Similarly, the second and third protonation equilibria of C-SNARF-1 have pKas near 3-4,(38) suggesting that the dye carries only a single positive charge in the low pH solutions. The lack of charge on the pore surface eliminates the possibility for coulombic interactions between the dye and pore surface, causing it to exhibit a more solution-like response.

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The emission ratio histogram also narrows and shifts to smaller values at high pH (8.9, see Figure 3). In this case, both the pore surface and the dye are expected to carry large negative charges. Thus, coulombic repulsion likely keeps the dye molecules relatively far from charged sites on the pore walls and its response follows more what would be expected from solution. Previous discussions of this narrowing at high pH have suggested it might reflect a buffering of the nanoscale environments in the films by silanol sites having majority pKas from 7-8(12, 59). This interpretation cannot be discounted in the present studies. The simulation data shown in Figure 4 provide a clear explanation for the apparent attenuation in the response of the dye to changes in the pH of the solution to which the films are exposed. Specifically, these simulations show that the pH within the pores remains largely invariant when exposed to low ionic strength solutions (i.e., diluted HCl) in the microchannels. The pH inside the mesopores is set by the surface silanols under these conditions and hence, the emission ratios observed in Figure 6 are all very similar, because the pH within the pores does not change much when the solution filling the microchannels is changed between pH 4 and 6.6. The simulations in Figure 4 show that the pH within the mesopores is only expected to change by 0.01 units across this pH range. Therefore, no change in emission ratios is expected under these conditions. Based on the discussion above, we can estimate the intrinsic acid concentration of the silica and the pKa of the silanol groups on the pore surfaces. Note that pH within the mesopores will remain constant until the pH of the HCl solution is less than the intrinsic pH inside the pores. The present experiments showed that the peak (or average) emission ratio falls between 1 and 2 when HCl solutions having pH = 3.2 were flowed through the microchannel (data not show). Thus, we estimate that the intrinsic pH value inside the SBA-15 pores is between 3.2 and 4. With the silanol

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group density mentioned above, the estimated pKa of the silanol groups inside the mesopores is between 7.1 and 8.7, which is consistent with previously reported values(54-56). Besides SBA15, this method for the estimation of acidity inside mesopores could be extended to other mesoporous materials utilized in catalysis or nanofluidic technology. Both the intrinsic pH inside solvent-filled pores and the pKa of acidic sites in these materials could be determined by this method.

Conclusion In this work, we sought to employ SM spectroscopic methods to make quantitative measurements of the local pH within the solution-filled pores of SBA-15 films. The dye CSNARF-1 was employed as a ratiometric fluorescent probe of the local pH. A microfluidic device placed over the silica film was utilized to ensure that the dye molecules being probed were present within the solution-filled pores of the film. The SM data were acquired as a function of the pH and ionic strength of solutions flowed through the channels of the microfluidic device. Histograms of the dye emission ratios in the solution-filled pores were found to be similar to those obtained previously in relatively dry films. Both the previous and present experiments produced bimodal distributions in the emission ratio at intermediate pH and low ionic strength. The ionic strength dependence of the dye emission ratio data revealed that coulombic interactions between the dye and charged sites on the silica pore surfaces likely caused changes in the dye response. The results reveal that the pH within the pores is controlled by the solutions flowing through the microfluidic channels only when [H+] (or [OH-]) in these solutions is greater than the intrinsic acid concentration of the silica pores, as set by the silanol sites on the pore surfaces. In these cases, emission ratios similar to those obtained from bulk solutions of the dye were obtained. For solutions of intermediate pH and low ionic strengths, in which the silanol sites control [H+]

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within the pores, very broad, bimodal emission ratio histograms were obtained. It was shown that these histograms became narrower and more monomodal when buffer solutions having higher ionic strengths were employed. The average emission ratios also exhibited a trend towards values obtained in bulk solution with increasing ionic strength. It was concluded that the broad emission ratio histograms and large emission ratios observed for C-SNARF-1 in solutions of intermediate pH and low ionic strength result from coulombic interactions between the C-SNARF-1 molecules and silanol-derived charged sites on the pore surfaces. Only those emission ratios that fall in the solution-like component of the distribution reflect the pH of the solution within the pores. These observations provide a better understanding of the spectroscopic response of pH-sensitive dye molecules within the tightly confined environments of mesoporous silica films. Importantly, the results obtained also provide new information on the mechanisms by which the pH within silica mesopores is set by the silanol groups on the pore surfaces and by protons contributed by external solutions. Specifically, it was demonstrated that only solutions of low pH or high ionic strength can modulate the pH within the nanopores. Exposure of the mesoporous silica to solutions of intermediate pH and low ionic strength has little impact on the pH within the mesopores and it is the ionization of surface silanol groups that sets the pH in these cases. The knowledge gained will ultimately aid in the understanding of the Brønsted acidity of mesoporous materials like those being developed for use in heterogeneous catalysis. The pKa of the silanols was estimated to fall between 7.1 and 8.7 based on the observations made in these studies. These studies will pave the way for more quantitative measurements of pH in other nano/mesostructured materials and biosystems using pH-sensitive probe dyes.

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Acknowledgements We gratefully acknowledge funding of this work by the U.S. National Science Foundation (CHE-1664664). The U.S. Department of Energy (DE-SC0002362) provided the microscope employed. Takashi Ito is thanked for providing access to the spectroscopic ellipsometer, Brian Grady is acknowledged for SAXS experiments, and Khanh Hoa Tran Ba and Dipak Giri are thanked for their help with the single molecule detection experiments.

Supporting Information Additional information on the methods, samples, control experiments and model employed are provided, free of charge, as supporting information at pubs.acs.org.

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