Wet-Chemical Enzymatic Preparation and Characterization of Ultrathin

Apr 19, 2014 - color camera (Leica). The CdSe/ZnS QDs solution was backfilled in the nanopores for imaging. For fluorescence imaging, a mercury lamp w...
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Wet-Chemical Enzymatic Preparation and Characterization of Ultrathin Gold-Decorated Single Glass Nanopore Haili He,†,‡ Xiaolong Xu,† and Yongdong Jin*,† †

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, Jilin P. R. China ‡ University of Chinese Academy of Sciences, Beijing 100039, P. R. China S Supporting Information *

ABSTRACT: The conical glass nanopore was modified through layer-by-layer electrostatic deposition of a monolayer of glucose oxidase, and then an ultrathin gold film was formed in situ through enzyme-catalyzed reactions. The morphology and components of single glass nanopore before and after ultrathin Au deposition were characterized by transmission electron microscopy (TEM) and energy-dispersive X-ray (EDX) analysis, respectively. In particular, the quenching of the quantum dots fluorescence in the nanopore tip zone further illustrated that the gold nanofilm was successfully deposited on the inner wall of the single glass nanopore. The Au thin films make the glass nanopores more biologically friendly and allow the nanopores facile functionalization of the surface through the Au−S bonds. For instance, the ionic current rectification (ICR) properties of the gold-decorated glass nanopores could be switched readily at different pHs by introducing different thiol molecules.

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polyimide (PI)), glass capillary nanopores have emerged recently as a cost-effective and versatile source of nanopores for biological detection.2,4,6,14,15 The advantages of the glassbased single nanopores over other nanopore configuration are obvious: they can be facilely obtained by pulling the capillaries in the laser puller in seconds with orifice diameters ranging from micrometers to nanometers, without requiring use of clean room facilities, elaborate nanolithographic, and TEMbased technique; and they are envisioned as a powerful means for integrated study and manipulation of single cells due to their unique device nanoarchitecture. To expand the realm of nanopore for biosensing, a variety of functionalization approaches for solid-state nanopores have been reported.6,13 Molecular or nanoscale control over the surface properties of nanopores can harness the interactions between nanopores and various analytes.3,16−19 However, most of approaches reported for nanopore modification, especially deposition of oxides and metals, usually lack good control over their thickness at a few nanometer scale, and in some ways the morphology is unknown.5 Although the common methods to metallize nanopores, such as wet-chemical electroless deposition, (dry) ion sputtering deposition, and electron beam evaporator, were succeeded in Au deposition in inner walls of ∼10-μm-long polymer membranes with typical pore sizes of

ver the past 2 decades, small holes in membranes or nanopipets with nanoscale apertures at very fine tips, called nanopores, have been explored for a variety of interesting applications in nanobiotechnology such as single-molecule biosensing,1 protein detection,2 and ultrafast, label-free DNA sequencing.3−8 These nanopores are precisely controlled structures with defined interfacial chemistry and offer an attractive platform for addressing a number of challenging questions in chemistry, biotechnology, and materials science.9 Biological pores (such as α-hemolysin, SP1 pore protein, or OmpG porin), as one of the most important type of the nanopores, have also been applied for the investigation of single biomolecules.2,5 In spite of the fact that biological nanopores have the advantage of a well-defined geometry and chemical structure with atomic precision, the fragility and sensitivity of the embedding lipid membrane restrain the suitability of them for more practical purposes.5 To date, various technical improvements have been made to improve nanopore resolution and stability; novel techniques and new forms of nanopores have been introduced to expand the utility of the technology.1,10−12 As compared to biological nanopores, synthetic solid-state nanopores have recently attracted a great deal of interest due to their flexible preparation (nanopore sizes are controllable through the fabrication process), excellent mechanical stability (stiffness), and especially the surface modifiability.7,13 Besides solid-state nanopores of silicon dioxide, silicon nitride, or polymer membrane (such as polyethylene terephthalate (PET), polycarbonate (PC) and © 2014 American Chemical Society

Received: December 15, 2013 Accepted: April 19, 2014 Published: April 19, 2014 4815

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Figure 1. Schematic representation of the preparation mechanism of ultrathin gold-decorated single conical glass nanopore by the GOx-catalyzed reduction of HAuCl4. To ensure that only the inner wall of the glass nanopipet was modified, the solutions for the modification were all backfilled and rinsed. This figure is not drawn to scale.

solution (3:1 98% H2SO4/30% H2O2) for ∼2 h to remove organic impurities. (Caution: piranha solution is a powerf ul oxidizing agent and reacts violently with organic compounds. It should be handled with extreme care.) The capillaries were rinsed thoroughly with deionized water and vacuum drying at 80 °C prior to use. The nanopores, with inner diameters of around 100 nm, were prepared by using a CO2 laser-based pipet puller (model P-2000, Sutter Instrument Co.) within 10 s, and a twoline program was used to pull borosilicate glass capillaries, with the following parameters: Heat = 420, Fil = 3, Vel = 30, Del = 220, Pull = 0 and Heat = 400, Fil = 2, Vel = 27, Del = 130, Pull = 250.15 Gold-Decorated Single Glass Nanopore Preparation. The freshly prepared glass nanopores were modified by layerby-layer adsorption of poly(ethylene imine) (PEI) and GOx. First, 0.1 mg/mL branched PEI (Mw = 10 000) aqueous solution was backfilled in the glass nanopore and centrifuged at 3000 rpm for 5 min to help the solution get to the very tip of the nanopore. After removal of excess PEI solution, the nanopore was baked at 50 °C for 1 h to stabilize the PEI coating. The nanopore was then filled with 5 mg/mL GOx solution (in 10 mM PBS, pH 7.4, GOx from Aspergillus niger, Type II, ≥15 000 units/g), followed by centrifugation at 3000 rpm for 5 min and then incubated at room temperature (25 °C) overnight. The nanopore was then filled with 10 mM PBS and centrifuged for at least 4 times to remove unfixed enzymes. Then, 1% HAuCl4 and 25 mM glucose solution (in 10 mM PBS) were mixed with the volume ratio of 1:1 and filled in the nanopore, followed by centrifugation at 3000 rpm for 10 min. And then the nanopore was incubated at room temperature overnight. The gold film decorated nanopore was then rinsed three more times with deionized water to remove unreacted HAuCl4 and glucose and then dried at room temperature for subsequent experiments. Modification of Gold-Decorated Single Glass Nanopore. The prepared gold-decorated nanopores were filled with 20 mM thiol molecule solutions−cysteamine hydrochloride (CysH), 3-mercaptopropionic acid (MPA), and cysteine (Cys), respectively. The nanopores were centrifuged at 3000 rpm for 10 min and then incubated at room temperature (25 °C) for 30 min. The nanopores were then filled with deionized water and centrifuged at least four times to remove excess thiol molecules and dried at room temperature for subsequent experiments. Characterizations. All ionic current measurements were performed using an Axopatch 200B amplifier (Axon Instruments) in voltage-clamp mode with the Digidata 1440A digitizer (Molecular Devices) and a PC equipped with pClamp 10.2 software (Molecular Devices). The I−V curves are recorded by sweeping the voltage from −500 to +500 mV. The applied voltage corresponds to the potential of the internal Ag/AgCl electrode versus the external Ag/AgCl electrode (as show in Figure S2d in the Supporting Information). Solutions

tens of nanometers,20−24 outside surfaces of thin SiN nanopores (pore sizes tens of nanometers and thickness 1 mm).14 Note that the typical aspect ratio of conical glass nanopores (∼104) is over 2 orders of magnitude bigger than that of polymeric nanopores (∼102),20−24 diffusion mass transfer kinetics (or process) in these two nanopore structures are quite different, rendering the inner wall of glass capillary nanopore (more like a 2D nanochannel) much more difficult to be metallized by either wet-chemical electroless deposition or (dry) sputtering or evaporation. We succeeded in the electroless deposition of a thin and smooth gold nanofilm on surface of planar glass slides recently27 but failed in controllable Au deposition on the internal surface of even largesized glass capillary nanopores (typical SEM image was shown in Figure S1 in the Supporting Information) due to the abovementioned issue. Although ultrathin Au-decorated functional (plasmonic) single glass nanopores with sizes down to tens of nanometers were envisioned to be promising in the nanopore’s ultimate usage for single molecule detection and DNA sequencing, attempts so far to deposit a thin and smooth Au layer on the inner wall of glass capillary nanopore have not been reported. Therefore, new preparation approaches and tactics toward high-quality and ultrathin Au films are highly desired and crucial to the field. Herein, we demonstrate a methodology to modify the working region of glass capillary nanopores with high-quality nanoscale gold thin films through mild surface-confined enzymatic reaction.28 As schematically shown in Figure 1, the glucose oxidase (GOx) anchored on the internal surface of nanopore could catalyze the oxidation of glucose with oxygen to gluconic acid and H2O2; then the generated H2O2 reduced HAuCl4 to Au0. During the reaction process, the AuCl4− ions coordinating to the amino groups of PEI (acting as nucleation sites) would be preferentially reduced to form small Au dots on the glass surface (rather than form free AuNPs in the solution), which acting as catalysts fast grow into a thin gold film covering the glass surface. Our method is simple and broadly expands the utility of nanopores for biological sensing: Dressing a glass capillary nanopore surface with Au thin films not only makes it more biologically friendly but further allows readily functionalize of the surface through the well-developed Au−S bonds.

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EXPERIMENTAL SECTION Fabrication of Glass Nanopores. Borosilicate glass capillaries with an outer diameter of 1 mm and an inner diameter of 0.58 mm (Sutter Instrument Co.) were used for all experiments. All glass capillaries used in the experiments were thoroughly cleaned by immersing in freshly prepared piranha 4816

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for I−V measurement are prepared by adding Milli-Q filtered water (Millipore) to a stock solution of 0.1 M KCl with 10 mM HEPES buffer at pH 7.0 unless otherwise indicated. The SEM images for the unmodified glass nanopores were taken with a XL30 field-emission scanning electron microscope at an accelerating voltage of 15 kV. To improve image quality, glass nanopores were coated with a thin layer of gold using an ion beam coater. TEM and high-angle annular dark-field scanning TEM (HAADF-STEM) were carried out by using a FEI TECNAI F20 EM with an accelerating voltage of 200 kV equipped with an energy dispersive spectrometer. TEM samples were prepared by placing a tip of the glass nanopore on a folding grid. Microscopic and fluorescence images were obtained with a DMI6000 B inverted microscope (Leica) and a DFC450 digital color camera (Leica). The CdSe/ZnS QDs solution was backfilled in the nanopores for imaging. For fluorescence imaging, a mercury lamp was used to provide narrowband excitation in the green range (546 ± 12 nm). An emission filter (600 nm ± 40 nm) was used to pass the Stokes-shifted fluorescence signals.

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RESULTS AND DISCUSSION Fabrication and Characterization of Glass Capillary Nanopores. The glass capillary nanopores were produced by laser-assisted pipet puller with inner diameters of around 100 nm (see Experimental Section).15 Figure S2a,b (see the Supporting Information) showed lateral and end-on view scanning electron miscopy (SEM) images of a typical glass capillary nanopore used in experiments of this report, respectively. The inner diameter at the tip was 120 nm. The typical current−voltage (I−V) curve of the glass nanopore in 0.1 M KCl, buffered with 10 mM HEPES (pH 7.0), was given as Figure S2c, Supporting Information. The inner diameter of the glass nanopore could be estimated through electrochemical measurements and using the pore diameter based on the equation, and the diameter was in good agreement with the diameter visible in the SEM image (Figure S2b, Supporting Information).3 The unmodified conical glass nanopore was charged and responded to a symmetric input voltage by exhibiting an asymmetric current output, which was referred to as ionic current rectification (ICR).29 The rectification resulted in a slight nonlinearity of I−V curve, and the degree of rectification was quantified as the ratio of absolute values of current recorded at a given positive voltage (+500 mV) and the same negative voltage (−500 mV). Also the logarithm of the ratio, or rectification coefficient r = ln |I+/I−|, was introduced to equally deal with positive and negative rectification.30 The rectification coefficient can be used as a parameter for monitoring the variation of glass nanopore’s electrical response with the introduction of a functionalized layer to the internal surface.31,32 Gold-Decorated Single Glass Nanopore Preparation. The native glass surface is negatively charged at neutral pH, and the unmodified glass nanopore surfaces induced a negative current rectification, with r = −0.66 (r < 0). The corresponding I−V curves of the functionalization process are given in Figure 2a. In order to prepare Au thin films on the internal surface of a glass nanopore, the negatively charged glass surface was first modified with a positively charged PEI monolayer by electrostatic interaction, as schematically depicted in Figure 1.

Figure 2. (a) I−V curves of single conical glass nanopore recorded in 0.1 M KCl (buffered with 10 mM HEPES, pH 7.0) prior to (black) and after (red) the immobilization of PEI monolayers. The diameter of the glass nanopore before the modification is about 71 nm. (b) The I− V curves of single glass nanopore recorded in 0.1 M KCl (buffered with 10 mM HEPES, pH 7.0) prior to (black) and after the immobilization of GOx and the formation of ultrathin gold films on nanopore internal surface. The diameter of the glass nanopore used for the modification is about 104 nm.

The successful anchoring of the polymer monolayer was confirmed by the changes in the I−V characteristics of the nanopore before and after the chemical modification.33 The inner wall physisorption of the positively charged PEI inverted the current rectification; in other words, the negative current became smaller and the positive current became larger, as the I−V curve shown in Figure 2a (with r = 0.43, r > 0). This is because the change of the polarity of the surface charge, the polyelectrolyte PEI bears positively charged amino groups and makes the internal surface positively charged at neutral pH, changing the dominant ionic species at the nanopore working region from cations to anions.32 This inversion of the current rectification suggests a successful coating of polycation monolayer on the nanopore internal surface. Further electrostatic deposition of GOx monolayers on the PEI coated nanopore surface reverted the current rectification to negative, for the GOx was negatively charged at neutral pH. The corresponding I−V curve showed that the rectification coefficient r = −1.10 (as shown in Figure 2b). Then, HAuCl4 and glucose were introduced in the GOx modified glass nanopore. AuCl4− ions would be coordinated to the amino groups of PEI (since the PEI layer may not be covered completely by GOx),34 and the H2O2 generated in situ from the enzymatic reaction could reduce AuCl4− to Au0 and lead to the deposition of gold thin films on the nanopore internal surface. 4817

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The I−V curve of the resultant gold-decorated glass nanopore also exhibited negative current rectification as r = −1.47 (Figure 2b). It is known that Cl− ions strongly adsorb to the Au surface, and when a gold-decorated glass nanopore is immersed in KCl solution, its net surface charge is therefore negative, ultimately leading to the negative current rectification.21,31 Characterization of Ultrathin Gold-Decorated Single Glass Nanopore. Since it is hard to characterize the inner wall of glass nanopores by SEM, in order to verify that the internal surface of single glass nanopore was successfully decorated by gold thin films, we tentatively characterized the morphology and the components of single glass nanopore before and after Au deposition by transmission electron microscopy (TEM) and energy-dispersive X-ray spectroscopy (EDX) analysis, respectively. To our knowledge, this is the first report to use TEM for morphological and elemental characterizations of glass capillary-based conical nanopores. As shown by a medium magnification TEM image in Figure 3a, the inner wall of the

(Figure 4). The contour profile of gold element distribution was exactly coinciding with the shape of the nanopore.

Figure 4. (a) Dark-field TEM images of a gold-decorated single conical glass nanopore. (b) The corresponding TEM elemental mappings of a gold-decorated single conical glass nanopore at the side wall (top panel) and tip orifice (bottom panel). Scale bars: 200 nm.

The as-prepared ultrathin Au nanofilms were robust and can be reused for nanopore experiments. The Au films will not be peeled off during rinsing and surface modification processes, and it is robust enough for low and medium magnification TEM measurements. However, the as-prepared ultrathin Audecorated glass capillary nanotips were unstable under strong electron beam irradiation when performing high magnification TEM, as shown in Figure S4 in the Supporting Information, which was totally deformed and the wall of the tip was destroyed. Both glass capillary nanotip melting and collapse (Figures 3a and 4a, cf. Figure S3a in the Supporting Information) and Au thin-film melting and reforming into closely packed small nanoparticles and lumps (Figure 3a, inset) were observed. This strictly limits the use of TEM for characterization of small sized bare and Au-decorated glass nanopores with diameter less than tens of nanometers, although they can be successfully fabricated and proved by traditional electrochemical characterizations. Taking into account all these factors, nanopores with larger diameters were chosen for the TEM characterization in this work, especially the TEM elemental mapping (the diameter was much larger as 390 nm, Figure 4) since the images were obtained by using high energy electron beam scanning. Although the accurate thickness of the thin Au layers is difficult to obtain by both TEM (due to 3D nanotubule feature of the glass capillary tip) and SEM (limited by its resolution) observations, the thickness of the as-prepared thin Au films (before high-energy electron beam irradiation to ruin it) was roughly estimated to be ∼2−5 nm from TEM measurements (Figure 3a and inset), calculated from the particle size and surface coverage of the melted and reformed Au island films (Figure 3a, inset). Fluorescence Microscopy Measurements. The success and potential plasmonic effect of Au thin-film decoration on the internal surface of glass nanopores was further confirmed by exploring use of fluorescence microscopy measurements. Gold nanostructures are known fluorescence quenchers.35,36 A fluorescent CdSe/ZnS quantum dots (QDs) solution was backed filled in the whole tip chamber of an unmodified (Aufree) and gold-decorated capillary nanopore, respectively. The fluorescence microscope image of the latter showed that the very tip of the nanopore “disappeared” as expected due to the effective fluorescence quenching of the filled-in QDs in close

Figure 3. (a) TEM image of an ultrathin gold-decorated single conical glass nanopore. The inset shows the local TEM image of the nanopore under higher magnification, showing melting of the ultrathin Au films. (b) EDX spectrum of a gold-decorated single conical glass nanopore.

resultant conical glass capillary tip was homogeneously covered by an ultrathin Au nanofilm, instead of formation of observable Au nanoparticles or chunks, as revealed by contrast homogeneity and remained some transparency, and compared with unmodified (Au-free) glass capillary tip (Figure S3a in Supporting Information). The EDX spectrum of the golddecorated nanopore exhibited distinct peaks of Au element compared with that of the unmodified nanopore (Figure 3b and Figure S3b in the Supporting Information, respectively). The chemical nature and homogeneity of the gold-decorated glass nanopore was further confirmed by TEM elemental mapping 4818

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proximity to Au nanofilms when tip pore size shrinks to a certain extent (Figure 5, right panel), in which strong coupling

Figure 5. Microscopic images (top) and fluorescence microscope images (middle) of the unmodified (Au-free) (left) and golddecorated (right) single conical glass nanopores. Bottom images are the overlay of both above.

between plasmonic Au and fluorescent semiconductor QDs occurs; whereas the unmodified (Au-free) nanopore was intact under fluorescence microscope since it has no effect on the fluorescence of QDs (Figure 5, left panel). Switchable Ion Current Properties of Thiol MoleculeModified Gold Nanopores. The gold film provides a friendly interface for versatile functionalization of the nanopores. After the conical glass nanopores were coated with ultrathin gold film, different thiol molecules, cysteamine hydrochloride (CysH), 3-mercaptopropionic acid (MPA), and cysteine (Cys), were introduced on its internal surface, rendering the gold-decorated nanopores with switchable ICR properties. The I−V curves of the gold-decorated nanopore before modification with thiol molecules at different pHs were given in the Supporting Information, Figure S5. The corresponding ICR ratio at pH 3.7, 5.8, 7.0, and 9.0 was 0.01, −0.51, −0.84, and −1.26, respectively, which had the same trend with the Au-free glass nanopore at different pHs (cf. Figure S6, Supporting Information). This is because the gold film on the glass nanopore surface was originally uncharged, but it could adsorb some Cl− ions and make the nanopore surface negatively charged, which property was similar to the unmodified glass nanopore.21 The I−V curves of the CysH-modified gold nanopore at different pHs are given in Figure 6a. At pH 3.7, the rectification coefficient of this nanopore was +0.17 (more positive than that of the gold-decorated nanopore at pH 3.7, which was 0.01, Figure S5 in the Supporting Information); and when the pH value was 5.8 or higher, the coefficient was also increased (and at pH 9.0, r = −1.20). It was because that the amino groups (−NH2) of the binding CysH were protonated under acid conditions and became positively charged ammonium groups (−+NH3), which made the nanopore positively charged. Then the nanopore preferentially rejected cations and transported anions and inverted the current rectification to positive.21,31 While the gold film on the nanopore surface might not be completely covered by the CysH molecules and could also adsorb some Cl− ions, which (partially) neutralizes the positive charge of −+NH3 and results in (partial) offset of rectification

Figure 6. I−V curves of gold-decorated single glass conical nanopores modified with (a) CysH, (b) MPA, and (c) Cys, respectively, at different pHs. The diameters of the nanopores used for the modification were (a) 104 nm, (b) 83 nm, and (c) 54 nm, respectively.

behaviors, especially when at pH 3.7, the corresponding I−V curve was nearly linear. When under alkali condition, CysH was deprotonated and in neutral form, thus the positive charge on the nanopore surface decreased and eventually disappeared. Taking these aspects into consideration, the net surface charge was negative and the nanopore was preferentially selective to cations; and therefore the ICR was observed and the coefficient was negative. In addition, we found that the concentration of KCl affects only the degree of ICR but not the positive or negative responses of the ICR behaviors of thiol moleculemodified nanopores, as previously discussed by Bard et al.29 When MPA molecules were self-assembled on the golddecorated nanopore, the rectification coefficients at different pHs showed all negative values. Especially at pH 9.0, the coefficient was as negative as −1.67, which was more negative than that of the gold-decorated nanopore at pH 9.0 (Figure S5 in the Supporting Information). The corresponding I−V curves 4819

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are shown in Figure 6b. This is reasonable because the carboxylic acid groups (−COOH) of the self-assembled MPA on the internal surface of Au nanopore is deprotonated to become negative carboxylates (−COO−) under alkali conditions and makes the nanopore more negatively charged and preferentially transports cations. However, at pH 3.7, the MPA was uncharged and the negative charge on the nanopore surface decreased, resulting in the decrease of the rectification coefficient to −0.20. We further investigate rectification behavior of the Cysmodified Au nanopore. The I−V characteristics of the Cysmodified Au nanopore at different pHs were investigated (Figure 6c). When under acid condition (pH 3.7), the ICR coefficient was +0.12, which was closed to (+0.17) that of the CysH-modified gold nanopore. This is due to that the −NH2 groups of the self-assembled Cys are protonated and become positively charged and the −COOH groups are neutrally charged at pH 3.7, which make the net surface charge positive and the current rectification positive. For the same reason as CysH-modified gold nanopore, the absorption of Cl− ions on the gold film made the charge on the nanopore surface neutralized and resulted in that the ion rectification phenomenon was unobvious and the I−V curve was nearly linear. Conversely, at pH 9.0, the −COOH groups were deprotonated to become negative −COO− and the −NH2 groups were uncharged, which make the interface negatively charged and the corresponding ICR coefficient changes to −1.50. On the basis of the above, the ICR properties of the golddecorated glass nanopores could be switched readily by introducing different thiol molecules. The gold interface provides a fascinating platform for the functionalization of the glass nanopore and which could be further applied in nano/ biotechnology and chemical science.

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CONCLUSION

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ASSOCIATED CONTENT

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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 work was supported by start-up funds from the Changchun Institute of Applied Chemistry of Chinese Academy of Sciences, the National Science Foundation of China (Grant No. 21175125), and the State Key Laboratory of Electroanalytical Chemistry (Grant No. 110000R387).

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In conclusion, we demonstrate an interesting methodology to prepare ultrathin gold films on the internal surface of the glass nanopore; the glass nanopore was modified by layer-by-layer electrostatic deposition of a monolayer of GOx, and then an ultrathin gold film was formed in situ through enzyme-catalyzed reactions. The successful preparation of the gold film was confirmed by the changes in the electrochemical I−V, TEM/ TEM-mapping characteristics of the nanopore before and after deposition of gold on its surface. In particular, the quenching of the QDs fluorescence in the nanopore tip zone further illustrated that the gold nanofilm was successfully deposited on the inner wall of the glass nanopore. Moreover, the gold film serves as a starting point to change the chemical characteristics of the nanopore surface by attaching different thiol molecules through Au−S coupling chemistry. This fundamental modification of the glass-based single nanopore forms the basis of a new platform for the applications in ultrasensitive detection and biosensing.

S Supporting Information *

SEM and TEM images, EDX spectrum, and I−V curves of the gold-decorated and unmodified Au-free glass nanopores. This material is available free of charge via the Internet at http:// pubs.acs.org. 4820

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