Controlled Formation of Thiol and Disulfide Interfaces - Langmuir

Nov 30, 2012 - The work reported herein describes the controlled creation of uniform thiol-functionalized siloxane-anchored self-assembled monolayers ...
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Controlled Formation of Thiol and Disulfide Interfaces Vlada Artel,†,‡ Reut Cohen,†,‡ Inbal Aped,†,‡ Maria Ronen,‡,§ Doron Gerber,‡,§ and Chaim N. Sukenik*,†,‡ †

Department of Chemistry, ‡Institute for Nanotechnology and Advanced Materials, and §Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel S Supporting Information *

ABSTRACT: The work reported herein describes the controlled creation of uniform thiol-functionalized siloxane-anchored selfassembled monolayers (SAMs) and their selective transformation into intramonolayer (bridging) disulfides. These disulfides provide for the efficient immobilization of (bio)molecules bearing pendant thiols or disulfides, with no need for added oxidant. The unambiguous development of this surface chemistry required analytical methods that distinguish thiol and disulfide moieties on a surface. Physical properties such as wetting and monolayer thickness do not suffice nor do routine spectroscopic techniques (e.g., XPS, IR). Therefore, a method for distinguishing and quantifying thiol and disulfide surface functionality on a monolayer array based on the reaction with 2,4-dinitrofluorobenzene (DNFB, Sanger’s reagent) is reported. DNFB readily reacts with thiol-SAMs (but not with disulfides) to form stable derivatives with distinctive IR, UV, and XPS signatures. Finally, the thiol−disulfide chemistry is applied to thiol-functionalized hybrid silica nanoparticles. These high-surface-area nanoparticles provide solid supports heavily loaded with thiol groups whose chemistry is also reported herein.

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INTRODUCTION Controlled biomolecule immobilization is essential to the creation of devices based on molecular arrays for screening and sensing.1 Molecules with pendant thiols are often immobilized by the interaction of their sulfhydryl groups with metal surfaces (e.g., Au, Ag, and Pt).2 Alternatively, electrophilically functionalized surfaces can provide for the covalent anchoring of thiols as thioethers.3,4 Another surface-anchoring strategy is to link surface thiols with those of the molecules to be immobilized. This kind of surface immobilization has advantages in terms of reversibility (by reduction) and in terms of disulfide exchange processes5 allowing for the equilibration of the anchored molecules into a most favorably packed array while still offering the benefits of covalent anchoring with controlled molecular orientation. To date, there are only a limited number of literature reports of disulfide-based anchoring.6−8 The work reported herein uses trichlorosilanes 1−5 (Scheme 1) to create uniform thioester-functionalized siloxane-anchored self-assembled monolayers (SAMs) that can be cleanly transformed into thiol-bearing SAMs by either hydrolysis or

reduction. The chemistry of the thiol-decorated SAMs is examined in terms of their reactivity with spectroscopic markers and in terms of their controlled conversion into intramonolayer (bridging) disulfides. This approach differs from the previously reported preparation of thiol/disulfide SAMs9,4,10,11 that does not distinguish between thiols and bridging disulfides or control their chemistry and/or interconversions. Given controlled disulfide formation, we can provide an efficient basis for the anchoring of either thiol- or disulfide-bearing molecules (including biomolecules). The work reported herein also addresses (i) the formation of thiol-decorated SAMs by various chemistries, including a photocleavage route that is significantly facilitated by the conjugation present in SAMs based on 3−5; (ii) the ability of surface thiols with different loading densities to form bridging disulfides within such functionalized arrays; and (iii) the application of the thiol/disulfide chemistry to hybrid silica nanoparticles made from trimethoxypropylthiol (Scheme 2). Extending this surface chemistry to such particles provides both high loading substrates and additional spectroscopic probes. A fundamental problem in developing this surface chemistry is the need for analytical methods that distinguish between interfacial thiols and disulfides. They have similar XPS binding energies and similar surface wetting properties. The thiol

Scheme 1. SAM Forming Compounds

Received: September 24, 2012 Revised: November 26, 2012 Published: November 30, 2012 © 2012 American Chemical Society

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rinsed with isopropanol, cleaned with CHCl3 and hexane (as described above), and dried under a stream of filtered nitrogen. Acid-Catalyzed Methanolysis of Thioester SAMs. In a reaction tube equipped with a small magnetic stirring bar and a reflux condenser, we placed a mixture of concentrated hydrochloric acid and methanol (1:9). The thioester-bearing substrates were placed in the tube, and the reaction mixture was held at reflux overnight. After the reaction cooled to room temperature, the substrates were withdrawn, rinsed with methanol, cleaned in CHCl3 and hexane (as described above), and dried under a stream of filtered nitrogen. Cyanide-Mediated Methanolysis of Thioester SAMs. 17 In a reaction tube equipped with a small magnetic stirring bar and a reflux condenser, we placed a solution of 100 mg of sodium cyanide in 10 mL of methanol. The mixture was heated to reflux to dissolve the salt, and then the thioester-bearing substrates were placed in the tube and refluxing continued for 25 min. After the reaction cooled to room temperature, the substrates were withdrawn from the mixture, rinsed with methanol, cleaned in CHCl3 and hexane (as described above), and dried under a stream of filtered nitrogen. Reaction of Thiol SAMs with 2,4-Dinitrofluorobenzene (2,4DNFB). Ethanol (pH 8, 10 mL) was prepared using added KOH. To this solution, 2,4-DNFB (100 μL) was added . A substrate bearing thiol-decorated SAMs was immersed in this solution for 5 min at room temperature. It was then withdrawn from the solution, rinsed with ethanol, cleaned in CHCl3 in a sonication bath for 6 min, soaked in warm hexane for 6 min, and dried under a stream of filtered nitrogen. This reaction could also be carried out by exposing a thiol SAM to 2,4DNFB vapors by placing 100 μL of 2,4-DNFB into the tip of an Eppendorf tube under (but not in contact with) a thiol-SAM-coated quartz wafer. The wafer was withdrawn after 15 min and washed with solvent (as described above). Regeneration of Thiol SAM from Its 2,4-DNFB Derivative. 18 The thiol groups can be restored by immersing the dinitrobenzenemodified substrates into a solution of 100 μL of β-mercapto-ethanol in 10 mL of ethanol at pH ∼8. After 24 h, they were withdrawn from the solution, rinsed with ethanol, cleaned in CHCl3 and hexane (as described above), and dried under a stream of filtered nitrogen. Conversion of Thiol SAM to Disulfide SAM by Reaction with I2. Thiol-SAM-coated wafers were placed in a solution of I2 (30 mg) in methanol (10 mL) for 5 min. They were then withdrawn from the solution, rinsed with ethanol, cleaned in CHCl3 and hexane (as described above), and dried under a stream of filtered nitrogen. Alternatively, the thiol-decorated substrate could be exposed to iodine vapors for 15 min under ambient conditions. Conversion of Disulfide SAM to Thiol SAM by Reaction with LiAlH4. Disulfide-coated wafers were placed in a LiAlH4 solution (1 M in THF) for 2 min. They were withdrawn from the solution, rinsed with dry THF, and soaked in 10% HCl(aq) for 1 h. The substrates were then cleaned in CHCl3 and hexane (as described above) and dried under a stream of filtered nitrogen. Anchoring p-Nitrothiophenol to Disulfide SAM by Disulfide Exchange. p-Nitrothiophenol (50 μL) was added to 10 mL of pH 8 ethanol. Disulfide-decorated wafers were soaked in this solution overnight. They were then withdrawn, cleaned in ethanol in a sonication bath for 6 min, washed in CHCl3 and hexane (as described above), and dried under a stream of filtered nitrogen. In Vitro Expression of Green Fluorescent Protein (GFP). GFP protein was in vitro pre-expressed using a cell-free TNT quick coupled transcription/translation system (Promega). The expression mixture (10.5 μL of the expression system and 2 μL of the GFP gene) was prepared and incubated on a heating block at 32 °C for 2.5 h. Anchoring of Antibody onto Disulfide SAM by Disulfide Exchange. Anti-GFP (green fluorescent protein) antibodies were immobilized via disulfide exchange chemistry onto SAMs bearing bridging disulfides. Briefly, polyclonal anti-GFP (Abcam) was spotted on the bridged disulfide-modified quartz slide and incubated overnight at room temperature, followed by washing with Hepes buffer. Next, the glass slide was completely covered with pre-expressed GFP, incubated for 30 min at room temperature, and washed with Hepes buffer. The GFP fluorescence signal resulting from the immobilized

Scheme 2. Sol-Gel Synthesis of Hybrid Alkyl Thiol−Silica Particles

content of the sol-gel particles is high enough to allow for an FTIR analysis of S−H bonds, but this is a weak signal that is not useful for the monolayer surfaces and is, even in the best case, a hard-to-quantify diagnostic. Therefore, we distinguish interfacial thiols and disulfides on the basis of their reaction with Sanger’s reagent (2,4-dinitrofluorobenzene, 2,4-DNFB), a reagent with minimal steric bulk that reacts with thiols12 but does not react with disulfides and provides derivatives with distinctive spectroscopic signatures (FTIR, UV, and XPS) that can be readily quantified even within a monolayer.

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EXPERIMENTAL SECTION

Materials. Reagents and solvents were obtained from SigmaAldrich, Acros Organics, Fluka, Bio-Lab Ltd., and Merck. The trichlorosilane thioesters used to create the SAMs described herein were all (1−5) prepared using published procedures,13−15 and their spectral properties were identical to those in the literature. Water was deionized and then distilled in an all-glass apparatus. Silicon wafers were obtained from Virginia Semiconductor (N type, undoped, ⟨100⟩, >1000 Ω·cm). Quartz substrates were obtained from Quarzschmelze Ilmenau. Monolayer Preparation. Silicon wafers (for ellipsometry and ATR-FTIR measurements) and quartz wafers (for UV and XPS measurements) were cleaned and activated as previously reported16 and used as substrates to deposit the siloxane-anchored SAMs based on 1−5. The SAMs were characterized by contact angle measurements on both substrates, by ATR-FTIR and ellipsometry on the silicon wafers, and by XPS and UV−vis spectroscopy on the quartz substrates.15 These characterization tools were applied to both the directly deposited SAMs and to those produced by in situ chemical transformations. In Situ Generation of Thiol Functionality. Thiol-decorated SAMs were generated from the thioester SAMs as follows. LiAlH4 Reduction of Thioester SAMs. Thioester-coated wafers were placed in a LiAlH4 solution (1 M in THF) for 2 min. They were withdrawn from the solution, rinsed with dry THF, and soaked in 10% HCl(aq) for 1 h. The substrates were then cleaned with CHCl3 in a sonication bath for 6 min, soaked in warm hexane for 6 min, and dried under a stream of filtered nitrogen. NaBH4 Reduction of Thioester SAMs. Ethanol (pH ∼8) was prepared by adding KOH. NaBH4 was dissolved in this solution (0.035 g in 10 mL). The thioester-bearing substrates were immersed in the NaBH4 solution for 90 min at 60 °C and then withdrawn from the solution and cleaned with ethanol, CHCl3, and hexane (as described above) and dried under a stream of filtered nitrogen. UV Photocleavage of Thioester SAMs in Oxygen-Free Isopropanol. Reagent-grade isopropanol (BIO LAB) was dried over 4 Å molecular sieves (Merck) and degassed under argon. The thioacetatebearing monolayer-coated substrates were placed in a quartz tube and purged with argon, along with 10 mL of oxygen-free isopropanol. The tube was then irradiated using a UV−vis photoreactor (Luzchem model LZC4) equipped with eight UV C (Ushio G8T5 UV−C 7J Hg) or UV A (Hitachi FL8BL-B) lamps for 4 h (temperature remained in the range of 24−28 °C). They were then withdrawn from the solution, 192

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GFP was measured using a fluorescence scanner (LS Reloaded, Tecan) with a 488 nm laser and a 535 nm filter. The median fluorescence intensities (after background subtraction) were calculated using GenePix software. The statistical analysis was done using the Mann−Whitney 2-tailed U test (n = 30). Synthesis and Derivatization of Hybrid Silica Particles. The procedure developed to prepare these particles combines the method reported for making thiolated microparticles19 with a standard sol-gel nanoparticle preparation.19,20 3-Mercaptopropyl trimethoxysilane (MPTMS, 3.724 g) and sodium dodecylbenzenesulfonate (SDBS, 0.0013 g) were added to water (30 mL) under vigorous stirring until the MPTMS droplets disappeared. NH3·H2O (0.5 mL) was added dropwise to the emulsion (pH 11.5), and the reaction mixture was held at 50 °C for 48 h. The colloidal dispersion obtained in this way was centrifuged, and the resulting precipitate was washed several times with water. Reactions of the thiol-bearing silica particles with iodine and/or with 2,4-DNFB were conducted in solution using the conditions described for the thiol-decorated SAMs but for longer times (1 h for iodine and 24 h for 2,4-DNFB).

two kinds of substrates are comparable in terms of their structure and reaction chemistry. The solution-based methodologies all gave complete acyl removal within minutes to hours. The relative merits of the various hydrolytic or reductive reagents are considered in the Discussion section. Of special note is the reductive photocleavage involving the irradiation of the thioester SAM through a thin layer of isopropanol. This approach to making thioldecorated surfaces affords the ability to control both the spatial definition of the thiols by using a photomask and the ability to control the rate of photocleavage by controlling both the light flux and wavelength. Similar to the reported15 wavelength dependence for the photo-oxidation of such surfaces, the photoreduction reported herein proceeds with longer-wavelength light in cases where the ester chromophore is conjugated (i.e., 3−5), with 4 being most effective. The reaction of the surface thiols with 2,4-DNFB could be followed by the appearance of nitroaryl signals at 1341, 1524, and 1593 cm−1 in the IR and by the new UV absorption at 338 nm. The regeneration of the free thiol surfaces (as evidenced by the disappearance of these signals) uses β-hydroxyethanethiol (Scheme 3). Figure 1 shows the IR and UV spectra of the thioacetate, thiol, and thiodinitrobenzene SAMs. Thickness, wetting, and XPS data for the variously functionalized SAMs are summarized in Table 2. Both the thiol SAMs based on 2−5 and their subsequent chemistry are essentially indistinguishable. For the two different chain lengths tested using SAMs based on 1 and 2, the thioacetate SAM is, as expected, slightly thicker and slightly more hydrophobic than the corresponding thiol. The increase of 0.5−0.6 nm after reaction with 2,4-DNFB is also reasonable. XPS of all surfaces shows the expected divalent sulfur. We note that whereas the ratio of the sulfur to nitrogen signal intensities in the 2,4-DNB derivative should be 1:2, the nitrogen signal has an unexpectedly large relative intensity (∼1:4). This may reflect a high cross section for the nitro group nitrogens and/or the fact that the nitrogens are more exposed than the sulfurs on the SAM surface. The removal of the 2,4-DNB group (from both C11 and C16 polymethylene tethers) and the regeneration of a thioldecorated SAM was achieved using β-mercapto-ethanol (Scheme 3). The ATR-FTIR spectra of the regenerated thiol surface showed a small (≤10%) decrease in the intensity of the methylene peaks that was perhaps due to the partial stripping of

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RESULTS Silicon and quartz wafers were coated with trichlorosilanes 1−5 to provide thioester-decorated SAMs with alkyl chains of either 11 or 16 methylenes. The acyl groups were removed by reduction (LiAlH4 or NaBH4) or solvolysis (methanol with either sodium cyanide or acid) or photocleavage. These processes are all described in the Experimental Section. They could each be followed on the basis of the disappearance of the spectroscopic signatures (Table 1) of the thioesters in the IR Table 1. Diagnostic IR and UV Peaks of SAMs Based on 1−5 SAMs based on 1−5

IR carbonyl (cm−1)

UV λmax (nm)

1 2 3 4 5

1695 1695 1662 1654 1660

231 231 203, 235, 273 254, 292 210, 245, 269

(on silicon) or UV (on quartz). We note that the original IR, UV, and XPS spectra of the SAMs and their derivatives (beyond those spectra included in this article) appear in the Supporting Information. The various thioesters all provided thiol-decorated SAMs with an anchoring chain of 11 methylenes, except for SAMs based on 1, which have 16 methylenes. The monolayers on the

Scheme 3. Thiol Reaction with 2,4-DNFB and the Removal of the DNB Group

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Figure 1. IR and UV spectra of thioacetate SAM based on 2 (blue), thiol (pink), and thiodinitrobenzene (red).

Table 2. Ellipsometry and Wetting Properties of Thioacetate, Thiol, and Dinitrobenzene SAMs thioacetate (TA) C16 thickness (±0.25 nm) contact angle adv/rec(deg) methylene peak position XPS(±0.2 eV)

2.4 80/78 2918 2850 S: 164.0,

C11 1.7 74/70 2922 2852 165.2

thiol C16 2.3 78/76 2918 2850 S: 164.0,

2,4-DNB C11

1.6 68/64 2922 2852 165.2

C16

regenerated thiol C11

2.9 2.1 75/70 60/55 2919 2924 2850 2853 S: 164.2 N: 406.4

C16 2.1 75/70 2920 2851 S: 164.0,

C11 1.5 62/58 2924 2852 165.2

2,4-DNB from regenerated thiol C16

C11

2.9 2.2 76/70 58/52 2920 2925 2851 2853 S:164.2 N: 406.4

solution or as iodine vapors) showed no significant changes in the ATR-FTIR or UV spectra or in the wetting properties or thickness of the SAM. The effectiveness with which iodine converts the surface thiols to disulfides (per Scheme 1) could be demonstrated by treating the substrate with 2,4-DNFB solution (as was done above for thiol SAMs). After the iodine treatment, no 2,4-DNB incorporation is observed (despite the fact that XPS shows the sulfurs to still be divalent and not oxidized to sulfonates). However, when the disulfide that had resulted from the iodine treatment is exposed to LiAlH4 (regenerating the thiols) and then to 2,4-DNFB, the resulting spectra were identical to those of a fresh thiol SAM reacted with 2,4-DNFB. Thiols and Disulfides on Hybrid Silica Particles. Thiolladen hybrid silica nanospheres have enabled us to observe the thiol functional groups and their chemistry directly by FTIR and to extend this chemistry to a high-capacity substrate. Using

the siloxane monolayer. The wetting properties and thickness of the regenerated thiol (Table 2) also suggest some small amount of monolayer damage, though the reaction of 2,4DNFB with the regenerated thiol gives a result that is still very similar to that obtained with the original thiol (Table 2). Overall, the extent of monolayer damage caused by these reactions is small. This is further confirmed by the fact that the methylene stretches in the IR do not shift as a result of the in situ reactions. That is, the stretches at ∼2920 and 2850 cm−1 have become the standard diagnostic for monolayer packing and order. The lower values seen for the longer chain tethers speak to their enhanced order.21 The lack of change in these values as a result of the various chemical transformations suggests that there is little or no change in monolayer packing due to the in situ chemistry. In Situ Generation of Bridging Disulfides. The reaction of thiol-functionalized surfaces with iodide (either in methanol 194

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expected 2,4-DNB IR signals at 1596 and 1528 cm−1 (Figure 3). Particles were treated with iodine to make disulfides. The intensity of the SH peak (normalized relative to the C−H signals) was consistently reduced by about 50%. This suggests that reaction with iodine converts approximately half of the thiol groups into disulfides but that this reaction is still more effective than reaction with 2,4-DNFB. The greater effectiveness of the reaction with iodine likely reflects its lower steric demand and the possibility that more widespread disulfide formation may be promoted by thiol−disulfide exchange. We also note that electron microscopy yields images comparable to that shown in Figure 2 regardless of whether the particles are reacted with 2,4-DNFB or with iodine; there is no evidence for particle aggregation or deformation under any of our reaction conditions. The reaction of the putative disulfide particles with 2,4DNFB (Scheme 4) did not show any further reduction in thiol signal nor did it show the appearance of the nitrobenzene signals at 1528 and 1596 cm−1. It seems that the readily accessible thiols have been converted to disulfides and neither those disulfides nor the remaining thiols react with 2,4-DNFB. Using Surface Disulfides to Anchor Molecules. The anchoring of molecules onto the disulfide surfaces described above can occur by two different kinds of exchange processes: with thiols or disulfides (Scheme 5). An example of each is reported below. The anchoring of a small-molecule thiol was observed using p-nitrothiophenol. The disulfide monolayer of compound 1 was reacted with p-nitrothiophenol in EtOH at pH ∼8, resulting in the appearance of IR peaks at 1520 cm−1 (aromatic ring) and at 1340 cm−1 (nitro) and a UV signal at around 324 nm (Figure 4). The measured thickness of the monolayer after the reaction was 1.8−1.9 nm, and the contact angle was 62 (adv)/58 (rec)°. It is interesting to compare the increase in SAM thickness of 0.2−0.3 nm obtained from this thiol−disulfide exchange process with the changes resulting from reaction with 2,4DNB (thickness increase of 0.5 nm). This difference is consistent with the expectation that in the exchange process half of the sulfurs have a nitrothiophenol attached, resulting in half the thickness increase. The other kind of molecular anchoring through disulfide exchange is when a preformed disulfide is immobilized by its interaction with a bridged disulfide surface (Scheme 5, right

3-mercaptopropyltrimethoxysilane (MPTMS), we prepared the particles (Scheme 2) whose SEM image is shown in Figure 2.

Figure 2. SEM image of hybrid silica particles made of MPTMS.

These submicrometer spherical silica particles are small enough (∼100 nm) and sufficiently functionalized so as to provide high functional group loading on a high-surface-area support. The thiol particles were reacted with 2,4-DNFB (Scheme 4). Given the larger quantity of reactive functional groups, we expected that elemental analysis might be informative. Although the error bars for the C, H, and S analyses were large, the incorporation of nitrogen is qualitatively clear: no nitrogen is detected in the original thiol particles whereas reaction with 2,4,DNFB shows nitrogen (1 to 2%). The particles also afforded a number of useful IR diagnostics. In particular, we now have a large enough SH concentration to see its signal at 2556 cm−1. Although this signal is broad and not particularly intense (Figure 3), it clearly shows the presence of SH groups in the particle. Efforts to monitor the reaction with 2,4-DNFB by IR also yielded useful results. Although the variability in the SH peak intensity prevents its use in the quantitation of the reaction with 2,4-DNFB, this reaction did result in the appearance of the

Scheme 4. In Situ Chemistry of Hybrid Thiol-Bearing Silica Nanoparticles

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Figure 3. FTIR of (a) original thiol particles (blue) and (b) thiol particles after reaction with 2,4-DNFB (red).

Scheme 5. Anchoring Thiols or Disulfides on a Disulfide Surface

cysteine amino acids). The immobilized antibody used was anti-GFP (green fluorescent protein). We chose not to use a secondary antibody for visualization because it could potentially react directly with the surface (as was the case for the primary antibody). Therefore, the immobilized antibody was visualized by incubation with in-vitro-expressed GFP followed by imaging with a fluorescent scanner. The use of in-vitro-expressed GFP achieved three goals. It allowed for the visualization of the immobilized antibody; it served as a control for nonspecific binding to the surface; and most importantly, it demonstrated the requisite chemistry for reaction with the anti-GFP antibody after immobilization. The results of this experiment and of the control experiment carried out with the original thioacetatefunctionalized SAM are shown in Figure 5. Although the image obtained (A) from the thioacetate SAM shows only weak fluorescence (perhaps due to the physisorbtion of a small amount of anti-GFP), the bridging disulfide surface shows a fluorescence (B) that is 2.6 times stronger (with p < 5 × 10−10).

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Figure 4. (A) ATR-FTIR and (B) UV−vis spectra of nitrothiophenol attached to a disulfide-functionalized monolayer.

DISCUSSION The controlled formation of interfacial thiols and their conversion to bridging disulfides within an interfacial array has been achieved. We have demonstrated the use of various approaches to thiol formation and the development of chemical

side). This was demonstrated using disulfide-functionalized monolayers of compound 1, which were reacted with an antibody containing disulfides (based on the thiols of its 196

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Figure 5. Fluorescence of anti-GFP/GFP on (A) thioacetate and (B) disulfide monolayers.

disulfide formation and the ability to create controlled concentrations of surface thiols remain important goals.

and spectroscopic probes that conclusively differentiate thiols from disulfides. Each of the various thioester cleavage procedures studied has its own advantages and disadvantages. Acid hydrolysis does not suffer from even the small amounts of monolayer stripping observed when using cyanide- or borohydride-based chemistry (basic conditions). There is no monolayer stripping with lithium aluminum hydride and the reaction is relatively quick (seconds/minutes), but the complete removal of residual aluminum salts from the monolayer surface is not always possible. Photocleavage is cumbersome in requiring that irradiation be done under an oxygen-free layer of isopropanol, but it affords unique control that can be used for spatially selective thiol formation and the light source can control (by intensity and/or wavelength) the rate of the cleavage process. In general, thiol-decorated surfaces can be taken for XPS analysis with no particular precautions, and the XPS results show no evidence of oxidized sulfurs (e.g., sulfonates). Furthermore, when these surface thiols are reacted with 2,4,DNFB, there is no change in the spectral intensity of the reaction products after the thiols have been exposed to air for a few minutes. We thus conclude that air oxidation of the thiols is not fast enough to complicate their short-term handling under ambient conditions. The long-term air stability of thioldecorated SAMs and/or particles as a function of time and/ or temperature has not yet been systematically examined. The reaction of thiol-bearing surfaces with 2,4-DNFB provides a useful assay that can be applied to both monolayers and thiol-laden nanoparticles. The controlled oxidation/ reduction of thiols/disulfides is also well established. The convenient use of iodine to make disulfides and the use of 2,4DNFB, both in solution and in the gas phase, to identify thiols while still being able to regenerate the original thiols are also important achievements.

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ASSOCIATED CONTENT

S Supporting Information *

Additional IR, UV, and XPS spectra. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the Israel Science Foundation (ISF) as part of the ISF Center of Excellence, by the Minerva Foundation, and by the Edward and Judith Steinberg Chair in Nanotechnology at Bar-Ilan University.

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REFERENCES

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CONCLUSIONS The control and analysis of the surface chemistry of thiols and disulfides and the potential for using bridging surface disulfides in biomolecule anchoring have been demonstrated. These concepts have been demonstrated both for ultrathin monolayer films and for high-surface-area nanoparticles. Nevertheless, the full scope and optimization of surface disulfide formation and disulfide-based biomolecule anchoring must still be explored. Issues such as the influence of monolayer packing on bridging 197

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dx.doi.org/10.1021/la303828s | Langmuir 2013, 29, 191−198