Supporting Information for. Resonantly Enhanced Nonlinear Optical Probes of Oxidized Multiwalled Carbon. Nanotubes at Supported Lipid Bilayers

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1 Supporting Information for Resonantly Enhanced Nonlinear Optical Probes of Oxidized Multiwalled Carbon Nanotubes at Supported Lipid Bilayers Alicia C. McGeachy, a Laura L. Olenick, a Julianne M. Troiano, a Ronald S. Lankone, b Eric Melby, c Thomas R. Kuech, c Eseohi Ehimiaghe, a,# D. Howard Fairbrother b, Joel A. Pedersen, c,d Franz M. Geiger a,* a Department of Chemistry, Northwestern University, Evanston, IL 60208, b Department of Chemistry, Johns Hopkins University, Baltimore, Maryland 21218, c Environmental Chemistry and Technology Program, University of Wisconsin, Madison, WI, 53706, d Department of Chemistry, University of Wisconsin, Madison, WI, 53706, United States. # Currently at Feinberg School of Medicine, Northwestern University, Chicago, IL Supporting Information 1. Second Harmonic Generation Theory. Two forms of SHG spectroscopy were used in this study: the SHG χ (3) technique, a nonresonant variant of SHG, and resonantly enhanced SHG spectroscopy. Both methods are described below, and a more thorough discussion of both techniques can be found in the literature. 1-5 SHG is a second-order coherent nonlinear optical spectroscopy in which two photons with frequency ω combine to form a single photon with frequency 2ω. Within the electric dipole approximation, SHG is forbidden in media in which the length of centrosymmetry extends further than the coherence length of SHG. 4, 6-8 However, at the interface between two phases, inversion symmetry is broken, resulting in an observable SHG signal. 7-8 Consequently, SHG spectroscopy is able to provide molecular-level resolution and 3-5, 9-11 surface selectivity. SHG spectroscopy is a particularly useful tool to study

2 biological interfaces, as the surface selectivity of the technique is higher than that of fluorescence or absorbance methods and it can be conducted in situ and under ambient conditions that are biologically relevant. 10 SHG intensity is a function of the second- and third-order nonlinear susceptibilities, χ (2) and χ (3). 5-7, 9, 11 The electric field, E, is directly proportional to the polarization at the second harmonic at the interfacial region, as summarized in Equation 1. 1, 9, P 2ω is the induced second-order polarization of the interface that results in the generation of said electric field, E SHG. The square of the electric field, denoted E SHG, is proportional to SHG intensity, I SHG. I SHG = E SHG P 2ω = χ (2) E ω E ω + χ (3) E ω E ω Φ (1) 0 The incident electric field is held constant, χ (2) and χ (3) are treated as constants and are related to the interface and Φ 0 is the interfacial potential. Simplification of Equation 1 results in Equation 2. E SHG P 2ω = A + BΦ 0 (2) As illustrated in Equation 1, the χ (2) term, arising from the aligned species second-order response resulting from the molecular hyperpolarizability, is independent of the interfacial potential generated by the charged surface. 9 The χ (3) term is directly influenced by interfacial potential or surface charge and is related to the polarization and orientation of water molecules that align as a consequence of the electric field generated by the charged interface. 3, 7, 9 Based on Equations 1 and 2, increasing the interfacial potential by increasing the magnitude of the negative charge would result in increases in SHG signal intensity. To determine that increases in SHG signal intensity upon adsorption of O- MWCNTs to supported lipid bilayers (SLBs) and silica surfaces were attributable to

3 large changes in interfacial potential, the incident wavelength was tuned to 800 nm (λ SHG =400 nm), away from the broad electronic transition that peaks at ~260 nm. Resonantly Enhanced SHG Spectroscopy. To study O-MWCNT adsorption to SLBs formed from 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-snglycero-3-phosphocholine (DOPC), and 9:1 mixtures of DOPC and 1,2-dioleoyl-3- trimethylammonium-propane (DOTAP), resonantly enhanced SHG was employed. Resonantly enhanced SHG, like the χ (3) technique, occurs when two photons at frequency ω combine to form a single photon at twice the frequency. When an adsorbate has an accessible electronic transition that is in resonance with the second harmonic frequency, the SHG signal intensity is dependent on the second order nonlinear susceptibility χ (2) as 10, 14 shown in Equation 3. I SHG = E SHG χ (2) E ω E ω 2 3 The second order susceptibility is the sum of both resonant and nonresonant contributions 10, 14 as demonstrated in Equation 4. E SHG χ (2) NR + χ (2) R e iδφ 2 4 Here, χ R (2) and χ NR (2) represent the resonant and nonresonant components, respectively, with ϕ being the phase factor between the two contributions. The resonant contribution, χ R (2) relates directly to the number of interfacial species in resonance, N, and the second-! order orientational average of the hyperpolarizability tensor, α (2) (Equation 5). χ R (2) = N! α (2) 5

4 Resonantly enhanced SHG experiments were conducted with incident wavelengths of ~600 nm (λ SHG = 300 nm) which overlaps with the π!π* electronic transition, and was used to determine the equilibrium constant and free energy of adsorption. The resonance enhancement arises from the increased polarizability of interfacial species when the SHG frequency, or fundamental frequency, approaches the resonant frequency of a molecular electronic transition. Thus, SHG spectroscopy can provide information with molecularlevel resolution. Based on equations 4 and 5, we expect the SHG signal to increase with increasing concentrations of O-MWCNT at the interface. Increases in SHG signal intensity are expected outcomes in both resonantly enhanced and potential-driven SHG experiments. Varying the incident wavelength allows us to determine the origins of the SHG signal increases. 2. SHG Control Experiments A. Power Studies. Power studies were conducted to determine the damage threshold of both the bilayer systems and the bare silica substrate in the presence of 2 mg/l O- MWCNT. After introducing the O-MWCNT to the flow cell, the SHG signal intensity was allowed to stabilize and then the pulse energy was reduced to 0.05 µj and then increased in increments of 0.05 µj up to 0.5 µj. Plots of SHG intensity as a function of incident pulse energy yield quadratic dependence, as expected (Figure 5A & B). Departures from the expected quadratic dependence indicate sample damage (e.g., highest points shown in Figure 5A). All SHG experiments were run at 0.40 ± 0.05 µj, below the threshold of thermal degradation.

5 B. Bandwidth and Polarization Studies As discussed in the main text, SHG bandwidth studies were performed to assure that the observed signals were indeed SHG and not due to fluorescence or other optical processes that might be produced by the particles. In these studies, the supported lipid bilayer was formed, rinsed with a Tris buffer solution, and then allowed to interact with O-MWCNTs (2 mg/l). Similarly, in experiments performed in the absence of SLBs, the O-MWCNT were allowed to interact with the bare fused silica surface. After allowing the SHG signal to stabilize, the incident wavelength was maintained at ~300 nm and the detected wavelength was scanned by changing the monochromator setting, with the expectation of generating a Gaussian lineshape. In the case of the SLBs formed from DOPC, DMPC, and 9:1 DOPC/DOTAP, and bare fused silica, polarization studies, in which the p- polarized incident light was held constant, and the output polarization was varied reveal that the SHG signal is well-polarized along the surface normal. 3. QCM-D Experiments We conducted QCM-D experiments to investigate the interaction of 7% and 12% O- MWCNTs with DMPC and DOPC bilayers. Except where otherwise noted, experiments were carried out by flowing 100 mm NaCl, 5 mm CaCl 2, ph 7.4 (10 mm Tris) until frequency and dissipation values stabilized. A solution of mg/ml phospholipid vesicles was introduced until stable values indicative of bilayer formation were achieved (Δf 25 Hz; ΔD ). Following formation, the bilayer was rinsed sequentially with vesicle-free buffer and buffer without CaCl 2. Following rinsing, O-MWCNT suspensions were flowed over the bilayer for 140 min to determine attachment. Due to the observed changes in effective hydrodynamic diameter with time (Figure 1E) in the

6 buffer solution, every 10 min we introduced O-MWCNTs freshly diluted from a stable stock suspension in ultrapure water. The attachment period was followed by a particlefree buffer rinse to assess attachment reversibility. 4. Adsorption of O-MWCNT to Bare Silica and SLBs A. Adsorption of O-MWCNT to bare silica at ph 7.4. O-MWCNT have been shown to adsorb to bare silica surfaces under circumneutral ph and at high ionic strength To confirm that we could indeed observe adsorption of the O-MWCNTs to silica by SHG spectroscopy under conditions that have already been shown to result in particle adsorption, we conducted a second set of experiments on a bare silica substrate. (Figure S1). Adsorption isotherms on bare silica (no bilayer present) show that as the concentration of O-MWCNT present in the flow cell increases, the observed SHG intensity also increases. This increase in I SHG suggests that O-MWCNTs are present at the silica/water interface (i.e., the O-MWCNT are binding to bare silica). After rinsing with buffer solution, flushing the flow system of any O-MWCNTs remaining in solution and presumably loosely adsorbed tubes, the SHG intensity remains largely unchanged within the signalto-noise of the SHG spectroscopy system indicating that O-MWCNT irreversibly adsorbed to the bare silica surface. O-MWCNTs have been shown to adsorb to silica substrates, under similar conditions of ionic strength and ph with similar trends. 16 The rationale for this trend is the same as for the experiments in which the bilayer was present at the interface. The presence of Na + screens the charges of both the negatively charged silica and the O-MWCNT reducing electrostatic repulsion and thereby reducing the barrier to particle attachment. The extent of O-MWCNT attachment, as quantified by

7 increase in I SHG, is comparable in the presence of PC-rich SLBs and bare silica. QCM-D control studies indicate that O-MWCNT do indeed adsorb to the underlying silica substrate, but that this adsorption is less than that of the adsorption of O-MWCNTs to SLBs formed on silica. However, we should emphasize that in the absence of any micron-scale defects in the SLB it is unlikely that the O-MWCNTs will adsorb to the underlying silica support in the bilayer experiments. B. SHG Adsorption Isotherms. As described in the main text, O-MWCNT have also been shown to adsorb to bilayers composed of zwitterionic 1,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC) and mixed lipid compositions containing cationic 1,2-dioleoyl- 3-trimethylammoniumpropane (DOTAP). Across all O-MWCNTs investigated with SLBs formed form DMPC, there is comparable adsorption to the SLB, regardless of the surface oxygen concentration (Figure S2). The adsorption isotherm for O-MWCNT (7%O) in the presence of an SLB formed from DMPC is presented in the main text. Here, we provide the SHG adsorption data, along with the Langmuir fit used to extract the equilibrium constant, for the adsorption of O-MWCNT (12% O) to the DMPC bilayer at 100 mm NaCl (Figure S3). The equilibrium constant from this fit (2.0± 0.6 L/µg) is comparable to that of O-MWCNTs with surface oxygen concentrations of 7% adsorbing to SLBs formed from DMPC under the same conditions. Here, we present the SHG signal intensity as a function of O-MWCNT concentration (7% O-MWCNTs) in Figure S4, with each marker representing an individual adsorption isotherm prior to normalization. In order to account for day-to-day variability in our SHG signal intensities which arise from small differences in alignment, stability, etc. (shown in Figure S4) we use the normalization process described in Troiano et al.. Briefly, we take the square root of the

8 SHG signal intensity, I SHG, and reference SHG signal gains to the SHG signal intensity associated with the SLB. After grouping neighboring concentrations of O-MWCNT, we normalize the SHG signal to the maximum signal gains (i.e. saturation coverage). QCM-D, as discussed in the main text, was used to track the adsorption of O- MWCNTs with surface oxygen concentrations of 7% and 12% to SLBs formed from DMPC at 100 mm NaCl (Figure S5). While large dissipation shifts prevent us from applying the Saurbrey equation and from extracting values for hydrated mass attached, the traces are used here qualitatively. QCM-D suggests that O-MWCNTs with higher surface oxygen concentration (12%) adsorb to a larger extent than O-MWCNTs with lower surface oxygen concentrations (7%). The role of ionic strength on the adsorption of O-MWCNT (7%O) to SLBs formed from DMPC was also explored. Though fractional increases in SHG signal intensity are evident at higher ionic strength (100 mm NaCl), no significant change in SHG signal intensity is observed at 1 mm NaCl (Figure S6). See main text for further discussion. 5. Variability in SLB/O-MWNCT interactions as observed by SFG Spectroscopy As mentioned in the main text, half of the SFG experiments exploring (3 out of 6 trials) the interaction of O-MWCNTs (7%O) with SLBs formed from DMPC at 100 mm NaCl did not result in any changes in SFG signal (Figure S7). A representative spectrum for the other three SFG experiments presented in Figure S7 were conducted with the same batch of O-MWCNTs. We suspect that this is due to batch-to-batch variability that is an inherent difficulty when working with nanomaterials. Though UV-Vis suggests that the two batches used in the SFG studies demonstrate similar aggregation behaviors, with the available techniques for evaluating the stability and characteristics of these materials in

9 suspension, we are unable to identify the difference between the two batches used for the SFG studies described herein. 6. MWCNT Preparation and Solution Characterization MWCNTs treated with 50%HNO 3 or 70%HNO 3 were first dispersed in HNO 3 (100mg MWCNTs/200mL of HNO 3 ) by sonication for 1 hour; the resulting suspension was then refluxed at 140 ⁰C for 90 minutes and allowed to cool prior to washing and purification. Oxidation with H 2 SO 4 /HNO 3 first required the addition of 100mg of MWCNTs to 8mL of a 3:1 mixture of H 2 SO 4 /HNO 3. This solution was then heated to 70 ⁰C for 8 hours without stirring. It was then allowed to cool prior to washing and purification. The O- MWCNTs were first rinsed and centrifuged (Unico, PowerSpin LX, five minutes, 4000 rpm) with deionized water for five cycles. The O-MWCNTs were then rinsed and centrifuged with 4M NaOH for five cycles, followed by rinsing and centrifuging with 4M HCl for five cycles. In the final step, the O-MWCNTs were rinsed with deionized water until the resistance of the supernatant was greater than 0.5 MΩ. After cleaning, the O- MWCNTs were dried and ball-milled to produce a uniform powder. The total oxygen content for each batch of O-MWCNTs produced was measured with X-ray photoelectron spectroscopy (XPS) using a PHI 5400 XPS (MgKα eV irradiation energy, 58.7eV pass energy, step size 0.125eV, data processed with CasaXPS, Teignmouth, UK). The level of oxidation was determined by XPS to be 6% and 7.4% for the MWCNTs oxidized with 50%HNO 3 and 70%HNO 3, respectively. Two different batches of oxidized MWCNTs were prepared using H 2 SO 4 /HNO 3 and determined to have 12.3% and 8% oxygen. To obtain stable suspensions for the experiments described below, several milligrams of dried O-MWCNTs were sonicated (Branson 1510 Ultrasonic Bath, 70W) in

10 HPLC-grade water overnight and subsequently centrifuged to remove any silica introduced by sonication and remaining carbon impurities. UV-Vis absorbance at 350 nm was used to determine the concentration and colloidal stability of O-MWCNT suspensions by taking advantage of the linear dependence of measured absorbance on O- MWCNT concentration Absorbance measurements were carried out using a Varian Cary 50 UV-Vis spectrophotometer. O-MWCNT suspensions with concentrations of either 1 mg/l or 200 µg/l were monitored in 100 mm NaCl, Tris buffer, and at a ph of 7.4 to determine their colloidal stability. UV-Vis analysis of the prepared suspensions carried out in a 1cm path length quartz cell and in a 5cm path length quartz cell gave the same results. For DLS measurements, O-MWCNT suspensions were placed into disposable cuvettes. Each time point represents one measurement collected for 10 seconds over 15 individual runs. For EPM measurements, O-MWCNT suspensions were placed into disposable folded capillary cells. For the SFG, SHG, and QCM-D studies, the stock O-MWCNT suspensions were vortexed for s to suspend any particles that might have settled during storage. The appropriate volume was removed from the stock solution and added to a clean scintillation vial containing Tris buffer solution. Serial dilution yielded solutions with concentrations ranging from 1 ng/l to 2 mg/l for SHG adsorption isotherms.

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13 Figure S1. Fractional increase in SHG signal intensity as a function of O-MWCNT concentration, in µg/l, in the presence of bare fused silica substrate at both 1 mm NaCl (open gray circle) and 100 mm NaCl (filled gray circle). Error bars are generated from the standard deviation of all data points at a given ionic strength (shown on last for clarity). Figure S2. SHG E-fields, referenced to the SHG signal generated from SLBs formed from DMPC prior to interaction with O-MWCNTs and normalized to the maximum E- field, as a function of O-MWCNT concentration, in µg/l, at 100 mm NaCl, 10 mm Tris, ph 7.4. O-MWCNTs with surface oxygen concentrations of 7%O (open black circle), and 12%O (filled triangle) are shown. Figure S3. SHG E-field as a function of O-MWCNT concentration (12% O), in µg/l collected at 300 nm, normalized to maximum E-field at high O-MWCNT concentration recorded at 100 mm NaCl, 298 K, ph 7.4 in the presence of a SLB formed from DMPC at 100 mm NaCl and referenced to the SHG signal from an SLB formed from DMPC at the silica/water interface (filled circles). The black solid line is a fit of the Langmuir adsorption model to the experimental data collected at 100 mm NaCl in the presence of a SLB formed from DMPC, specifically of the form θ = K L C/(1+K L C), where K L is the apparent equilibrium attachment constant, C is the concentration of O-MWCNT in suspension (µg/l), and θ is the relative SHG E-field. Figure S4.SHG signal intensity as a function of O-MWCNT concentration (7% O), in mg/l collected at 300 nm, normalized to maximum SHG signal intensity at high O- MWCNT concentration recorded at 100 mm NaCl, 298 K, ph 7.4 in the presence of a SLB formed from DMPC at 100 mm NaCl and referenced to the SHG signal from an

14 SLB formed from DMPC at the silica/water interface. Each marker represents a different trial. Figure S5. Shifts in frequency upon introduction of 1 mg/l O-MWCNT with surface oxygen concentrations of 7% (black solid) and 12% (gray solid line) at 100 mm NaCl and ph 7.4 (10 mm Tris) to supported lipid bilayers composed of DMPC, and rinsing with 100 mm NaCl buffer solution at ph 7.4 as determined by QCM-D measurements. Dashed lines indicate changes in aqueous conditions. (Frequency data for the 5 th harmonic are shown). Figure S6. Fractional increase in SHG signal intensity as a function of O-MWCNT concentration, in µg/l, in the presence of SLB formed from DMPC at both 1 mm NaCl (open black circle) and 100 mm NaCl (filled black circle). Error bars are generated from the standard deviation of all data points and is only showed on the last points for clarity. Figure S7. ssp-polarized SFG spectra of supported lipid bilayers formed from DMPC (green), DMPC after interaction with 1 mg/l O-MWCNT with surface oxygen concentration of 7%O (gray) and after rinsing with 100 mm NaCl buffer solution (blue). Three characteristic peaks at ~2960, ~2900, and ~2870 cm -1 are associated with C-H oscillators associated with the alkyl chains of the SLB formed from DMPC.

15 Fractional Increase in ISHG [a.u.] mm NaCl 1 mm NaCl O-MWCNT [µg/l] Figure S1.

16 7% O 12% O Normalized E SHG [a.u.] O-MWCNT [µg/l] Figure S2.

17 0.001 Normalized and Referenced E SHG [a.u.] O-MWCNT [µg/l] Figure S3.

18 200 ISHG [a.u.] O-MWCNT [mg/l] Figure S4.

19 Figure S5.

20 Fractional Increase in I SHG [a.u.] mm NaCl 1 mm NaCl O-MWCNT [µg/l] Figure S6.

21 Figure S7.