Supporting Information Effect of Non-Ionic Surfactants on Dispersion and Polar Interactions in the Adsorption of Cellulases onto Lignin Feng Jiang, Chen Qian, Alan R. Esker and Maren Roman, * Macromolecules Innovation Institute, Department of Chemistry, and Department of Sustainable Biomaterials, Virginia Tech, Blacksburg, Virginia 24061, United States Scheme S1. Phosphitylation of hydroxyl groups by CTMDP S1
Table S1. Surface tensions, γ l, of the test liquids and their polar and dispersive components, γ p l and γ d l, respectively. 1 test liquid γ l p (mn m 1 ) γ l d (mn m 1 ) γ l (mn m 1 ) water 51 21.8 72.8 diiodomethane 0 50.8 50.8 formamide 19 39 58 Table S2. Average static contact angles of the three test liquids on untreated and Tween 80- treated lignin substrates a test liquid OL KL MWL untreated treated untreated treated untreated treated water 65º ± 1º 60º ± 0º 62º ± 1º 58º ± 0º 54º ± 1º 52º ± 1º diiodomethane 24º ± 1º 32º ± 0º 22º ± 1º 33º ± 1º 18º ± 1º 28º ± 1º formamide 27º ± 1º 27º ± 0º 25º ± 0º 25º ± 1º 26º ± 1º 19º ± 1º a Data are means ± one standard deviation of three measurements. Table S3. Surface concentrations from SPR of adsorbed Tween 80 (ГTween 80) and cellulase (ГCellulase) adsorbed on untreated and Tween 80-treated SAM substrates after buffer rinse a substrate G Tween 80 (mg m 2 ) untreated G Cellulase (mg m 2 ) treated SAM-CH 3 1.38 ± 0.35 2.1 ± 0.3-0.03 ± 0.02 SAM-OH 0.08 ± 0.04 0.33 ± 0.03 0.05 ± 0.01 SAM-COOH 0.07 ± 0.04 2.8 ± 0.03 2.66± 0.01 a Data are means ± one standard deviation of three measurements. S2
Table S4. Surface roughnesses of the SAM substrates before and after Tween 80 and cellulase adsorption. a substrate surface roughness (nm) bare gold 1.1 ± 0.1 SAM-CH 3 2.2 ± 0.2 untreated SAM-CH 3 after exposure to cellulase solution 7.7 ± 0.4 Tween 80-treated SAM-CH 3 1.5 ± 0.1 Tween 80-treated SAM-CH 3 after exposure to cellulase solution 1.3 ± 0.1 SAM-OH 1.8 ± 0.1 untreated SAM-OH after exposure to cellulase solution 3.2 ± 1.2 Tween 80-treated SAM-OH 1.5 ± 0.2 Tween 80-treated SAM-OH after exposure to cellulase solution 2.0 ± 0.4 SAM-COOH 1.4 ± 0.1 untreated SAM-COOH after exposure to cellulase solution 0.8 ± 0.2 Tween 80-treated SAM-COOH 1.3 ± 0.1 Tween 80-treated SAM-COOH after exposure to cellulase solution 1.0 ± 0.3 a Values were measured over 5 μm 5 μm areas Table S5. Surface concentrations, Г QCM and Г SPR, and water contents of the adsorbed cellulase layers on the SAM substrates. substrate Г QCM (mg m 2 ) Г SPR (mg m 2 ) water content (%) SAM-CH 3 4.1 2.3 43.5 SAM-OH 1.1 0.3 72.7 SAM-COOH 6.1 2.6 57.9 S3
Table S6. Mass increases on untreated and Tween 80-treated lignin substrates after 10 min and 4 h of exposure of the substrates to a cellulase-containing buffer solution a,b,c substrate untreated substrates mass increase (mg m 2 ) Tween 80-treated substrates 10 min 4 h 10 min 4 h OL 6.0 ± 0.3 13.9 ± 0.5 5.4 ± 0.3 16.5 ± 1.2 KL 5.5 ± 0.5 9.5 ± 0.4 1.3 ± 0.1 11.9 ± 0.6 MWL 3.7 ± 0.3 17.5 ± 0.9 1.4 ± 0.2 6.8 ± 0.1 a determined by viscoelastic modeling of Δf and ΔD for the 3 rd and 5 th overtones. The density of the adsorbed surface layer was fixed at 1100 kg m 3. 2 b Data are means ± one standard deviation of three measurements. c The mass of the adsorbed cellulase layer includes the mass of incorporated water. Figure S1. Quantitative 31 P NMR spectra of (a) OL, (b) KL, and (c) MWL after phosphitylation with CTMDP. The internal standard (I.S.) peak stems from phosphitylated e-hndi. S4
Figure S2. Representative (a) (Δf n /n), (b) DD curves (5 th overtone), and (c) Dθ sp for Tween 80 adsorption onto SAM substrates: (red ) SAM-CH 3, (blue r) SAM-OH, (black ) SAM-COOH. Arrows indicate a switch in liquid feed. Figure S3. (Δf n /n) curve (5 th overtone) for Tween 80 adsorption onto the OL substrate, illustrating Δ(Δf n /n) I, Δ(Δf n /n) II, and Δ(Δf n /n) III. Arrows indicate a switch in liquid feed to the QCM-D flow modules. S5
Figure S4. Representative (a) (Δf n /n) and (b) DD curves (5 th overtone) for PEG adsorption onto lignin substrates: (red ) OL, (blue r) KL, and (black ) MWL. Arrows indicate a switch in liquid feed to the QCM-D flow modules. Frequency Dissipation 0 (a) 12 (a) -20 10 Untreated -40-60 -80-100 Tween 80-treated ( f n /n) /Hz Dx10 6 8 6 4 2 0 0 1 2 Time /h 3 4 0 1 2 Time /h 3 4 Figure S5. Representative (Δf n /n) (a) and DD (b) curves (5th overtone) from cellulase adsorption experiments on untreated and Tween 80-treated lignin substrates: OL substrate (), KL substrate (Δ), and MWL substrate ( ). Arrows indicate a switch in liquid feed to the QCM-D flow modules from cellulase solution to buffer. S6
Figure S6. Representative (a-c) (Δfn/n) (5th overtone), (d-f) ΔD, and (g-i) Dθsp for cellulase adsorption onto SAM substrates (red ) before and (blue r) after Tween 80 treatment: (a,d,g) SAM-CH3, (b,e,h) SAM-OH, and (c,f,i) SAM-COOH. Arrows indicate a switch in liquid feed. S7
Figure S7. Representative fit of the decreasing section of a (Δf n /n) curve (5 th overtone) for cellulase adsorption onto a lignin substrate (untreated OL substrate): (red) experimental data, (blue) fit with eq 2. Figure S8. Representative adsorbed mass vs. time curves for cellulase adsorption onto untreated (open symbols) and Tween 80-treated (filled symbols) lignin substrates: (a) OL, (d) KL, and (c) MWL. Arrows indicate a switch in the liquid feed to the QCM-D flow modules from cellulase solution to buffer. The mass of the adsorbed layer was determined by viscoelastic modeling of Δf and ΔD for the 3 rd and 5 th overtones. S8
Figure S9. Representative (a) (Δfn/n) and (b) DD curves (5th overtone) for cellulase adsorption onto PEG-treated lignin substrates: ( ) OL, (r) KL, and ( ) MWL. Arrows indicate a switch in liquid feed to the QCM-D flow modules from cellulase solution to buffer. Figure S10. Representative AFM height images: (a) Bare gold surface, (b) SAM-CH3, (c) cellulase-exposed SAM-CH3, (d) Tween 80-treated SAM-CH3, (e) cellulaseexposed Tween 80-treated SAM-CH3, (f) SAM-OH, (g) cellulase-exposed SAMOH, (h) Tween 80-treated SAM-OH, (i) cellulase-exposed Tween 80-treated SAMOH. The scan size and z-scale for all images are 5 μm 5 μm and 30 nm, respectively. S9
EXPERIMENTAL SECTION S1.1. Preparation and characterization of self-assembled monolayers (SAMs) Gold QCM-D (Q-sense AB, gold, 5 MHz) and SPR (Reichert, gold) sensor chips were cleaned by exposure of the gold surface to piranha solution for 40 min, rinsed with DI water and dried in a stream of nitrogen. Immediately upon drying, the sensor slides were placed in a 1 mm solution of thiol (1-dodecanethiol (98%), 11-mercapto-1-undecanol (99%), or 12-mercaptododecanoic acid (96%), all from Sigma-Aldrich) in ethanol for at least 24 hours. When they were needed, the SAM substrates were retrieved from the thiol solution, rinsed with ethanol for the removal of thiol molecules, rinsed with DI water, dried in a stream of nitrogen, and immediately characterized in terms of contact angle and surface roughness. The contact angles of the SAM substrates were determined from sessile drop contact angles, measured with a video-based contact angle goniometer (FTA200, First Ten Angstroms, Inc.). 1-dodecanethiol-treated gold substrates (SAM-CH3) had a contact angle of 102 ± 5. In contrast, gold substrates treated with 11-mercapto-1-undecanol (SAM- OH) had contact angle of 9.6 ± 1.5. According to the literature, the static contact angles for SAM- CH, SAM-OH and SAM-COOH are 114.1 ± 0.4, 8.6 ± 2.4 and 4.3 ± 1.2. 3 The measured static contact angles were reasonably close to the values reported in the literature. As expected, the contact angle results indicated that the SAM-CH 3 substrates were hydrophobic, whereas the SAM-OH substrates were hydrophilic. S1.2. Monitoring adsorption by quartz crystal microbalance with dissipation monitoring (QCM-D) A 1 M sodium citrate buffer (ph=4.5) was prepared by dissolution of a predetermined amount of citric acid monohydrate and sodium hydroxide in DI water. 1 mm Tween 80 solution, used in the experiments, was prepared by mixing of 2 ml of 50 mm Tween 80 solution in DI water with S10
5 ml of 1 M sodium citrate buffer, followed by dilution with DI water to 100 ml. 50 mm sodium citrate buffer solution (ph=4.8) was prepared by twenty fold dilution of 1 M sodium citrate buffer 20 times by volume with DI water. In the remainder of this chapter, the term buffer solution refers to 50 mm sodium citrate buffer. Cellulase solution was prepared by dilution of 0.5 g Celluclast 1.5 L with 100 ml buffer solution. A Q-Sense E4 QCM-D system was used for measurement of the "wet mass" of cellulase adsorbed on the SAMs. The flow rate for all solutions was set at 0.25 ml min 1 and the temperature of the solutions was 22.00 ± 0.02 C. The contact angles of the SAM substrates were determined before the slides were placed into the QCM-D flow modules. For measurements of cellulase adsorption onto untreated SAMs, an initial buffer solution was introduced until a flat baseline was obtained, upon which it was switched to cellulase solution. After 40 min of cellulase adsorption, the buffer feed was restored for removal of reversibly adsorbed cellulase from the SAM. For measurements of cellulase adsorption on Tween 80-treated SAMs, once the flat based line was obtained, the initial buffer solution was replaced by 1 mm Tween 80 solution for 23 min. Then, the buffer solution was restored for 20 min for rinsing of loosely bound Tween 80 molecules off of the substrates. Finally, a 40 min cellulase solution feed was introduced for cellulase adsorption, followed by a buffer rinse. S1.3. Monitoring adsorption by surface plasmon resonance (SPR) A Reichert SR7000 SPR spectrometer was used for measurement of the "dry mass" (or "optical mass") of cellulase adsorbed onto the SAMs. The flow rate for all solutions was set at 0.25 ml min 1 and the temperature of the solutions was 22.00 ± 0.02 C. The contact angles of the SAM substrates were determined before the slides were placed into the SPR flow modules. S11
The buffer, Tween 80, and cellulase solutions used in SPR experiments were the same as the solutions used in QCM-D experiments. All solutions were degassed for at least 8 h before being introduced into the flow cell. A switch valve was used to switch solution feeds and care was taken when switching solutions to avoid introducing bubbles into the flow cell. Measurements of cellulase adsorption were carried out in the same manner as the QCM-D experiments. The change in surface plasmon resonance angle, θ sp, was recorded and used for the calculation of surface concentration (Г SPR ) in units of mg m 2. S1.4. Atomic force microscopy (AFM) An Asylum Research AFM (MFP-3D-BIO, Asylum Research) was used for AFM imaging. During AFM measurements, height images were obtained under ambient conditions (22 C, 50% humidity) with OMCL-AC160TS standard silicon probes (Olympus Corp.). The roughness of height images was determined with the MFP-3D software as the root mean square value of all collected height values in the scanned area. References 1. Good, R. J.; Chaudhury, M. K.; van Oss, C. J., Theory of adhesive forces across interfaces 2. Interfacial hydrogen bonds as acid-base phenomena and as factors enhancing adhesion. In Fundamentals of adhesion, Lee, L.-H., Ed. Plenum Press: New York, 1991; pp 153-179. 2. Dutta, A. K.; Nayak, A.; Belfort, G. Viscoelastic properties of adsorbed and cross-linked polypeptide and protein layers at a solid liquid interface. J. Colloid Interface Sci. 2008, 324, 55 60. 3. Tsai, M. Y.; Lin, J. C. Surface characterization and platelet adhesion studies of selfassembled monolayer with phosphonate ester and phosphonic acid functionalities. Journal of Biomedical Materials Research 2001, 55, 554-565. S12