Supporting Information

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1 Supporting Information Label-Free Detection of Tear Biomarkers Using Hydrogel Coated Gold Nanoshells in a Localized Surface Plasmon Resonance-Based Biosensor Heidi R. Culver a,b,, Marissa E. Wechsler a,b,, Nicholas A. Peppas a,b,c,d,e * a Institute for Biomaterials, Drug Delivery, and Regenerative Medicine, b Department of Biomedical Engineering, c McKetta Department of Chemical Engineering, d Division of Molecular Pharmaceutics and Drug Delivery, College of Pharmacy, and e Department of Surgery and Perioperative Care, Dell Medical School, The University of Texas at Austin, Austin, TX, 787, United States Current: Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, CO, 800, United States Authors contributed equally Table of contents: Details of silica-gold nanoshell (AuNS) synthesis... Table S. DLS analysis of AuNS synthesis and modification... Figure S. Dependence of LSPR shifts on protein isoelectric point (pi)... Figure S. ELISA analysis of lactoferrin and lysozyme concentrations in human tears... Figure S. Effect of ionic strength, ph, and temperature on PNM swelling... Table S. Quantitative analysis of LSPR shifts for all protein concentrations tested in human tears... Figure S. LSPR shifts for individual vs. simultaneous protein variation... Figure S. Representative LSPR spectra for biomarker binding... 6

2 Details of silica gold nanoshell (AuNS) synthesis Nanopure water (80 ml), N NaOH (. ml), and THPC stock solution (%, ml) were combined in a 0 ml beaker and stirred for minutes. HAuCl (9.7 mm, 6.7 ml) was quickly added to the rapidly stirring THPC solution. After the color stabilized, the resultant gold colloids (AuNPs) were stored at C for at least two weeks prior to subsequent steps. Seeds for growth of silica gold nanoshells (AuNSs) were prepared by adsorbing aged AuNPs to 0 nm aminated silica NPs. The aminated silica NPs ( wt% in ethanol) were dispersed by probe sonication (hydrodynamic diameter ~80 nm, PDI<0.). Then, the dispersed silica NPs (70 L) were added to a 0-mL tube containing AuNPs (0 ml) and M NaCl ( ml), vortexed for 60 seconds, and mixed end-over-end for 8-7 hours. On the same day, potassium carbonate (0 mg) and HAuCl (9.7 mm, ml) were combined in nanopure water ( L) and allowed to age. After 8-7 hours, the seeds were collected and washed by two rounds of centrifugation (000g, 0 min), re-suspension in water, and probe sonication. The washed seeds were then diluted to an optical density of ~0. ( = 0 nm, 00 μl in 96 well plate). To determine the optimal ratio of aged HAuCl to seeds, small-scale concentration sweeps were performed in FisherBrand Premium. ml polypropylene tubes (Cat No ). AuNSs did not form well in other tubes. In these sweeps, the volume of aged HAuCl was kept constant (00 L) but the volume of the seed suspension was varied from L. Formaldehyde (7% solution, L, 0. m filtered) was added and the mixture was vortexed for 0 s. The optimal ratio was the one that gave the best peak-to-trough ratio (~) with an absorbance maximum (i.e., LSPR wavelength) around nm. Once the optimal ratio was determined, AuNS synthesis was scaled up to 0 ml batches. All reagent solutions (i.e., aged HAuCl, seed, and formaldehyde) were measured by mass rather than volume to ensure batch-to-batch consistency. After synthesis, AuNSs were centrifuged (000g, 0 min), re-suspended in a 0.0% potassium carbonate aqueous solution (0 mg in L nanopure water), and centrifuged again (000g, 0 min). AuNSs were suspended in the 0.0% potassium carbonate aqueous solution and stored at C until use. Table S. DLS for AuNS synthesis and modification Sample D h ZP (mv) Silica a Bare b PMAO-g-PEGMA b PNM b a Measurements performed after probe sonication. b Measurements performed after sonication in water. Values reported as mean SD (n=).

3 Figure S. Dependence of LSPR shifts on protein isoelectric point (pi). Gaussian fits to LSPR peaks of absorbance spectra with increasing concentrations of (a) ovalbumin (pi ~.), (b) myoglobin (pi ~7.0), or (c) hen egg white lysozyme (pi ~.) in 0.X PBS. Figure S. ELISA standard curves for analysis of lactoferrin and lysozyme concentrations in human tears.

4 D h X PBS (ph 7.) X PBS (ph 7.) 0.X HBS (ph.) X HBS (ph.) Temperature ( C) Figure S. Effect of ionic strength, ph, and temperature on PNM swelling. PNM nanogel swelling at ph 7. (i.e., in PBS, blue shades) was more sensitive to changes in ionic strength (as evidenced by the significant difference between hydrodynamic diameter in 0.X vs. X PBS) than nanogel swelling at ph. (i.e., in HBS, red shades), but was less sensitive to temperature. Table S. Quantitative analysis of LSPR shifts for all protein concentrations tested in diluted human tears. PBS HBS λlspr λlspr λlspr Both λlspr λlspr Lactoferrin Lysozyme Lactoferrin Lysozyme 6. ± ± ±. ± 0.7 ±.9 0 ±.6 6 ±. 7 ±.8 ±. 6 ±. 6 ±. 8 ±.8 6 ±. ±.6 ±. ±.8 ±. ± ±. ±. ±. ±.7 6 ±.0 6 ±.8 96 ±. ± 6. 9 ±. 6 ±. 0 ±. ±. 6 0 ±. 9 ±. ±. ±.8 0 ±. ±. 6 ±.6 ±.0 ±.8 9 ±. 0 ±.0 6 ±.6 6 ±. ±.8 6 ±.0 ±.9 ±.0 7 ±.0 76 ±. 6 ± 6.0 ±. ±. ±.8 7 ±. 96 ±.0 7 ±. ±.8 ±. ±. 8 ± ±.6 9 ±.0 9 ±.0 ±. ±.0 0 ±.7 [Protein] (μg/ml) λlspr Both

5 Figure S. LSPR shifts for individual vs. simultaneous protein variation. Black bars represent results from when lactoferrin was varied while keeping baseline lysozyme; white bars represent when lysozyme was varied while keeping baseline lactoferrin; red bars represent the summation of the black and white bars, which corresponds to hypothetical shifts if there were no competition and sufficient binding sites for both proteins; and gray bars represent LSPR shifts measured when the concentrations of both proteins were varied simultaneously. (a) In PBS, both proteins contribute to the LSPR signal at low concentrations ( 96 μg/ml), as evidenced by the fact that the sum of the LSPR shifts for lysozyme and lactoferrin alone (red) was not significantly different from the actual LSPR shifts observed (gray). However, at higher concentrations ( 6 μg/ml), the measured shifts in LSPR for simultaneous variation (gray) were not significantly different than those for lysozyme alone (white), suggesting preferential binding of lysozyme. (b) In HBS, there was high cross-reactivity, as evidenced by the binding saturation at low concentrations and the fact that shifts in LSPR were similar when protein concentrations were varied simultaneously (gray) to when the concentrations were varied individually (black and white) for most concentrations above 96 μg/ml. Data are presented as mean standard deviation (n, Dunnett s test, * p < 0.0, p < 0.0, # p< 0.00).

6 a Extinction Efficiency Lactoferrin Lysozyme Both μg/ml 7 μg/ml μg/ml μg/ml 9 μg/ml μg/ml μg/ml μg/ml 9 μg/ml μg/ml μg/ml Normalized extinction Figure S. Representative LSPR spectra for biomarker binding. (a) Raw extinction spectra (top) and normalized spectra in the region used for Gaussian fitting (bottom) in HBS diluted tears. (B) Raw extinction spectra (top) and normalized spectra in the region used for Gaussian fitting (bottom) in PBS. 6