Supporting Information MATERIALS AND METHODS Materials Polycaprolactone with an average molecular weight of 40 kDa was obtained from Polysciences, Inc. Anhydrous N,N-dimethylformamide (DMF), mouse laminin, Tween 20, N-(3Dimethylaminopropyl)-N’-ethylcarbodiimide (EDC), N-hydroxy succinimide (NHS), 2-(Nmorpholino)ethanesulfonic acid (MES), and sodium dodecyl sulfate (SDS) were obtained from Sigma-Aldrich. Dichloromethane and phosphate buffered saline (PBS) were obtained from Fisher Scientific. Fabrication of electrospun fibers A 20 wt % solution of PCL was prepared by dissolving PCL in a 50:50 (w/w) mixture of anhydrous DMF and dichloromethane and stirring at RT overnight. PCL fibers were electrospun using a custom built set-up consisting of a syringe pump (Aladdin AL-1000) and a rotating 2” wide mandrel. The mandrel was connected to a motor (Bodine NSH-12R) with a speed controller (Minarik Electric Company SH-14), allowing the mandrel to rotate between 0 and 8000 rpm. A 5-mL syringe was filled with polymer solution and fed through a 21 gauge stainless steel needle at flow rates of 1-5 mL/h with an applied potential of 18.5 kV. The gap between the needle and the collector was fixed at 7 inches and the collector was set to an applied potential of -3 kV. Fibers were dried under vacuum at RT overnight before characterization and functionalization. Electrospun fiber modification For the covalent attachment of protein to the fibers, vacuum dried PCL fibers were cut to fit in 24 well plates and plasma treated in air using an inductively coupled radio-frequency (RF) plasma cleaner (Harrick PDC-32-G) for 5 min at a RF power of 18 W to introduce carboxylic functionalities to the surface of the fibers. Plasma treated fibers were then immersed in a MES buffer (pH = 6) containing of 5 mg/mL EDC and 5 mg/mL NHS for 1 h at RT. Fiber mats were then rinsed with MES buffer and incubated in a 50 µg/mL laminin solution overnight at 4 °C. The protein solution was removed and the mats were washed using the following methods: (1) No rinse, (2) 0.05% Tween 20 solution in PBS with gentle shaking for 30 min RT, or (3) 5% SDS with gentle shaking for 1 h at 50 °C. Mats (except for method 1) were then washed thoroughly with PBS and deionized water and dried for characterization. Two types of control samples were also included in the study: unmodified PCL fibers and plasma-treated PCL fibers that were not reacted with EDC/NHS. These samples were also evaluated with methods outlined above. Chemical Characterization Surface compositional analysis was performed using a Kratos Axis Ultra 165 X-ray photoelectron spectroscopy (XPS) system equipped with a hemispherical analyzer. Sampling areas of 1 mm x 0.5 mm were irradiated with a 100 W monochromatic Al Kα (1486.7 eV) beam and take-off angle of 90°. The XPS chamber pressure was maintained between 10−9 and 10−10 Torr. Elemental high resolution scans were conducted with a 20 eV pass energy for the C 1s, O 1s and N 1s core levels. A value of 284.6 eV for the hydrocarbon C 1s core level was used as the calibration energy for the binding energy scale. Data was processed using Casa XPS software. All reported atomic percentages are the average of n = 2 measurements on a minimum of 3 replicated samples. RESULTS Mats composed of PCL + plasma + NHS chemistry that were not rinsed retained the highest amount of laminin protein with an average nitrogen-to-carbon ratio (N/C) of 19.75 ± 0.04 (Figure S1). Rinsing the mats with 0.05% Tween 20 removed 49% of the protein, compared to the unrinsed control sample. However, using a strong surfactant (SDS) removed 74% of the protein compared to the unrinsed sample, leaving an XPS N/C ratio of 5.1 remaining. Figure S1. Atomic nitrogen-to-carbon ratios of electrospun polycaprolactone (PCL) fibers with attached laminin (LN) as determined by x-ray photoelectron spectroscopy. Key: Cov-LN = fibers were plasma treated and reacted with EDC/NHS and soaked in laminin to covalently attach laminin, plasma-LN = fibers were plasma treated and soaked in laminin, PCL-LN = unmodified fibers were soaked in laminin, Tween =fibers were rinsed in 0.05% Tween 20 surfactant in PBS, SDS = sodium dodecyl sulfate. Error bars represent mean ± standard deviation. The plasma-treated PCL mats that were not reacted with EDC/NHS had a lower amount of protein on the unrinsed sample with an N/C ratio of 11.68 ± 0.06. Rinsing these samples with 0.05% Tween removed only 24% of the protein compared to the unrinsed control sample, suggesting these samples had less physically adsorbed protein compared to the fiber mats reacted with EDC/NHS. Rinsing the mats with SDS removed 85% of the protein compared to the unrinsed control sample, leaving 1.7 (XPS N/C ratio) remaining. Thus, without the EDC/NHS coupling reaction, only one-third of the protein remained after rinsing with SDS compared to the samples that underwent the covalent coupling reaction. The PCL control sample had similar amounts of protein on the unrinsed sample with a XPS N/C ratio of 17.02 ± 0.08. Rinsing with 0.05% Tween removed 93% of the protein. The fiber diameter was also investigated before and after covalent protein attachment (Figure S2). There was no significant change in the diameter or morphology (PCL diameter =155 ± 70 nm (n = 50), PCL + LN diameter = 138 ± 90 nm (n = 54)). Figure S2. Scanning electron micrograph of electrospun PCL fibers. (A) Unmodified PCL fibers, (B) PCL fibers with covalently attached laminin protein. Scale bar denotes 5 µm. DISCUSSION Sodium dodecyl sulfate is known to unfold proteins and interfere with the forces responsible for the physical adsorption of protein to surfaces. For these reasons, it is often employed as a test to determine whether proteins are covalently attached or physically adsorbed to surfaces. In the experiments conducted, a large portion of the protein attached to the surfaces was removed upon rinsing with SDS. In fact, approximately 75% of the protein was removed compared to the norinse controls, and 50% compared to the 0.05% Tween-rinsed samples. Thus, it is evident that the 0.05% Tween rinsed samples, which were used in cell culture assays, had both covalently attached and physically adsorbed proteins. While SDS is a superior surfactant for protein removal, since it denatures proteins, it cannot be used to prepare samples for cell culture experiments. The plasma-treated mat reacted with NHS had more laminin protein attached than the plasmatreated mats not reacted with NHS (XPS N/C=19.8 ± 0.04 vs. 11.7 ± 0.06 for laminin). Since the majority of protein attached was physically adsorbed, the effect of NHS on the no-rinse sample was not expected to be significant. In addition, the PCL control no-rinse sample also had a lower amount of protein than the plasma-treated mat reacted with NHS. This is most likely attributed to the contact angle or wettability of the surfaces. The surface of the plasma-treated mat was extremely wettable, with a contact angle of less than 5º due to the high concentration of oxygencontaining species introduced from the plasma treatment. The native PCL fiber mat was very hydrophobic, with a contact angle of 124º ± 6.1º (n = 12). The NHS reacted plasma-treated mat has a contact angle in between the two extremes due to introduction of carbon on the surface from the NHS group (25º ± 5.3º, n = 5), which may have better enabled protein adsorption.