Fluorescence-Based Assessment of Plasma-Induced Hydrophilicity in Microfluidic Devices via Nile Red Adsorption and Depletion

We present a simple method, called fluorescence-based assessment of plasma-induced hydrophilicity (FAPH), that enables spatial mapping of the local hydrophilicity of surfaces normally inaccessible by traditional contact angle measurement techniques. The method leverages the change in fluorescence of a dye, Nile Red, which is adsorbed on an oxygen plasma-treated surface, and its correlation with the contact angle of water. Using FAPH, we explored the effect of microchannel geometries on the penetration distance of oxygen plasma into a microchannel and found that entrance effects prevent uniform treatment. We showed that these variations have a significant impact on cell culture, and thus the design of cell-based microfluidic assays must consider this phenomenon to obtain repeatable and homogeneous results.

To obtain a baseline for cell morphology and adhesion in microchannels, native (non-NRadsorbed) PS substrates were passively bonded to cured PDMS layers with microchannels. Prior to bonding, PS substrates were exposed to three different levels of oxygen plasma treatment: (i) highly hydrophilic (<15˚), (ii) moderately hydrophilic (30-60˚), and (iii) hydrophobic (~85˚, untreated PS). Cured PDMS layers containing microchannels were then passively bonded to the PS substrates to create enclosed channels for cell culture. As a positive control for HUVECs, a fourth surface treatment involved coating the PS with fibronectin (FN) at 0.1 mg/mL to promote cell adhesion via attachment to adsorbed matrix proteins that are typically found in endothelial basement membranes 1 .
To characterize the effect of plasma treatment patterns on cell adhesion and growth in microchannels, native (non-NR-adsorbed) PS substrates were passively bonded to cured PDMS layers with microchannels to create enclosed conduits. Plasma treatment was applied to each sample at 2 mbar, 50% power, and for 50 seconds. The PDMS was then removed, and cells were seeded over the entire exposed PS surface.

Cell Imaging and Analysis
Images of cell stains of phalloidin (green) and nuclear Hoechst dye (blue) were acquired with a 10x objective, and overlaid using ImageJ. All cell analysis and quantification was performed using ImageJ, where images were converted to binary images using the threshold function.
Binary images were used to quantify overall area coverage and number of cells in each microchannel condition. To quantify penetration distance with cell, fluorescence intensity along channel length was averaged across channel width, and the resulting intensity profile was analyzed to determine the location where the intensity reached half of its maximum.

Cell Adhesion: The Need for in situ Measurements
The need to develop a fast, convenient, and informative method for determining surface hydrophilicity within microchannels was motivated in part by observations of inadequate cell adhesion and spreading in microchannels in cases when the plasma treatment was not optimized.
Further, little is known about how plasma treatment occurs in confined environments though plasma in microchannels has been used to perform analog computing 2 . To illustrate how surface conditions, after oxygen plasma treatment, can affect cell adhesion, we cultured two established 4 adherent cell types in microchannels with PS substrates, human umbilical vein endothelial cells (HUVECs) (Figure S4A), and NIH-3T3 fibroblasts (FBs) (Figure S4B), quantified the number of adhered cells and the cell spreading area (Figure S4C), and normalized this data against their respective control conditions (Figure S4D), as a metric of compatibility with the surface. For HUVECs, the control surface was PS with FN coating at 0.1 mg/mL, and for FBs, the control surface was untreated PS.