Blocking Protein Adsorption in Microfluidic Chips by a Hydrophobin Coating

Abstract

Microfluidics allows the miniaturization of biochemical analyses. Small dimensions reduce sample and reagent consumption and enhance reaction rates. A downside is that high surface-to-volume ratios increase the unspecific binding of proteins to the substrate material. The resulting sample loss and reagent depletion decrease the sensitivity and specificity of protein-based assays, especially if low concentrations are analyzed. Here, we introduce the hydrophobin coating of microfluidic chips made of cyclic olefin copolymers (COC). The recombinant hydrophobin H*Protein B self-assembles into stable monolayers on hydrophobic surfaces, making them hydrophilic and thus reducing hydrophobic interactions between the chip surfaces and proteins. The substrate and sealing layers of the microfluidic chip were simply dip-coated and subsequently assembled by thermodiffusion bonding, which renders our coating procedure compatible with mass fabrication. Contact angle measurements and atomic force microscopy were used to evaluate the effect of high temperatures (107 °C) on COC substrates coated with H*Protein B. The efficiency of the protein-repellent coating was evaluated by depletion experiments with bovine serum albumin, human serum, and cerebrospinal fluid in microfluidic chips. Protein recovery was investigated down to protein concentrations of 0.3 μg/mL. Recoveries of 90% were observed with total protein amounts of 10 ng, even for microfluidic channels up to 835 mm in length and with a cross section of 80 μm × 230 μm in a COC 6013/8007 foil. For comparison, only 30 and 60% of the protein was recovered in uncoated microfluidic channels with lengths of 835 and 128 mm, respectively. The longterm stability of the hydrophobin-coated chips for 8 weeks was demonstrated.

Assessing Surface Adsorption in Cyclic Olefin Copolymer Microfluidic Devices Using Two-Dimensional Nano Liquid Chromatography-Micro Free Flow Electrophoresis Separations

“…The existence of protein adsorption in COC devices is well established. − However, nLC-μFFE separations provide unique contextual insight on the temporal dynamics of adsorption in a continuous-flow system. The relatively low protein adsorption on COC surfaces is appropriate for many μFFE applications, especially those that rely primarily on lateral, electrophoretic separation, which is not affected by surface interactions.…”

“…Previous surface characterization studies have demonstrated that hydrophilic treatments successfully reduce adsorption in COC devices. ,− Surface-treated COC devices exhibit similar or lower adsorption of proteins and hydrophobic small molecules than devices made from other surface-treated polymers. However, these studies rely on single-time point measurements of analyte retention or recovery after a period of incubation.…”

“…Microfluidic devices fabricated in COC often undergo surface treatments to introduce hydrophilic groups that reduce these interactions . Common surface functionalization approaches include plasma treatments, ,− UV/ozone oxidation, − and hydrophobic chemical coatings introduced by UV photografting, − dip-coating, or dynamic coating …”

Toward the Development of Rapid, Specific, and Sensitive Microfluidic Sensors: A Comprehensive Device Blueprint

“…This is most commonly observed in devices fabricated with hydrophobic materials such as PDMS or PMMA that have high affinity toward hydrophobic residues in the analyte proteins . This nonspecific protein adsorption can be significantly reduced by increasing the hydrophilicity of the microchannel walls by means of chemical modification or by using blocking agents that repel proteins and other molecules. − The most common blocking agents used to reduce nonspecific protein absorption range from protein-based agents such as bovine serum albumin (BSA) and casein from dry milk , to chemical agents such as Tween-20 and omniphobic fluorinated silanes. , …”

Patterning of COC Polymers by Middle‐Energy Ion Beams for Selective Cell Adhesion in Microfluidic Devices

“…Advantages of surface modifications include the ability to control cell attachment, [23] improved biocompatibility, [24,25] and tailored functionality for specific applications. [26] These modifications can be achieved through various approaches, such as low-pressure plasma treatment like oxygen, argon, and nitrogen plasma, [27][28][29][30] UV/ozone treatment, [31] electron beam treatment, [32] laser structuring, [33,34] chemical functionalization including self-assembled monolayers, [35][36][37][38] and physical patterning. [39][40][41] However, each method has its advantages and disadvantages, particularly when considering mass production in microfluidic device fabrication.…”