Air Plasma-Enhanced Covalent Functionalization of Poly(methyl methacrylate): High-Throughput Protein Immobilization for Miniaturized Bioassays

Miniaturized systems, such as integrated microarray and microfluidic devices, are constantly being developed to satisfy the growing demand for sensitive and high-throughput biochemical screening platforms. Owing to its recyclability, and robust mechanical and optical properties, poly(methyl methacrylate) (PMMA) has become the most sought after material for the large-scale fabrication of these platforms. However, the chemical inertness of PMMA entails the use of complex chemical surface treatments for covalent immobilization of proteins. In addition to being hazardous and incompatible for large-scale operations, conventional biofunctionalization strategies pose high risks of compromising the biomolecular conformations, as well as the stability of PMMA. By exploiting radio frequency (RF) air plasma and standard 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) chemistry in tandem, we demonstrate a simple yet scalable PMMA functionalization strategy for covalent immobilization (chemisorption) of proteins, such as green fluorescent protein (GFP), while preserving the structural integrities of the proteins and PMMA. The surface density of chemisorbed GFP is shown to be highly dependent on the air plasma energy, initial GFP concentration, and buffer pH, where a maximum GFP surface density of 4 × 10^-7 mol/m² is obtained, when chemisorbed on EDC-NHS-activated PMMA exposed to 27 kJ of air plasma, at pH 7.4. Furthermore, antibody-binding studies validate the preserved biofunctionality of the chemisorbed GFP molecules. Finally, the coupled air plasma and EDC-NHS PMMA biofunctionalization strategy is used to fabricate microfluidic antibody assay devices to detect clinically significant concentrations of Chlamydia trachomatis specific antibodies. By coupling our scalable and tailored air plasma-enhanced PMMA biofunctionalization strategy with microfluidics, we elucidate the potential of fabricating sensitive, reproducible, and sustainable high-throughput protein screening systems, without the need for harsh chemicals and complex instrumentation.