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

Abstract

Miniaturized systems, such as integrated microarray and microfluidic devices, are constantly being developed to satisfy the growing demand for sensitive and highthroughput 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 1ethyl-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/m 2 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.

Improved Osteogenesis of Selective-Laser-Melted Titanium Alloy by Coating Strontium-Doped Phosphate With High-Efficiency Air-Plasma Treatment

“…This evidence demonstrates that air-plasma treatment can significantly improve the biocompatibility of inorganic implants and may be conducive for osteogenesis. After air-plasma treatment, hydroxyl radicals are added to the treated surface (Tsougeni et al, 2015;Kurzyp et al, 2017;Sathish et al, 2019;Wieland et al, 2020;Zhang et al, 2020), further reducing water contact angle shown in Figure 4, which forms an adsorption effect with adhesion proteins and enhances biocompatibility. Hydrophilicity is critical for the bioactivity of bone tissue implants and can improve cell adhesion and proliferation.…”

Antibody Micropatterned Lubricant‐Infused Biosensors Enable Sub‐Picogram Immunofluorescence Detection of Interleukin 6 in Human Whole Plasma

“…Antibody embedded lubricant‐infused surfaces were created on PMMA, a low‐priced polymer with many advantages such as optical transparency, durable chemical and mechanical properties, and recyclability. [ 57,58 ] PMMA surfaces were first oxygen plasma treated to induce hydroxyl groups on the surface, and then fluorosilanized via chemical vapor deposition (CVD) of trichloro(1 H ,1 H ,2 H ,2 H ‐perfluorooctyl)silane followed by heat treatment at 90 °C to promote the hydrolysis and condensation reactions. Immediately after the plasma treatment, the hydrophilicity of the surface can cause adsorption of water molecules on the surface.…”

Proof‐of‐concept modular fluid handling prototype integrated with microfluidic biochemical assay modules for point‐of‐care testing

“…The microfluidic chlamydia assay module was fabricated by first covalently immobilizing MOMP (50 µg/mL) onto PMMA substrates, using our developed air-plasma enhanced bio-functionalization strategy. 6 Next, double-sided adhesive tapes were cut to form microchannels using a carbon dioxide laser cutter (VLS 3.50, Universal Laser Systems, USA), and bonded to the MOMP-functionalized PMMA strips on one side and IPA-cleaned PMMA strips on the other side to complete the microfluidic device ( Figure 1B). The bare surfaces of the PMMA microchannels were finally blocked with 1% weight/volume of bovine serum albumin, to prevent nonspecific adsorption of proteins, and attached to the upper portion of the FHD using M3 bolts to complete the assembled prototype.…”

“…The microfluidic chlamydia assay module was fabricated by first covalently immobilizing MOMP (50 µg/mL) onto PMMA substrates, using our developed air‐plasma enhanced bio‐functionalization strategy 6 . Next, double‐sided adhesive tapes were cut to form microchannels using a carbon dioxide laser cutter (VLS 3.50, Universal Laser Systems, USA), and bonded to the MOMP‐functionalized PMMA strips on one side and IPA‐cleaned PMMA strips on the other side to complete the microfluidic device (Figure 1B).…”