title: Evaluation of different nonspecific binding blocking agents deposited inside poly(methyl methacrylate) microfluidic flow-cells.
abstract: Poly(methyl methacrylate) (PMMA) flow-cells containing microwells were deposited with different nonspecific binding blocking agents, namely, bovine serum albumin (BSA), cationic lipid (DOTAP:DOPE) and diethylene glycol dimethyl ether (DEGDME). Water contact angle (WCA) and atomic force microscope (AFM) measurements were carried out to confirm the successful depositions of BSA, DOTAP, and DEGDME onto the PMMA surfaces. Fluorescent intensity measurements were performed to evaluate the degree of nonspecific adsorption of Cy5-labeled anti-IgG proteins onto plain and oxygen plasma-treated (PT) PMMA flow-cells as well as PMMA flow-cells deposited with different above-mentioned blocking agents. We then employed a label-free detection method called total internal reflection ellipsometry (TIRE) to evaluate the stability of the deposited blocking agents inside the PMMA flow-cells. It was found that, while DOTAP:DOPE was the best agent for blocking the nonspecific adsorption, it could be removed from the PMMA surfaces of the flow-cells upon rinsing with phosphate buffered saline (PBS) and later deposited back onto the Au-coated glass sensing substrate of the TIRE. The removal of the blocking agents from PMMA surfaces and their deposition onto the sensing substrate were further manifested by measuring the kinetics and the amount of adsorbed anti-α-hCG proteins. Overall, the dry DEGDME coating by plasma-enhanced chemical vapor deposition (PECVD) showed very good blocking and excellent stability for subsequent assay inside the microwells. Our results could be useful when one considers what blocking agents should be used for PMMA-based microfluidic immunosensor or biosensor devices by looking at both the blocking efficiency and the stability of the blocking agent.
authors.affiliation: Biomedical Diagnostics Institute (BDI), Dublin City University, Dublin, Republic of Ireland. nam.le@csiro.au
authors.forename: Nam Cao Hoai
authors.initials: NC
authors.lastname: Le
authors.forename: Vladimir
authors.initials: V
authors.lastname: Gubala
authors.forename: Ram P
authors.initials: RP
authors.lastname: Gandhiraman
authors.forename: Stephen
authors.initials: S
authors.lastname: Daniels
authors.forename: David E
authors.initials: DE
authors.lastname: Williams
mesh_terms: D000327:Adsorption; D000818:Animals; D055910:Antibodies, Immobilized / Q000737:chemistry / Q000276:immunology; D002417:Cattle; D006063:Chorionic Gonadotropin / Q000276:immunology; D005026:Ethylene Glycols / Q000737:chemistry; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry; D006801:Humans; D008738:Methyl Ethers / Q000737:chemistry; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D010714:Phosphatidylethanolamines / Q000737:chemistry; D019904:Polymethyl Methacrylate / Q000737:chemistry*; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000737:chemistry; D013499:Surface Properties; D014835:Volatilization
chemical_list: C410262:1,2-dioleoyl-glycero-3-phosphatidyl ethanolamine; D055910:Antibodies, Immobilized; D006063:Chorionic Gonadotropin; D005026:Ethylene Glycols; D005229:Fatty Acids, Monounsaturated; D008738:Methyl Ethers; D010714:Phosphatidylethanolamines; D000644:Quaternary Ammonium Compounds; D012710:Serum Albumin, Bovine; D019904:Polymethyl Methacrylate; C007391:diglyme; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane