Real-time QCM-D immunoassay through oriented antibody immobilization using cross-linked hydrogel biointerfaces.
This report presents the development of pre-cross-linked and in situ cross-linked polyethyleneimine-carboxymethylcellulose antibody immobilization platforms for real-time QCM-D immunoassay of sepsis-related biomarkers. These platforms differ significantly from recent trends in QCM-based assays, a rapidly expanding field given the affordability and sensitivity of the transduction system, by providing ultrafast biointerface deposition through cross-linking of polysaccharides. Using rhIL-1ra (17 kDa), a known sepsis biomarker, for development, various immunoassay modifications to increase sensitivity were investigated, including the use of Protein A, Protein G, and anti-IgG Fc specific antibody capture ligands for oriented antibody immobilization, higher-frequency QCM-D crystals, and amplification using secondary antibodies. The optimized assay employs Protein A oriented immobilization on pre-cross-linked polymer and secondary antibodies to achieve a detection limit of 25 ng/mL on 5 MHz crystals. Assay repeatability using the optimized chemistry is robust, with no loss in 100 ng/mL antigen detection over 20 cycles of the 10 min sandwich assay. Nonspecific adsorption of human serum albumin, as characterized by ToF-SIMS, is minimal and negligible for the pre-cross-linked and in situ cross-linked compositions, respectively.
Carrigan
Shawn D
SD
Department of Biomedical Engineering, McGill University, 3775 University Street, Duff Medical Building, Room 316, Montreal, Quebec, Canada.
Scott
George
G
Tabrizian
Maryam
M
D000906:Antibodies; D000941:Antigens / Q000032:analysis*; D001666:Binding Sites, Antibody; D003432:Cross-Linking Reagents; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D007118:Immunoassay / Q000379:methods*; D011791:Quartz; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D018629:Spectrometry, Mass, Secondary Ion / Q000379:methods; D018474:Weightlessness Simulation
D000906:Antibodies; D000941:Antigens; D003432:Cross-Linking Reagents; D011791:Quartz; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate