title: Development of fluorescent polymerization-based signal amplification for sensitive and non-enzymatic biodetection in antibody microarrays.
abstract: Antibody microarrays are a critical tool for proteomics, requiring broad, highly sensitive detection of numerous low abundance biomarkers. Fluorescent polymerization-based amplification (FPBA) is presented as a novel, non-enzymatic signal amplification method that takes advantage of the chain-reaction nature of radical polymerization to achieve a highly amplified fluorescent response. A streptavidin-eosin conjugate localizes eosin photoinitiators for polymerization on the chip where biotinylated target protein is bound. The chip is contacted with acrylamide as a monomer, N-methyldiethanolamine as a coinitiator and yellow/green fluorescent nanoparticles (NPs) which, upon initiation, combine to form a macroscopically visible and highly fluorescent film. The rapid polymerization kinetics and the presence of cross-linker favor entrapment of the fluorescent NPs in the polymer, enabling highly sensitive fluorescent biodetection. This method is demonstrated as being appropriate for antibody microarrays and is compared to detection approaches which utilize streptavidin-fluorescein isothiocyanate (SA-FITC) and streptavidin-labeled yellow/green NPs (SA-NPs). It is found that FPBA is able to detect 0.16 + or - 0.01 biotin-antibody microm(-2) (or 40 zmol surface-bound target molecules), while SA-FITC has a limit of detection of 31 + or - 1 biotin-antibody microm(-2) and SA-NPs fail to achieve any significant signal under the conditions evaluated here. Further, FPBA in conjunction with fluorescent stereomicroscopy yields equal or better sensitivity compared to fluorescent detection of SA-eosin using a much more costly microarray scanner. By facilitating highly sensitive detection, FPBA is expected to enable detection of low abundance antigens and also make possible a transition towards less expensive fluorescence detection instrumentation.
authors.affiliation: Department of Chemical and Biological Engineering, ECCH 111, UCB 424, University of Colorado, Boulder, CO 80309, USA.
authors.forename: Heather J
authors.initials: HJ
authors.lastname: Avens
authors.forename: Christopher N
authors.initials: CN
authors.lastname: Bowman
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D001672:Biocompatible Materials / Q000737:chemistry*; D001710:Biotin / Q000737:chemistry*; D004801:Eosine Yellowish-(YS) / Q000737:chemistry; D004983:Ethanolamines / Q000737:chemistry; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D007700:Kinetics; D053758:Nanoparticles / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D040081:Protein Array Analysis / Q000379:methods*; D040901:Proteomics / Q000379:methods; D019809:Streptavidin / Q000737:chemistry
chemical_list: D000906:Antibodies; D001672:Biocompatible Materials; D004983:Ethanolamines; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D011108:Polymers; C008430:N-methyldiethanolamine; D001710:Biotin; D019809:Streptavidin; D016650:Fluorescein-5-isothiocyanate; D004801:Eosine Yellowish-(YS)