AVS 50th International Symposium
    Biomaterial Interfaces Monday Sessions
       Session BI-MoM

Invited Paper BI-MoM3
Immobilized Microarrays of Capture Agents for Bioassay: A Return to the Past for Protein Surface Stability?

Monday, November 3, 2003, 9:00 am, Room 307

Session: Protein-Surface Interactions
Presenter: D.W. Grainger, Colorado State University
Authors: D.W. Grainger, Colorado State University
P. Gong, Colorado State University
M. Lochhead, Accelr8 Technology Corporation
S. Metzger, Accelr8 Technology Corporation
Correspondent: Click to Email
Microarrays of antibodies, nucleic acids, and antigens all encounter problems with prolonged bioactivity and desired capture sensitivity in immobilized formats. Surface chemistry is used to produce high target capture activity (high signal sensitivity) with low non-specific binding (noise). These surfaces can exhibit shelf-life problems limited, for example, by intrinsic hydrolysis of amine-reactive coupling chemistry (active esters, aldehydes) even under protective conditions. Reactive commercial array surfaces targeting amine-reactive nucleic acids or proteins have been regenerated in situ using N-hydroxysuccinimide to re-activate amine reactivity, improving functionalization of the commercial surfaces and improving immobilization of amine-terminated probes above original capacity. XPS and ToF-SIMS results for surface re-derivatization are correlated with DNA probe immobilization and target capture efficiencies. In a second effort, contact printed immobilized antibodies against a probe analyte on commercial polymer microarraying surfaces (70-micron spots) were assayed for model target capture (goat IgG) in sandwich immunoassay with fluorescently labeled secondary antibodies in full goat serum, imaged by fluorescence scanning. Off-array noise and on-array signal were compared as a function of printed antibody concentration. Despite masking with prescribed protocols (e.g., BSA or polymer masking), assay signal:noise was markedly improved on a non-masked three-dimensional polymer hydrogel commercial chemistry. Last, commercial arraying surfaces were used to exploit nucleic acid amplification (PCR reaction)on-array, capture fluorescently labeled target amplicons with printed probes, and rinse away all PCR reaction reagents in a single-step assay without prior separations or compromise to signal:noise performance. This provides substantial advantages in time and effort should sufficient signal:noise be achieved without costly, tedious PCR separation steps.