David Finnskog

Macro/Nano-Structured Silicon as Solid Support for Antibody Arrays: Surface Design, Reproducibility, and Assay Characteristics Enabling Discovery of Kallikrein Gene Products for Prostate Disease Diagnostics

To facilitate high-throughput biomarker discovery and high-density protein-chip array analyses of complex biological samples, a novel macro-and nanoporous silicon surface for protein microarrays was developed. The surface offers three-dimensional surface enlarging properties and spot confinement, enabling both high sensitivity bioassays and design of high density arrays. Reproducible manufacturing of the protein chip surface was accomplished as demonstrated by the low imprecision when standard IgG bioassays were performed at 100 pM antigen level on a series of protein chips scanned at widely different locations within a silicon wafer, as well as between different wafers from two different manufacturers. The relative standard deviation (RSD) of fluorescence spot intensity within an array on a chip was less than 20%. Mean spot intensity RSD was 19% for all 25 microarray chips in the study. Within-manufacturer-lot RSDs in chips from either manufacturer were <15% of mean spot intensity. The detection limit and dynamic range of the novel protein chip surface were examined to evaluate whether they match criteria required in a search for novel biomarkers such as for prostate cancer. Monoclonal IgG against prostate-specific antigen (PSA) was arrayed on the porous silicon chips. These were subsequently incubated in serum samples containing widely different levels of fluorescence-labeled PSA. Detection of PSA in serum at concentrations from 0.7 ng/mL (26 pM) up to 104-fold higher levels verified assay characteristics required in the search for prostate biomarkers (e.g., kallikrein gene products) at clinically relevant levels.To facilitate high-throughput biomarker discovery and high-density protein-chip array analyses of complex biological samples, a novel macro-and nanoporous silicon surface for protein microarrays was developed. The surface offers three-dimensional surface enlarging properties and spot confinement, enabling both high sensitivity bioassays and design of high density arrays. Reproducible manufacturing of the protein chip surface was accomplished as demonstrated by the low imprecision when standard IgG bioassays were performed at 100 pM antigen level on a series of protein chips scanned at widely different locations within a silicon wafer, as well as between different wafers from two different manufacturers. The relative standard deviation (RSD) of fluorescence spot intensity within an array on a chip was less than 20%. Mean spot intensity RSD was 19% for all 25 microarray chips in the study. Within-manufacturer-lot RSDs in chips from either manufacturer were <15% of mean spot intensity. The detection limit and dynamic range of the novel protein chip surface were examined to evaluate whether they match criteria required in a search for novel biomarkers such as for prostate cancer. Monoclonal IgG against prostate-specific antigen (PSA) was arrayed on the porous silicon chips. These were subsequently incubated in serum samples containing widely different levels of fluorescence-labeled PSA. Detection of PSA in serum at concentrations from 0.7 ng/mL (26 pM) up to 104-fold higher levels verified assay characteristics required in the search for prostate biomarkers (e.g., kallikrein gene products) at clinically relevant levels.