Protein and Nucleic Acid Detection by Rolling Circle Amplification on Gel-based Microarrays

Microarrays are becoming the platform of choice for the analysis of complex genomes, transcriptomes and proteomes. For a number of applications, however, sample or analyte abundance constraints limit the usefulness of microarrays. Rolling circle amplification (RCA) has previously been shown to be a signal amplification method that is useful in these applications on glass microarrays. This report describes use of RCA for multiplexed detection of nucleic acids and proteins on 3-dimensional, porous microarrays (CodeLink(TM)). Assays combined a sandwiched immunoassay with RCA signal amplification of associated haptens, achieving sensitivities of 0.1 pg/ml for IL6, IL8, MIP-1[beta], and EGF. A similar RCA strategy was utilized in a genotyping assay on CodeLink(TM) microarrays that provided three-log enhancement of signal intensity. RCA assays on CodeLink(TM) microarrays were rapid, and utilized low-volumes, suggesting that performance of RCA universal signal amplification on CodeLink(TM) microarrays may find useful applications in multiplexed measurements, rare biomolecule detection, and small sample analysis.