Microarray‐based kinetic colorimetric detection for quantitative multiplex protein phosphorylation analysis

Commonly used colorimetric detection applied to protein microarrays with enzymatic signal amplification leads to non‐linear signal production upon increase in analyte concentration, thereby considerably limiting the range and accuracy of quantitative readout interpretation. To extend the detection range, we developed a kinetic colorimetric detection protocol for the analysis of ELISA microarrays designed to measure multiple phosphorylated proteins using the platforms ArrayTube™ and ArrayStrip™. With our novel quantification approach, microarrays were calibrated over a broad concentration range spanning four orders of magnitude of analyte concentration with picomolar threshold. We used this design for the simultaneous quantitative measurement of 15 phosphorylated proteins on a single chip.