Nitrocellulose Compartmentalized Linker Array for Highly Sensitive Three-Dimensional Antibody Microarray-Based Multiplex Bioassays

Microarrays are a powerful tool for creating multiplex bioassays but remain unavailable to most biomedical and biological research laboratories due to the high cost of the equipment needed to fabricate them. Moreover, conventional methods of microarray fabrication can cause the loss of bioactivity of the delicate bioreagents, thus compromising the assay performance. We have developed a nitrocellulose compartmentalized linker array (nCLA) technology that creates 3D antibody microarrays by simply pipetting microliter antibody solutions into compartments prepatterned with nitrocellulose microarrays through which the antibodies are self-assembled into microarrays via binding onto nitrocellulose. To form the nitrocellulose microarray with the background region deactivated, a selective background deactivation technique has been developed. As a proof of concept, three cancer-related proteins, EGFR, TNF-α, and GM-CSF, were measured using nCLA in a multiplexed sandwich immunoassay. Low picograms per milliliter limits of detection were achieved in both antigen-spiked PBS and human serum, demonstrating its potential in biomedical research. The nCLA provides a new method to fabricate membrane-based, microarray-printer-free, highly sensitive, and scalable bioassays. It maintains reagent bioactivity and forms antibody microarrays via simple pipetting, making the powerful 3D microarray technology widely available to the biomedical research community.