Hydrogel droplet microarrays with trapped antibody-functionalized beads for multiplexed protein analysis

Abstract

Antibody microarrays are a powerful tool for rapid, multiplexed profiling of proteins. 3D microarray substrates have been developed to improve binding capacity, assay sensitivity, and mass transport, however, they often rely on photopolymers which are difficult to manufacture and have a small pore size that limits mass transport and demands long incubation time. Here, we present a novel 3D antibody microarray format based on the entrapment of antibody-coated microbeads within alginate droplets that were spotted onto a glass slide using an inkjet. Owing to the low concentration of alginate used, the gels were highly porous to proteins, and together with the 3D architecture helped enhance mass transport during the assays. The spotting parameters were optimized for the attachment of the alginate to the substrate. Beads with 0.2 µm, 0.5 µm and 1 µm diameter were tested and 1 µm beads were selected based on their superior retention within the hydrogel. The beads were found to be distributed within the entire volume of the gel droplet using confocal microscopy. The assay time and the concentration of beads in the gels were investigated for maximal binding signal using one-step immunoassays. As a proof of concept, six proteins including cytokines (TNFα, IL-8 and MIP/CCL4), breast cancer biomarkers (CEA and HER2) and one cancer-related protein (ENG) were profiled in multiplex using sandwich assays down to pg mL(-1) concentrations with 1 h incubation without agitation in both buffer solutions and 10% serum. These results illustrate the potential of beads-in-gel microarrays for highly sensitive and multiplexed protein analysis.

Alginate-Based Biomaterials for Regenerative Medicine Applications

“…Recently, the use of a tissue engineering approach for developing a 3D high throughput screening assay for drug screening and diagnostic devices has been of great interest [142,143,144,145]. An alginate hydrogel has been utilized as a 3D platform for microarray systems as well as surface micro-patternings.…”

Hydrogel microparticles for biosensing

“…A wide range of hydrogel chemical compositions have been explored for DNA or protein microarrays, in particular polyacrylamide [2, 16, 17], polyethylene glycol [18–20], and alginate [21] derivatives. Several methods to functionalize the gels have been explored, ranging from in situ functionalization at the time of synthesis to post-synthesis functionalization utilizing functional groups in the gel [22].…”

Macroporous hydrogel micropillars for quantifying Met kinase activity in cancer cell lysates

“…Hydrogels provide a three-dimensional hydrated environment more similar to the native protein environment than a two-dimensional structure and previously have been used to quantify enzyme activity 27–31 . Macroporous hydrogel arrays also enhance mass transport rates, increasing both the sensitivity and kinetics of the assay 30–32 . The use of whole cell lysate during the assay eliminates the need for kinase purification and permits measurement of kinase activity and inhibition of that activity in the normal cellular milieu.…”