Spatial Separation of Microbeads into Detection Levels by a Bioorthogonal Porous Hydrogel for Size-Selective Analysis and Increased Multiplexity.

Multiplex detection techniques are emerging within the fields of life science research and medical diagnostics where it is mandatory to analyze a great number of molecules. The detection techniques need to be highly efficient but often involve complicated and expensive fabrication procedures. Here, we present the immobilization and geometric separation of fluorescence-labeled microbeads for a multiplex detection in k levels. A compound of differently sized target molecules (DNA, proteins) is channeled into the respective detection levels by making use of a hydrogel as a size selective filter. The immobilized microbeads (10-20 μm) are considerably larger than the pores of the hydrogel network and therefore stay fixed at the well bottom and in higher elevations, respectively. Small biomolecules can diffuse through the pores of the network, whereas medium-sized biomolecules pass slower and large molecules will be excluded. Besides filtering, this method discriminates the used microbeads into k levels and thereby introduces a geometric multiplexity. Additionally, the exclusion of large entities enables the simultaneous detection of two target molecules, which exhibit the same affinity interaction. The hydrogel is formed through the combination of two macromonomers. One component is a homobifunctional polyethylene glycol linker, carrying a strained alkyne (PEG-BCN) and the second component is the azide-functionalized dendritic polyglycerol (dPG-N3). They react via the bioorthogonal strain-promoted azide alkyne cycloaddition (SPAAC). The hydrogel creates a solution-like environment for the diffusion of the investigated biomolecules all the while providing a stable, bioinert, and surface bound network.