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

Abstract

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.

Hydrogels and Their Role in Biosensing Applications

“…[155] Further composite material approaches include the integration of micro-or nanoparticles into the hydrogel matrix and the addition of fluorescent or chromogenic components for optical detection. [148,[156][157][158] Many bioreceptors were immobilized into or onto the matrix for the detection of a specific target. Such bioassays have wide applications as diagnostic tools in healthcare, and they have traditionally been performed by laboratory methods (e.g., PCR or ELISA).…”

“…Human motion Electrical Adhesion [ 125] Agarose L-lactate Potentiometric Encapsulation [ 140] Functional Hydrogels PEG, hPG Oligonucleotide, Fab, Ab Fluorescence Covalent, amide bond [ 148] PANI/PNIPAAm BSA IR Molecular imprinting [ 150] methods to immobilize the probe molecule onto various base materials, called substrates. However, not all substrates work with all detection techniques, and not all immobilization methods work on all substrates.…”

“…Adapted with permission. [148] Copyright 2019, American Chemical Society.…”

“…This is especially advantageous in the preparation of a multiplex assay since it enables storage under dry conditions. [148] The combination of these macromonomers crosslinked by SPAAC into a hydrogel was also used for molecular imprinting. The method of molecular imprinting is a more elaborate version of encapsulation because it shows shape selectivity toward the imprinted biomolecule and therefore, does not need a probe.…”

A multiplex microchamber diffusion assay for the antibody-based detection of microRNAs on randomly ordered microbeads

“…We used our in-house developed VideoScan platform [17] as a readout platform, which is based on a fully automated multispectral fluorescence microscope. For this, we have developed multiplex assays for the imaging platform to detect and quantify biomolecules including short protein or DNA sequences [18] and (auto)antibodies [19].…”