Abstract
Encoded hydrogel microparticles synthesized via flow lithography have drawn attention for multiplex biomarker detection due to their high multiplex capability and solution-like hybridization kinetics. However, the current methods for preparing particles cannot achieve a flexible, rapid probe-set modification, which is necessary for the production of various combinations of target panels in clinical diagnosis. In order to accomplish the unmet needs, streptavidin was incorporated into the encoded hydrogel microparticles to take advantage of the rapid streptavidin–biotin interactions that can be used in probe-set modification. However, the existing methods suffer from low efficiency of streptavidin conjugation, cause undesirable deformation of particles, and impair the assay capability. Here, we present a simple and powerful method to conjugate streptavidin to the encoded hydrogel microparticles for better assay performance and rapid probe-set modification. Streptavidin was directly conjugated to the encoded hydrogel microparticles using the aza-Michael addition click reaction, which can proceed in mild, aqueous condition without catalysts. A highly flexible and sensitive assay was developed to quantify DNA and proteins using streptavidin-conjugated encoded hydrogel microparticles. We also validated the potential applications of our particles conducting multiplex detection of cancer-related miRNAs.
Highlights
Encoded particle-based assays have attracted considerable attention for biomolecule detection due to their fluid-phase kinetics with respect to target–probe interaction, which has better efficiency than the solid-phase kinetics of planar arrays [1]
We confirmed that the conjugated streptavidin remained stable for at least five weeks, indicating the suitability of adopting the aza-Michael addition click reaction to conjugate streptavidin in encoded hydrogel microparticles (Figure S3, Supplementary Materials)
Using thethe unreacted double bonds remaining in the encoded hydrogel microparticles after thethe fabrication process, directly conjugated streptavidin to the encoded hydrogel microparticles fabrication process, wewe directly conjugated streptavidin to the encoded hydrogel microparticles viavia aza-Michael addition click reaction
Summary
Encoded particle (bead)-based assays have attracted considerable attention for biomolecule detection due to their fluid-phase kinetics with respect to target–probe interaction, which has better efficiency than the solid-phase kinetics of planar arrays [1]. Substantial efforts have been made to adapt the streptavidin–biotin system to encoded hydrogel microparticles to achieve a highly flexible probe-set modification in addition to the high assay performance For this purpose, acrylate-modified streptavidin was incorporated into the monomer and conjugated during the particle synthesis [25]. We presented a novel method of streptavidin conjugation to the encoded hydrogel microparticles to fully exploit the high assay capability and achieve a rapid probe-set modification This goal was accomplished through the aza-Michael addition click reaction that involves the formation of bonds between primary amine groups of streptavidin and unreacted double bonds in the encoded hydrogel microparticles under a mild reaction condition. We demonstrated the potential applications of our particles by performing multiplex detection of cancer-related miRNAs
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