Direct Conjugation of Streptavidin to Encoded Hydrogel Microparticles for Multiplex Biomolecule Detection with Rapid Probe-Set Modification

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.

Direct functionalization of cell‐adhesion promoters to hydrogel microparticles synthesized by stop‐flow lithography

“…The detailed calculation procedures and related datas were presented in the supporting information and Figure S2. These unreacted acrylate groups are highly reactive with various functionalized molecules through chemical reactions, including Michael addition reactions 10,16,24 . In particular, considering that various cell‐adhesion promoters (e.g., PLL, fibronectin, collagen, and dopamine) contain amine groups in their molecular structures, the aza‐Michael addition of cell‐adhesion promoter molecules to microparticles can be directly achieved without requiring the preconjugation of linker molecules 2 …”

“…Considering that there are highly abundant amine groups in the PLL structures, we induced the aza‐Michael addition reaction between electrophilic α,β‐unsaturated double bonds in the particles and nucleophilic nitrogen donor groups in the PLL (Figure 1B). 24,31 Among diverse acrylate‐based conjugation chemistries, the aza‐Michael addition reaction has distinct advantages, including commercial availability, no requirement for catalyst, and low productivity of reactive by‐products during polymerization 32 . To confirm the conjugation of the PLL to particles by a proposed manner, we used PLL labeled with fluorescein isothiocyanate (PLL‐FITC) to qualitatively observe and quantitatively analyze the fluorescent images and signals of attached PLL in the particles.…”

“…To confirm the conjugation of the PLL to particles by a proposed manner, we used PLL labeled with fluorescein isothiocyanate (PLL‐FITC) to qualitatively observe and quantitatively analyze the fluorescent images and signals of attached PLL in the particles. We selected a fluorescent labeling method for the cell‐adhesion promoter since it has powerful advantages for molecular conjugation analysis in hydrogel microparticles, such as the visualization of distributed molecules and the facile quantification of the loaded amount of the molecules by fluorescent intensity 14,21,22,24 …”

“…Accordingly, we characterized the amount of conjugated PLL‐FITC in unit particles by fluorescent signals based on the pH of the incubation buffer and incubation time during the aza‐Michael reaction process. First, we optimized the pH of the incubation solution considering that pH is one of the critical factors for efficient aza‐Michael addition reactions as it affects the nucleophilicity of electron donors 24 . We screened optimal pH conditions from 7 to 10 using the fluorescent signals of the particles conjugated with the PLL‐FITC for 4 h (Figure 2A).…”

“…To avoid using additional linker molecules and complicated preconjugation steps, the direct conjugation of cell‐adhesion promoter molecules containing amine groups can be an alternative by inducing the aza‐Michael reaction between unreacted acrylate groups in the particles and the nucleophilic amine groups in the cell‐adhesion promoters. Recently, there has been high interest in utilizing unreacted acrylate groups in PEG crosslinkers during polymerization for a high‐performance and simple bio‐conjugation process, in which the remnant acrylate groups enable Michael addition reactions with amine and thiol groups in various biomolecules 10,16,24,25 . It has already been demonstrated that the unreacted acrylate group‐based functionalization methods have powerful advantages, such as the high conjugation capacity, 25 simplicity of process because of the unnecessity of linker molecules, 26 and prevention of the aggregation of biomolecules inside the gel structures 16 .…”