Linker-free antibody conjugation for sensitive hydrogel microparticle-based multiplex immunoassay

Hyun Jee Lee, Ju Yeon Kim, Yoon Ho Roh, Sun Min Kim, Ki Wan Bong

Research output: Contribution to journalArticlepeer-review

10 Citations (Scopus)


Multiplex immunoassay, or the simultaneous detection of multiple proteins in a single sample, is expected to enable a new level of protein analysis across diverse disciplines, such as medical diagnostics and biomarker discovery. A bead-based assay using graphically encoded hydrogel microparticles synthesized using stop flow lithography has been a promising platform because of its high multiplex capacity and its superior sensitivity and dynamic range compared to the enzyme-linked immunosorbent assay (ELISA). The functionalization of these particles has been dependent on the use of a heterobifunctional linker to conjugate the capture antibodies on the hydrogel. However, the linker chemistry, which is based on linking the primary amine groups of antibodies with acrylate functional groups on the hydrogel monomer, is vulnerable to hydrolysis in aqueous conditions and can potentially damage the antigen binding region of the antibody. In this work, we introduce a new antibody conjugation method that avoids the use of the linker and further enhances the sensitivity of hydrogel microparticle-based immunoassays. Disulfide bonds in antibodies are reduced to liberate free thiols, which can directly bond with the double bonds remaining in the hydrogel after particle synthesis. We characterize the optimal reduction of antibodies for producing the highest detection signal and demonstrate an average two-fold improvement in sensitivity compared to the linker-dependent antibody conjugation method. Lastly, we validate the accuracy and specificity of the multiplex assays with particles conjugated with antibodies using the linker-free method.

Original languageEnglish
Pages (from-to)6712-6720
Number of pages9
Issue number22
Publication statusPublished - 2019 Nov 21

Bibliographical note

Funding Information:
This work was supported by the Engineering Research Center of Excellence Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2016R1A5A1010148) and the Next-Generation Biogreen 21 Program funded by Rural Development Administration of Republic of Korea (No PJ013158). This research was also supported by the Basic Science Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2018R1D1A1B07046577 and NRF-2017R1D1A1B03029883).

Publisher Copyright:
© 2019 The Royal Society of Chemistry.

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Environmental Chemistry
  • Spectroscopy
  • Electrochemistry


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