Point-of-care vertical flow immunoassay system for ultra-sensitive multiplex biothreat-agent detection in biological fluids

Jasmine Pramila Devadhasan, Alexander Jarrett Summers, Jian Gu, Stanley Smith, Baiju Thomas, Ali Fattahi, James Helton, Sujata G. Pandit, Marcellene Gates-Hollingsworth, Derrick Hau, Kathryn J. Pflughoeft, Douglas C. Montgomery, Supriya Atta, Tuan Vo-Dinh, David AuCoin, Frederic Zenhausern

Research output: Contribution to journalArticlepeer-review


This paper presents simple, fast, and sensitive detection of multiple biothreat agents by paper-based vertical flow colorimetric sandwich immunoassay for detection of Yersinia pestis (LcrV and F1) and Francisella tularensis (lipopolysaccharide; LPS) antigens using a vertical flow immunoassay (VFI) prototype with portable syringe pump and a new membrane holder. The capture antibody (cAb) printing onto nitrocellulose membrane and gold-labelled detection antibody (dAb) were optimized to enhance the assay sensitivity and specificity. Even though the paper pore size was relaxed from previous 0.1 μm to the current 0.45 μm for serum samples, detection limits as low as 0.050 ng/mL for LcrV and F1, and 0.100 ng/mL for FtLPS have been achieved in buffer and similarly in diluted serum (with LcrV and F1 LODs remained the same and LPS LOD reduced to 0.250 ng/mL). These were 40, 80, and 50X (20X for LPS in serum) better than those from lateral flow configuration. Furthermore, the comparison of multiplex format demonstrated low cross-reactivity and equal sensitivity to that of the singleplex assay. The optimized VFI platform thus provides a portable and rapid on-site monitoring system for multiplex biothreat detection with the potential for high sensitivity, specificity, reproducibility, and multiplexing capability, supporting its utility in remote and resource-limited settings.

Original languageEnglish (US)
Article number114796
JournalBiosensors and Bioelectronics
StatePublished - Jan 1 2023


  • Biothreat agents
  • Colorimetric assay
  • Microarray fabrication
  • Multiplex analysis
  • Vertical flow device

ASJC Scopus subject areas

  • Biotechnology
  • Biophysics
  • Biomedical Engineering
  • Electrochemistry


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