Detection and Quantification of Label-Free Infectious Adenovirus Using a Switch-On Cell-Based Fluorescent Biosensor

Miguel Ricardo Guerreiro, Daniela Filipa Freitas, Paula Marques Alves, Ana Sofia Coroadinha

Research output: Contribution to journalArticlepeer-review

11 Citations (Scopus)


Reliable and fast viral detection and quantification protocols are a requirement for the advance of basic research and clinical approaches with wild type or recombinant viruses. However, available cell-based assays are either time-consuming or require labeled viral particles, which may alter virus biology or pose safety issues in clinical applications. Since adenoviruses constitute a major healthcare burden but also, when engineered, widely used vectors in vaccination and gene and oncolytic therapies, herein we developed a genetically encoded switch-on fluorescent biosensor consisting of a cyclized Green fluorescent protein - cVisensor - with an adenoviral protease cleavable site as a switch. After initial sensor optimization (35% increase in performance), whole-cell biosensors were established - by stably expressing cVisensor in mammalian cells - and used for live-cell monitoring of adenovirus infection as the intracellular biosensor is specifically activated by the viral protease. A rapid flow cytometry-based bioassay using cVisensor cells was established 48 h postinfection, showing an estimated limit of detection of 105 infectious particles/mL, in-line with previously reported flow cytometry assays requiring labeled virus, and significantly faster than standard plaque-forming assays requiring up to 14 days. cVisensor was also successfully applied in the detection of HIV-1 protease activity, validating its wider potential for the detection of other viruses. Overall, this work presents a fast and easy method for detection and quantification of label-free infectious virus, allowing the establishment of new biosensing platforms for basic research in virology and biotechnological applications of recombinant virus biopharmaceuticals.

Original languageEnglish
Pages (from-to)1654-1661
Number of pages8
JournalACS Sensors
Issue number6
Publication statusPublished - 28 Jun 2019


  • Cell-based bioassay
  • Fluorescent biosensor
  • Intein
  • Label-free
  • Protease
  • Quantification
  • Virus


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