Letter pubs.acs.org/ac
Aptamer-Based Viability Impedimetric Sensor for Viruses Mahmoud Labib,† Anna S. Zamay,†,‡ Darija Muharemagic,† Alexey V. Chechik,† John C. Bell, and Maxim V. Berezovski*,†
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Department of Chemistry, University of Ottawa, 10 Marie Curie, Ottawa, Ontario K1N 6N5, Canada Institute of Molecular Medicine and Pathological Biochemistry, Krasnoyarsk State Medical University, 1 P. Zheleznyaka Street, Krasnoyarsk 660022, Russia § Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 501 Smyth Road, Ottawa, Ontario K1H 8L6, Canada ∥ Jennerex Inc., 450 Sansome Street, 16th Floor, San Francisco, California 94111, United States ‡
S Supporting Information *
ABSTRACT: The development of aptamer-based viability impedimetric sensor for viruses (AptaVISens-V) is presented. Highly specific DNA aptamers to intact vaccinia virus were selected using cell-SELEX technique and integrated into impedimetric sensors via self-assembly onto a gold microelectrode. Remarkably, this aptasensor is highly selective and can successfully detect viable vaccinia virus particles (down to 60 virions in a microliter) and distinguish them from nonviable viruses in a label-free electrochemical assay format. It also opens a new venue for the development of a variety of viability sensors for detection of many microorganisms and spores.
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anaerobic microorganisms.6 However, restriction of detection to aerobes represents the major disadvantage of these methods. Electrochemical impedance spectroscopy (EIS) is a technique generating more and more interest for biologists as it has a less destructive effect on the measured biological interactions because it is performed at a very narrow range of small potentials and is label-free.7,8 This method was successfully used to monitor viable bacteria,9,10 yeast,11 and parasites12 by measuring the permittivity of the medium containing them. However, the measured signals suffer from interference of the electric properties of the background electrolyte. This effect is frequency-dependent and cannot be reliably subtracted.13 There has been a renewed interest in the use of vaccinia virus (VACV) vaccines as a defense against the deliberate release of variola virus, as an act of bioterrorism.14 This has now led to rebuilding of vaccine stocks and created an urge for development of rapid and sensitive methods for detection of viable VACV for quality control purposes. Moreover, owing to the increasing number of zoonotic poxvirus infections in humans, differentiation between potentially infectious Orthopoxvirus (OPV) species, such as VACV, Monkeypox virus or Cowpox virus, is required.15 In this work, DNA aptamers specific to live vaccinia virus (VACV, Jennerex Inc., Ottawa, Canada) were selected based on Cell-SELEX,16,17 from a 80nt DNA library containing a 40nt random region, 5′-CTC CTC
tandard methods used to assess the presence of microorganisms (bacteria and viruses) and to determine whether they are viable or not involve the use of specific enrichment media to separate, identify, and count viable cells. This process is time-consuming and requires bacterial growth media or cell cultures in case of viruses. The most popular cell culture-based method is the plaque-forming assay. Viral plaque assays can determine the number of plaque forming units (PFU) in a virus sample. A viral plaque is formed when a virus infects a cell within a fixed cell monolayer. Plaque formation can take 3−14 days, depending on the virus being analyzed. Regular spectrophotometric assays with dye exclusion or metabolic transformation by live cells cannot be applied on viruses due to their very small sizes (
