Detection of avian influenza virus by fluorescent DNA barcode-based immunoassay with sensitivity comparable to PCR† Cuong Cao, a Raghuram Dhumpa, b Dang Duong Bang, b Zohreh Ghavifekr, b Jonas Høgberg b and Anders Wolff * a Received 13th August 2009, Accepted 30th November 2009 First published as an Advance Article on the web 15th December 2009 DOI: 10.1039/b916821b In this paper, a coupling of fluorophore-DNA barcode and bead-based immunoassay for detecting avian influenza virus (AIV) with PCR-like sensitivity is reported. The assay is based on the use of sandwich immunoassay and fluorophore-tagged oligonucleotides as representative barcodes. The detection involves the sandwiching of the target AIV between magnetic immunoprobes and barcode-carrying immunoprobes. Because each barcode-carrying immunoprobe is functionalized with a multitude of fluorophore-DNA barcode strands, many DNA barcodes are released for each positive binding event resulting in amplification of the signal. Using an inactivated H16N3 AIV as a model, a linear response over five orders of magnitude was obtained, and the sensitivity of the detection was comparable to conventional RT-PCR. Moreover, the entire detection required less than 2 hr. The results indicate that the method has great potential as an alternative for surveillance of epidemic outbreaks caused by AIV, other viruses and microorganisms. Introduction Avian influenza viruses (AIV), belonging to the Orthomyxoviridae family, have attracted global concern due to the potential pandemic threat for human health and enormous economic losses. It has killed millions of poultry and hundreds of people not only in Asia but also throughout Europe and Africa. 1–3 To control the epidemic diseases, there has been a surge of interest in sensitive, specific and rapid detection of AIV. Several decades ago, traditional viral detection methods such as Madin-Darby canine kidney (MDCK) cell culture, 4 comple- ment fixation (CF), 5 or hemagglutinin-inhibition (HI) 6 were mostly used. These approaches are laborious and time- consuming (e.g., up to 4–10 days for the viral culture). Furthermore, in some cases the techniques are insufficient due to lack of specificity. Recently, reverse transcriptase PCR (RT-PCR) has emerged as the most sensitive method for AIV detection and pathotyping. 3,7 RT-PCR involves an extraction of viral nucleic acids (RNA), an in vitro reverse transcription process to synthesize cDNA from the viral RNA, followed by an enzymatic amplification and detection of the cDNA. Although very sensitive, specific, and much faster than the aforementioned procedures, RT-PCR also has some drawbacks such as a complicated procedure, high cost, and high false positive rate arising from cross contaminations between samples. Furthermore, it still requires a day and experienced personnel to obtain results. Immunoassays could be an alternative for detection of AIV via immunoreactions between surface antigens (nucleoproteins, matrix proteins) of the AIV and their developed antibodies. Many studies have been devoted to the development of enzyme- linked immunosorbent assay (ELISA), 7–10 immunochromato- graphic strip test, 1,11 or microsphere immunoassay (MIA) for detection of AIV. 12 Although these immunoassays have been shown to be rapid, inexpensive to perform and possible to automate, they have lower sensitivity compared to those obtained from RT-PCR. 8 Bio-barcode immunoassay, proposed by Nam et al., is the only approach for detection of biological targets that can obtain PCR-like sensitivity without the enzy- matic amplification. 13,14 Typically, the bio-barcode immunoassay utilizes two types of particles: (1) a magnetic microparticle (MMP) functionalized with antibody (primary antibody) which is to capture and isolate the target analyte from the sample solution, and (2) another particle (gold nanoparticle, polystyrene or silica microparticle) anchored with secondary antibodies, which is specific to the same target, and double-stranded DNA. 13–16 Only one strand of the double-stranded DNA is covalently immobilized onto the secondary particle probe, and after the sandwiching immunoreaction, the complementary DNA strand can easily be released by increasing the temperature. The DNA surrogates for the target of interest and is therefore called a DNA bio-barcode. The surrogate DNA bio-barcode can subsequently be detected by PCR, DNA microarrays, colori- metric assays, or fluorophore-based assays. Unlike other conventional sandwich immunoassays, where the signal intensity is limited by number of antigenic valences for specific binding of reporter-tagged antibody, each particle is functionalized with a multitude of DNA strands and thus many DNA barcodes are released for each positive binding event resulting in amplification of the assay. Although the bio-barcode amplification assays have a DTU-Nanotech, Department of Micro and Nanotechnology, Technical University of Denmark, DTU Building 345 East, DK-2800 Kongens Lyngby, Denmark. E-mail: Anders.Wolff@nanotech.dtu.dk b DTU-VET, Laboratory of Applied Micro-Nanotechnology, Department of Poultry, Fish, and Fur Animals, The National Veterinary Institute, Technical University of Denmark, Hangovej 2, DK-8200 Aarhus N, Denmark † Electronic Supplementary Information (ESI) available: Compared images before and after dissociating Cy5-biobarcodes from the immunocomplexes; and high resolution image of the Cy5-DNA barcode arrays. See DOI: 10.1039/b916821b This journal is ª The Royal Society of Chemistry 2010 Analyst, 2010, 135, 337–342 | 337 PAPER www.rsc.org/analyst | Analyst