Rapid detection of dengue virus in serum using magnetic separation and fluorescence detection Won-Suk Chang,{ a Hao Shang,{ a Rushika M. Perera, b Shee-mei Lok, b Dagmar Sedlak, b Richard J. Kuhn b and Gil U Lee* a Received 18th July 2007, Accepted 30th November 2007 First published as an Advance Article on the web 20th December 2007 DOI: 10.1039/b710997k A magnetophoretic fluorescence sensor (MFS) has been developed to rapidly detect dengue virus in serum at a sensitivity that was approximately three orders of magnitude higher than conventional solid phase immunoassays. UV inactivated type 2 dengue virus was first reacted with a mixture of superparamagnetic and fluorescent microparticles functionalised with an anti-type 2 dengue virus monoclonal antibody in 10% fetal calf serum. The magnetic particles were separated from the serum based on their magnetophoretic mobility, and dengue virus was detected by the co-localization of magnetic and fluorescent particles at a specific point in the flow chamber. The MFS was capable of detecting dengue-2 virus at 10 PFU ml 21 with a reaction time of 15 min. The MFS demonstrated a high specificity in the presence of yellow fever virus, a closely related flavivirus, which also did not produce any detectable increase in background signal. The improved performance of this technique appears to result from the rapid kinetics of the microparticle reaction, improved signal-to-noise ratio resulting from magnetophoretic separation, and rapid fluorescent particle detection. These results suggest that the MFS may be useful in early stage diagnosis of dengue infections, as well as other diseases. Introduction It is becoming increasingly clear that the global community is vulnerable to the rapid spread of infectious diseases, as we have seen several viruses mutate, jump between species, and spread through our integrated economy. 1–3 Early stage detection in patients could enhance recovery and also limit the spread of the disease. Therefore, there is a particular need for rapid, sensitive, and inexpensive point-of-care sensors that are capable of simultaneously identifying multiple pathogens in complex samples such as blood. The development of superparamagnetic microparticles has made it possible to rapidly and efficiently separate cells from complex mixtures, such as fermentation broths and culture media, in a manner that does not require complicated equipment. 4,5 In this technique the magnetic particles are coated with antibodies (or other receptors) that react with specific ligands on the cells of interest and are allowed to reaction with the sample. A magnet is then used to separate the magnetic particles and the specific cell type of interest in a single step. Magnetic separation is exceptionally efficient because most biological materials are not susceptible to magnetic fields. 6,7 It has also proven to be a very effective means of identifying specific cell lines, as demonstrated by the report that the genotype of 7–22 fetal cells has been identified in 16 ml of maternal blood. 8 Magnetophoresis is a separation process in which both hydrodynamic and magnetic fields are used to separate a magnetic material from an aqueous solution on a continuous basis. In high volume, continuous industrial applications, high-gradient fields have been used to collect large quantities of magnetic material from suspensions. 9–11 Highly efficient rare cell purification has also been achieved using high- gradient, continuous magnetic separation. 12,13 Recently, superparamagnetic microparticles have been separated in microfluidics systems using magnetophoresis. 14,15 In such assays, the particles travel at the same velocity with the buffer in a laminar flow regime and a perpendicular magnetic field is applied to deflect the particle flow to collect the particles in different wells. By independently tuning the scale of the two forces the particles can be sorted into different regions based on their size, magnetic susceptibility, and loading of magnetic materials. Alternatively, an inductive magnetic field can be produced by a wire to direct magnetic material to a central well, whereas the non-magnetic material was separated into two side streams. 16 In this report, we demonstrate magnetophoretic separation and fluorescence detection of type 2 dengue virus. We chose to study dengue, a member of the flavivirus genus of enveloped RNA viruses, as it is one of the most significant mosquito- borne viral pathogens, given the impact of the recent resurgence of dengue fever and dengue hemorrhagic fever. 17 There are four dengue virus serotypes namely dengue 1, 2, 3, and 4. Severe dengue hemorrhagic fever can develop in children and adults experiencing a second dengue virus infection with a serotype different from that of their first dengue virus infection. Currently, dengue is identified using immunocolorimetric assays that detect the immunoglobulin M a Schools of Chemical and Biomedical Engineering, Forney Hall, Purdue University, West Lafayette, IN 47907, USA. E-mail: gl@ecn.purdue.edu; Tel: +1-765-494-0492 b Department of Biological Sciences, Lilly Hall, Purdue University, West Lafayette, IN 47907, USA { These authors contributed equally. PAPER www.rsc.org/analyst | The Analyst This journal is ß The Royal Society of Chemistry 2008 Analyst, 2008, 133, 233–240 | 233