A nucleic acid-based electrochemical biosensor for the detection of influenza B virus from PCR samples using gold nanoparticle-adsorbed disposable graphite electrode and Meldola’s blue as an intercalator† Seyma Aydinlik, a Dilsat Ozkan-Ariksoysal, * a Pinar Kara, a A. Arzu Sayiner b and Mehmet Ozsoz a Received 15th March 2011, Accepted 5th May 2011 DOI: 10.1039/c1ay05146f In the presented study, a novel method is introduced that demonstrates the electrochemical detection of influenza B virus based on DNA hybridisation. The detection utilised gold nanoparticles (AuNPs) and Meldola’s Blue (MDB), which is utilised as an intercalator label. The developed methodology, combined with a disposable pencil graphite electrode (PGE) and differential pulse voltammetry (DPV), was performed using both synthetic oligonucleotides and polymerase chain reaction (PCR) amplicons. The electrochemical oxidation response of guanine (approximately +0.1 V) and the voltammetric reduction signal of MDB (approximately 0.2 V) were measured before and after hybridisation reactions between a single strand DNA probe and its complementary target strain (synthetic target or denatured PCR samples). Before the immobilisation of the synthetic DNA probe of influenza type B virus, the transducer surface was interacted with AuNPs solution using a simple wet adsorption method. AuNP immobilisation was confirmed with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) to characterise the recognition surface of the genosensor. After the interaction between the PGE and AuNPs, a thiol-linked DNA probe was immobilised onto the nanoparticle-covered surface. When hybridisation occurred between the probe and its synthetic targets or specific PCR products, the highest MDB signal was observed. The probes were also challenged with equal quantities of non-complementary DNA at the PGE surface for the determination of biosensor selectivity. AuNP-coated electrodes showed high sensitivity and selectivity, specifically in real samples for the detection of the hybridisation reaction. The results obtained in the presented study indicated that the electrode surface area could be enhanced with AuNPs. The detection limit of the genosensor was found to be 54 picomoles for the synthetic target and 3.3  10 7 molecules for the real samples (PCR) in 30 mL of sample volume. Future prospects and analytical performance of the sensor is briefly discussed. 1. Introduction The influenza virus has been known to be a primary cause of respiratory infections in both adults and children for a long period of time. The virus has different antigenic types such as A, B, and C, which are grouped according to the differences in matrix proteins. Among these types, type A causes avian influ- enza in poultry, whereas types B and C cause human epidemic diseases. These viruses can significantly increase the rate of morbidity or even mortality in adults. In addition, influenza can cause economic loss through medical costs and labour loss. 1 The classical method used for the diagnosis of influenza is viral culture, which has been generally accepted as the ‘‘gold stan- dard’’. However, this method requires time (2 to 14 days), and its clinical value is limited. 2 Other techniques used for the identifi- cation of influenza viruses include immunofluorescence staining (IFA), 3 enzyme immunoassay (EIA), 4 RT-PCR, 5 DNA micro- arrays, 6 and TaqMan-based real-time PCR. 7 The detection of influenza in infected cells using IFA 8 has been widely used; however, this technique can take from 2 h to 1 day. Additionally, intact cells, highly skilled researchers, and special equipment are required. EIA is a rapid (15 min to 1 day) technique that can be a ‘‘near patient’’ test, but disadvantages include its high cost and its inability to recover the virus. One of the most sensitive and specific molecular techniques is PCR, which has been used for identification of the types of influenza viruses. Although PCR has been reported as being highly sensitive and allows for further molecular analysis, it is an expensive method and requires private kits and special a Department of Analytical Chemistry, Faculty of Pharmacy, Ege University, 35100 Bornova-Izmir, Turkey. E-mail: dilsat.ariksoysal@ege. edu.tr; Fax: +0090 232 388 52 58; Tel: +0090 232 311 13 53 b Department of Microbiology, Faculty of Medicine, Dokuz Eylul University, Inciralti-Izmir, Turkey † Electronic supplementary information (ESI) available. See DOI: 10.1039/c1ay05146f This journal is ª The Royal Society of Chemistry 2011 Anal. Methods Dynamic Article Links C < Analytical Methods Cite this: DOI: 10.1039/c1ay05146f www.rsc.org/methods PAPER Downloaded by Ege Universitesi on 22 June 2011 Published on 21 June 2011 on http://pubs.rsc.org | doi:10.1039/C1AY05146F View Online