Facilitating the indirect detection of genomic DNA in an electrochemical DNA biosensor using magnetic nanoparticles and DNA ligase Roozbeh Hushiarian a, c, * , Nor Azah Yusof b, c, ** , Abdul Halim Abdullah b , Shahrul Ainliah Alang Ahmad b , Sabo Wada Dutse b, d a Institute of Bioscience, Universiti Putra Malaysia, 43400, UPM Serdang, Selangor, Malaysia b Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400, UPM Serdang, Selangor, Malaysia c Institute of Advanced Technology, Universiti Putra Malaysia, 43400, UPM Serdang, Selangor, Malaysia d Department of Science Laboratory Technology, Hussaini Adamu Federal Polytechnic, Kazaure, Nigeria article info Article history: Received 2 August 2015 Received in revised form 12 October 2015 Accepted 12 October 2015 Available online 21 October 2015 Keywords: Iron oxide magnetic nanoparticle DNA biosensor DNA hybridization DNA ligase Electrochemical analysis abstract A common problem in applying biosensors for the detection of genomic DNA is detecting short se- quences in large amounts of long double stranded DNA. A gold electrode modified with a conductive nanocomposite, poly(3,4-ethylene-dioxythiophene), and gold nanoparticles was functionalized with 2,6- Pyridinedicarboxylic acid. Immobilization of a 20-mer DNA probe as the bioreceptor was successfully carried out via a peptide bond on the surface of the modified electrode. Two segments of 15 and 20 base probes were designed and named as Capture and Reporter probes respectively. The 20-mer Reporter probe was complementary to the bioreceptor and the 15-mer Capture probe was designed to bind on to the surface of the iron oxide magnetic nanoparticles. A 35-base Target DNA complementary to the Capture and the Reporter probes was used as Template in the ligation process, with the ligation between the Reporter and Capture probes mediated by T4 ligase. Iron oxide magnetic nanoparticles functionalized with carboxylic groups on their surface synthesized in a new method were attached to the 15-mer Capture probe. After the denaturation of the final ligation product, the separation of the attached probes was carried out using 5 G permanent magnets in a three step washing procedure in TE buffer. The hybridization of the DNA bioreceptor and the Reporter probe attached to the Capture probe-Fe 3 O 4 was monitored via oxidation and reduction of the new redox marker (ruthenium complex) intercalated into the double helix. This technique was found to be reliably repeatable. The indirect detection of genomic DNA using this method is significantly improved and showed high efficiency in small amounts of samples with the detection limit of 5.37  10 14 M. © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction The design of the electrochemical biosensor described in this paper could equally have been applied to any genomic DNA but the white rot fungus, Ganoderma boninense was selected in this study. This is a pathogen, which causes both basal and upper stem rot in oil palms and presents a major threat to a highly lucrative industry in the south East Asia. G. boninense has a devastating effect on a plantation; it causes direct loss of the stands and reduces the yield of the palms, creating the need for earlier replanting [1]. As soon as young palms show symptoms of the disease they inevitably die within a year or two while older trees survive only a few years longer [2]. As well as trying a multitude of methods of control, extensive work has already been carried out on the early detection of G. boninense [3]. After experimenting with PCR [4], researchers * Corresponding author. Institute of Bioscience, Universiti Putra Malaysia, 43400, UPM Serdang, Selangor Malaysia.. ** Corresponding author. Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400, UPM Serdang, Selangor Malaysia. E-mail addresses: hushiarian@gmail.com (R. Hushiarian), azahy@upm.edu.my (N.A. Yusof), halim@upm.edu.my (A.H. Abdullah), ainliah@upm.edu.my (S.A.A. Ahmad), swdutse@yahoo.com (S.W. Dutse). Contents lists available at ScienceDirect Analytical Chemistry Research journal homepage: www.elsevier.com/locate/ancr http://dx.doi.org/10.1016/j.ancr.2015.10.004 2214-1812/© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Analytical Chemistry Research 6 (2015) 17e25