Talanta 85 (2011) 1927–1932 Contents lists available at ScienceDirect Talanta j ourna l ho me page: www.elsevier.com/locate/talanta Electrochemical genosensors for the detection of Bonamia parasite. Selection of single strand-DNA (ssDNA) probes by simulation of the secondary structure folding Valeria Narcisi a , Marcello Mascini b,∗ , German Perez c , Michele Del Carlo b , Pietro Giorgio Tiscar a , Hideko Yamanaka d , Dario Compagnone b a Department of Comparative Biomedical Sciences, University of Teramo, Italy b Department of Food Science, University of Teramo, 64023, Italy c Department of General Chemistry, University of Havana, Cuba d Department of Analytical Chemistry, São Paulo State University (UNESP), Brazil a r t i c l e i n f o Article history: Received 17 February 2011 Received in revised form 27 June 2011 Accepted 7 July 2011 Available online 18 July 2011 Keywords: Electrochemical genosensors ssDNA probes Screen-printed electrodes Bonamia exitiosa Bonamia ostreae Folding simulation hybridisation prediction a b s t r a c t A post-PCR nucleic acid work by comparing experimental data, from electrochemical genosensors, and bioinformatics data, derived from the simulation of the secondary structure folding and prediction of hybridisation reaction, was carried out in order to rationalize the selection of ssDNA probes for the detection of two Bonamia species, B. exitiosa and B. ostreae, parasites of Ostrea edulis. Six ssDNA probes (from 11 to 25 bases in length, 2 thiolated and 4 biotinylated) were selected within different regions of B. ostreae and B. exitiosa PCR amplicons (300 and 304 bases, respectively) with the aim to discriminate between these parasite species. ssDNA amplicons and probes were analyzed separately using the “Mfold Web Server” simulating the secondary structure folding behaviour. The hybridisation of amplicon–probe was predicted by means of “Dinamelt Web Server”. The results were evaluated consider- ing the number of hydrogen bonds broken and formed in the simulated folding and hybridisation process, variance in gaps for each sequence and number of available bases. In the experimental part, thermally denatured PCR products were captured at the sensor interface via sandwich hybridisation with surface- tethered probes (thiolated probes) and biotinylated signalling probes. A convergence between analytical signals and simulated results was observed, indicating the possibility to use bioinformatic data for ssDNA probes selection to be incorporated in genosensors. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Ostrea edulis is the native European oyster species representing in the past an economically important oyster production in several European countries. Considering the importance of the commer- cial exchanges of live mollusc stocks around the world and the associated risk of disease spread from affected to free areas, the zoosanitary control of transfers is essential [1]. It is thus necessary to establish the health status of the oyster production areas and to characterize the pathogens. Bonamia sp. is a well-known parasite of the flat oyster, O. edulis, causing worldwide significant oyster loss [2]. Bonamiosis is mainly caused by B. ostreae and B. exitiosa: the first one is diffused in the Northern hemisphere while B. exitiosa is known to infect oysters in the Southern hemisphere. Neverthe- less, in the last years, B. exitiosa was found in European waters ∗ Corresponding author. Tel.: +39 0861266912. E-mail address: mmascini@unite.it (M. Mascini). [1,3]. To identify the proper Bonamia sp., a restriction fragment length polymorphism (RFLP) assay is necessary (official method) after a PCR reaction with genus specific primers (Bo-BoAS). Other approaches have been also reported [4,5]. Despite the interesting performances obtained, the equipment necessary for these tech- niques is expensive, highlighting the need for a more convenient and flexible instrumental alternative [6,7]. DNA biosensors are analytical devices resulting from the inte- gration between DNA sequence-specific probe with a signal transducer and representing a good alternative for species identifi- cation [7–13]. These sensors based on the interfacial hybridisation of the sample to surface-immobilised probes, have been reported to identify differences in amplicon primary sequence discriminating even single nucleotide polymorphism [10,13,14]. Since the speci- ficity of the hybridisation reaction is essentially dependent on the bio-recognition properties of the capture oligonucleotide, design of this probe is undoubtedly the most important pre-analytical step [14–17]. Folding of amplicon and probes is an important fac- tor to consider due to the conformational degrees of freedom, 0039-9140/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2011.07.019