Enzyme-amplified electrochemical hybridization assay based on PNA, LNA and DNA probe-modified micro-magnetic beads Serena Laschi, Ilaria Palchetti ⁎, Giovanna Marrazza, Marco Mascini Dipartimento di Chimica, Università di Firenze, Via della Lastruccia 3, 50019 Sesto Fiorentino, Italy abstract article info Article history: Received 22 December 2008 Received in revised form 27 February 2009 Accepted 27 February 2009 Available online 11 March 2009 Keywords: DNA Locked Nucleic Acid Peptide Nucleic Acid Electrochemical biosensor Magnetic beads Horseradish peroxidase In the present study, we investigated the properties of PNA and LNA capture probes in the development of an electrochemical hybridization assay. Streptavidin-coated paramagnetic micro-beads were used as a solid phase to immobilize biotinylated DNA, PNA and LNA capture probes, respectively. The target sequence was then recognized via hybridization with the capture probe. After labeling the biotinylated hybrid with a streptavidin–enzyme conjugate, the electrochemical detection of the enzymatic product was performed onto the surface of a disposable electrode. The assay was applied to the analytical detection of biotinylated DNA as well as RNA sequences. Detection limits, calculated considering the slope of the linear portion of the calibration curve in the range 0–2 nM were found to be 152,118 and 91 pM, coupled with a reproducibility of the analysis equal to 5, 9 and 6%, calculated as RSD%, for DNA, PNA and LNA probes respectively, using the DNA target. In the case of the RNA target, the detection limits were found to be 51, 60 and 78 pM for DNA, PNA and LNA probes respectively. © 2009 Elsevier B.V. All rights reserved. 1. Introduction The use of nucleic acids as biorecognition elements in biosensor design represents an exciting area in analytical chemistry [1]. In particular, in the recent developments of nucleic acids hybridization biosensors, significant progress has been made toward rapid and accurate detections of specific DNA or RNA sequences [2,3]. The basis for these nucleic acid hybridization devices is the strong interaction between two complementary nucleic acid strands. Accordingly, these devices rely on the immobilization of a single stranded DNA molecule, called capture probe, for hybridizing with the complementary target strand in a given sample. As with other types of biosensors, high selectivity is crucial for the success of hybridization biosensors [4]. The selectivity of nucleic acid hybridization assays depends primarily on the selection of the probe and then of the hybridization conditions. Thus, the design of the capture probe is undoubtedly the most important pre-analytical step [4]. Remarkable sequence specificity has been achieved recently using modified nucleic acids [4]. Many types of modifications have been introduced into native nucleic acids with high affinity toward DNA or RNA, such as peptide nucleic acid (PNA) and locked nucleic acid (LNA). PNA was developed [4–6] as a new oligomer having high affinity hybridization with single-strand DNA. PNA is a nucleic acid analogue of DNA, in which the phosphate backbone of DNA is replaced with a structurally homomorphous pseudopeptide backbone. PNA, having a backbone that is structurally homomorphous to the sugar–phosphate backbone of RNA and DNA, has been shown to hybridize with complementary strands of DNA or RNA, following Watson–Crick base- pairing rules, either in what has been called a parallel or an antiparallel fashion, i.e. with the N-terminus of PNA located adjacent to the 5′-end or the 3′-end of the oligonucleotide strand, respectively. A PNA strand can also hybridize with another complementary PNA strand to form a helical duplex. PNA forms a B-like helix with DNA and an A-like helix with RNA. The neutral backbone of PNA implies a lack of electrostatic repulsion between the PNA and DNA strands (compared to that existing between two negatively charged DNA oligomers) and hence a higher stability of PNA/DNA duplex. LNA was prepared as an ideal oligomer for recognition of RNA [7– 10]. LNA is a nucleic acid analogue of RNA, in which the furanose ring of the ribose sugar is chemically locked by the introduction of a methylene linkage between O2 and C4′. The covalent bridge effectively ‘locks’ the ribose in the N-type (3-endo) conformation that is dominant in A-form DNA and RNA. This conformation enhances base stacking and phosphate backbone pre-organization and results in improved affinity for complementary DNA or RNA sequences. Further, LNA residues confer a relative degree of nuclease resistance, both by exo- and endonucleases. In the present study, we investigated the properties of PNA and LNA capture probes in the development of an enzyme-amplified electrochemical hybridization assay. In particular, in order to avoid adsorption of non-specific nucleic acid sequences or enzymatic labels Bioelectrochemistry 76 (2009) 214–220 ⁎ Corresponding author. Tel.: +39 0554573323; fax: +39 0554573397. E-mail address: ilaria.palchetti@unifi.it (I. Palchetti). 1567-5394/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bioelechem.2009.02.012 Contents lists available at ScienceDirect Bioelectrochemistry journal homepage: www.elsevier.com/locate/bioelechem