In situ evaluation of gemcitabine–DNA interaction using a DNA-electrochemical biosensor Rafael M. Buoro a,b , Ilanna C. Lopes a , Victor C. Diculescu a , Silvia H.P. Serrano b , Liseta Lemos c , Ana Maria Oliveira-Brett a, ⁎ a Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade de Coimbra, 3004-535 Coimbra, Portugal b Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo, 05508-000 São Paulo, Brazil c Serviços Farmacêuticos, Centro Hospitalar e Universitário de Coimbra (CHUC), 3000-075 Coimbra, Portugal abstract article info Article history: Received 14 February 2014 Received in revised form 9 May 2014 Accepted 30 May 2014 Available online 17 June 2014 Keywords: Gemcitabine DNA Guanine Electrochemical DNA-biosensor Interaction mechanism The electrochemical behaviour of the cytosine nucleoside analogue and anti-cancer drug gemcitabine (GEM) was investigated at glassy carbon electrode, using cyclic, differential pulse and square wave voltammetry, in different pH supporting electrolytes, and no electrochemical redox process was observed. The evaluation of the interaction between GEM and DNA in incubated solutions and using the DNA-electrochemical biosensor was studied. The DNA structural modifications and damage were electrochemically detected following the changes in the oxida- tion peaks of guanosine and adenosine residues and the occurrence of the free guanine residues electrochemical signal. The DNA–GEM interaction mechanism occurred in two sequential steps. The initial process was indepen- dent of the DNA sequence and led to the condensation/aggregation of the DNA strands, producing rigid struc- tures, which favoured a second step, in which the guanine hydrogen atoms, participating in the C–G base pair, interacted with the GEM ribose moiety fluorine atoms. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Nucleoside analogs of nucleobases are a pharmacological class of compounds with cytotoxic, immunosuppressive and antiviral proper- ties [1], and the pyrimidine nucleoside analogs are relevant derivatives effective in cancer treatment. Gemcitabine (GEM), 2,2-difluorodeoxycitidine, Scheme 1A, is a nucleoside analogue of cytidine, Scheme 1B, and plays a major role in the treatment of bladder and breast [1–4] cancer, and when combined with doxorubicin in hepatic [5], non-small cell lung and pancreatic cancer [6–8]. Due to its lipophilic characteristic, GEM is easily transported inside the cell by nucleoside membrane transporters where it is phosphorylat- ed and then competes with cytidine derivatives in the DNA synthesis. A high concentration of GEM triphosphate inhibits cytidine triphosphate (CTP) synthetase, and cytidine monophosphate (CMP) deaminase, which maintain CTP in low concentrations [1] and assures greater avail- ability of GEM triphosphate. GEM mainly exerts its biological activity by two mechanisms. The first pathway corresponds to the incorporation of GEM into DNA triggering the mechanism of DNA repairing, but the enzyme responsible for the base excision is not capable of removing GEM and replication stops [1]. In the second pathway blocking of DNA synthesis, through the inhibition of ribonucleotide reductase occurs, im- peding the synthesis of the new strand. GEM compared to the other drugs used on cancer treatment, in mono- and combined chemotherapies, is well tolerated among patients, with acceptable side effects and toxicity [5–8]. Due to the positive aspects of nucleoside analogs in several types of cancer treatment, their analytical determination is an important issue. Electroanalytical methods have been previously used for the characterization of purine nucleosides analogues, such as claribidine [9], clorafabine [10] and fludarabine [11], and the influence of the structural differences in the ribose moiety on their electrochemical behaviour investigated. HPLC- MS and UV–vis spectrophotometry were also applied for the determina- tion and quantization of GEM [12–14]. The interaction of some purine nucleosides with DNA has been pre- viously studied in incubated solutions and with a DNA-electrochemical biosensor [9–11]. Interaction of the damaging agent with DNA caused changes into the properties of the DNA recognition layer and this effect was quantified electrochemically [15–18]. It has been shown that the nucleoside analog caused dsDNA structural modifications in a time- dependent manner, but no DNA oxidative damage [9,10]. Although the DNA damage mechanism by nucleoside analogues ap- parently involved secondary biochemical reactions there are studies Bioelectrochemistry 99 (2014) 40–45 ⁎ Corresponding author. Tel./fax: +351 239 835295. E-mail address: brett@ci.uc.pt (A.M. Oliveira-Brett). http://dx.doi.org/10.1016/j.bioelechem.2014.05.005 1567-5394/© 2014 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Bioelectrochemistry journal homepage: www.elsevier.com/locate/bioelechem