Elsevier

Biosensors and Bioelectronics

Volume 51, 15 January 2014, Pages 170-176
Biosensors and Bioelectronics

DNA probe modified with 3-iron bis(dicarbollide) for electrochemical determination of DNA sequence of Avian Influenza Virus H5N1

https://doi.org/10.1016/j.bios.2013.07.026Get rights and content

Highlights

  • Oligonucleotide probes bearing 3-iron bis(dicarbollide) label is suitable for electrochemical determination of DNA derived from AIV type H5N1.

  • Changes in redox activity of Fe(III) centre of redox label was used as analytical signal tracing hybridization process.

  • E - genosensor was suitable for discrimination of PCR products with different location of the complementarity region in fM concentration range.

Abstract

In this work, we report on oligonucleotide probes bearing metallacarborane [3-iron bis(dicarbollide)] redox label, deposited on gold electrode for electrochemical determination of DNA sequence derived from Avian Influenza Virus (AIV), type H5N1. The oligonucleotide probes containing 5'-terminal NH2 group were covalently attached to the electrode, via NHS/EDC coupling to 3-mercaptopropionic acid SAM, previously deposited on the surface of gold. The changes in redox activity of Fe(III) centre of the metallacarborane complex before and after hybridization process was used as analytical signal. The signals generated upon hybridization with targets such as complementary or non-complementary 20-mer ssDNA or various PCR products consisting of 180–190 bp (dsDNA) were recorded by Osteryoung square-wave voltammetry (OSWV). The developed system was very sensitive towards targets containing sequence complementary to the probe with the detection limit estimated as 0.03 fM (S/N=3.0) and 0.08 fM (S/N=3.0) for 20-mer ssDNA and for dsDNA (PCR product), respectively. The non-complementary targets generated very weak responses. Furthermore, the proposed genosensor was suitable for discrimination of PCR products with different location of the complementarity region.

Introduction

The electrochemical genosensors have gained strong interest in the field of monitoring of environmentally and medical relevant samples because of low equipment requirements, short time of analysis and low costs (Chang et al., 2011, Drummond et al., 2003).

Different approaches for detection of the probe-analyte hybridization processes have been applied in various genosensors. One of them is the approach based on changes in electrochemical activity of nucleobases upon the hybridization events. This concept has been proposed by Palecek (2009) and Palecek and Bartosik (2012). The other idea is based on changes of electrochemical parameters of the interface: electrode/aqueous solution caused by hybridization reaction (Malecka et al., 2012; Zhang et al., 2008). This category includes biosensors originally developed by Umezawa et al., in which mechanism of analytical signal generation is based on ion-channel mimetic system (Umezawa and Aoki, 2004).

One of the unfavorable features of genosensors based on nucleobases electroactivity is rather weak analytical signals. Whereas, the ion-channel mimetic genosensors, in which the redox marker molecules are present in solutions, demand well ordered, free of pinholes monolayers deposited on the electrode surfaces, which very often is difficult to fulfill. One of the ways to avoid this problem is fixing the redox molecule on the surface of the electrode. The oligonucleotide probes having the steam loop structure, modified with redox active label has been reported (Fan et al., 2003, Rai et al., 2012). As a consequence of hybridization event, self-assembled monolayer (SAMs) are characterized by precisely defined structure, more rigid in comparison with the structure of probe immobilized on the surface of electrode (Ceres and Barton, 2003, Gooding et al., 2003). Such a change causes increase of distance between redox active labels covalently attached to the probe from the surface of electrode. In consequence, the increase of electrons transfer resistance was observed (Anne and Demaille, 2006, Aoki et al., 2010, Farjami et al., 2011, Lai et al., 2006, Lubin et al., 2009, Pavlovic et al., 2008, Yang and Zhang, 2010). Such a system is called “on–off” mode of the electrochemical signal.

The most frequently used redox active labels is ferrocene (Anne and Demaille, 2006, Aoki et al., 2010, Chatelain et al., 2012, Zhao et al., 2012, Zhuang et al., 2013) as well as methylene blue (Abi and Ferapontova, 2012, Farjami et al., 2011, Farjami et al., 2012, Ferapontova and Gothelf, 2009, Kang et al., 2009, Kang et al., 2012, Lai et al., 2006, Liu et al., 2010, Lubin et al., 2009, Patterson et al., 2010, Pavlovic et al., 2008, Ricci et al., 2010, Yang and Zhang, 2010). Applicability of these labels is based on their reversible redox properties in the appropriate potential windows. However, genosensors containing probes modified with ferrocene or methylene blue are not totally free from the non-specific adsorption when applied in complex media, such as for example, blood serum (Ferapontova and Gothelf, 2009).

Working in this very vivid research field, we decided to use an alternative type of redox label, namely the chemically and biologically stable complex of carborane – Fe(III) with well-defined electrochemical properties (Corsini et al., 2006, Jelen et al., 2009, Olejniczak et al., 2009, Olejniczak, 2011). Metallacarboranes are vast family of metallocene-type complexes consisting of at least one carborane cage and one or more metal cation. Carborane clusters are versatile and efficient ligands for cation of metals such as Co, Fe, Ni, Cr, Re, Al, Au, Cu, Ir, Mn, Pt, and others. This allows for incorporation of variety of metals with different properties into nucleic acids and their components. An additional advantage of metallacarboranes is susceptibility to redox potential tuning via particular derivatization of the boron cluster ligands (González-Cardoso et al., 2010, Grimes, 1982). The metallacarboranes have been applied for modification of target ssDNA (Ziółkowski et al., 2012).

Here we present the approach in which the gold electrodes were modified with oligonucleotide probe with 3-iron bis(dicarbollide) redox active complex, an alternative and probably more stable redox labels in comparison with already reported. Moreover, the novelty of the approach presented is affiliated with the attachment of the redox label at the “foot” of the oligonucleotide probe, very close to the electrode surface. To the best our knowledge, such system was not presented in the literature till now.

The new type of genosensor destined for determination of the genetic material of the H5 subtype of Avian Influenza Virus (AIV) is illustrated in Scheme 1.

Section snippets

Reagents and materials

3-mercaptopropionic acid (HS(CH2)2CO2H), EDC: N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (C8H17N3 HCl), NHS: N-Hydroxysuccinimide (C4H5NO3), MES: 2-(N-Morpholino)ethanesulfonic acid (C6H13NO4S), sodium perchlorate (NaClO4), sodium chloride (NaCl), potassium phosphate monobasic (KH2PO4), trifluoroacetic acid (CF3COOH), ammonia (NH3), ethanolamine (NH2(CH2)2OH) were purchased from Sigma-Aldrich (Poznań, Poland). KOH, H2SO4, ethanol, methanol were obtained from ABChem, Gliwice,

Results and discussion

Recent reports showed that influenza can spread rapidly and cause a range of illness among humans, therefore the early detection of this virus is highly needed, especially-because the H5 subtype is highly pathogenic (Amano and Cheng, 2005). The detection of the influenza virus is based on non-labelling techniques, such as surface plasmon resonance (SPR) or quartz crystal microbalance (QCM) (Amano and Cheng, 2005, Wang and Li, 2013). However, these techniques demand quite sophisticated

Conclusions

A novel genosensor, in which 3-iron bis(dicarbollide) redox label was attached at the “foot” of the oligonucleotide probe, very close to the electrode surface, displayed good selectivity and sensitivity towards complementary targets, 20-mer ssDNA and PCR products derived from Influenza Virus, type H5N1. As a sensing technique, Osteryoung square-wave voltammetry was applied and the changes in redox activity of Fe(III) centres were used as analytical signal generated by hybridization event. The

Acknowledgments

This work was supported by the Innovative Economy Program, Grant no. POIG.01.01.01-14-007/08, Grant no. N N204 531739 to ABO and ZJL, and Institute of Animal Reproduction and Food Research of Polish Academy of Sciences, Olsztyn, Poland. Partial contribution of the Statutory Fund of IMB PAS is also gratefully acknowledged.

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