Biosensors and Bioelectronics 26 (2010) 12051217

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Biosensors and Bioelectronics

journal homepage: www.elsevier.com/locate/bios

Review

Electrochemical DNA hybridization sensors applied to real and complex biological samples
J.P. Tosar, G. Branas, J. Laz
Analytical Biochemistry Unit of the Nuclear Research Center, Faculty of Science, Universidad de la Repblica, Montevideo, Uruguay

a r t i c l e

i n f o

a b s t r a c t
DNA hybridization biosensors, also known as genosensors, are analytical devices for the detection of specic DNA target sequences in solution, upon hybridization of the targets with complementary probes immobilized on a solid substrate. Electrochemical genosensors hold great promise to serve as devices suitable for point-of-care diagnostics and multiplexed platforms for fast, simple and inexpensive nucleic acids analysis. Although a lot of progress has been made in the past few years, the performance of genosensors in complex biological samples has been assayed in only a small fraction of published research articles. This review covers such a group of reports, from the year 2000 onwards. Special attention is played in the nature and complexity of the samples and in the way matrix effects were treated and specicity controls were performed. 2010 Elsevier B.V. All rights reserved.

Article history: Received 15 April 2010 Received in revised form 30 July 2010 Accepted 17 August 2010 Available online 24 August 2010 Keywords: DNA biosensor Real samples Genomic DNA RNA PCR

Contents 1. 2. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1205 Detection of RNA samples without target amplication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1209 2.1. Detection of ribosomal 16S/18S rRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1209 2.2. Detection of specic mRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1210 2.3. Detection of microRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1211 Detection of DNA samples without target amplication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1212 3.1. Detection of genomic DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1212 3.2. Detection of sequences inserted into plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1213 Detection of PCR products obtained with a low number of PCR cycles and electrochemical real-time PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1213 Detection of PCR amplicons obtained with more than 20 PCR cycles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1214 5.1. PCR detection with reagent-less methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1215 5.2. PCR detection with reagent-based approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1215 Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1216 Future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1216 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1216 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1217

3.

4. 5.

6. 7.

1. Introduction In light of the progress made during the 1990s, DNA biosensors and gene chips offered, by the beginning of the XXIst century, considerable promise for obtaining sequence-specic information in a faster, simpler and cheaper manner compared to traditional

Corresponding author at: Centro de Investigaciones Nucleares, Facultad de Ciencias, Mataojo 2055, 11400 Montevideo, Uruguay. E-mail address: jlaiz@cin.edu.uy (J. Laz). 0956-5663/$ see front matter 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2010.08.053

hybridization assays (Wang, 2000). Today, 10 years after, we may ask how close is the eld of electrochemical DNA hybridization biosensors to make an impact on the society by bringing new and revolutionary technologies to the medical clinic and the patient bedside. Some authors expect that, with further development and resources, portable electrochemical devices will speed up the diagnosis of certain diseases such as cancer, playing an important and signicant role in the transition towards point-of-care diagnostics (Wang, 2000). DNA hybridization biosensors with electrochemical detection have a very important strength which is the ease of

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implementation in a miniaturized and automated device. The consolidated success obtained with glucose amperometric biosensors opens the question if similar hand-held analyzers may be designed to detect specic nucleic acid sequences in unprocessed biological samples. Many different strategies have been described for the electrochemical detection of nucleic acids. They rely on the intrinsic electrochemical properties of DNA or in the concentration of electroactive species near the electrode, either before or after hybridization. The different methods are usually classied into two main categories: label-free or label-based approaches, depending on the nature of the electrochemical signal. Label-free methods imply that the signal arises from the hybridization event by itself, without the need of concentrating any special electroactive species on the electrode surface, apart from target DNA. Labelbased approaches rely on the use of other electroactive molecules which can discriminate between single-stranded DNA (ssDNA) and double-stranded hybrids (dsDNA), or are concentrated on the electrode surface only after target DNA is present. A slightly different classication divides the literature into reagent-less (Fig. 1) and reagent-based methods (Fig. 2). The concept behind this classication is not strictly the same and we will focus on it for practical reasons. A reagent-less method avoids the need of introducing a reagent during the protocol, and then washing the reagent out. For this reasons, they are easier to automate than reagent-based methods which may be time-consuming or more expensive. Intrinsic electrochemical properties of DNA, i.e., oxidation of guanine moieties, can be exploited for sequence-specic detection of DNA. However, both target sequences and DNA probes usually contain guanine residues, making it difcult to assign the guanine oxidation signal to the probes or to the targets. This problem was solved by replacing guanine residues in the immobilized probes by inosine (Wang et al., 1998), a base which is not naturally present in DNA but can also base-pair with cytosine, as guanine

does (Fig. 1A). Since DNA is a polyanionic molecule, hybridization usually results in considerable changes in the electrochemical properties of the interface, which can be measured by different electrochemical methods (Fig. 1B). Fan et al. (2003) rst described the use of immobilized partially self-complementary DNA probes labeled with an electroactive reporter as the hybridization sensing element (Fig. 1C). On hybridization, the distance between the label and the electrode is signicantly altered due to conformational changes in the probes, leading to a measurable signal change. It should be noticed that although this method is label-based, it is still reagent-less, as the label is bound to the probes and there is no need of a labeling step in the protocol. Reagent-based methods usually rely on electroactive species which bind specically to either ssDNA (Fig. 2A) or dsDNA (Fig. 2B). Probably the most widely used of such a kind of redox indicators is methylene blue (MB). Many reports have shown how the reduction current of MB is diminished after hybridization, as MB binds specifically to free guanine bases present in ssDNA (Yang et al., 2002). However, it is also shown that MB can bind dsDNA with high afnity, by either electrostatic, groove or intercalative binding (Zhou et al., 2008). For this reasons, many reports seem contradictive, as MB reduction signal may increase or decrease after hybridization depending on the experimental conditions. Besides, as target DNA sequences are usually larger than oligonucleotide probes, hybridization both increases the amount of dsDNA and ssDNA near the electrode surface (Fig. 1A), yielding a difcult interpretation of the results. Apart from the immobilized probes and the hybridized target sequences, a third redox-labeled reporter sequence may be exogenously added. If the reporting sequence is complementary to the target in a different region to the sequence which is recognized by the probes, then detection can be performed by what is called a sandwich approach. One advantage of this kind of strategies is that they are very insensitive to non-specic cross-hybridization. Sandwich approaches are also very useful to concentrate redox enzymes

Fig. 1. Reagent-less methods for the electrochemical detection of DNA hybridization. (A) Detection by guanine oxidation. Guanine-free oligonucleotides probes may be designed by replacing guanine (red circles) with inosine (yellow circles). (B) Detection by monitoring changes in the electrochemical properties of the interface after hybridization. Oligonucelotide probes are shown in red, and complementary DNA targets are represented in green. The different distribution of anions and cations after hybridization represents measurable changes in the electrochemical properties of the interface. (C) Hybridization-induced conformational changes of redox-labeled selfcomplementary DNA probes. (For interpretation of the references to color in this gure legend, the reader is referred to the web version of the article.)

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Fig. 2. Reagent-based methods for the electrochemical detection of DNA hybridization. Oligonucelotide probes are shown in red, and complementary DNA targets are represented in green. (A) Redox indicators with afnity towards single-stranded DNA (blue arrowheads). (B) Redox indicators with afnity towards double-stranded DNA (red ellipses). Some of such indicators may have electrocatalytic properties towards guanine oxidation. (C) Signal amplication by concentration of redox enzymes through sandwich hybridization assays. Labeled reporter probes are shown in blue. Enzyme-conjugated antibodies specic to the labeled reporter are shown in orange. The enzymes substrate is denoted by the letter S, which is later converted to an electroactive product, P. (For interpretation of the references to color in this gure legend, the reader is referred to the web version of the article.)

near the electrode after hybridization (Fig. 2C). Enzymes catalyze conversion of many substrate molecules to electroactive products, which are oxidized or reduced at the electrodes surface. In this way, substrate molecules can be regenerated, leading to an amplication of the signal. Some other strategies were also described, as the use of silver and gold nanoparticles (Steel et al., 1998), but most of the articles reviewed herein fail into one of the schemes summarized in Figs. 1 and 2. A rule of thumb is that, when describing a new detection method or introducing a new variation, initial experiments should be carried out with synthetic oligonucleotides which are complementary and not-complementary to the immobilized probes (Fig. 3A). These types of experiments are necessary since they serve as the proof of concept that the immobilization and detection method which is being investigated does work, and that it is able to discriminate between complementary targets and unspecic DNA sequences. Furthermore, ultrasensitive detection at femtomolar levels has been achieved by many authors using synthetic oligonucleotides (Baur et al., 2010; Liu et al., 2008; Zhang et al., 2004, 2006; Hansen et al., 2006). Although these results are encouraging, the performance of the genosensor with a real complex biological sample may be entirely different. Indeed, results cannot be extrapolated to the use of more complex samples without the inclusion of adequate controls, as the matrix in which the target sequence is embedded may give a background signal which should be taken into consideration. Some of the problems which may be encountered when changing the experimental conditions from synthetic oligonucleotides in solution to a complex biological sample are schematized in Fig. 3. For instance, the target sequence may be present, but hybridiza-

tion is impaired because the matrix components can block the electrode surface leading to a false negative result (Fig. 3B). Otherwise, matrix components may bind the electrode surface with high afnity, either by cross-hybridization with the probes or by unspecic adsorption. If these unspecic binding resist the washing steps, then false positives may also appear (Fig. 3C). Hybridization may take place normally, but matrix components may be able to suppress the analytical signal for a variety of reasons (Fig. 3D). Especially true when working with RNA, the target sequence may be degraded by enzymes present in the sample (Fig. 3E). The importance of analyzing the performance of electrochemical genosensors with a variety of complex biologically relevant samples has been addressed by other authors (Luong et al., 2008; Sadik et al., 2009). Only a very small fraction of research articles in the eld of electrochemical genosensors have gone beyond the use of synthetic oligonucleotides and evaluated the performance of the system in real samples. The actual scope of this review is to address progress achieved in this direction. A hierarchical classication of the most common types of samples used in the articles reviewed herein is given in Fig. 4. This classication takes into consideration both the strategies used for sample pre-processing and the complexity of the resulting matrix. As the same type of sample may be obtained by different methods, the degree of complexity is actually variable. It may be argued that a method which has a good performance with the most complex samples (a crude cell extract, for example) is robust. We will use the target to interference ratio as a means to illustrate the analytical challenge of detecting a specic target sequence in a sample which contain an excess of other sequences and molecules which we will generally group as interferences.

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Fig. 3. Matrix effects can affect the performance of a genosensor when performing detection with real biological samples. Studies made with pure synthetic complementary and non-complementary oligonucleotides in solution (A) are compared with different situations which can arise when incubating the electrodes in biological material (BE). Immobilized probes are represented in blue, complementary targets are painted green and interferences are shown in red. The grey circles represent free electroactive dsDNA intercalators. Intercalators which have bound to DNA are shown as black circles. (For interpretation of the references to color in this gure legend, the reader is referred to the web version of the article.)

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Fig. 4. Different types of biological material which can be used for target detection using genosensors. Upper samples are less processed and more complex. The target to interference ratio increases downwards.

2. Detection of RNA samples without target amplication Only a very small percentage of genomic DNA codes for proteins. However, it is well known that a very large fraction of DNA is actively transcribed to RNA (Gerstein et al., 2007). Thus, signicant fractions of RNA species present in a cell extract are non-coding RNAs distinct from mRNAs. Researchers are not only interested in the detection and quantication of protein-coding RNAs. Ribosomal RNAs are highly abundant and conserved molecules which can be used for species identication without the need of in vitro nucleic acid amplication steps. Regulatory RNAs such as miRNAs can regulate the expression of a large subset of genes and are implicated in many human pathologies included cancer (Esquela-Kerscher and Slack, 2006). Many times the aim of the analysis is not focused in host gene expression but in the detection of pathogens. It should then be considered that many viruses are RNA viruses, and carry their genetic information in the form of RNA molecules. 2.1. Detection of ribosomal 16S/18S rRNA Ribosomal 16S RNA (16S rRNA) forms part of the smaller subunit of the prokaryotic ribosome (in eukaryotes, the smaller ribosome subunit contains a larger 18S rRNA). Since it is functionally constant, shows a mosaic structure of conserved and more variable regions, occurs in all prokaryotic organisms, has limited intragenomic heterogeneity between multiple 16S rRNA operons and is present in a very high copy number in cell extracts (Coenye and Vandamme, 2003), it is widely used as a target molecule in genosensor articles in which prokaryotic organisms are detected at the RNA level. The high copy number is a decisive factor, since it enables detection without prior in vitro amplication. In the year 2001, Gau et al. designed a microelectromechanical (MEMS) system capable of detecting 1000 Escherichia coli (E. coli) cells without PCR amplication. Bacteria cultures were lysed and an amount of biotinylated oligonucleotides was added to the crude cell extract in order to perform solution-phase hybridization between the ssDNA probes and bacterial rRNA. Fluorescein-conjugated

detector probes, complementary to a different sequence of the bacterial rRNA, were also added. Thus, an ssDNArRNAssDNA sandwich hybrid was formed. An amount of this mixture was placed on the streptavidin-coated working electrodes of the MEMS detector array. By doing so, the solution-formed sandwich hybrid was immobilized due to the biotinstreptavidin interaction. After washing, anti-uorescein antibodies conjugated to peroxidase were added. A second washing step eliminated unbound antibodyenzyme conjugates. The substrate of the enzyme (H2 O2 ) was then introduced, together with the redox soluble mediator 3,3 ,5,5 -tetramethylbenzidine (TMB), which can carry electrons from the electrode to the oxidized form of the enzyme, and vice versa. 20 s after addition of substrate, the electrical current corresponding to the reduction of oxidized TMB was registered amperometrically. The authors compared the electrical signals obtained after different dilutions of an E. coli lysate with that obtained after incubation with an extract from Bordetella bronchiseptica, an unrelated bacterium. They found that the electrical current from as low as 1000 E. coli cells was more than twice that of 2.5 105 cells of B. bronchiseptica (Gau et al., 2001). With the exception that the probe/target/reporter sandwich hybrid was formed in solution in this case, the overall strategy is the same to that schematized in Fig. 2C. It should be noticed that the nature of the sample in the above mentioned report was a crude cell extract, the highest type of sample in terms of complexity of those shown in Fig. 4. The background signal was dened using another crude extract obtained upon lysis of an average number of cells from an unrelated organism. Thus, the background signal accounts not only for the unspecic adsorption of enzymes and substrates and the possible cross-hybridization with rRNA from a different organism, but also serves to estimate the effect of unspecic adsorption of different components of the sample on the analytical signal (matrix effect). A very similar approach was followed by Sun et al. (2005) for rapid, species-specic detection of uropathogens based on the sequence of their 16S rRNA. In this case, the uorescein-conjugated detector probes where hybridized in solution with the bacterial lysates, and the mixture was deposited on the working electrodes of a 16-sensor array developed by GeneFluidics Inc. (Montrey Park,

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CA). The working electrodes were coated with streptavidin, to which 35-mer biotinylated capture probes were immobilized. The authors compared the signal obtained with capture and detection probes specic to E. coli 16S rRNA, after incubation with lysates from different uropathogenic bacteria. The signal corresponding to the E. coli lysate was about 20 times higher than when incubating with lysates from Proteus mirabilis, Klebsiella oxytoca, Pseudomonas aeruginosa, Klebsiella pneumoniae and Enterobacter cloacae. The negative samples gave electrical signals very similar to the negative control, without bacterial nucleic acids. Thus, they showed that it was possible to electrochemically discriminate E. coli from different uropathogens in non-amplied crude cell extracts. The authors showed that detection could be carried out in 40 min and at room temperature, although the signal to background ratio was higher at 65 C (Gau et al., 2005; Sun et al., 2005). Gabig-Ciminska et al. (2004) described an electric chip for detection and quantication of nucleic acids, and evaluated the detection of 16S rRNA in a total RNA extract from a E. coli culture. The biotinylated capture probes were immobilized on strepatividin-coated magnetic beads. After incubation with the sample, a digoxigenin (DIG)-labeled detection probe was added, which was complementary to a different region of the target. Upon completion of hybridization, the magnetic beads were separated using an external magnet, washed, and exposed to anti-DIGALP conjugates (ALP stands for alkaline phosphatase). p-Aminophenol phosphate (pAPP) was added, which was hydrolyzed by bounded ALP, yielding an electroactive product which could be detected amperometrically after transfer to the electric chip (Gabig-Ciminska et al., 2004). LaGier et al. (2005) also developed an electrochemical RNA hybridization assay for detection of E. coli, based on its 16S rRNA sequence. Probe-modied magnetic beads were used, and incubated with either RNA extracts from E. coli (from 108 colony forming units, CFU) or E. coli cell lysates (from 109 CFU). Magnetic beads were separated and washed after hybridization, and treated with sulfuric acid in order to hydrolyze nucleic acids and release nucleotides into solution. Guanine nucleotides were accumulated at a graphite electrode and were afterwards detected by differential pulse voltammetry (DPV; the guanine oxidation method is schematized in Fig. 1A). A blank run without E. coli RNA gave the background signal corresponding to the guanine bases present in the oligonucleotide probes. The authors also incubated magnetic beads not conjugated to DNA probes with total E. coli RNA extracts, in order to show that there was negligible non-specic adsorption to the beads. The capture probe was designed to bind not only E. coli 16S rRNA but also rRNA from other species belonging to the family Enterobacteriacae. Indeed, all Enterobacteriacae species tested gave positive and similar signals, while P. aeruginosa and Enterococcus fecalis (non-enterobacteria) gave signals indistinguishable from the background (LaGier et al., 2005). A sandwich hybridization assay was developed by Metes et al. (2005) for detection of the toxic dinoagellate (algae) Alexandrium ostenfeldii. A biotinylated capture probe and a digoxigenin (DIG)labeled detection probe were designed against A. ostenfeldii 18S rRNA. The capture probe was immobilized to the surface of a disposable avidin-coated sensor chip. Peroxidase was concentrated at the electrode surface by conjugation to anti-DIG antibodies. The redox mediator p-aminodiphenylamin (ADPA) was used in this case, and its reduction current was measured amperometrically using a hand-held device. The hybridization conditions were optimized changing the salt concentration, the hybridization temperature, and evaluating heat fragmentation of the rRNA and the addition of helper oligonucleotides which hybridize to other segments of the 18S rRNA and help to keep the rRNA in an opened conguration. Total RNA extraction from algae cultures was performed, and the signal intensities of three different A. ostenfeldii strains were compared to different Alexandrium species and found

to be much higher in the three cases. Furthermore, the two species which gave a weak but measurable signal were those closer to A. ostenfeldii in phylogenetic tree analysis (Metes et al., 2005). A portable, multi-probe and semi-automated device using a similar detection system was later designed for taxon specic detection of toxic algae (Diercks et al., 2008). Again, the probe specicity tests were done comparing laboratory strains with closely related species, in order to avoid false positives and to guarantee that only desired strains were detected. We have stated above that incubation with extracts from similar but unrelated organisms may serve as an adequate negative control in order to account for the background signal given by unspecic adsorption of reactants and matrix components. However, when the aim is to perform species-specic detection, comparisons should be made between the closest neighbors in the phylogenetic tree, as the appearance of a false positive signal is more probable to arise from cross-hybridization (Fig. 3C). Probes should be carefully designed so that they are 100% complementary to the target sequence in the desired specie/s, but they contain a reasonable number of mismatches towards the target sequence in its closest neighbors (Sun et al., 2005; LaGier et al., 2005). 2.2. Detection of specic mRNAs The copy number of a specic mRNA in a particular cell type varies drastically from an mRNA to another, and depends on a series of factors which are usually related to the molecular function of the protein for which that mRNA codies. In general, the copy number of a particular mRNA in a cell extract is much smaller than the copy number of ribosomal rRNA molecules, although some viral mRNAs or mRNAs expressed from articially introduced expression vectors can also be transcribed to high intracellular levels. Thus, detection of specic mRNAs without prior amplication is a challenging task, due to the low target to interference ratio which would probably exist. In the year 2004, Xie et al. described a method for detection and quantication of breast cancer susceptibility genes by microarrays with electrochemical detection (Xie et al., 2004a). The authors immobilized 24-mer oligonucleotides in each spot of an 8 8 sensor array, which were complementary to the sequences of breast cancer susceptibility genes such as p53, HSP90, BRCA1 and Histone H4, plus a house-keeping gene (GAPDH) used for normalization. mRNA was extracted from both healthy and cancer human breast tissues and total mRNA was labeled with cisplatincoupled biotin conjugates. Labeled mRNA was hybridized to the array surface and incubated with avidinglucose oxidase and then the surface was overcoated with a cationic redox polymer containing osmiumbipyridine complexes. This type of redox polymers were previously shown to interact and electrochemically activate anionic enzymes such as glucose oxidase (Xie et al., 2004b). The oxidation current of glucose in solution was detected amperometrically at 0.36 V vs. Ag/AgCl. By comparing glucose oxidation currents in 20 ng mRNA using oligonucleotide probes complementary and not-complementary to p53, the authors demonstrated the specicity of the assay. The sensitivity was evaluated by addition of a specic mRNA into 50 ng of total mRNA. It was possible to unambiguously detect gene expression differences of less than 1-fold. The authors also validated their method by comparing their results with another conventional non-electrochemical method for gene expression quantication (ribonuclease protection assays) obtaining very good agreement for the ve genes that were tested. The lowest detectable amount of a specic gene which could be detected was found to be around 800 copies in 1.5 ng of mRNA. There are several aspects in this seminal work that we think should be highlighted. We have previously shown examples of how

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specicity of a given method may be addressed, by the use of control experiments with extracts from similar but unrelated samples (e.g., extracts from different organisms, different cell types, or different experimental conditions). In this case, the authors kept the same sample but changed the sequence of the probe. Shifting the bases of the probe in order to design an irrelevant sequence with the same base composition may be the best way of evaluating the matrix effect. Testing the same probes in a similar but unrelated sample may give complementary information, provided that the samples are similar enough in composition and that there is independent evidence that the target nucleic acid is absent or expressed at negligible levels. These kinds of negative controls are conceptually different than testing the genosensor in extracts derived from close evolutionary related species. As we have stated before, that kind of experiments are used for demonstrating the possibility of performing species-specic detection, since the control sample is expected to express the target sequence in similar levels, though containing at least one mismatches. Another aspect that we would like to highlight is the way in which the sensitivity of the assay was evaluated. Serial dilutions of a pure synthetic complementary oligonucleotide are not very informative because they do not take the matrix effect into consideration. When informing the detection limit, it is much more accurate and analytically relevant to express the minimum number of copies/grams/moles of the target sequence that could be detected in a given amount of total mRNA, because that illustrates the target to interference ratio. Tansil et al. (2005) also reported a nucleic acid biosensor approach for detection of the oncogene p53 (also called TP53) in mRNA extracted from rat liver tissues, without a RT-PCR step. The electroactive intercalator PIND-Ru was used in this case. PIND-Ru intercalates very strongly to dsDNA and can be electrochemically oxidized at 0.62 V vs. Ag/AgCl. The authors also showed how guanine bases in the target sequences were catalytically oxidized at that potential by PIND-Ru, since the anodic current at 0.62 V increased almost linearly with the guanine content in the targets (this type of detection strategy is schematized in Fig. 2B). The selectivity of the biosensor was evaluated in 1.0 g mRNA, comparing the signal obtained with probes containing perfectly matched sequences to probes which contained one-base mismatch to the p53 mRNA. The current increment for the one-base mismatched was only about 40% of that of the perfectly matched sequence (Tansil et al., 2005). Since the sample is the same for both comparisons, it can be stated that the sensor has a very good specicity and is sensible to single base mismatches. There are many reports that intend to show that a given genosensor is sensible to single base mismatches by simply changing incubation in a pure aqueous solution of synthetic targets by a pure aqueous solution of synthetic one-base mismatched oligonucleotides. As performed by Tansil et al. (2005), when working with real samples the sequence of the probe should be changed in order to introduce the mismatches, except samples from patients known to contain single-nucleotide polymorphisms (SNPs) in the target gene are available. 2.3. Detection of microRNAs microRNAs (miRNAs) are non-coding RNAs of 2124 nucleotides, which are processed from an endogenous 70nucleotide hairpin RNA precursor. They are evolutionarily conserved molecules and are thought to have important functions in various biological mechanisms (Hutvagner and Simard, 2008). As other families of regulatory small RNAs, they recognize their target mRNAs by sequence-specic base pairing and trigger their degradation or silence their translation. As key regulators of gene expression, miRNAs are also linked to the onset and development

of several diseases including cancer. Some miRNAs can act as tumor suppressors, while others can act as oncogenes, either directly or indirectly (Esquela-Kerscher and Slack, 2006). Due to their small size, mature miRNAs cannot be directly assayed by conventional RT-PCR, and more laborious procedures are necessary for miRNA detection and quantitation. However, they have the typical size of oligonucleotide probes used for hybridization biosensors, so in theory many of the detection methods previously discussed could be adapted for miRNA detection. A detailed review on miRNA detection and quantication including electrochemical-based methods was recently published (Hunt et al., 2009). Gao and Yang reported in 2006 a miRNA assay employing electrocatalytic nanoparticle tags. RNA extracts were treated with periodate, which reacts with the 3 end ribose of RNA molecules and yields 3 end dialdhydes. After hybridization and washing, isoniazid-capped OsO2 nanoparticles were introduced and brought to the electrode through a condensation reaction with the 3 end of periodate treated miRNAs. The resulting electrode exhibited electrocatalytic activity toward the oxidation of hydrazine, drastically reducing its oxidation overpotential. By performing amperometry at a low applied potential in the presence of hydrazine, it was possible to detect specic miRNAs in total RNA extracts of HeLa cells (Gao and Yang, 2006). In the absence of the miRNA target, the nanoparticle tags were removed during the washing steps and no oxidation of hydrazine was registered. A very similar approach was followed in two reports published by Gao and Yu in 2007. Instead of OsO2 nanoparticles, two different transition metal complexes were used as tags for direct labeling of RNAs. The transition metal complexes used were covalently ligated to the 3 end of miRNAs (Gao and Yu, 2007a) or coordinated to their purine bases (Gao and Yu, 2007b). The electroactive tags were concentrated on the electrode surface by sequence-specic hybridization and uptake of the tags due to unspecic miRNAs was removed by extensive washing. In both reports, the tags showed excellent catalytic activity towards the oxidation of ascorbic acid (Gao and Yu, 2007a) or hydrazine (Gao and Yu, 2007b), shifting the oxidation potential negatively by as much as 600 mV and 850 mV, respectively, and greatly enhancing the oxidation peak current. Amperometrical detection was performed at low applied potentials, for the same reason explained in the previous paragraph. Total RNA was extracted in both reports from HeLa cells, and the RNA extracts were enriched in miRNAs and other short RNA species using a centrifugal lter device with a regenerated cellulose membrane permeable to molecules less than 50,000 g/mole. In both cases, three different miRNAs were quantied. The results were normalized to total RNA and compared with normalized values obtained by northern blot (the gold standard for miRNA validation and quantitation). Very similar results were obtained for all miRNAs tested. The lowest amount of total RNA needed for successful detection was about 10 ng, which is very low compared to the amount needed for northern blots assays, typically in the micrograms range. Since the labeling reactions are not miRNA-specic, it is reasonable to suppose that most RNAs in the sample are going to be labeled. Thus, extensive washing after hybridization is a necessary and very important step. Otherwise, false positives would be introduced. A nanogapped microelectrode-based array for miRNA detection on the basis of conductance measurements was reported by Fan et al. (2007). Peptide nucleic acid (PNA) probes were immobilized in 300-nm gaps between a pair of interdigitated microelectrodes. Hybridization was performed by incubating with total RNA extracts from HeLa and lung cancer cells. After hybridization with target miRNAs, an acidic solution of aniline, horseradish peroxidase and H2 O2 was introduced. The enzyme oxidizes aniline monomers and cationic aniline molecules polymerized around the nega-

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tively charged miRNA/PNA hybrids. By doping with HCl vapors, polyaniline was expanded to form a disordered conductive net. The formation of such a conductive net depends on the presence of negatively charged miRNAs hybridized to the uncharged PNA probes, and can be easily detected by monitoring changes in the conductivity of the solution between the microelectrodes (Fan et al., 2007).

3. Detection of DNA samples without target amplication 3.1. Detection of genomic DNA If a genosensor is designed to detect a specic gene in a genomic DNA extraction, the target to interference ratio will vary signicantly according to the size of the genome (which is a property of the species), but it will be very low even if the sample does not contain any other material apart from the genomic DNA. However, some genes are present at high copy numbers in the genome due to gene duplication events, and conserved sequences within duplicated genes may be used for design of capture probes. Another possibility is to design oligonucleotide probes which can hybridize to regions of the genome which are conserved but do not codify for proteins. In fact, intergenic regions account for 75% of the human genome (Lander et al., 2001; Venter et al., 2001), many of which is under evolutionary selection (Gerstein et al., 2007). An important fraction of intergenic regions is composed of different types of repeated elements such as satellite DNA, minisatellites, microsatellites, and tandem repeats, which consist of blocks of DNA bases which are repeated in tandem a variable number of times in the genome. These may serve as convenient sequences to use as targets, since they are naturally amplied. The same happens, for example, with rRNA genes (rDNA), which are present in high copy numbers in the genome, and are suitable for species discrimination. The target to interference ratio is signicantly higher if detection of a multi-copy sequence such as 16S rDNA is intended in an organism with a small genome such as E. coli, rather than detection of a single-copy gene in genomic DNA extractions of vertebrate cells, for example. Mascini et al. (2005) described a label-free method for identication of mammalian species (bovine and sheep) in DNA extracts obtained from unprocessed tissues, based on the sequences of satellites DNA and on the guanine oxidation signal. According to Pech et al., 29% of the total bovine genome consists of satellite DNA (Pech et al., 1979). Due to the high copy number of the target sequence, it was possible to perform detection without prior PCR amplication. The report served as a proof of concept that species identication with electrochemical label-free detection in unamplied DNA extracts was possible. The authors stated that rapid characterization of food samples to determine whether or not they contain unwanted species seems suitable using this technique (Mascini et al., 2005). Probes were carefully designed in order to hybridize with regions within satellite DNA which showed the least degree of similarity between different mammalian species. This enabled to perform species-specic detection by avoiding cross reactivity between different samples. Inosine-modied, guanine-free probes were used as previously described. In that way, the appearance of the guanine oxidation peak could be assigned to the hybridized targets, though unspecically adsorbed nucleic acids would also contribute to the guanine oxidation signal. This fact should be considered when performing detection based on the guanine oxidation signal in real samples. Puried genomic DNA was fragmented with the restriction enzyme EcoRI. This step aimed to reduce the size of DNA fragments in order to avoid steric hindrance during hybridization. DNA sam-

ples were thermally denaturalized by dipping the vial containing the sample in a boiling water bath at 95 C for 5 min, followed by 1 min in an ice-water bath. A drop of this solution was deposited on the probe-modied working electrode for 10 min and square-wave voltammetry was performed in order to detect the appearance of the guanine oxidation signal. The authors tested the selectivity of the sensor using bovineand sheep-specic probes, while incubating with genomic DNA extracted from bovine and porcine tissues. Thus, three types of negative controls were included: bovine probes with porcine extracts, sheep probes with bovine extracts, and sheep probes with porcine extracts. The three negative controls did not differ signicantly from each other and presented signals much lower than those obtained when the bovine probes and the bovine samples were used (a guanine peak area increase of more than 4-fold was observed for sample concentrations of 2080 g/mL). Arora et al. (2007) reported an electrochemical sensor based on the signal of methylene blue, for detection of E. coli based on a 5 biotin-labeled probe specic to E. coli, immobilized on a Pt disk electrode via avidin-modied polyaniline (PANI). E. coli genomic DNA (0.01 ng/ L) was used as the sample, but detection was also achieved in total cell extracts from E. coli cultures. Furthermore, the sensor could be reused 57 times at temperatures of 3045 C (Arora et al., 2007). Reusability is an important factor to consider when electrodes are made from expensive material such as Pt. The same group published another report in which genomic DNA from Mycobacterium tuberculosis could be detected within 1 min of hybridization time using NH2 -modied peptide nucleic acid (PNA) probes covalently immobilized to PANI/Au electrodes. Again, methylene blue was used for detection and reusability of the electrodes was also studied (Prabhakar et al., 2008). In both reports, genomic DNA was fragmentized by sonication. In 2009, another report from the group of Bansi Malhotra was published, in which a specic gene from the etiologic agent of Gonorrhoea (the Gram negative bacterium Neisseria gonorrhoeae) was detected in DNA extracted directly from patients endocervical and urethral swabs. 20-Mer biotinylated probes specic to the Opa gene from N. gonorrhoeae were immobilized on avidinmodied PANI/ITO electrodes (Singh et al., 2009). Detection was performed using differential pulse voltammetry (DPV), by monitoring the redox behavior of methylene blue. When incubating the electrodes with N. gonorrhoeae genomic DNA extracted from patients or from N. gonorrhoeae cultures, a signicant decrease of the methylene blue signal was observed. Genomic DNA extracted from the relative bacterium Neisseria meningitides and from the non-related Gram negative bacterium E. coli were used as controls of specicity. These negative controls generated an increase rather than a decrease of the methylene blue signal. Detection was also performed on the basis of the guanine oxidation signal. In this case, a signicant increase of the guanine peak was observed when incubating with N. gonorrhoeae DNA, while it decreased after incubation with the negative samples (Singh et al., 2009). We would like to highlight the criteria used for the election of the target sequence. The Opa genes codify for surface proteins important for bacterial attachment to different cell types encountered during infection and are present in high copy number in the N. gonorrhoeae genome. A single strain can harbour up to 12 Opa genes in its genome (Hauck and Meyer, 2003) and there is near-perfect identity in DNA sequence over approximately 80% of the length of the coding sequence (Dempsey et al., 1991). Thus, the election of a conserved sequence within a multi copy gene increases the target to interference ratio in the sample without the need of any articial amplication steps, increasing the analytical performance of the method. Another article from the Malhotras group aimed at detection of E. coli using methylene blue as the electroactive hybridiza-

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tion indicator was published in 2009 (Solanki et al., 2009). Once again, a multi-copy sequence was used, corresponding to the genes which codify for 16S rRNA. Nanostructured zirconium oxide was synthesized and deposited onto ITO coated glass plate, and probes were attached to the surface of the electrode via afnity of NanoZrO2 for phosphate. A decrease of the methylene blue signal was observed (as expected) with complementary oligonucleotides, but an increase of the signal appears after incubation with E. coli genomic DNA. The authors say that the increase of the methylene blue signal after incubation with genomic DNA is a consequence of the length of DNA, which introduces more guanine bases leading to the accumulation of methylene blue onto the electrode surface. This explanation is reasonable, but in such case the same should have been observed in the article published by Singh et al. (2009) described above. Anyway, it is not clear that the increase in the methylene blue signal is a consequence of sequence-specic hybridization since genomic DNA from a non-related bacterium was not included as a control. Recently, authors from the same group have also used the voltammetric signal of methylene blue for detection of N. meningitides genomic DNA (Patel et al., 2010). Recently, Siddiquee et al. (2009) reported an electrochemical DNA biosensor for the detection of a specic gene of the fungus Trichoderma harzianum in DNA extracts obtained from cultured isolates. 3-Mercaptoproponic acid self-assembled monolayers (MPA-SAMs) were deposited on gold disk electrodes, which were later modied with thiol derivatized oligonucleotide probes. Methylene blue was accumulated onto the modied electrodes after hybridization and cyclic voltammetry was performed by scanning the potential from 1.50 to +1.50 V vs. Ag/AgCl. A cathodic peak was observed around 0.5 V vs. Ag/AgCl and the absolute value of the amplitude of such a peak decreased when incubating with genomic DNA extracted from isolates of different Trichoderma species in the following order: T. harzianum > T. inhamatum > T. aureoviride > T. virens > T. longibrachiatum > T. koningii. Since T. harzianum and T. infantum are closely related, and since the signal from those species is almost twice the signal from T. aureoviride and 45 times the signal from the rest, the sensor seems to be species-specic (Siddiquee et al., 2009). It is important to know the nature of the analytical signal which is used for an accurate interpretation of the results. The authors say that the increase in the level of the voltammetric reduction signals of methylene blue reects the extent of hybrid formation (Siddiquee et al., 2009). However, a cathodic peak at the same potential (about 0.5 V) was also observed when incubating probemodied electrodes without methylene blue. Thus, the analytical signal may not necessary correspond to methylene blue reduction. Whatever the case, it seems to reect the extent of hybrid formation as the highest peak current was found when incubating with genomic DNA from the target organism T. harzianum. 3.2. Detection of sequences inserted into plasmids Plasmids are extra-chromosomal short molecules of doublestranded DNA, usually circular, which occur naturally in bacteria. They can be easily puried from cultures due to their small size in comparison to genomic DNA, and commercial kits for plasmid purication are also available. Plasmids used in molecular biology are present in high copy numbers inside the cells which incorporate them, and selection is applied to force the cells to keep replicating the desired plasmids, or otherwise die. Thus, plasmid preparations are often very pure and contain no signicant numbers of other nucleic acids besides the plasmid of interest. RNA is co-puried in conventional plasmid preparations, but RNase A is usually added in order to degrade it. As plasmids are covalently closed circular double-stranded DNA molecules, they may be opened by restriction digestion and heat denatured to facilitate hybridization to the

biosensor. In such a case, the strand of the insert which is complementary to the probe is the target sequence, while the other strand and all the rest of the plasmid backbone may be regarded as possible interfering material. Hejazi et al. (2008) reported the construction of a label-free electrochemical sensor for detection of human interleukine-2 (hIL-2) DNA insert into the pET21a(+) expression vector. Capture probes were immobilized on pencil graphite electrodes by applying a constant positive potential. As the DPV signal of guanine oxidation was used as the detection method, the 20-mer oligonucleotide probes were designed so that they only bared one guanine moiety. Plasmids were heat denatured at 95 C for 5 min and immediately placed on ice, and about 72 ng were pipetted onto the electrode surface. Electrochemical sensing was performed afterwards and an increase of the guanine peak was observed. Specicity was tested using the non-recombinant plasmid pET21a(+). Since the backbone of the plasmid is 5438 bp long and the hIL-2 insert is only 401 bp, 93% of the sequences present are identical between the positive sample and the negative control. Electrode activation and washing times were optimized in order to maximize the ratio between the guanine signal arising from specic hybridization and the noise observed when incubating with the non-recombinant plasmid (Hejazi et al., 2008).

4. Detection of PCR products obtained with a low number of PCR cycles and electrochemical real-time PCR End-point PCR is often performed in clinical or molecular biology laboratories in order to ensure that the desired sequence has been amplied to a degree which can be easily detected by agarose gel electrophoresis. For achieving this, a large number of PCR cycles are needed (2040). Amplication is exponential but reaches saturation after a certain number of cycles. An important consequence of this is that the original concentration of the target DNA cannot be adequately estimated by end-point PCR, as the target DNA is amplied until saturation, and reaches levels which are almost independent of its original concentration. Real-time PCR or quantitative PCR (Q-PCR) is a method which permits to detect and quantify a specic DNA sequence in a given sample, based on the polymerase chain reaction. Q-PCR needs a sensitive method which can detect DNA in very low concentrations at the end of each PCR cycle. The cycle number in which a reliable signal indicating the presence of DNA rst appears is correlated to the original concentration of the sequence which is being amplied. Different types of uorescent indicators or uorescencelabeled probes are usually used, and special thermocyclers capable of reading uorescence intensity are needed. This section covers different articles in which specic DNA sequences amplied by few PCR cycles (less than 15) could be detected. We think it is important to distinguish between detection of PCR amplicons using a low or high number of PCR cycles for different reasons. In rst place, few PCR cycles generate an enrichment of the target sequence but the target to interference ratio in the sample still remains low (Fig. 3). Secondly, a method which is able to detect sequences slightly amplied has a competitive advantage over conventional agarose gel electrophoresis with ethidium bromide staining. Last, such a method can be implemented for electrochemical real-time PCR, what may eventually have some advantages over uorescence-based real-time PCR, which requires expensive equipment and reagents. The rst electrochemistry-based real-time PCR technique for sequence-specic nucleic acid detection was described in 2006 by Yeung et al. Capture probes were immobilized on ITO electrodes and the electrodes were subjected to different cycles of a nonconventional solid-phase PCR reaction. Fc-labeled dUTP was added

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to the reaction mixture. Uracil is a nucleotide naturally found in RNA but not in DNA molecules, but DNA polymerases can incorporate uracil if it is included in the reaction mixture in the form of dUTP. After hybridization, the temperature was raised and DNA polymerases started the elongation process. Immobilized probes that were hybridized to larger DNA molecules served as primers and Fc-labeled uracil was incorporated to the probes during the elongation process. Thus, immobilized probes were elongated in each PCR cycle, incorporating more redox label as the number of PCR cycles increased (Yeung et al., 2006a). Later on, the same authors published another article in which the same method was further studied. They found that for a high concentration of template DNA in the unamplied material (3 106 copies/ L) the onset thermal cycle, dened as the cycle where the analytical signal begins to be distinguishable from the background, was much lower (just 35 PCR cycles) than that of the uorescencebased counterparts. However, the onset thermal cycle increased considerably as the DNA initial concentration was lowered down. As electrochemical sensing of the electrodes interfered with the PCR reaction, the authors decided to only scan the electrodes once during the whole reaction. Using different electrodes and setting reactions in parallel, real time analysis was performed, though each individual electrode was scanned in only one determined thermal cycle (Yeung et al., 2008). In the same year, Lermo et al. (2008) described another method towards electrochemical quantitative PCR. In this case, the eaeA gene of pathogenic E. coli was used as the target sequence. PCR was performed and stopped after 5, 10, 15, 20, 25 or 30 thermal cycles. The methodology described has the particularity that it is not strictly a genosensor-based approach as no capture probe is immobilized in the electrode surface. Instead, PCR primers were labeled with biotin (forward primers) or digoxigenin (DIG; reverse primers), and the double-tagged PCR amplicons were immobilized after completion of the reaction on avidin-modied electrodes. anti-DIG antibodies conjugated to horseradish peroxidase (HRP) were added, and after washing steps to remove unbound enzyme, hydrogen peroxide was added together with hydroquinone as a redox mediator. Hydroquinone is oxidized to quinone by HRP, and the electrical current corresponding to quinone reduction was registered amperometrically. In a second set of experiments, double-tagged PCR amplicons were immobilized in straptavidincoated magnetic beads, which were concentrated using a magnet onto the surface of a graphite-epoxy biocomposite (Lermo et al., 2008). Comparative studies were performed using standard Q-PCR based on TaqMan uorescent probes and agarose gel electrophoresis. For a DNA template concentration of 2 ng/ L, the onset thermal cycle was 10 and 13 for magnetic and non-magnetic electrochemical strategies, respectively, 15 for gel electrophoresis and 17 for TaqMan Q-PCR. The detection limit was dened as the lowest DNA template concentration which gave a cathodic current signicantly higher than that which arises from a blank experiment (no DNA added to the PCR reaction mixture), for a given number of PCR cycles. For the magnetic bead strategy, the authors report detection limits of 0.45 ng/ L, 4.5 pg/ L and 0.45 pg/ L template DNA at 10, 15 and 20 PCR cycles, respectively. When performing immobilization of double-tagged amplicons to avidin-coated electrodes, detection limits were 4.5 ng/ L, 0.45 ng/ L and 4.5 pg/ L, respectively (Lermo et al., 2008). Electrochemical real-time PCR microuidic devices were developed by Fang et al. (2009), where both the amplication of the target sequence and subsequent electrochemical detection of the PCR amplicon were realized simultaneously at selected PCR cycles in the same device. PCR mixture containing template DNA was loaded in a chamber present in the microdevice and pumped along the chip following a serpentine arrange of microchannels. The chip

contained three thermoelectric coolers which were responsible of generating three zones in the chip with different temperatures, corresponding to the temperatures needed for PCR reaction. Due to the loops present in the microchannels, the PCR mixture was pumped several times across zone 1 (denaturation), 2 (annealing) and 3 (extension), and a total of 26 PCR cycles were included in the chip. 11 sets of electrochemical detection stations were fabricated on the chip. Each station comprised a platinum working, counter and pseudo-reference microelectrode connected to an external potentiostat by an insulating wire. Electrochemical detection stations were placed at only some selected PCR cycle numbers: 5, 8, 10, 12, 14, 16, 18, 20, 22, 24 and 26. This mechanism of in situ electrochemical detection of PCR amplicons is based on the DNA intercalation of methylene blue. Square-wave voltammetry measurements were performed at each detection station and the signal corresponding to the reversible oxidation of methylene blue at the platinum electrodes was registered. As the number of PCR cycles increases, more double-stranded DNA is present in solution at the end of each extension step. Accordingly, more methylene blue binds DNA and so the number of indicator molecules which can suffer reversible oxidation at the electrode surface is reduced. As expected, it was observed that the peak intensities decreased with the PCR cycle number. Peak intensities vs. PCR cycle number were plotted to obtain the threshold cycle (Ct), which is the number of cycle in which the electrochemical signal reaches a particular threshold. Comparisons were made with conventional uorescence-based real-time PCR using SYBR Green. The Ct values for both the electrochemical and uorescence-based assays decreased linearly with the increase of the input target quantity (Fang et al., 2009) but Ct values were about 7 cycles lower for conventional uorescence detection. Neither probes nor PCR amplicons were immobilized on the electrodes in this method, so this report is not strictly based on genosensor technology. Nevertheless, it is important to highlight that the method is able to electrochemically detect PCR amplicons amplied by a low number of cycles (5, 8 10, 12. . .), enabling both quantitative and real-time PCR.

5. Detection of PCR amplicons obtained with more than 20 PCR cycles Starting from a very complex sample which may eventually contain very small amounts of DNA from different organisms together with a variety of other compounds, PCR can amplify a fragment seizing less than a hundred to more than a thousand base-pairs long. Amplication may be performed to such high levels that a single and intense DNA band of the expected size may be seen after agarose gel electrophoresis and ethidium bromide staining. If this type of PCR product is going to be used for detection using a genosensor, the target to interference ratio approximates the value of 1, as the strand which is complementary to the probe will be the target stand while the complementary strand may act as the interference. Thus, it may happen that a detection method which works efciently for detection of PCR amplicons may not have such a performance with other types of samples with lower target to interference ratios. If both forward and reverse primers are not included in similar concentrations in the PCR reaction mixture, then asymmetric PCR takes place. Due to the excess of one of the primers, one DNA strands is preferentially amplied over the other, and the PCR product after a large number of cycles gets mainly single-stranded. This avoids the need of heat denaturing before hybridization with the probe-modied electrode. Furthermore, if amplication is efcient, the target to interference ratio may acquire values greater than 1. Asymmetric solution-phase and solid-phase PCR are used

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for electrochemical detection in a variety of reports (Duwensee et al., 2009; Loaiza et al., 2009; Wu et al., 2010; Yeung et al., 2006b). Nucleic acid sequence-based amplication (NASBA) is a isothermal amplication technique for RNA sequences distinct from conventional retrotranscription and PCR (RT-PCR) which was used by some authors for rapid detection of viable microorganisms and spores (Baeumner et al., 2003, 2004; Hartley and Baeumner, 2003). 5.1. PCR detection with reagent-less methods Pournaghi-Azar et al. (2008) described the detection of PCRamplied (35 cycles) human interleukin-2 (hIL-2) coding DNA sequence without further purication of the PCR product. The selectivity of the sensor was assessed with negative control PCR samples and seven different non-complementary PCR products corresponding to 16S rDNA from different bacteria. Pencil graphite electrode (PGE) was used as the working electrode in a classical three electrode system. Before immobilization, an electrochemical pretreatment was carried out and DNA probes were immobilized on the section of the electrode at a constant positive applied potential. Electrodes were rinsed for 300 s with washing solution after incubation with the samples. The guanine oxidation signal was followed by anodic differential pulse voltammetry (ADPV). Selectivity was studied using seven non-complementary PCR samples corresponding to 16S rDNA of different bacteria. Noncomplementary products were about four times longer than the specic product, and a blank PCR sample (without DNA) was also analyzed in order to examine the effect of the PCR components such as primers and dNTPs on the guanine oxidation signal. Magnetic beads and guanine oxidation were combined in the work by Erdem et al. (2005) for detection of HBV amplicons. Inosine-substituted biotinylated capture probes were attached to streptavidin-coated magnetic beads and the complexes were magnetically separated after hybridization and treated with an alkaline solution in order to denaturalize the hybrids. Magnetic beads were taken off and a carbon pencil electrode was dipped in the solution containing the target DNA. Adsorption of DNA was performed by applying +0.5 V vs. Ag/AgCl for 5 min. The guanine oxidation signal was measured by DPV and unrelated PCR products were used as negative controls. In another work from the same group, Ariksoysal et al. (2005) reported a label-free electrochemical hybridization genosensorbased on guanine oxidation signal for the detection of hepatitis B virus genotype on the development of Lamivudine resistance. Later on, Kara et al. (2007) used inosine-substituted 18-mer probes for detection of Hepatitis B Virus (HBV). Probes were covalently bounded to modied pencil graphite electrodes via a 5 NH2 (CH2 )6 link. Hybridization was followed by monitoring the guanine oxidation signal with DPV. The highest response was obtained with the probe sequence which was complementary to the middle section of the amplicons, owing to its stabilization and prevention of duplex formation. Berganza et al. (2007) developed a microdevice for detection of PCR-amplied Escherichia coli (E. coli) 0157:H7 based on self-assembled monolayer (SAM) technology. A mixed SAM with a blocking molecule was used in order to prevent non-specic adsorption. Cyclic Voltammetry (CV) measurements of potassium hexacyanoferrate (K3 Fe(CN)6 ) were used for detection. Although the method is indirect as the signal arises from the electrochemical activity of hexacyanoferrate, the reagent is included in the electrolyte throughout the assay, without the need of introducing the reagent at some particular point and then wash out the excess. Thus, this kind of strategy can be considered reagent-less and is schematized in Fig. 1B. Hybridization induces changes in the electrochemical properties of the interface, and these changes are

indirectly measured by characterizing the electrodes with a redox soluble indicator such as hexacyanoferrate. Selectivity was evaluated using synthetic oligonucleotides, Negative PCR sample (without DNA) and unrelated PCR products were not included in the analysis. We insist on the importance of these kinds of controls, as they are the only possible way of evaluating matrix effects, and the effects of amplied and relatively long non-complementary DNAs. Yang et al. (2009) made use of the synergistic effect between polyaniline nanobers and multi-walled carbon nanotubes (MWNT) to detect PCR amplicons corresponding to the NOS gene of one kind of genetically modied soybean. DNA hybridization was monitored by Electrochemical Impedance Spectroscopy (EIS) using K3 Fe(CN)6 /K4 Fe(CN)6 redox couple An increase in the electrode transfer resistance after hybridization with complementary PCR products was detected. Again, unrelated and negative PCR samples were not analyzed. Detection of PCR-amplied samples by means of hybridizationinduced conformational changes of labeled probes (Fig. 1C) was used by Lai et al. (2006), from the groups of Kevin Plaxco and Alan J. Heeger. A methylene blue-labeled self-complementary probe was covalently attached to a gold electrode and exposed to the PCR product containing the target sequence. After hybridization, conformational changes in the probe displaced the redox label from the proximity of the electrode, and a decrease in the methylene blue signal was registered. An asymmetric PCR protocol was used, enabling enrichment in the single-stranded complementary sequence, what considerably improves the target to interference ratio of the sample. Regeneration of the electrode could be easily achieved by washing with distilled water. Based on the same strategy, an integrated microuidic disposable monolithic chip was later designed, capable of performing PCR amplication, enzymatic single-stranded DNA generation and electrochemical detection (Ferguson et al., 2009). 5.2. PCR detection with reagent-based approaches Enzyme-linked electrochemical detection of PCR products amplied with more than 20 cycles using sandwich hybridization assays (schematized in Fig. 2C) was performed by many authors (Aitichou et al., 2004; Farabullini et al., 2007; Bettazzi et al., 2008; Berti et al., 2009; Miranda-Castro et al., 2009; Tang et al., 2009). Double-tagged PCR (achieved by tagging both PCR primers) enables to immobilize PCR amplicons after solution-phase PCR, and to concentrate redox enzymes after immobilization by means of the second tag (Marques et al., 2009; LaGier et al., 2007; Lermo et al., 2007; Yean et al., 2008). Another double-tagged PCR strategy was used by Bonanni et al. (2009), with the exception that detection was performed by recording impedance spectra after concentration of gold nanoparticles. It should be noticed that these kinds of strategies are not based on immobilized probes and hybridization with complementary single-stranded DNA. Thus, care should be taken in order to discard the presence of unspecic PCR amplication products, which would not be discriminated. A hybridization-based sensor using immobilized probes and single tagged PCR amplicons was described by Carpini et al. (2004). Instead of using redox enzymes to generate an electroactive compound, Won et al. (2008) used a biotin-tagged dsDNA intercalator (anthraquinone) to concentrate streptavidin-HRP on the electrode surface, in order to yield an insoluble product and measure the increase in the interfacial resistance. An impedimetric DNA sensor for single-nucleotide polymorphism was described by Akagi et al. (2006), based on enzymatic ligation between the immobilized probes and a second biotin-tagged detection probe. Hybridization with perfectly matched PCR amplicons was a pre-requisite

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Fig. 5. Histogram representing the number of articles which were reviewed herein according to their publication year. Articles in which PCR amplicons with more than 20 PCR cycles are detected are shown in blue, articles in which PCR amplicons with less than 20 PCR cycles are detected (including electrochemical real-time PCR reports) are shown in red, reports in which target detection was performed in unamplied DNA and RNA are shown in green and orange, respectively. (For interpretation of the references to color in this gure legend, the reader is referred to the web version of the article.)

reproducible results when upgrading from experiments with synthetic oligonucleotides to the use of real samples. However, it may be possible that more interdisciplinary research may be needed, involving physics, engineers, chemists, molecular biologists and medics, in order to develop integrated and portable devices suitable for detection of real-life samples. The second conclusion should be the importance of using adequate negative controls when analyzing the performance of the sensors with real samples. Selectivity of the sensors should be studied by changing the sequence of the probe and testing with exactly the same sample. Alternatively, samples with a very similar composition may be used, provided there is independent evidence that the target sequence is absent. It is relatively common to study selectivity with pure solutions of complementary and non-complementary oligonucleotides, and then incubate the electrodes with a complex sample which contains the sequence of interest. However, the rst experiment is not a validation of the second, as the matrix effect is not taken into consideration. 7. Future perspectives Electrochemical DNA hybridization sensors are acquiring very interesting and promising applications in relation to parallel detection of multiple targets, implementation into miniaturized and automated devices suitable for point-of-care diagnostics and rapid screening of multiple unprocessed samples. Nowadays, uorescence-based microarray platforms are extensively used for massively parallel analysis of gene expression between different experimental conditions, and other applications. However, as high-throughput next-generation sequencing technologies may continue getting increasingly affordable, uorescence-based microarrays may become less useful. Electrochemical microarrays may then nd a niche if they are able to combine in situ multiplex analysis with speed and low cost. There are certain companies which offer microarray chips compatible with an electrochemical read-out. CombiMatrix developed the ElectraSense platform, a user-friendly microarray reader that electrochemically measures up to 12,000 probes in less than 25 s (www.combimatrix.com). The methodology was used by an independent research group to differentiate pandemic (H1N1) 2009 from seasonal inuenza (Straight et al., 2010). GeneFluidics is another company which offers electrochemical sensing technology (www.geneuidics.com). Industrial development of electrochemical microarrays and integrated DNA chips requires research and development in mainly three aspects. One aspect is related to the miniaturization, automation and integration of the nished device, which usually incorporates microuidics technology for injection of the samples, reagents, and buffers. A second aspect regards the transduction of the hybridization reaction into a measurable electrochemical signal. Some general methods were described in Figs. 1 and 2, but much more are present in the literature. Last, the performance of the sensor or microarray with real biological samples is also a cornerstone if the device is intended for real-life applications. Although progress is evident, more research is needed in order to satisfy the demands and provide a reliable, robust and sensible technology for rapid and automated nucleic acid analysis. Acknowledgments We would like to thank the staff of the Nuclear Research Center and the Faculty of Science for their support and helpful advices. Specially, we acknowledge everyday support from our team of the Analytical Biochemistry Unit and from our colleagues from the Laboratorio de Biomateriales (Faculty of Science). J.P.T is a research

for efcient ligation and subsequent detection by impedance spectroscopy. Apart from redox enzymes, electroactive compounds with intrinsic afnity for either ssDNA or dsDNA are widely used (Fig. 2A and B). As it was seen in many reports described in the previous sections, methylene blue is also widely used for PCR amplicon detection (Lin et al., 2010; Meric et al., 2002, 2004; Yang et al., 2008; Zhang et al., 2008; Zhou et al., 2008). Other redox indicators used include daunomycin (Marazza et al., 2001), ferrocenium hexauorophosphate (Ye et al., 2003), Meldola Blue (Kara et al., 2004), 2-nitroacridone (Chen et al., 2008), anthraquinonemonosulfonic acid (Luo et al., 2010) and Heoechst33258 (Ahmed et al., 2010). 6. Concluding remarks Although the eld of electrochemical genosensors has grown considerably in the past 10 years and a great number of different detection methods and strategies are available, still most research articles are based on the use of pure solutions of synthetic oligonucleotides to address relevant analytical parameters such as sensitivity and selectivity of the detection assay. We have argued that this gives only preliminary information as the performance of the sensor may be entirely different in presence of biologically relevant samples, where the target sequence is present within a relatively large excess of non-complementary nucleic acids, proteins, organic molecules, salts and other undened components. We have analyzed the small fraction of the literature in which complex biological samples were used for evaluating performance of different electrochemical genosensors. More emphasis was placed on the most unprocessed and complex samples, which include crude cell lysates, genomic DNA and RNA extracts, since those samples offer the lowest target to interference ratios and are more analytically challenging. Focus was also placed on those articles describing detection of PCR products amplied with a low number of cycles, since such methods may be suitable for implementation in electrochemical devices for quantitative and/or real-time PCR. The present review offers two main conclusions. In rst place, it seems clear that the number of research articles involving complex biological samples is very low in comparison with the total number of articles in the eld which are published every year, although growth is evident (Fig. 5). This may reect difculties in obtaining

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fellow from the Agencia Nacional de Investigacin e Inovacin (ANII). References

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