Journal of
Electroanalytical Chemistry Journal of Electroanalytical Chemistry 567 (2004) 29–37 www.elsevier.com/locate/jelechem
Rigid carbon composites: a new transducing material for label-free electrochemical genosensing Arzum Erdem
a,c,* ,
M. Isabel Pividori
b,c
, Manel del Valle c, Salvador Alegret
c,*
a
Faculty of Pharmacy, Analytical Chemistry Department, Ege University, 35100 Bornova, Izmir, Turkey Facultad de Bioquımica y Ciencias Biologicas, Universidad Nacional del Litoral, 3000 Santa Fe, Argentina Grup de Sensors i Biosensors, Departmant de Quımica, Universitat Autonoma de Barcelona, 08193 Bellaterra, Catalonia, Spain b
c
Received 9 June 2003; received in revised form 22 October 2003; accepted 29 October 2003 Available online 20 February 2004
Abstract A rigid carbon–polymer composite material as a transducer for the electrochemical determination of label-free DNA based on differential pulse voltammetry (DPV) is reported. Graphite–epoxy composites (GEC) have an uneven surface allowing DNA, oligonucleotides and free DNA bases to be adsorbed using a simple and fast wet-adsorption procedure. In contrast with other transducers commonly used for electrochemical genosensing, the oxidation potentials are much lower when GEC is used. Free guanine base is oxidized at +0.35 V while adenine oxidation occurs at +0.63 V (vs AgjAgCl). Cytosine and inosine free bases show no peaks within the experimental potential range. The oxidation of DNA guanine moieties occurs at a potential of +0.55 V while DNA adenine bases are oxidized at +0.85 V. A novel label-free hybridization genosensor using GEC as an electrochemical transducer for the specific detection of a sequence related with Salmonella spp. is also reported. This approach relies on the wet adsorption of the 23-mer inosine-substituted probe. The extent of hybridization onto the GEC surface between the probe and the target has been determined by using the oxidation signal of guanine coming from the target in connection with DPV. DNA hybridization has been determined in a target concentration of 10 lg/ml in 15 min of hybridization time. The hybridization event has also been detected in co-existing salmon testes DNA (stDNA) as interference. The features of this device are discussed and compared with state-of-the-art of label free DNA detection methods. Ó 2003 Elsevier B.V. All rights reserved. Keywords: Graphite–epoxy composite; Label-free electrochemical genosensor; DNA; Adenine; Guanine; Salmonella spp.
1. Introduction DNA biosensor technologies are currently under intense investigation owing to their great promise for rapid and low-cost detection of specific DNA sequences in human, viral and bacterial nucleic acids [1,2]. As the sequencing of the human genome continues, the mutations responsible for numerous inherited human disorders have now been mapped [3,4]. Pathogens responsible for disease states, bacteria and viruses, are also detect*
Corresponding authors. Tel.: +90-232-343-4000, ext. 5131; fax: +90-232-388-5258 (A. Erdem), Tel.: +34-93-581-2118; fax: +34-93-5812379 (S. Alegret). E-mail addresses:
[email protected] (A. Erdem),
[email protected] (S. Alegret). 0022-0728/$ - see front matter Ó 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jelechem.2003.10.049
able via their unique nucleic acid sequences and interest in their detection continues to grow [5]. The easy fabrication of DNA modified surfaces, which are reproducible, stable and selective to complementary DNA sequences, is crucial in the development of emerging molecular technologies such as DNA chips and simple diagnostic devices for detecting a few sequences of target DNA. Electrochemical DNA biosensors for the detection of DNA sequences may greatly reduce the assay time and simplify the assay protocol. They also provide the basis for a fast, cost-effective and simple diagnosis of inherited or infectious diseases [5– 10]. They can be employed for determining early and precise diagnoses of infectious agents in various environments [2]. These devices can be exploited for monitoring sequence-specific hybridization events directly
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[11] or by DNA electroactive indicators (metal coordination complexes, etc.), which form complexes with the nitrogenous bases of DNA [12–17]. Oligonucleotides labeled with enzymes [18–21] have been employed in hybridization detection protocols. Enzyme labeling based on biotin and streptavidin interaction confers sensitivity and flexibility allowing ease of production for commercial kits using less toxic reagents. Additionally, a hybridization detection scheme based on ferrocene modified adenine moieties was reported [22]. The use of inosine-labeled (guanine-free) probes and the appearance of the guanine signal upon hybridization with the target opened up a new field in the electrochemical research. This procedure eliminated the external labels and shortened the assay time. Guanine was reported to be the most redox active nitrogenous base in DNA. The chemical mechanism of the oxidation of guanine was reported in detail [23]. Since then, many reports used the oxidation signal of guanine. For example, Wang et al. [24] described a label-free electrochemical DNA biosensor, which involves the immobilization of inosine-substituted (guanine-free) probe on carbon paste electrode (CPE) and the detection of hybrid by using the appearance of the guanine oxidation signal of the target in connection with chronopotentiometric stripping analysis (PSA). Additionally, Wang et al. [25] attached biotinylated inosine-substituted oligonucleotides on streptavidin coated magnetic beads. Magnetic separation greatly eliminated the nonspecific adsorption effects. Thorp and co-workers [11,26] reported a catalytic guanine oxidation protocol with a indium tin oxide electrode using ruthenium complexes as oxidation catalysts. Palecek and co-workers [27] reported the oxidation signals of guanine and adenine at low concentrations of DNA and PNA by applying chronopotentiometry and voltammetry at pyrolytic graphite electrode (PGE). A pencil graphite biosensor for label-free electrochemical detection of hybridization was also reported by Wang et al. [28]. Rigid conducting graphite–polymer composites and biocomposites have been used extensively by Alegret and co-workers for electrochemical biosensing [29,30]. Additionally, the ideal transducing properties of graphite–epoxy composites (GECs) [19,31] and biocomposites (GEB) [32] to develop electrochemical genosensors based on enzyme labeling have been previously reported. In addition to ease of preparation, GECs present numerous advantages over more traditional carbon-based materials: higher sensitivity [19,30], robustness and rigidity. The mechanical properties of GEC permit the design of genosensors in different configurations and the regeneration of the surface by a simple polishing procedure. Additionally, GECs have an uneven surface where DNA can be adsorbed using simple adsorption proce-
dures [33] avoiding the use of procedures based on previous activation/modification of the surface transducer and subsequent immobilization [18] which are tedious, expensive and time-consuming. In this study, the use of GEC as an electrochemical transducer for the determination of ss and dsDNA, oligonucleotides and free DNA bases, based on differential pulse voltammetry (DPV) is reported for the first time. The oxidation signals of both guanine and adenine DNA moieties are studied using GEC as a transducer. Additionally, the utility of GEC for label-free hybridization genosensing is illustrated for the specific detection of a sequence related with Salmonella spp. [34–36]. Traditional cultural procedures for isolating and identifying Salmonella spp. in foods require 3–4 days to provide presumptive results and an additional 1–2 days for further biochemical confirmation. Accelerated Salmonella spp. detection procedures would allow the food industry to reduce ware-housing costs and permit increased testing of both food ingredients and final products. The insertion sequence IS200 is a transposable element of some 700 bp [34], being present in more than 90% of the pathogenic or foodpoisoning isolates of Salmonella spp. [35,36]. IS200 has been used as a suitable sequence to identify isolates of Salmonella with relatively high accuracy [37–40]. To the best of our knowledge, the detection of a sequence related on Salmonella spp. by using the oxidation signal of guanine without any modifications in the native bases or any external labeling has not been reported yet.
2. Experimental 2.1. Equipment The oxidation signals of ssDNA and dsDNA, oligonucleotides, and free DNA bases were investigated by using DPV with an AUTOLAB PGSTAT 30 electrochemical analysis system (Eco Chemie, The Netherlands). A three-electrode set-up was used comprising a platinum auxiliary electrode (Crison 52-67 1, Spain), a double junction AgjAgCl reference electrode (Orion 900200) with 0.1 M KCl as the external reference solution and a working electrode based on a GEC. All potentials were referred against the AgjAgCl reference electrode. 2.2. Reagents Free DNA bases (adenine, guanine, cytosine, and inosine) were purchased from Sigma. Salmon testes DNA (stDNA), polynucleotides as poly(dA)–poly(dT) and poly(dA), were purchased from Sigma. dT(50) was
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supplied by TIB-MOLBIOL (Berlin, Germany) as well as the 23-mer synthetic oligonucleotides (the target and its complementary probe). Their base sequences are: IS200 Target: 0 0 5 -GCC GAA GAT GAG TGT GTC GAG TT-3 Inosine-substituted probe: 0 0 5 -AAC TCI ACA CAC TCA TCT TCI IC-3 All DNA stock solutions (100 mg/l) were prepared with sterilized and deionized water and kept frozen. More dilute solutions of probe were prepared using 0.50 M acetate buffer containing 20 mM NaCl, pH 4.80 (ABS). More dilute solutions of target were prepared using 50 mM phosphate buffer containing 20 mM NaCl, pH 7.4 (PBS). Free DNA base stock solutions (1000 mg/l) of cytosine, inosine and adenine were prepared in ABS. As guanine is not completely soluble in ABS, saturated guanine solutions in ABS were employed. Other chemicals were of analytical reagent grade was supplied from Sigma. The in-house sterilized and deionized water was used in all solutions.
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in ABS was employed. After immobilization, the free DNA base-modified GEC electrode was washed with ABS (pH 4.8) for 5 s. The oxidation signals of all free DNA bases (cytosine, inosine, guanine and adenine) were measured by using DPV in ABS by scanning from +0.10 to +1.20 V at an optimized pulse amplitude of 100 mV. Repetitive measurements were carried out by polishing the surface and repeating the above procedure. 2.6. Detection of adenine moieties in ss and dsDNA
Graphite powder and epoxy resin were hand-mixed in a 1:4 (w/w) ratio. The resulting paste was placed to a depth of 3 mm in a cylindrical PVC sleeved body (6 mm i.d.) with an electrical contact. The composite material was cured at 40 °C for 1 week [29,41]. Before each use, the surface of the GEC electrode was wetted with twice distilled water and then thoroughly smoothed with abrasive paper and then with alumina paper (polishing strips 301044-001, Orion). The reproducibility of the construction of the sensors based on the GEC electrode and the polishing procedure have been reported previously [19].
The hybridization procedure, using synthetic polynucleotides, consisted of the following steps: Poly(dA) immobilization. 20 ll of 15 lg/ml poly(dA) in ABS was added to the pretreated GEC electrode surface by simple wet-adsorption and then the immobilization of probe was allowed to proceed for 15 min. After immobilization, the poly(dA) modified GEC electrode was washed with ABS (pH 4.8) for 5 s. Hybridization with dT(50). 20 ll of 15 lg/ml dT(50) solution in PBS (pH 7.4) was added to the poly(dA)modified GEC electrode surface. The hybridization was allowed to proceed for 15 min. The hybridmodified GEC electrode was dipped into PBS for 5 s for washing. As a control assay, the immobilization of poly(dA)– poly(dT) and dT(50) was performed as described previously for poly(dA). In this case, no hybridization was performed. Electrochemical measurements. In all cases, the oxidation signal of adenine was measured by using DPV in ABS by scanning from +0.30 V to +1.20 V at a pulse amplitude of 100 mV. Repetitive measurements were carried out by polishing the surface and repeating the above assay format.
2.4. Electrochemical pretreatment of GEC electrodes and DPV measurements
2.7. Full coverage study based on adenine and guanine signals
In all cases, the GEC transducer was pretreated by applying +1.20 V for 1 min in PBS. Both guanine and adenine oxidation signals were detected using DPV. The oxidation peak height after baseline fitting was used as the analytical signal. The raw data were treated using the Savitzky and Golay filter (level 2) of the General Purpose electrochemical software (GPES) of Eco Chemie (The Netherlands) with a moving average baseline correction, using a ‘‘peak width’’ of 0.04.
The procedure consisted of the IS200 target immobilization on the GEC electrode surface. 20 ll of IS200 target from 1 to 120 lg/ml in ABS was added to the pretreated GEC electrode. The wet-adsorption of the target was allowed to proceed for 15 min. After immobilization, the target modified GEC electrode was washed with ABS (pH 4.8) for 5 s. In all cases, the oxidation signals of adenine and guanine were measured by using DPV in ABS by scanning from +0.30 to +1.20 V at a pulse amplitude of 100 mV.
2.3. Construction of the GEC electrode
2.5. Voltammetric determination of free DNA bases 20 ll of 20 lg/ml free DNA base solution in ABS was added to the pretreated GEC surface by simple wetadsorption and then it was allowed to proceed for 15 min. In the case of guanine, 20 ll of saturated solution
2.8. Label free hybridization genosensor for the detection of Salmonella spp. based on guanine signal The procedure of hybridization detection is shown in Fig. 1 and consisted of the following steps [42]:
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Fig. 1. Schematic representation of the label-free electrochemical genosensor based on GEC. (1) Electrochemical pre-treatment step. (2) Wet-adsorption immobilization step of an inosine substituted probe on the GEC electrode. (3) Hybridization step. (4) Voltammetric transduction step based on guanine oxidation signal.
Inosine-substituted probe immobilization. The same procedure was followed as described previously for poly(dA), but in this case at a concentration of 60 lg/ml. Hybridization with the target. 20 ll of target solution in PBS (pH 7.4) from 1 to 180 lg/ml was added to the inosine substituted probe-modified GEC electrode. The hybridization was allowed to proceed for 15 min. The hybrid modified GEC electrode was dipped into blank PBS for 5 s. Electrochemical measurements. In all cases, the oxidation signal of guanine was measured by using DPV in ABS by scanning from +0.30 to +1.20 V at a pulse amplitude of 100 mV. Repetitive measurements were carried out by polishing the surface and repeating the above assay format.
3. Results and discussion 3.1. Oxidation of free DNA bases In Fig. 2, the oxidation signals coming from free bases, adenine (A), guanine (G), cytosine (C) and inosine (I) are shown. These signals were obtained for a concentration level of 20 lg/ml except in the case of guanine, in which a saturated solution in ABS was employed. As shown in Fig. 2, the guanine and adenine give well-defined oxidation peaks when using GEC as a transducer. While guanine oxidation occurs at +0.35 V, adenine is oxidized at +0.64 V. Cytosine and inosine show no peaks in this potential range, as expected. In
Fig. 2. Differential pulse voltammograms for the oxidation signals of free DNA bases in ABS at a concentration of 20 lg/ml. (A), adenine; (G), guanine; (C), cytosine; (I), inosine. A blank in ABS is also shown (B).
contrast to other transducers, previously reported and commonly used for electrochemical genosensing, the oxidation potentials of free DNA bases are markedly lower for GEC. In the case of a pretreated glassy carbon electrode (GCE) [43] free guanine base is oxidized at +0.8 V while adenine at +1.1 V. Moreover, the oxidation potentials at the pretreated GCE are slightly more negative and better defined than those obtained at a bare non-pretreated GCE [43]. Nevertheless, Zen et al. [44] have reported the oxidation of free guanine and adenine bases with a bare GCE at +1.05 and +1.25 V, respectively. In a NafionÒ coated GCE, adenine and guanine are oxidized at the same potential as GCE, but showing higher signals [44]. However, the same authors [44] have reported a shift in potential compared to GCE, guanine
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and adenine being oxidized at +0.92 and +1.1 V respectively, at a chemically modified GCE (with Nafionruthenium oxide pyrochlore). Additionally, Wang and Kawde [45] have reported the oxidation of guanine and adenine using a pencil graphite electrode at +0.87 and 1.18 V, respectively. The shifts in potential previously reported for the GEC compared with other transducers [43–45] are approximately 0.6 and 0.5 V for guanine and adenine, respectively. These shifts in potential towards the negative direction are clear evidence that both bases – guanine and adenine – are better oxidized using GEC as a transducer. This fact will be discussed in Section 3.3. The lower over-potential and the sharper and betterdefined current responses obtained with GEC indicate an acceleration of the electron transfer reaction compared with the other reported transducers [43–45]. Moreover, the shifts between the adenine and guanine oxidation peaks are higher (+0.31 V) than those obtained with other transducer [44] when using a GEC. The GEC is thus capable of a better discernment between free adenine and guanine bases. 3.2. Detection of adenine moieties in ss and dsDNA Fig. 3 shows the oxidation signal of (a) poly(dA) and (b) dT(50). As can be seen, the oxidation signal of DNA based on adenine occurs at +0.85 V while dT(50) shows no signal. When adenine DNA moieties are analyzed, a shift in the potential of 0.2 V is observed compared to free adenine base. Fig. 3 also shows the oxidation signal of the preformed dsDNA poly(dA)– poly(dT) (c). In this last case, smaller signals are obtained compared to poly(dA) alone. As adenine moieties are hybridized, these bases are less available for oxidation [44]. The label-free hybridization procedure relies on the hybridization of the immobilized poly(dA) with dT(50), and the results are shown in Fig. 3(d). Comparing the
Fig. 3. Differential pulse voltammograms for the oxidation signals of adenine in ABS at (a) 15 lg/ml of poly(dA), (b) 15 lg/ml of dT(50), (c) 15 ppm of preformed poly(dA–dT) and (d) 15 lg/ml of poly(dA) hybridized with 15 lg/ml of dT(50).
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signals coming from the hybrid poly(dA)–dT(50) (d) generated on the surface of GEC electrode with those obtained with the preformed dsDNA poly(dA)– poly(dT) (c), the signal is higher for the hybrid poly (dA)–dT(50) than for poly(dA)–poly(dT). This difference can be explained by the fact that poly(dA) is a 260 mer, and a complete match with dT(50) is not possible. As a result, free adenine bases in poly(dA)–dT(50) are still available for oxidation yielding a higher adenine signal in comparison with the preformed-completematch hybrid poly(dA)–poly(dT). However, in both cases, smaller signals are obtained compared to that for poly(dA) alone. 3.3. Oxidation of adenine and guanine moieties in DNA: full coverage study In order to avoid non-specific adsorption in a labelfree hybridization genosensor, full coverage of the GEC electrode surface needs to be addressed. This study was performed by immobilizing the IS200 target on the GEC electrode surface in different concentrations such as 0, 1, 5, 15, 30, 60, 90 and 120 lg/ml. The results in Fig. 4(A), (a) and (b), show the oxidation signal of both guanine and adenine DNA moieties, occurring at +0.55 and +0.85 V, respectively. As can be seen from Fig. 4(A), with an increment of the IS200 target concentration, the guanine (a) and the adenine (b) signals increased up to 60 lg/ml. After this concentration, a slight reduction of the electrochemical signal was observed. This decrease in the signal, related to higher quantities of DNA, can be explained either by steric hindrance or by non-specific self-association. Both effects compete with ssDNA adsorption on the transducer, thus decreasing the signal. Nevertheless, Fig. 4(A) demonstrates that full coverage of the GEC transducer was achieved with 60 lg/ml of IS200 target. This concentration of 60 lg/ml was then used to perform full coverage of the GEC transducer when an inosine-substituted probe was immobilized onto the GEC. Fig. 4(B) shows that a linear relationship is achieved when the logarithm of IS200 target concentration is plotted against the oxidation signal of guanine (a) and adenine (b). In both cases, a linear range was obtained from 0 to 60 lg/ml of DNA target giving regression coefficients of 0.996 and 0.995, respectively. As the number of guanine bases in the DNA target (9) is almost double the number of adenine moieties (5), the guanine oxidation signal is 1.8 times higher than that for adenine. ssDNA detection can thus be achieved with similar sensitivity using both guanine and adenine base moieties. Three subsequent experiments for the detection of the guanine signal gave reproducible results. The mean guanine signal of 24.75 lA obtained from a 30 lg/ml
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Fig. 4. (A) Calibration plot for guanine (a) and adenine (b) oxidation signals with the increment of the IS200 target concentration on a GEC transducer. (B) Logarithm of IS200 target concentration against the guanine (a) and adenine (b) oxidation signal of the IS200 target. The guanine signal was obtained at +0.55 V, while the adenine signal occurs at +0.85 V vs AgjAgCl.
IS200 target sequence gave a RSD value of 7.23% (n ¼ 3). The more surprising fact is that the oxidation potentials for guanine and adenine DNA moieties are considerably lower using GEC as a transducer than those previously reported for other transducers such as carbon paste (+1.01 and +1.20 V) [24,42], pencil graphite (+1.05 and +1.25 V) [45], pretreated or non pretreated glassy carbon (+0.80 and +1.11 V) [46], and pyrolytic graphite (+1.12 and +1.37 V) [27] electrodes. The shift in potentials is almost 0.5 V for guanine and 0.4 for adenine DNA moieties compared to classical transducers such as carbon paste [24,42] or pencil graphite electrodes [45]. Moreover, compared with pretreated or non-pretreated GCE [46], the GEC transducer is able to distinguish between free bases and DNA-bound bases reflecting the behavior of the free (monomeric) nucleobases [47]. The shift to lower potential is 0.2 V for free guanine and adenine compared to DNA moiety counterparts. This effect was also reported at a pencil graphite electrode for guanine but the shift is almost negligible for adenine [45]. The electrochemical behavior of free or DNA-bound bases at a GEC transducer demonstrates that adenine and guanine are more easily and efficiently oxidized compared to other previously reported transducers such as carbon paste [24,42], glassy carbon [46], pencil graphite [45] or pyrolytic graphite [27] electrodes. The shifts in potentials towards the negative direction, as well as the lower over-potential and the sharper and better-defined current responses obtained with the GEC, are clear evidence of an increased electron transfer reaction compared with other reported transducers [24,27,42–46]. This electrochemical behavior can be explained because of the mixed nature of the GEC, formed by 80%
of epoxy resin and 20% of graphite powder, whereas in classical carbonaceous transducers the composition is mostly graphite. In carbon-based materials, DNA is mainly stabilized on the transducer by electrostatic interactions via the negatively charged hydrophilic sugarphosphate backbone with bases oriented towards the solution [48]. Due to the mixed nature of the GEC, DNA–GEC interactions are different from those of other carbonaceous materials. As in the case of carbonbased transducers, it is possible that DNA was stabilized on the GEC by means of electrostatic interactions with the graphite within the polymeric matrix, but also by means of hydrophobic interactions with the non-polar epoxy resin. These hydrophobic interactions would play a crucial roll in the peculiar electrochemical behavior of DNA and monomeric guanine and adenine bases in GEC material. Kauffmann et al. [49] have reported the effect of altering the content of carbon black in a polymeric matrix. They also have observed sizeable shifts of working potentials. 3.4. Label-free hybridization genosensor for the detection of Salmonella spp. based on guanine signal The synthetic IS200 specific sequence for Salmonella spp. was used for the label-free hybridization genosensors. For the first time, a GEC electrode is employed as a novel transducer for label free electrochemical genosensing. The appearance of the guanine oxidation signal coming from the IS200 target after its hybridization with the complementary inosine-substituted probe previously immobilized on the GEC electrode surface was used as the analytical signal. The oxidation signal of guanine was observed at +0.55 V (Fig. 5(b) and (c)). No guanine signal was observed with the inosine-substituted probemodified GEC electrode (Fig. 5(a)). Wang et al. [24]
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Fig. 5. Differential pulse voltammograms for the oxidation signals of guanine with a full coverage inosine-substituted probe modified GEC electrode after hybridization with (a) PBS (no IS200 target), (b) 10 lg/ ml IS200 target, (c) 30 lg/ml IS200 target.
have reported a peak at +0.77 V, associated with the oxidation of the inosine probe residue. This peak has not been observed in our studies. At a surface coverage concentration of the inosinesubstituted probe of 60 lg/ml immobilized on the GEC electrode, the IS200 target concentration was increased from 0 to 180 lg/ml (0, 1, 5, 10, 30, 60, 90, 120, 150, 180) for a hybridization time of 15 min. From 0 to 5 lg/ml, no guanine signal coming from the target was observed. However, it is possible to detect hybridization based on the guanine signal at a concentration level such as 10 lg/ ml of IS200 target sequence (Fig. 5(b)). Fig. 6 shows that a linear relationship is achieved when the logarithm of IS200 target concentration is plotted against the oxidation signal of guanine. The linear range for the quantification of IS200 target is observed from 30 to 150 lg/ml giving regression coefficients of 0.996. The detection limit estimated from S/N ¼ 3, corresponds to 13.27 fmol/ml target concentration using the
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Fig. 7. Differential pulse voltammograms for the oxidation signals of guanine with a full coverage inosine-substituted probe modified GEC electrode after hybridization with (a) PBS (no IS200 target), (b) 30 lg/ ml stDNA, (c) 30 lg/ml IS200 target, (d) a mixture of 30 lg/ml IS200 target and 30 lg/ml stDNA.
GEC transducer modified with 60 lg/ml of inosinesubstituted probe. Compared to the detection limits obtained with other electrochemical transducers such as CPE based on label-free electrochemical detection for DNA hybridization, the detection limit obtained with GEC was much lower than those previously reported [24,42]. 3.5. Selectivity and non-specific adsorption studies Fig. 7 shows the results for the selectivity study. As shown comparatively in Fig. 7(c) and (d), the hybridization without stDNA (c) occurs to the same extent as in the presence of stDNA (d) as possible interference. Fig. 7 also shows the effectiveness of the GEC surface coverage with the inosine probe. The non-specific adsorption of stDNA onto the GEC surface is eliminated after its modification with the inosine probe at a full coverage concentration (a) since no signal is obtained when only stDNA is added in the hybridization solution (b).
4. Conclusion
Fig. 6. Calibration plot for the guanine oxidation signal against the logarithm of the IS200 target concentration at the hybridization step. The inosine-substituted probe was kept at a full coverage concentration level of 60 lg/ml on the GEC electrode surface, with increasing concentration of the IS200 target during hybridization.
The development of new transducing materials, whose preparation is simple and suitable for mass industrial fabrication, while also possessing a high sensitivity and lower detection range for the analysis of DNA, is a key issue in the current research efforts for electrochemical genosensing. The utility and versatility of GEC-based materials for label-free electrochemical genosensing is demonstrated for the first time. GEC electrodes have an uneven surface where DNA, free DNA bases and synthetic oligonucleotides can be adsorbed using a simple wet-adsorption procedure. This
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technique is the simplest immobilization method and the easiest to automate, avoiding the use of procedures based on previous activation of the surface transducer and subsequent immobilization, which are tedious, expensive and time-consuming [18]. Hybridization of target DNA onto the surface of the GEC electrode is performed successfully, demonstrating that DNA bases of the probe are available for hybridization after its adsorption into this material in contrast with mercury electrodes [48,50]. Both oxidation signals of guanine and adenine moieties in DNA can be followed with a GEC electrode as the transducer using DPV with similar sensitivity. As the inosine base is able to hybridize with cytosine, but shows no oxidation signal, inosine substituted (guanine-free) probes can be used for label-free hybridization genosensing following the target guanine oxidation signal, as reported previously by other groups. However, the guanine (and also adenine) moieties are more easily oxidized on GEC compared to other carbon-based transducers previously reported [24,27,42– 47]. Because of this surprising shift in potentials of 0.5 V, some electrochemical interferences coming from real samples could be avoided. Non-specific adsorption of DNA different from the target can be completely eliminated by full probe coverage of the GEC surface. In comparison with the conventional carbonaceous transducers, the reported sensitivity is significantly higher when using GEC. Additionally, the GEC electrode is able to discriminate better between free bases and their DNA moiety counterparts since free bases are easily oxidized on the GEC by a shift of almost 0.2 V. In this contribution, we demonstrate the utility of the label-free electrochemical genosensor based on a GEC electrode for the detection of short fragments of Salmonella spp. by using the GEC electrode. Such use of electrochemical DNA inosine labeled probes can decrease the time and cost of Salmonella spp. screening without any extra labeling. The appearance of the guanine signal enables the monitoring of hybridization in shorter times. The needs for external indicators such as carcinogenic antitumor drugs, metal complexes and organic dyes have all been eliminated. Further improvements may be achieved by using several probes from different regions, in connection with multi-electrode array and multiple hybridization events. The reported procedure is simple, economical and provides rapid detection of Salmonella spp. GEC material is thus a promising material for the design of more effective DNA hybridization genosensors, which will further be useful in DNA microchip systems. Future work by this group will focus on its application on PCR samples to detect Salmonella spp., mainly in the food industry, by using the GEC electrode as a novel transducer.
Acknowledgements This work has been supported by the Turkish Academy of Sciences, in the framework of the Young Scientist Award Program (KAE/TUBA-GEBIP/2001-28). Financial support for this work is provided by the Spanish Ministry of Science and Technology (McyT), Madrid, Spain, through projects PPQ2001-1950 and BIO2000-0681, and by the Department dÕUniversitats, Recerca i Societat de la Informaci o (DURSI), Government of Catalonia, Barcelona.
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