Anal. Chem. 2005, 77, 3351-3356
DNA Electrochemical Sensor Based on Conducting Polymer: Dependence of the “Signal-On” Detection on the Probe Sequence Localization Steeve Reisberg, Benoıˆt Piro, Vincent Noe 1 l, and Minh Chau Pham*
Laboratoire Interfaces-Traitements-Organisation et Dynamique des Syste` mes (ITODYS), Universite´ Paris 7-Denis Diderot, associe´ au CNRS, UMR 7086, 1, rue Guy de la Brosse, 75005 Paris, France
We show in this work that it is possible to make selective direct electrochemical hybridization detection of a target strand onto a probe strand immobilized on a conducting polymer modified with a quinone group, which presents cation-exchange properties. This leads to a “signal-on” detection, a unique behavior in comparison to similar systems described in the literature. It is shown that this system is efficient for various probe and target lengths (10-30 bp) and can discriminate a single mismatch. To go further in comprehension of the detection mechanism, a systematic study of the electrochemical response versus the probe sequence localization onto the immobilized strand is performed. For example, a 30-bp target strand is divided into three shorter 10-bp sequences (A-C, respectively), and we investigate the successive hybridization of these 1/3 strands onto the 30-bp probe strand. It is shown that one probe strand can be used to address several shorter targets. In recent years, DNA electrochemical sensors have gained growing interest in the literature.1 Among these sensors, on one hand, one finds transduction schemes using electroactive labels added in solution (i.e., intercalation compounds), electroactive labels on DNA targets, enzyme-modified targets, etc.2 These methods are obviously inadequate for in vivo diagnosis or rapid tests. A nice development of labeled DNA concerns nanoparticlebased electrochemical detection, which dramatically increases the detection sensitivity.3-5 On the other hand, label-free transduction schemes are based on the electroactivity of DNA6 or on modification of the electrochemical impedance of the system.7 Electronically conducting polymer (ECP)-modified electrodes are good candidates as smart substrates, i.e., as substrates onto which DNA probes can be covalently grafted8 and whose redox * Corresponding author. Tel: +33-1-44276961. Fax: +33-1-44276814. E-mail:
[email protected]. (1) Palecek, E. Talanta 2002, 56, 809-819. (2) Drummond, T. G.; Hill, M. G.; Barton, J. K. Nat. Biotechnol. 2003, 21, 11921199. (3) Fojta, M.; Havran, L.; Vojtiskova, M.; Palecek, E. J. Am. Chem. Soc. 2004, 126, 6532-6533. (4) Wang, J.; Liu, G.; Jan, M. R. J. Am. Chem. Soc. 2004, 126, 3010-3011. (5) Wang, J. Anal. Chim. Acta 2003, 500, 247-257. (6) Palecek, E. In Encyclopedia of Electrochemistry; Bard, A. J., Stratsman, M., Eds,; Vol. 9. Biopolymers, Wiley-VCH: Weinheim, Germany, 2002. (7) Southeyrand, E.; Cloarec, J. P.; Martin, J. R.; Wilson, C.; Lawrence, I.; Mikkelsen, S.; Lawrence, M. F. J. Phys. Chem. B 1997, 101, 2980-2985. 10.1021/ac050080v CCC: $30.25 Published on Web 04/12/2005
© 2005 American Chemical Society
properties are modulated following hybridization. Nice examples exist in the literature.9-14 Interpretations of the detection process are based upon ion-exchange hindering or polymer structure reorganization. All these works report a “signal-off” detection. Our approach takes the opposite course. Indeed, as it is systematically stated that an anion-exchange process is involved upon hybridization, we chose a cation-exchange redox group (quinone) as the immobilized redox-active label. We recently described the construction of such a reagentless hybridization sensor,15 based on differential pulse voltammetric measurements in the quinone electroactivity domain. Herein, a new approach is used to investigate for the first time to our knowledge the “signal-on” detection of DNA/ECP interactions. It is first shown that this modified electrode is highly selective versus the oligonucleotide (ODN) target strand. Obviously, a random sequence is discriminated, but also double-mismatches and single-mismatch sequences. In a second step, to have a better understanding of the transduction phenomena, which are not really described in the literature until now, an original approach has been used. The initial target strand has been divided into two or three separate shorter segments, and the resulting electrochemical changes were recorded versus the target sequence localization onto the probe strand. The results show that, by using one probe strand, it is possible to address several shorter targets. EXPERIMENTAL SECTION Chemicals. N′-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were provided by Sigma. PBS (0.137 M NaCl; 0.0027 M KCl; 0.0081 M Na2HPO4; 0.00147 M KH2PO4, pH 7.4) was from Gibco. Aqueous solutions were made with bidistilled or ultrapure (Millipore) water. Juglone and 1-naphthol were purchased from Fluka, and thioglycolic acid from Acros. Acetonitrile was supplied by Aldrich (HPLC (8) Livache, T.; Roget, A.; Dejean, E.; Barthet, C.; Bidan, G.; Teoule, R. Nucleic Acids Res. 1994, 2915-2921. (9) Korri-Youssoufi, H.; Garnier, F.; Srivastava, P.; Godillot, P.; Yassar, A. J. Am. Chem. Soc. 1997, 119, 7388-7389. (10) Korri-Youssoufi, H.; Makrouf, B. Anal. Chim Acta 2002, 469, 85-92. (11) Wang, J.; Jiang, M.; Fortes, A.; Mukherjee, B. Anal. Chim. Acta 1999, 402, 7-12 (12) Thompson, L. A.; Kowalik, J.; Josowicz, M.; Janata, J. J. Am. Chem. Soc. 2003, 125, 324-325. (13) Emge, A.; Bauerle, P. Synth. Met. 1999, 102, 1370-1373. (14) Lassalle, N.; Vieil, E.; Correia, J. P.; Abrantes, L. M. Synth. Met. 2001, 119, 407-408. (15) Pham, M. C.; Piro, B.; Tran, L. D.; Ledoan, T.; Dao, L. H. Anal. Chem. 2003, 75, 6748-6752.
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grade). All other reagents used were of analytical grade. Oligonucleotides were synthesized by MWG Biotech and are detailed in Table 1 and Table 4. Electrochemical Apparatus. For electrochemical experiments, a conventional one-compartment, three-electrode cell was employed. An EG&G 263A potentiostat was used with the Echem software (Ecochemie). The working electrode was a glassy carbon disk (Tokaı¨) of 0,07-cm2 area. The auxiliary electrode was a platinum grid and the reference electrode a commercial SCE. Construction of the Reagentless Hybridization Sensor. (a) Electrosynthesis. The electrochemical synthesis of poly(JUGco-JUGA) films was carried out by electrooxidation of a mixture of 5 × 10-2 M juglone (JUG) + 5 × 10-3 M 5-hydroxy-3-thioacetic1,4-naphthoquinone (JUGA, synthesized in our laboratory) + 2 × 10-3 M 1-naphthol + 0.1 M LiClO4 in acetonitrile, on a glassy carbon electrode, under dried argon atmosphere, by potential scans from 0.4 to 1 V versus SCE during 50 cycles at 50 mV‚s-1. The polymer obtained has been thoroughly characterized by XPS and FT-IR spectroscopy, and its structure is given below.
(b) ODN Probe Immobilization. Poly(JUG-co-JUGA)-coated glassy carbon electrodes were dipped into a solution containing 0.1 µM probe (SHORT, GEM, and GEMC, Tables 1 and 4), 1.5 × 10-2 M EDC (3 × 10-5 mol, 1 equiv), and 3 × 10-2 M NHS (6 × 10-5 mol, 2 equiv) in distilled water at 37 °C. After 20 h, the ODN immobilization reaction was stopped by removing the films from this solution. Then, the electrode was washed with distilled water (5 min) and after with PBS (2 h at 37 °C) in order to remove noncovalently bound ODN. In addition, phosphate groups contained in PBS are able to react on NHS-activated ester. Washing with PBS can then remove these unreacted ester groups. Hybridization. For hybridization experiments, solutions containing 0.1 µM target (HIV, CHIV, MHIV, M′HIV, 2MHIV, A, B, C, RAND, RANDA; see Tables 1 and 4) in PBS (pH 7.4) was used. A freshly prepared electrode is used for each hybridization. The electrode bearing the probe strand was dipped in the target solution for 2 h at 50 °C (above the melting temperatures of mismatching double strands) and cooled slowly to room temperature (25 °C). Then, the electrode was washed with PBS for 1 h at 37 °C in order to remove the nonhybridized strands. These steps were performed under aerated media. Square Wave Voltammetry (SQW). In the potential domain (-0.8 V; 0 V), the quinone group presents a well-defined electroactivity corresponding to the quinone/hydroquinone couple. The hybridization events were detected by recording the modification of the redox process of this quinone group, using SQW, during an anodic scan from -0.8 V up to 0 V. The following parameters were used: pulse height 50 mV, scan increment 2 mV, frequency 12.5 Hz, potential domain (-0.8; 0V) versus SCE. The medium 3352 Analytical Chemistry, Vol. 77, No. 10, May 15, 2005
Table 1. ODN Name, Function, and Sequence name
function
GEM
probe
SHORT HIV MHIV M′HIV 2MHIV RAND RANDA A B
probe target mismatch mismatch mismatch random random 1/2-target 1/2-target
sequence 5′-TCGCACCCATCT-CTCTCCTTCTAGCCT-3′C7NH2 5′-TCCTCTCCTTCTAGCCT-3′C7NH2 3′-CGTGGGTAGAGAGAGGAAGA-5′ 3′-CGTGGGTAAAGAGAGGAAGA-5′ 3′-CGTGGGTAGAGAGATGAAGA-5′ 3′-CGTGGGTAAAGAGATGAAGA-5′ 3′-ATCCATGCATTCCGCCTAAG-5′ 3′-TCCGCCTAAG-5′ 3′-GAGAGGAAGA-5′ 3′-CGTGGGTAGA-5′
was PBS (concentrations given in the Chemicals section), bubbled with argon for 40 min before and during SQW measurements. For each experiment, the SQW scans were repeated until stabilization of the signal. For that, four SQWs are performed within 40 min. The signal is considered as stable because its variation never exceeds 10%. All electrochemical experiments were conducted at 25 °C. In the following experiments, we will consider the changes of current intensities at ∼ -500 mV, i.e., for the main oxidation peak. The reproducibility of these experiments is as follows. Grafting leads to a current decrease in 7 of 10 cases (i.e., 70%); The remaining 30% are considered as aberrant values and removed. This is probably due to the grafting step, which is difficult to control. Conversely, hybridization (with complementary, random, or mismatch strands) give a reproducibility of 90%. The whole results represent ∼100 SQW experiments. RESULTS AND DISCUSSION Hybridization with 10-bp Probe and Target. SQW was performed on a poly(JUG-co-JUGA)-modified electrode onto which the SHORT probe (10 used bp, 5 bp as spacer after the 3′-aminoC7 modifier, and 2 unused bp on the 5′-end to avoid edge effect) is grafted (sequence detailed in Table 1 ). Targets (10 used bp) as A (full complementary) and RANDA (random) are then added (Figure 1). Curve 1 corresponds to the SQW response of a poly(JUG-co-JUGA)-modified electrode onto which the SHORT probe is grafted. If we consider the current for the main oxidation peak
Figure 1. SQW response of a poly(JUG-co-JUGA)-modified GC electrode onto which the SHORT probe sequence is grafted (9, curve 1), after addition of A (1, curve 2) or RANDA (O, curve 3) targets. [target] ) 0.1 µM. SQW conditions described in the Experimental Section. S ) 0.07 cm2. Medium, PBS. The background current at -0.2 V is subtracted.
rel var (%) )
Figure 2. SQW response of a poly(JUG-co-JUGA)-modified GC electrode onto which the GEM probe is grafted (curve 1, 9), and after addition of HIV (curve 2, 1), MHIV (curve 3, O), RAND (curve 4, 4), M′HIV (curve 5, [), and 2MHIV (curve 6, g) targets. Same conditions as Figure 1. Table 2. SQW Responses after Hybridization on the GEM Probe with 20-Base Targetsa target response ( 10% a
HIV 100
MHIV 19
M′HIV 3
2MHIV -8.5
RAND 20
Relative variations are calculated from formula 1.
at -500 mV, the results are very clear. As shown, the RANDA target sequence does not lead to any measurable current change (curve 3), whereas A leads to a signal increase (curve 2). The results demonstrate that this 10-bp sequence is accessible for hybridization. Then, experiments were extended to 20-bp probe and target sequences. Hybridization with 20-bp Probe and Target. SQW was performed on a poly(JUG-co-JUGA)-modified electrode onto which the GEM probe (20 used bp) is grafted. The 20-bp targets are added (Figure 2): HIV (full complementary), MHIV, and M′HIV (single-mismatch sequences), 2MHIV (double-mismatch sequence) or RAND (random). Curve 1 corresponds to the SQW response of a poly(JUG-co-JUGA)-modified electrode onto which the GEM probe is grafted. Addition of the single-mismatch sequence M′HIV (mismatch positioned near the 3′-end of the probe, curve 5) does not lead to any significant signal change. For the other single-mismatch sequence MHIV (mismatch positioned near the 5′-end of the probe, curve 3), as for the random sequence RAND (curve 4), the current increases slightly but corresponds to only 20% of the current recorded for the fullcomplementary HIV (curve 2). The double-mismatch sequence 2MHIV leads to a slight current decrease (curve 6). However, variations below 10% (in relative value), considering the standard deviation, are not significant. The results are summarized in Table 2. The current changes were calculated as follows. If i0 is the SQW main peak current (at ∼ -500 mV) recorded for the GEM-grafted film, i the peak current due to the considered target strand and imax the maximum peak current response over all the target strands, the relative current variations are defined as
i - i0 × 100 imax - i0
(1)
As an example of calculation, the RAND response was calculated as follows: 6.9 - 5.5/12.5 - 5.5 × 100 ) 20% (imax corresponds here to the HIV response. See Table 2). These results demonstrate the high selectivity of the response versus the target addition, including for mismatch sequences, wherever the mismatch may be (in the first part or the second part of the target strand). For example, the result with M′HIV demonstrates that the first 10 probe bp (near the 3′-end of the probe, i.e., near the polymer surface) are sensitive to a single mismatch. Experiments were extended to a 30-bp probe strand and various target sequences. Hybridization with 30-bp Probe and Target. Experiments were performed on the poly(JUG-co-JUGA)-modified electrode onto which the GEMC probe (30 used bp) is grafted. The 30-bp (full complementary, CHIV), 20-bp (HIV, MHIV, M′HIV, 2MHIV, RAND), and 10-bp (C) probes were added (Figure 3). The results are summarized in Table 3. As shown, the response to the 30-bp full complementary target CHIV gives the highest signal (100%), followed by the 20-bp complementary sequence HIV (76%), and the 10-bp complementary strand C (64%). This seems to indicate that the current response depends on the target length (Figure 3a). The selectivity on this 30-bp probe was not tested with 30-bp but with 20-bp sequences (RAND, MHIV, M′HIV, 2MHIV). As stated in Table 2, the random and the mismatch sequences are discriminated, considering that the responses due to the mismatches or random sequences are much lower than the one due to the complementary strand HIV. At this stage, it is shown that all the areas of the probe sequence are accessible to hybridization. To progress in the understanding of the transduction phenomena, these experiments were extended as follows. Signal Dependence upon the Probe Sequence Localization. (a) Target Divided into Two Shorter Sequences. The 20base target strand (HIV) was divided into two separate 10-bp strands, A and B, respectively (Table 1). The two half-strands were added successively using a 20-bp probe (GEM), under the same conditions as for HIV target (Figure 4). Current changes were calculated as explained for Table 2. It is very interesting to remark that hybridization of A first leads to a current decrease (∼ -28%), and then hybridization of B leads to a current increase (up to 98%), which is near that obtained with the HIV strand. When B is added first, the signal reaches 100%, and hybridization of A leads to a signal decrease (down to 70%). This last result could be due to a slight dehybridization of the B strand during hybridization of A. In both cases, it appears that the B strand, which hybridizes near the 5′-end of the probe sequence (i.e., far from the electrode surface), gives a higher signal. It is therefore probably possible to know the position of the hybrid onto the probe strand. However, at this stage, it is not possible to ascertain whether a different signal indicates a different position of hybridization or a different amount of hybridized target. This experiment was extended to the 30-bp GEMC probe, with target divided into three 10-bp target strands A-C (Table 4). Analytical Chemistry, Vol. 77, No. 10, May 15, 2005
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Figure 3. SQW responses of a poly(JUG-co-JUGA)-modified GC electrode onto which the GEMC probe is grafted (curve 1, 9), after addition of CHIV (curve 2, 1), HIV (curve 3, open circles), C (curve 4, 3), and RAND (curve 5, 0) (a), or after addition of CHIV (curve 6, 1), MHIV (curve 7, 0), M′HIV (curve 8, O), and 2MHIV (curve 9, 3) (b). Same conditions as Figure 1.
Figure 4. SQW response of a poly(JUG-co-JUGA)-modified GC electrode onto which the GEM probe is grafted (curve 1, 9), after addition of B (curve 2, 1), and A (curve 3, 3) a), or A (curve 4, 4) and B (curve 5, 1) (b) Same conditions as Figure 1. Table 4. ODN Name, Function, and Sequencea name
Table 3. SQW Responses after Hybridization on the GEMC Probe with 30-, 20-, and 10-Base Targetsa target response ( 10% target response ( 10% a
CHIV 100 MHIV 4
HIV 76 M′HIV 13
C 64 2MHIV -21
RAND 7.8
Relative variations are calculated from formula 1.
(b) Target Divided into Three Shorter Sequences Using a 30-bp Probe. Experiments were conducted as described in (a), for three 1/3-strands. As an example, results for the A, B, C and C, A, B sequences are reported in Figure 5. The whole results are presented in Figure 6. These results back up those obtained with the A and B 1/2-strands. Indeed, the most intense variation is obtained in all cases for addition of the C strand (1/3-strand far from the film surface). The system is less sensitive to the B strand, and the lowest response is obtained with the A strand. When added after B and C, A tends to decrease the current. As explained formerly, this could be due to a slight dehybridization of the former sequences during hybridization with A. 3354 Analytical Chemistry, Vol. 77, No. 10, May 15, 2005
function
GEMC
probe
CHIV
target
A B C
1/3-target 1/3-target 1/3-target
a
sequence 5′-TCCCTCATAGTC-GCACCCATCTCTCTCCTTCTAGCCT-3′C7NH2 3′-GGAGTATCAG-CGTGGGTAGAGAGAGGAAGA-5′ 3′-GAGAGGAAGA-5′ 3′-CGTGGGTAGA-5′ 3′-GGAGTATCAG-5′
Relative variations are calculated from formula 1.
As it was suggested in (a), these results show that it is possible to know which part of the probe strand (near the 5′-end, near the 3′-end, or in the middle) is addressed for hybridization. Beyond, these results can be used to propose a transduction mechanism, as discussed below. (c) Interpretation. Hybridization with Probe and Target of the Same Length. In previous works dealing with direct electrochemical hybridization detection on conducting polymers,where a “signal-off” detection was observed, assumptions were based on ion exchange hindering or polymer reorganization.9-14,16-18 ODN strands are polyanionic molecules. These polyanionic oligomers
Figure 5. Same experiments as in Figure 3, with a poly(JUG-coJUGA)-modified GC electrode onto which the GEMC probe is grafted (curve 1, 9), after addition of A (curve 2, 2), then B (curve 3, O), then C (curve 4, 1) (a), or C (curve 5, 1), then A (curve 6, 2), then B (curve 7, O) (b). Same conditions as Figure 1. Responses are summarized in Figure 6.
Figure 6. Bars deduced from the SQW responses of a poly(JUGco-JUGA)-modified GC electrode onto which the GEMC probe is grafted, after addition of A (white), B (gray), or C (black), in various orders. Same conditions as Figure 1. Relative variations are calculated from formula 1.
attract cations from the solution (here Na+ or K+; H+ is not significantly present at pH 7), that form a positively charged shield around the ODN strand. As a consequence, ODN molecules carry a very high charge density. It is commonly admitted that the ionic environment influences ODN hybridization (ionic force, but also charged surfaces);20,21 It seems equally justified to consider that
the high charge density carried on ODN can influence its environment, i.e., in our case, the polymer/solution interface. The literature on related works describes such interactions on polypyrrole, polyaniline, or polythiophene-modified electrodes.1,9-14,16-18 In these cases, with p-doping polymers, which exchange anions, hybridization is systematically followed by a current decrease (“signal-off”). Publications generally admit that it is due to a modification of the anion-exchange process. In our case, the poly(JUG-co-JUGA) film exchanges cations (via the quinone group, which has been demonstrated elsewhere). This opposite process perhaps can explain the opposite response, i.e., “signal-on, upon hybridization, if we consider that the electrochemical response is due to an ion-exchange modification. Besides, we believe that conformation changes of the ODN strands induce a modification of the polymer/solution interface and, therefore, a modification of the electrochemical response. ODN conformations have already been discussed to explain the electroactivity changes of an electroactive group tethered to a probe strand or a target strand,12,19 immobilized on an electrode surface. Single-stranded ODN (ssODN) are supposed to behave as classical polymer chains. This means that, in the vermicular model, they have a contour length (L), a persistence length (p), and a rigid segment length l ) L/N, with N the number of nucleobases. It is generally admitted that l equals to 0.43 nm for a ssODN, and 0.34 nm for a double-stranded ODN (dsODN). Therefore, L varies slightly between dsODN and ssODN (LdsODN ) LssODN × 0.8), but p varies dramatically. Indeed, pdsODN ≈ 50 nm,22,23 whereas pssODN varies between 0.8 and 8 nm, as a function of the ionic strength. Herein, the salt concentration is ∼0.15 M. In this case, one finds pssODN ≈ 1 nm.22,23 p has to be compared to L to give a concrete idea of the strand conformation. If p . L, the strand is straight and rigid, whereas if p , L, the ODN strand behaves as a coil. We consider three different strands of 10, 20, and 30 bases, onto which a C7 chain and 7 nucleobases are added as spacers (Tables 1 and 4). Therefore, with l ) 0.43 nm, L10, L20, and L30 equal 8.2, 12.5, and 16.8 nm, respectively, for ssODN. As a consequence, as pdsODN . L, these ODN can be considered as a straight cylinder when hybridized (in this model, the double helix is assimilated to a cylinder of 1 nm in radius and LdsODN in length).24 In contrast, ssODN are usually assumed as coils, more or less folded depending on the ratio L/p (the 30-bp ODN is more folded than the 10-bp one). This is also quantified by the gyration radius RG, with RG ) (Lp/3)1/2. This gives RG10, RG20, and RG30 of 1.65, 2.04 and 2.37 nm, respectively. These values of gyration radii are interesting. Indeed, they are higher than the dsODN radius (in the cylindrical model, 1 nm).24 Therefore, conformation (16) Garnier, F.; Korri-Youssoufi, H.; Srivastava, P.; Mandrand, B.; Delair, T. Synth. Met. 1999, 100, 89-94. (17) Cha, J.; Han, J. I.; Choi, Y.; Yoon, D. S.; Oh, K. W.; Lim, G. Biosens. Bioelectron. 2003, 18, 1241-1247. (18) Kertesz, V.; Whittemore, N. A.; Chambers, J. Q.; McKinney, M. S.; Baker, D. C. J. Electroanal. Chem. 2000, 493, 28-36. (19) Anne, A.; Bouchardon, A.; Moiroux, J. J. Am. Chem. Soc. 2003, 125, 111211 (20) Chan, V.; McKenzie, S. E.; Surrey, S.; Fortina, P.; Graves, D. J. J. Colloid Interface Sci. 1998, 203, 197-207. (21) Vainrub, A.; Montgomery Petitt, B. Biopolymers 2003, 68, 265-270. (22) Tinland, B.; Pluen, A.; Sturm, J.; Weill, G. Macromolecules 1997, 30, 57635765. (23) Smith, S. B.; Cui, Y.; Bustamante, C. Science 1996, 271, 795-799. (24) Frank-Kamenetskii, M. D. Phys. Rep. 1997, 288, 13-60.
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changes (from ssODN to dsODN) during hybridization modify the shape of these objects, therefore, their steric hindrance at the film/solution interface. This could explain the electroactivity changes observed in our system. Hybridization with Targets Shorter Than the Probe. As shown on Figure 6, the current response upon hybridization of the first target strand depends strongly on its position onto the probe strand. This is not directly explained by the conformation modification (RG) detailed above. However, the accessibility of each segment onto the probe strand probably depends on the conformation of the probe. For an ODN strand that is grafted via its 5′-end, it is a straightforward hypothesis to consider that the nucleobases near the anchoring point are less accessible (more hindered, less mobile) than the ones that are farther (more mobile). For instance, hybridization of the A strand (near the 5′end) gives a weak signal because this segment is probably hindered by the probe folding and is less mobile. Hybridization of the B strand (more far from the 5′-end than A) gives a stronger signal, and hybridization of C gives the strongest signal, considering that it is more mobile, therefore more accessible for hybridization. In other words, hybridization is probably less efficient closer to the polymer surface. CONCLUSION It was shown herein that hybridization of a target ODN onto a probe strand immobilized on a poly(JUG-co-JUGA)-modified
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electrode generates a modification of the SWV response that could be considered as “signal-on”, by contrast to most similar work on conducting polymers. This could be explained by the original redox group used, i.e., quinone-hydroquinone, which is a cationexchanging group. It was shown that the system is able to detect selectively hybridization with 1-30-bp probe or target strands and can discriminate one mismatch on the target sequence. Target strands have been divided into two and three shorter strands, and hybridization of these shorter sequences suggested that it is possible to know which part of the probe strand is addressed for hybridization. These results are very promising because they suggest that one probe sequence can be used to address several shorter targets. Work is in progress to study thoroughly this feature. ACKNOWLEDGMENT We thank the French Ministry of Research, ACI Health Technologies, 00B0098. S.R. thanks the French Ministry of Research for a Ph.D. grant.
Received for review January 14, 2005. Accepted March 17, 2005. AC050080V
