Development of Innovative and Versatile Polythiol Probes for Use on

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Development of Innovative and Versatile Polythiol Probes for Use on ELOSA or Electrochemical Biosensors: Application in Hepatitis C Virus Genotyping Myriam Lereau,†,‡,○ Chantal Fournier-Wirth,*,†,○ Julie Mayen,‡ Carole Farre,§ Albert Meyer,‡ Vincent Dugas,§ Jean-François Cantaloube,∇ Carole Chaix,*,§ Jean-Jacques Vasseur,‡ and François Morvan*,‡ †

Laboratoire TransDiagSécurité Transfusionnelle et Innovation Diagnostique, Etablissement Français du Sang Pyrénées-Méditerranée, Montpellier, 34184, France ‡ Département des Analogues et Constituants des Acides Nucléiques, Institut des Biomolécules Max Mousseron, UMR 5247 CNRS Université Montpellier 1 Université Montpellier 2, Montpellier, 34095, France § Département Laboratoire des Sciences Analytiques, Institut des Sciences Analytiques, UMR 5280 CNRS Université de Lyon, Université Lyon 1, Villeurbanne, 69100, France ∇ Laboratoire Emergence et Co-évolution Virale, Etablissement Français du Sang Alpes-Méditerranée, Marseille, 13005, France S Supporting Information *

ABSTRACT: The aim of this study was to develop versatile diagnostic tools based on the use of innovative polythiolated probes for the detection of multiple viruses. This approach is compatible with optical enzyme-linked oligosorbent assay (ELOSA) or electrochemical (biosensors) detection methods. The application targeted here concerns the rapid genotyping of Hepatitis C virus (HCV). HCV genotyping is one of the predictive parameters currently used to define the antiviral treatment strategy and is based on the sequencing of the viral NS5b region. Generic and specific NS5b amplicons were produced by real-time polymease chain reaction (RT-PCR) on HCV(+) human plasma. Original NS5b probes were designed for genotypes 1a/1b, 2a/2b/2c, 3a, and 4a/4d. Robust polythiolated probes were anchored with good efficacy on maleimide-activated microplates (MAM) and gold electrodes. Their grafting on MAM greatly increased the sensitivity of the ELOSA test which was able to detect HCV amplicons with good sensitivity (10 nM) and specificity. Moreover, the direct and real-time electrochemical detection by differential pulse voltammetry enabled a detection limit of 10 fM to be reached with good reproducibility. These innovative polythiolated probes have allowed us to envisage developing flexible, highly sensitive, and easy-to-handle platforms dedicated to the rapid screening and genotyping of a wide range of viral agents.

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combination of a lack of proof-reading activity by the viral RNA-dependent-RNA polymerase encoded by the NS5b region and a high level of viral replication.12 Six major genotypes have been identified so far, as well as more than 80 subtypes.13 In France, genotype 1a/1b is the most frequent (57%), followed by genotypes 3 (20.8%), 2 (9.3%), 4 (8.9%), 5 (2.7%), 6 (0.2%) and, last, mixed genotypes (0.9%).14 Treatment of chronic Hepatitis C has, for over 10 years, been based on the combination of pegylated interferon alpha (peg IFN-α) and ribavirin. HCV genotyping is used as a predictive parameter to define the antiviral treatment strategy. In this context, genotyping tests are useful to more accurately predict the

he inherent physicochemical stability and highly specific probe/target recognition properties displayed by nucleic acids have made them the most targeted molecules for in vitro detection and typing of different organism strains and allowed the elaboration of robust and sensitive DNA biosensors. The application of such biosensors within systems devoted to pointof-care analyses requires that they must be cost-effective, rapid, sensitive, and easy to handle. Few well-documented reviews have been recently published that compare the performance of DNA sensors.1−9 In this context, developing a direct and highly sensitive detection system is a great challenge that holds great expectations.10 The Hepatitis C virus (HCV) is a major cause of liver disease for the estimated 130 million people worldwide chronically infected.11 HCV is an enveloped RNA virus belonging to the Flaviviridae family, genus Hepacivirus, and presents a high degree of genetic variability, which is explained by the © XXXX American Chemical Society

Received: June 28, 2013 Accepted: September 9, 2013

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dx.doi.org/10.1021/ac401941x | Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

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sensitivity. Osmetech have commercialized an electrochemical system based on the use of four-ferrocene modified probes for the simultaneous detection of 298 sequences.49 We recently reported a strategy to introduce several ferrocene moieties into an oligonucleotide.50 It uses α-anomeric oligonucleotides exhibiting the ferrocene groups on some phosphodiester linkages, since it has been shown that the resulting modified α-oligonucleotides hybridize with a complementary strand in parallel orientation with a lower destabilization than corresponding ferrocenyl oligonucleotides with natural β-anomery. In the current study, the ferrocenes were introduced into the α-oligonucleotides using α-nucleoside ferrocenylpropyl phosphoramidites (see below). Thiolated probes are often used to graft on gold electrodes in electrochemical biosensors. Most commercially available compounds to introduce thiol functions into oligonucleotides lead to a 3′ or 5′ monointroduction using the corresponding solid support or phosphoramidite. However, it is well-known that the thiol monolayer is only moderately stable on a gold surface. Indeed, the Au−S bond tends to oxidize in air and media,51 tends to decompose with increasing temperature in an aqueous solution,52 and has a narrow potential window within which it is stable, which limits the usefulness of this chemistry for biosensors. Two publications have reported the use of a “trebler” phosphoramidite,53 allowing the introduction of three alcohol functions that were further elongated with a monothiol phosphoramidite52 or use of a phosphoramidite adamantanebased trihexylthiol,54 both of which lead to an oligonucleotide with only three thiols and exclusively at its 5′-end. Recently, an alternative was reported with the use of cyclodithioerythritol, which was converted to a phosphoramidite or a solid support.42 Thus, for each incorporation of these compounds, two thiol functions are introduced. Theoretically, by this strategy, it is possible to introduce as many thiols as desired into an oligonucleotide at the 3′- or 5′-end. However, in our hands, the coupling exhibited poor efficiency, despite the 10 min coupling time according to the manufacturer’s recommendations leading to a low yield when synthesizing an oligonucleotide with four thiols. This limitation prompted us to design a new phosphoramidite, 3, bearing a protected thiol function (see Scheme S1 in the Supporting Information (SI)) that could be introduced several times into oligonucleotides at the 3′- or 5′end, leading to polythiolated oligonucleotides. In the present study, we aimed to develop two sensitive and specific detection methods based on these polythiolated probes for direct HCV genotyping in the NS5b region. These robust and innovative polythiolated probes were anchored with good efficacy on both maleimide-activated microplates (MAM) and on gold electrodes. Their analytical performance for HCV genotyping was evaluated by ELOSA and DPV. To the best of our knowledge, this is the first time that the performances of probes of similar sequence in ELOSA and electrochemical sensor have been compared.

effectiveness of the antiviral response, dictate the duration of therapy, and determine the dosage of ribavirin.15 Several assays are currently commercially available for the detection and/or quantification of HCV RNA.16,17 Over the last two decades, enzyme-linked oligo-sorbent assay (ELOSA) has emerged as a new method to detect nucleic acids.18−23 This technique is based on the immobilization of an oligonucleotidic probe onto a surface and on the detection of multiple target/probe hybridization events. ELOSA has already been applied to retrovirus identification,23 HIV quantification22 or HCV genotyping.20 Biosensor devices for the qualitative and quantitative detection of HCV by bioelectric recognition assays,24 surface plasmon resonance,24 and piezoelectric response25 have been published over the past decade. During this period, the development of electrochemical DNA biosensors based on base-pair recognition of DNA probes has received increasing attention.3,26−28 For the detection of HCV, Ahour et al. developed an electrochemical sensor based on the measurement by differential pulse voltammetry (DPV) of Methylene Blue intercalation upon hybridization,29 and Park et al. reported using an impedimetric sensor.30 Dastagir et al. developed a field-effect transistor with the use of HCV sequences grafted on single-walled carbon nanotubes (SWNTs).31 The detection limit reached by these methods was described to be in the range of 100 pM to 1 pM. Electrochemical detection is well-adapted for the development of cost-effective, single-use, and portable diagnostic systems,32,33 and of multidetection microarrays potentially associated with multiple step reaction-integrated systems known as lab-on-chip systems. Riccardi et al. have described a label-free detection scheme for short sequences based on conducting-modified polypyrrole films deposited at a microelectrode surface.34 Synthetic HCV-1 genotype 18-mer DNA probe designed according to the sequence of 5′NCR was immobilized on electrodes and exposed to a solution of mismatched oligonucleotides (244-mer HCV type 2a/2c, 2b, and 3). The HCV-1 DNA probe showed no unspecific interactions in the presence of the 4-bp to 6-bp mismatched sequences. HCV detection methods based on site-specific DNA cleavage by BamHI endonuclease of thionine-35 or ferrocene-36 labeled 21-mer probes hybridized with a HCV 21-mer target have been reported by Cai et al., where they monitored the variation of voltammetric signal before and after endonuclease incubation. This technique has been further improved by combining gold nanoparticles, allowing the detection of HCV1b cDNA (244-mer) with a detection limit of ∼3.1 × 10−22 M.37 In a general manner, electrochemical sensors are based on the change of an electrical parameter at the electrode/ electrolyte interface. Signal variation upon hybridization of the immobilized probes with the sequence-specific complementary target DNA can be measured either by impedance38,39 or by voltammetry with the use of redox labels that either bind to the double-stranded DNA40,41 or are covalently tethered to one of the DNA strands.42 Ferrocene is often used as an electrochemical label in molecular diagnostics, because it affords a sharp and intense redox signature and electrochemically responds in a potential range compatible with the use of biomolecules in solution (−200 mV to +600 mV). Furthermore, the high sensitivity of ferrocenes toward their surrounding medium is of great interest for developing detection methods.43,44 In the literature, different strategies have been investigated using ferrocene as a redox marker.45−48 However, the use of single-ferrocene probes limits their

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MATERIAL AND METHODS HCV Targets. The HCV amplicons were obtained in the NS5b viral region from sequencing HCV-positive blood donors plasma samples and produced by RT-PCR as fully described in the Supporting Information (SI). Synthetic 15-mer and 105-mer oligonucleotides were supplied by Eurogentec (Angers, France). Sequences are presented in the SI. Synthesis of the Thiolated Phosphoramidite 3, the α2′-Deoxynucleoside Cyanoethyl or Ferrocenyl PhosB



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Figure 1. (A) Structure of polythiolated 1a/1b probes bearing 1−8 thiol groups (SH). Y = T/C. (B) ELOSA study of the response as a function of the number of thiol groups. The hybridization of 15-mer and 105-mer 1a/1b synthetic targets was tested by ELOSA. Nonspecific 3a targets were used as negative controls. [Legend: ⌀, no probe; HB, hybridization buffer.]

phoramidites and the Oligonucleotidic Probes. The phosphoramidites were obtained and used as described in the SI. Oligonucleotides were synthesized on a 1-μmol scale by standard phosphoramidite chemistry, using a DNA synthesizer (see the SI). Diagnostic Assays. The probe/target hybridization was first evaluated by ELOSA. The DELFIA streptavine-europium assay was achieved on a Victor Instrument (Perkin−Elmer, Inc., Waltham, MA, USA). Then, the electrochemical assays were performed in a conventional three-electrode single-compartment cell. For analysis, the signal was recorded by DPV within a potential range from −200 mV to 300 mV (scan rate = 20 mV s−1). Protocols are described in the SI.

removed the acyl protections on the nucleobases and on the thiols and hydrolyzed the succinyl linker. This protocol avoided the Michael addition between the free thiols and acrylonitrile. After evaporation, the modified oligonucleotides were purified by C18 reverse-phase HPLC and stored lyophilized at −20 °C. Before performing ELOSA, 5 nmol of probes were resuspended into Milli-Q water and tris(2-carbonylethyl)phosphine (TCEP) was added to reduce disulfide bonds. The resulting polythiolated probes specific for the HCV 1a/ 1b genotype were grafted onto maleimide-activated microwells. Hybridization was tested using 15-mer and 105-mer synthetic targets. As shown in Figure 1B, no signal was observed without probe (denoted as “⌀”), without target (denoted as “HB”) or with nonspecific targets (tetrathiolated probe for genotype 3a). The specific signal (1a/1b) was increased with the concentration of targets and with the number of thiols. Moreover, it reached a plateau from the 4-thiolated probe with the 15-mer target whereas it continued to increase slightly with the 105mer target. Since four-thiol functions were enough to observe high signals with both short and long synthetic targets by ELOSA, even at low concentrations (10 pM), the experiments performed thereafter used a maximum of four thiols on the probes. Study of the Optimal Number of Thiol Groups Allowing the Highest Grafting Stability on a Gold Surface. Three oligonucleotides bearing a ferrocenyl moiety at the 5′-end and 1, 2, or 4 thiol groups at the 3′-end were synthesized (Figure 2A). The synthesis was carried out starting from an universal solid support 1,3-propanediol onto which phosphoramidite 3 was introduced (1, 2, or 4 times), a 13-mer sequence was elongated and one ferrocene was introduced using ferrocenepropyl α-thymidine phosphoramidite. Electrochemical detection of the ferrocenyl group at the 5′-end permitted the calculation of the density of the grafted oligonucleotide (see the SI). Deprotection was performed as described above, and the three constructions were purified by C18 reverse-phase highperformance liquid chromatography (HPLC). The resulting

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RESULTS AND DISCUSSION Synthesis of the Polythiolated Probes. The different oligonucleotides were synthesized using a DNA synthesizer by standard phosphoramidite chemistry using commercially available reagents, cyanoethyl α- or β-nucleoside phosphoramidites, or ferrocenylpropyl α-nucleoside phosphoramidites. The thiol phosphoramidite 3 was introduced either at the 3′- or at the 5′ end (see the SI). ELOSA To Determine the Optimal Number of Thiol Groups for the Highest Probe Sensitivity. Five constructions exhibiting 1, 2, 4, 6, or 8 thiol groups at the 5′-end of a sequence targeting 1a/1b subgenotypes were synthesized according to standard phosphoramidite chemistry using standard ancillary reagents and phosphoramidites (Figure 1A). Starting from 1 μmol of a dC solid support, a 1a/1b DNA complementary sequence was further elongated with four thymidines, used as a spacer, before one, two, four, six, or eight thiol phosphoramidites 3 were introduced. A two-step protocol was used for deprotection. First, a treatment with 10% piperidine in acetonitrile was performed for 15 min, allowing the removal of the cyanoethyl groups by β-elimination and the resulting acrylonitrile was removed by washing with acetonitrile. Second, treatment with concentrated aqueous ammonia C



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the oxidation wave was reported after the successive treatments, and we compared the stability of the different structures. We found that the tetrathiol anchor significantly improved the temperature stability of the system. This structure allowed five regenerations with no strong loss of signal. After 5 min of immersion in water at 80 °C, the tetrathiol maintained 80% of its initial signal. Conversely, under the same conditions, monothiol and dithiol maintained only 50% and 20% of signal, respectively. Thus, an efficient regeneration of the biochip was permitted at a high temperature (80 °C), even after hybridization with long DNA targets. We were surprised to observe that the dithiol was less stable than the monothiol probe at high temperatures. One explanation could be that the dithiol linker maintains a strong ability to reform the more thermodynamically favorable intramolecular disulfide bond at the expense of the Au−S bond. Our data are concordant with the thermal instability of monothiol and dithiol analogues on gold surface found in a previous study on multidentate monolayer film stability.55 The DNA/DNA hybridization detection principle emerged over the last two decades to detect nucleic acids. The DNA probe generally contains one chemical function with which it anchors to the support. This is usually an amino group at an extremity (5′ or 3′), which allows a covalent immobilization on a carboxyl-coated surface,56 or a thiol group permitting the grafting onto a gold surface. To our knowledge, our study is pioneering in that the same probe thiol chemistry was used for optical and electrochemical detection. We have shown that anchoring with more than one thiol group to maleimideactivated and gold surfaces enhances the signal obtained upon target hybridization. In a similar way, Raddatz et al. reported the synthesis of new phosphoramidite building blocks and their use for the modification of oligonucleotides with hydrazide functions:57 they demonstrated an improved efficiency of branched hydrazide oligonucleotides compared with monohydrazide oligonucleotides and standard amino-modified oligonucleotides at immobilizing DNA on NHS ester surfaces. As our tetrathiolated probes permitted a stable immobilization on gold and displayed a high level of sensitivity at detecting the target using ELOSA, we decided to conduct further experiments with these probes. Synthesis of the Tetrathiolated Probes Targeting 2a/2c, 2b, 3a, and 4a/4d Genotypes. Genotype-specific β probes

Figure 2. (A) Structure of 5′-monoferrocene 3′-thiolated oligonucleotides bearing 1−4 thiol groups. (B) Grafting stability on gold surface as a function of the number of thiol groups. The variation in peak current intensity was recorded on gold electrodes functionalized by (1) the monothiol probe (n = 1), (2) the dithiol probe (n = 2), and (3) the tetrathiol probe (n = 4), after incubation (immersion) in water at 80 °C.

monoferrocene thiolated-oligonucleotides were immobilized onto a gold electrode and their stability was monitored via cyclic voltammetry (CV). The regeneration of biochips is of great interest, first to confirm the recognition event by recovering the initial signal after denaturation and, second, to reuse the biochip for several experiments. To test the regeneration potential of our grafted gold surface, we mimicked the regeneration step by incubating the gold electrode in deionized water at 80 °C for 1 min, followed by a waiting period during which the signal stabilized in a phosphate buffer (PBE). The operation was repeated five times, and the signal intensity was monitored by cyclic voltammetry (CV). On Figure 2B, the maximum intensity of

Figure 3. ELOSA response of probe 3a for the detection of different target lengths. The 3a β linear 4-thiolated probe was tested with 15-mer and 105-mer synthetic targets and with long (401nt) and short (143nt) HCV amplicons from two plasma samples (identified here as “1” and “2”). Long amplicons were diluted 100 times and short amplicons were used at dilutions of 1/100 and 1/1000. Nonspecific 1a/1b targets were used as negative controls. [HB = hybridization buffer.] D



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Figure 4. HCV genotyping by ELOSA using the four genotype-specific probes (1a/1b, 2a/2b/2c, 3a, and 4a/4d) with HCV amplicons. Different long generic amplicons (401nt) were diluted by a factor of 100 and tested for hybridization with the four different probes. 15-mer synthetic targets were used as controls, and nonspecific targets were used as negative controls. [HB = hybridization buffer.]

not been purified, may be entirely different. Another point concerns the dilution at which the amplicons are tested in the majority of studies: amplicons are usually tested at low dilutions from 1/3 to 1/40,18,19,22,58−61 compared to our study in which, despite the amplicons being diluted by a factor of 100, a strong signal was observed. Application of the ELOSA Protocol to HCV Genotyping. Long generic amplicons (401nt) from different plasma samples were produced by classical real-time polymerase chain reaction (RT-PCR) for 1a, 1b, 2a, 2b, 2c, 3a, 4a, and 4d subtypes and tested for hybridization by ELOSA with the tetrathiolated probes specific for 1a/1b, 2a/2b/2c, 3a, and 4a/4d genotypes. As shown in Figure 4, the probes designed in this study were highly specific for one genotype as the three other nonspecific signals were all negative. This system allowed the typing of long (401nt) generic HCV amplicons diluted by a factor of 100, corresponding to a concentration of 10 nM. ELOSA has already been applied to HCV genotyping in the 5′-NCR region.20 Moreover, two oligonucleotide arrays based on the DNA/DNA hybridization principle were reported on HCV genotyping in the 5′-NCR61 and NS5b60 regions. In these studies, the amplicons were produced using semi-nested60 or nested PCR20,61 and were used at dilutions of 1/3,60 1/10,61 and 1/50.20 Our sensitive ELOSA detection approach avoids the use of two successive runs of PCR and, thus, the high risk of cross-contamination. Evaluation of Probe/Target Hybridization by Electrochemistry. We then wanted to evaluate the ability to determine HCV genotype by electrochemistry. To this end, we first studied the hybridization stability of the ferrocenyl α-probe with the complementary β-sequence by melting temperature experiment (Tm) and their detection response by ELOSA. In a second part, the influence of the position of the ferrocene moiety versus the thiol groups was studied and, finally, the influence of the number of ferrocene residues on the electrochemical response was determined. Melting Temperature Study. Alpha 1a/1b oligonucleotides exhibiting a 5′-tetrathiol T4 and 0, 1, 3, or 5 ferrocenepropyls on the 5′-part of the sequence were synthesized by phosphoramidite chemistry on a DNA synthesizer (Figure 5A). Note that α-probes hybridize to β-

were synthesized as described above with four thiol groups and a T4 spacer at their 5′-end for ELOSA study: 5′-β Thiol4-T4 [PROBE SEQUENCE]-3′. Probe sequences are given in Table S1 in the SI. Study of the Target Size by ELOSA. We demonstrated that a long sequence (105-mer) hybridized to 1a/1b probe could be efficiently visualized by ELOSA (Figure 1B). We further studied the possibility of detecting amplicon sequences of different sizes, one short (143nt/191nt) and one long (401nt). The tetrathiolated probe specific for the HCV 3a genotype was grafted onto maleimide-activated microwells via the four thiol functions. Hybridization was tested using specific and nonspecific targets: 15-mer and 105-mer synthetic targets and amplicons of different sizes produced from two different plasma samples. As shown in Figure 3, no signal was observed with the nonspecific targets (1a/1b): 15-mer and 105-mer synthetic targets, and the short (191nt) and long (401nt) amplicons. In contrast, the specific signals (3a) were high whatever the target, even at the dilution of 1/1000 for the short amplicons. This suggests that the ELOSA method is a sensitive technique able to detect short as well as long targets, even at low concentrations. To our knowledge, the majority of DNA/DNA hybridization studies performed to date have been based on longer probes in the range of 18−36nt21,22,58 and/or shorter synthetic targets or amplicons in the range of 80−270nt18,21,22,58 expected to facilitate the hybridization between the probe and the target. As reported in the review of Tosar et al., when describing a new detection method or introducing a new variation, initial experiments are carried out with synthetic oligonucleotides that are strictly complementary or not to the immobilized probes.3 These types of experiments are necessary, since they serve as the proof-of-concept that the immobilization and detection method that is being investigated works, and that it is able to discriminate between complementary targets and unspecific DNA sequences. Furthermore, ultrasensitive detection at femtomolar levels has been achieved by many authors using synthetic oligonucleotides.3 Although these results are encouraging, the performance of the DNA/DNA hybridization sensor or genosensor at detecting targets in a real-life and complex biological sample, such as a PCR product which has E



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Figure 5. (A) Structure of tetrathiolated 1a/1b α-probes bearing 0, 1, 3, or 5 ferrocene residues. (B) ELOSA with ferrocenyl α-probes for probe validation for the electrochemical assay. 15-mer and 105-mer synthetic targets were tested for hybridization by ELOSA. Nonspecific 3a targets were used as negative controls. [Legend: ⌀, no probe; Fc, ferrocene; HB, hybridization buffer.]

complementary sequences, according to a parallel orientation.62 Each probe was mixed with the complementary 15-mer βsequence for hybridization and melting curves were recorded and Tm values and thermodynamic parameters were determined (see Table S2 in the Supporting Information). The α:βduplex without ferrocene residue displayed a Tm value of 63.1 °C. We observed a nonproportional destabilization of the duplexes containing ferrocene moieties with a slight destabilization of −0.8 °C for one residue but −7.0 °C and −18.7 °C for 3 and 5 residues, respectively, corresponding to a decrease in Tm of 2.33 and 3.74 °C, respectively, per modification. This result was surprising since, in a previous study in which ferrocenepropyl was introduced through a phosphoramidate linkage, we observed a slight stabilization of +0.1 °C per modification.50 These data suggest that phosphotriester and phosphoramidate linkages confer different behaviors to the corresponding duplex. Comparison between α- and β-Probes by ELOSA. The 1a/1b α-tetrathiolated probes with an increasing number of ferrocene residues were tested by ELOSA using 15-mer and 105-mer synthetic targets. Comparisons were made with the 1a/1b β-tetrathiolated probe. As shown in Figure 5B, signals were similar between the α- and β-probes with 15-mer targets. However, hybridization with the 105-mer targets was 10-fold lower with the α probe. In order to assess the effect of the presence and number of ferrocene(s) in the 15-mer sequence on the hybridization efficiency, three 1a/1b α-probes were synthesized with 1, 3, and 5 ferrocenyl groups at the 5′-end of the 1a/1b sequence. The DELFIA signal was found to strongly decrease as the number of ferrocenes increased with a complete abrogation of hybridization when 5 ferrocenes were in the sequence. This experiment allowed us to validate the good chemical synthesis of the different probes before testing them in electrochemistry. Finally, taking into account the ELOSA results, the probe containing 5 ferrocenes was not tested further in electrochemistry.

Application in a HCV Biological Setting: Study of the Optimal Number of Ferrocene Groups Allowing Maximum Duplex Stability by Cyclic Voltammetry (CV). The assays were performed with HCV 1a/1b and 3a sequences, since these genotypes are the most frequent in France. Considering the ELOSA findings of the α-probe containing five ferrocenes strongly destabilizing the hybridization event, experiments were conducted with α HCV 1a/1b linear 5′tetrathiolated probes containing 1 or 3 ferrocenes at the 3′-end (Figure 6A). In agreement with the destabilizing ability of ferrocene on hybridization, our first experiments using the above ferrocenyl α-probes (Figure 5A) with the thiol groups close to the ferrocene residues at the 5′-end were not sensitive enough likely due to the ferrocene environment at the single strand level and at the duplex level being too close despite the presence of the T4 spacer (data not shown). The influence of the number of ferrocenes grafted to the 1a/ 1b tetrathiol α-probe on the electrochemical assay was studied (Figure 6B). Tests at different concentrations of the DNA targets 1a/1b and 3a were performed to study the signal variation upon hybridization and to determine the detection limit. We observed that 3 ferrocenes grafted to the probe (3Fc) improved the sensitivity of the assay. The experiment run with the 3-ferrocene tetrathiol α-probe and the complementary target at 10 fM gave a positive response upon hybridization, with a signal above the detection threshold (0.05) determined as the average value of 4 negative assays plus 3 times the standard deviation. A signal variation superior to this value was considered as a positive response. The same assay run with the 1Fc probe at 10 fM was not sensitive enough; however, at 100 fM, a high signal was observed. These data confirm the great interest of increasing the number of ferrocenes grafted to the probe for achieving a sensitive electrochemical assay. It is worth noting that tests were performed in a complex medium made with nonspecific DNA to avoid false positive results. The response of our system does not appear to be quantitative, since F



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Figure 6. (A) Structure of 1a/1b and 3a α-probes with the tetrathiol linker at the 5′-end and 1 or 3 ferrocene residues at the opposite extremity. (B) Electrochemical sensitivity of 1a/1b probes as a function of the number of ferrocenyl groups. Hybridization assays were performed with 10 pM, 100 fM, and 10 fM concentrations of 1a/1b and 3a targets and recorded by DPV, on gold electrodes functionalized with the 1a/1b tetrathiol probe modified with 1 or 3 ferrocenes (Fc). (C) Comparison between 1a/1b and 3a genotype-specific probes: hybridization assays were performed with 100 fM, 10 fM, and 1 fM concentrations of 3a and 1a/1b targets and recorded by DPV, on gold electrodes functionalized with the 1a/1b and 3a tetrathiol probes modified with 3 ferrocenes (Fc).

was noted with the noncomplementary target. In conclusion, overall, these data allow us to conclude that the detection limit reached with the electrochemical sensor was 10 fM. This very good detection limit is the result of the experimental design and optimization of the probes. The best structure was with a tetrathiol linker at the 5′-end and 3 ferrocenes bound to the phosphate internucleosidic linkage of the α-sequence at the 3′ end.

the assay achieved at 10 pM of target gives a similar or lower variation of signal than the one at 100 fM. Our hypothesis is that, at the high concentration of 10 pM of target, an electrostatic effect of the negatively charged target induces a slight increase of current that counters the decrease due to the binding. We have previously reported this phenomenon in a study on ferrocenyl alpha oligonucleotides as probes used in solution.50 A second experiment was performed to validate the specificity of the assay (Figure 6C). The two α-probes 1a/1b and 3a containing 3 ferrocenes were grafted on the gold electrode of the sensor and target concentrations from 100 fM to 1 fM were tested. Hybridization with the complementary target led to a positive DPV signal variation for 100 fM and 10 fM. No positive signal was visualized at 1 fM. No considerable change in the DPV signal (variation inferior to 0.05 in ratio)

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CONCLUSION The polythiolated probes developed in this study displayed an increased sensitivity in both in vitro ELOSA on maleimideactivated plates and electrochemical assays on gold electrodes. The detection limit was greatly improved. Long synthetic targets (105-mers) were detected by ELOSA at a concentration G



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of 10 pM. This system allowed the genotyping of long generic HCV amplicons (401nt) diluted 100 times, corresponding to a mean concentration of 10 nM, and our system does not require the use of heminested or nested PCR. A detection limit of 10 fM with 105nt synthetic targets was reached by the use of the electrochemical sensor in a complex medium with good reproducibility. These preliminary data demonstrate that the sensitivity of electrochemical detection is 1000 fold higher than that obtained by optical ELOSA assays. The common polythiol linker of the probes provides an unprecedented improvement to the performances of both assays. Future perspectives will concern developing a multiplexed format for the electrochemical device to enable the execution of numerous assays in parallel with real-time and direct detection. These robust polythiolated probes allow us to envisage developing flexible and highly sensitive platforms dedicated to viral screening or genotyping. This study represents a key preliminary step before the integration of these innovative tools in miniaturized format, which will open up the pathway toward a future micro/ nanoscale lab-on-chip.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +33 (0)4 67 14 49 61 (F.M.), +33 (0)4 37 42 35 57 (C.C.), +33 (0)4 67 61 64 56 (C.F.-W.). Fax: +33 (0)4 67 04 20 29 (F.M.), +33 (0)4 67 61 64 57 (C.F.-W.). E-mail: [email protected] (F.M.), [email protected] (C.C.), [email protected] (C.-F.-W.). Notes

The authors declare no competing financial interest. ○ The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.

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ACKNOWLEDGMENTS This work was supported by the French “Agence Nationale pour la Recherche” (ANR-09-PIRI-0023 VirProbe), LyonBioPole and Eurobiomed. The authors thank the LAAS-CNRS for providing the gold material used for the electrodes. F.M. is a member of Inserm.

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Development of Innovative and Versatile Polythiol Probes for Use on

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