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Amplification Strategy Using Aggregates of Ferrocene-Containing Cationic Polythiophene for Sensitive and Specific Electrochemical Detection of DNA Patricia Harding Lepage,† Regis Peytavi,‡ Michel G. Bergeron,‡ and Mario Leclerc*,† †
Canada Research Chair on Electroactive and Photoactive Polymers, Departement de Chimie, Universite Laval, Quebec City, Quebec, Canada, G1V 0A6 ‡ Centre de recherche en infectiologie, Centre hospitalier universitaire de Quebec (Pavillon CHUL), Universite Laval, Quebec City, Quebec, Canada G1V 4G2
bS Supporting Information ABSTRACT: We report a new electrochemical amplification strategy for an ultrasensitive electrochemical detection of DNA sequences using aggregates composed of a water-soluble, ferrocene-functionalized polythiophene. A two-step hybridization is performed at one addressing surface with PNA capture probes whereas the electrochemical detection is done on an electrode nearby. Specific and quantitative detection of DNA targets with a detection limit of 4 10 16 M (about 4 zeptomoles or about 2500 copies of oligonucleotides) was achieved.
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any efforts have been made worldwide to develop and improve electrochemical methods for nucleic acid analyses since these technologies are amenable to portable and affordable devices for point-of-care applications. The goal behind these studies is to develop fast, simple, and cost-efficient methods for evaluating specific molecular hybridization of label-free DNA. Despite many advances in this field, there still is a challenge of finding new approaches to improve the selectivity and sensitivity of the DNA detection platform detection to respond to the demands and needs for modern biomedical research applications and medical diagnostics. Strategies based on conjugated polymers for the transduction of hybridization events into an electrical signal have been reported.1 17 In most cases, the detection relies on a modification of the electrical properties of an oligonucleotide-functionalized conjugated polymer following hybridization with its complementary nucleic acid target.18 The main drawbacks of these approaches are (1) a low signal-to-noise ratio caused by the steady presence of the polymer adsorbed onto the substrate and (2) a decrease of the electrical signal after specific recognition of the target.19 Cationic polythiophenes offer unique DNA recognition capabilities when complexed in aggregates.20 27 Moreover, cationic polymers are well-known to spontaneously assemble with DNA through electrostatic interactions to yield polyelectrolyte complexes. This process is influenced by different variables such as pH, temperature, and ionic strength. The effects of these parameters on polycation/polyanion interactions are relatively well understood28 and abrupt changes of these parameters can be used to promote the dissociation of polyelectrolyte assemblies “on demand”.29,30 r 2011 American Chemical Society
Along these lines, we have previously reported different solid state electrostatic methods based on neutral peptide nucleic acid (PNA) capture probes and an electroactive, cationic, watersoluble polythiophene transducer bearing one ferrocene (Fc) moiety per monomer unit. The use of PNA probes immobilized onto a solid support by self-assembly is of great use to investigate a sample containing different DNA targets as its neutral skeleton does not bind cationic polythiophene and unbound DNA species can easily be discarded by rinsing steps following hybridization. This also allows the deposition of distinct probes at different locations in close proximity, opening the door to multiplex detection. These simple and rapid methodologies have enabled the detection of 50 nanomoles of unmarked DNA targets31 and the detection of 75 femtomoles of human r-thrombin.32 Yet, like most electrochemical approaches, this level of sensitivity is not sufficient for developing a detection system without prior target amplification. Signal amplification can be performed by various ways. One direct approach is to increase the amount of immobilized capture probes and hence increase the electrical signal. Fang et al. reported that the use of gold nanoparticle-modified electrodes instead of a planar gold electrode led to a 103-fold amplification.33 Other strategies based on the attachment of a large number of reporters per hybridization event are probably the most promising. In addition, reported approaches involving silver enhancement,34 36 biometallization,37 gold nanoparticles,38 42 enzymatic amplification coupled to redox polymers,43 ferrocene-capped gold Received: March 21, 2011 Accepted: September 20, 2011 Published: September 20, 2011 8086
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Analytical Chemistry nanoparticle/streptavidin conjugates,44,45 biobarcode,46 49 and quantum dots48,50,51 have recently been at the center of numerous studies. On the basis of these considerations, we propose here a new electrochemical amplification strategy for the ultrasensitive detection of DNA using aggregates composed of water-soluble, ferrocene-functionalized polythiophene. The approach consists of a quantitative sandwich type assay in which DNA targets are captured by PNA probes and DNA/polymer aggregates act as transducers. The electrochemical detection is done on a nearby bare electrode after aggregates have been released from the PNA array and fragmented. A preconcentration process of the ferrocenyl groups is performed by square wave stripping voltammetry to further increase the sensor performance. This methodology exploits the unique combination of the large number of electroactive moieties reporting each hybridization event, indebted to the polymeric nature of our transducer and the DNA recognition capabilities of cationic polythiophenes
Figure 1. Chemical structure of polymer 1.
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in the aggregate complex. It combines the sensitivity and ease of electrochemical detection methods to the greater hybridization affinity of PNA toward DNA thereby allowing detection of DNA targets with a detection limit of 4 zmol (2500 copies in 10 μL) along with an excellent selectivity against mismatched DNA.
’ RESULTS AND DISCUSSION In all experiments, a water-soluble cationic polythiophene bearing a ferrocene (Fc) substituent (Figure 1) was employed as the electrochemical mediator. As previously reported,31,32 a square-wave voltammetry (SWV) study of polymer 1 adsorbed on gold electrodes reveals two oxidation processes. The first oxidation peak at 490 mV versus Ag/AgCl is well-defined and attributed to the reversible oxidation of the ferrocene (Fc) group to ferrocenium (Fc+). A second weak and broad oxidation signal at 690 mV versus Ag/AgCl is related to the reversible oxidation of the polythiophene backbone. The polymer is not sequence specific, but when coupled to single-stranded DNA to form an electrostatic complex, it shows unique recognition properties. Therefore, Fc oxidation is used as an electroactive reporter of the hybridization process. Consequently, following the strategy depicted in Figure 2, PNA capture probes are first spotted on the surface of a microarray SuperAldehyde glass slide. Covalent coupling between PNA capture probes and the substrate occurs through
Figure 2. Detection strategy: the hybridization of a DNA target is followed by hybridization of a fluorescent and electroactive aggregated complex acting as reporter. The surface is then rinsed to remove any unbound species. After ultrasonication of the PNA surface, the resulting solution containing hybridized aggregates is collected and electrochemical detection is done on a nearby electrode by square wave stripping voltammetry. The current associated to oxidation of the ferrocenyl group is used to reveal hybridization. 8087
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Figure 3. Solid state fluorescence for PNA probes and perfect match Cy3-modified DNA targets hybridized for 1 h at 37, 40, and 45 °C in a 4, 6, and 8 PBS buffer. Three arrays (composed of two rows of three spots) of PNA capture probes were manually deposited on each microscope slide substrate. A self-sticking hybridization chamber was used to hybridize different targets; the capture probe is the same for all conditions. λEx = 550 nm; λEm = 570 nm; PMT/laser power: 100%.
Schiff base aldehyde amine chemistry. After probe grafting, the modified slide is incubated with a DNA target (49-mer) to allow recognition. After hybridization, the slide is carefully washed to remove unbound DNA species and dried. In parallel, DNA labeled oligonucleotides complementary to another region of the target (double recognition) are mixed in an equal charge ratio with the cationic conductive polythiophene bearing ferrocenyl groups to form neutral duplexes (coacervates). The slide is incubated with the mixture containing the duplexes. Due to the unique recognition capabilities of these duplexes, hybridization occurs only between the binary complex and its cDNA target bound to the PNA probe. If no DNA was hybridized on the first step, the PNA probe and the duplex will not bind since the complementary sequence is missing. After incubation, the slide is carefully washed to remove any unbound species. High selectivity is obtained with this method because we introduce a second hybridization process to discriminate false positives. In a subsequent step, the aggregates captured by the PNA probes are released from the surface and fragmented by ultrasonication in the presence of a chaotropic agent at an increased temperature. The supernatant fluid containing released fragments is collected and detected on a nonfunctionalized gold electrode by square-wave stripping voltammetry (SWSV). SWSV is a technique offering superior resolution and current sensitivity due to a combination of a preamplification step and a negligible contribution of charging current. The presence of an oxidation signal at 690 mV versus Ag/AgCl (saturated KCl) reference electrode31 (or at 590 mV versus a silver wire pseudoreference electrode) will confirm the presence of the polymer and, hence, occurrence of the hybridization between the target in aggregate form and the probe. The rationale behind the choice to release aggregates from the surface and perform detection on a nonfunctionalized electrode rather than addressing PNA probes can be explained by two reasons. Even though it was suggested that the conjugated
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Figure 4. Hybridization of 15-mers PNA probe with different 49-mer DNA targets; (a) perfectly complementary, (b) 3 mismatches, and (c) 15 mismatches. Each panel is composed of a microarray of 64 spots organized in eight rows of eight spots. Each pair of rows was printed by two print-tips mounted in parallel on the array printer. PNA capture probe is the same for all conditions. Hybridization was performed for 1 h at 22 °C in (6 SSPE/0.1%PVP/30% formamide) buffer. λEx = 550 nm; λEm = 570 nm; PMT/laser power: 100%. Raw fluorescence data are available in the Supporting Information.
polymer backbone could play a role in electron shuttling from a remote electroactive moiety to the electrode,32 the electron shuttling loses its efficiency as the distance between the redox center and the electrode surface increases. Therefore, only ferrocenyl groups located in the close vicinity of the electrode would generate an oxidation current and the advantage of having a large number of redox reporters in the aggregate would be lost. In addition, the current that can be collected by an electrode is limited by the chemical passivation or fouling of its surface. The tethered PNA probes would consequently limit the sensibility and also affect reproducibility. To optimize this strategy, different hybridization buffers commonly reported in the literature were studied to determine the most appropriate medium for the two distinct hybridization steps of our system. For the hybridization between 49-mer DNA target with the 15-mer PNA probe, the first buffer studied was phosphate-buffered saline (PBS). Tested concentration varied from 4 to 8 at temperatures ranging between 37 and 45 °C. A Cy3-modified DNA target was used to monitor the hybridization process by solid state fluorescence. Figure 3 shows that hybridization of a perfectly matched DNA target occurs at 37 °C regardless of the ionic strength of the solution. More importantly, it shows that this short double helix is highly sensitive to temperature as it is not stable over 37 °C. It is not unusual for short nucleotides strands to exhibit low melting points as the short double helix possesses a limited number of noncovalent stabilizing interactions in comparison to a double helix composed of longer strands. The temperature dependence and insensibility to stringency confirms that probe/target interactions are governed by specific Watson Crick base pairings rather than electrostatic binding. We therefore decided to use a buffer composed of 6 SSPE, 0.1% PVP, and 30% formamide, a highly stringent composition allowing specific hybridization at room temperature52 8088
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Figure 6. Fluorescence intensity observed for detection of 4 10 9 M different single-strand DNA targets by double hybridization using SWSV. (a) Perfectly matched target, (b) noncomplementary oligonucleotide target, and (c) negative control (hybridization solution with no DNA). λEx = 550 nm; λEm = 570 nm; PMT/laser power: 100%. Error bars represent standard deviation of the mean for an array of 64 spots. Values biased by more than 2SD were not considered for statistical analysis. Figure 5. Double hybridization of 15-mer PNA probe with a 49-mers DNA target exhibiting (a) 0 mismatch (b) 3 mismatches, and (c) 15 mismatches. Each panel is composed of a microarray of 64 spots organized in eight rows of eight spots. Each pair of rows was printed by two print-tips mounted in parallel on the array printer. PNA capture probe is the same for all conditions. The first hybridization step was performed for 1 h at 22 °C in (6 SSPE/0.1%PVP/30% formamide) buffer. The second hybridization step was performed for 1 h at 37 °C in 0.3 mM Triton X100. λEx = 550 nm; λEm = 570 nm; PMT/laser power: 100%.
(see Figure 4). We observed specific hybridization (differentiation between perfectly complementary and 15-mismatch targets) but poor discrimination of triple nucleotide polymorphisms. The difference of fluorescence intensity between each pair of rows originates from intrinsically known limitations of the microarray technology. Principal sources of spot intensity variability within and between arrays are the location on the array and printing process variation. Nonuniformity of the coating over the slide surface and the fact that microarray slides are not anatomically flat are the factors impacting the most data precision. In this study, differences between the two print-tips mounted in parallel on the array printer also caused differences between rows of the same condition. For the second hybridization step, the first buffer used was PBS. After incubation at 37 °C, nonspecific adsorption was observed but no hybridization occurred. It is believed that the preaggregation step between DNA and the cationic polymer modifies the electrostatic properties of the complex which in turn modifies its ability to hybridize with PNA. Meanwhile, Bazan et al.53 showed that Tween 20, a surfactant, can help hybridization between PNA and DNA. In our hands, the use of 10% Tween 20 as the hybridization buffer permitted molecular hybridization but little improvement of mismatch discrimination. Increasing the stringency by lowering the percentage of Tween 20 to 5% reduced the hybridization efficiency and increased the noise. The use of Triton X-100 reduced solution, a nonionic surfactant, as the hybridization buffer provided more intense and specific signals. Triton X-100 not only provides an effective shielding effect but also solubilizes polymer/ssDNA aggregates complexes which facilitate hybridization with PNA/ssDNA
complexes. The optimal concentration of Triton was found to be 0.3 mM. We believe partial hydrophobicity of Triton X-100 alters the balance between competitive electrostatic and hydrophobic forces, which in turn affects the stability of complexes participating in the hybridization process (PNA/ssDNA probe target pair, polymer/ssDNA aggregated transducer, and PNA/dsDNA/polymer). At lower Triton concentration, negative charges of PNA/ ssDNA probe target complexes are incompletely screened in the absence of salts. Electrostatic interactions favor aggregates adsorption rather than specific hybridization. At higher concentrations, Triton molecules are present in sufficient quantity to start competing with ssDNA to associate with the polymer, which is promoted by their common partial hydrophobic nature. Since polymer/ssDNA stability originates from electrostatic forces (ssDNA acting as a counterion for polymer cationic charges), the insertion of Triton molecules in the aggregate structure (and consequent increase of polymer/ssDNA intermolecular distances) destabilizes the aggregates structure and eventually ruptures. Specific DNA recognition by aggregates occurs only if their structure remains intact. The density of surface probes was optimized by varying the concentration of the PNA in the grafting solution. The concentration yielding the optimal S/N ratio and discrimination against mismatches is 0.75 μM. On the basis of these results, Figure 5 presents the solid state fluorescence observed after double hybridization of PNA with different 49-mer DNA targets in 0.3 mM Triton X-100. In the presence of a complementary target, the spots exposed to Cy3-aggregates yield a significant fluorescence signal (Figure 5a). Two control experiments were done to verify the specificity of detection. A target containing 3 mismatches generated little fluorescence intensity under the same conditions (Figure 5b) while a nonspecific target containing 15 mismatches generated no signal (Figure 5c). The absence of cross-hybridization reveals a good specificity of detection. In the final step, the aggregates captured by the DNA target hybridized to PNA probes have to be released from the surface and fragmented (see Figure 2). To optimize the conditions to 8089
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Figure 7. Current observed for detection of 4 10 9 M different single-stranded DNA targets by double hybridization using SWSV. (a) Perfectly matched target, (b) noncomplementary oligonucleotide target, and (c) negative control (hybridization solution with no DNA). The figure does not include error bars; each waveform is a single reading of supernatants collected over each 64-spot array presented in Figure 6.
induce a release of the bound aggregates, we studied the influence of ultrasonication, as well as the use of a chaotropic salt on the aggregate size by dynamic light scattering (DLS). With a mean diameter of approximately 1100 nm and a width of about 260 nm, the volume of the polymer/Cy-3 DNA aggregates is estimated to be 5.6 109 nm3. Therefore, these data suggest that aggregates contain approximately 40 polymer strands. Considering that the polymer chain is composed of approximately 10 monomer units, this corresponds to approximately 400 ferrocenyl groups per aggregate, i.e., a possible amplification factor of 400 per hybridization event. Additional DLS experiments have revealed that the mean diameter of aggregates suspended in a 0.5 M phosphate buffer is reduced by 30% after 30 min of ultrasonication. Moreover, the transfer of aggregates to a 0.1 M lithium perchlorate (LiClO4) solution induce a 70% reduction of mean diameter compared to that observed in the biological buffer. When combined with ultrasonication, LiClO4 further reduces the mean diameter of aggregates by 80%. The use of a more concentrated LiClO4 solution (1M) induced the precipitation of aggregates by a salting-out effect. We then believe that, in combination with ultrasonication and high temperatures, the increase of ionic character of the solution containing aggregates introduces destabilizing interactions that promote the dissociation of the polymer from DNA, leading to the disruption of the binary complex. Therefore, the aggregates captured by the DNA target hybridized to the PNA probes were released from the surface and fragmented by ultrasonication in 0.1 M LiClO4 at 65 °C. The supernatant fluid containing released fragments was collected and detected on a nonfunctionalized gold electrode by SWSV. The presence of an oxidation signal at 590 mV versus a silver wire pseudoreference electrode confirmed the presence of the polymer and, hence, that the hybridization between the aggregates used as the reporter and the DNA target did occur. In order to determine how the optical and electrochemical methods were correlated, simultaneous optical and electrochemical detections were performed. SuperAldehyde microarray slides were selected as substrate for their superior covalent coupling efficiency through aldehyde surface chemistry. PNA capture probes were spotted on glass slides substrate using an arrayer to obtain reproducible spots. Each slide was composed of three microarrays of 64 spots organized in eight rows of eight spots printed by two print-tips mounted in parallel on the array printer. PNA capture probe was the same for all conditions;
self-sticking hybridization chambers were used to hybridize different targets. It should be noted that this slide configuration could easily be adapted for multiplexing using different capture probes for each array. After incubation, the surface was monitored using solid state fluorescence to reveal hybridization and discrimination of mismatched targets. The same slide was then submitted to the release and fragmentation of bound aggregates. After collection and concentration of aggregates on the gold electrode, electrochemical measurements were performed, and the two results were compared. In order to compare numerical results from different slides, experimental conditions were printed side by side with a negative control on the same array. In order to compensate for variations occurring over the course of the print-run, the fluorescence intensity of each condition was determined by computing the mean of all spots for that condition, excluding data point biased by more than 2SD. Experimental results were then normalized against the negative control. This method allows discriminating bias arising from experimental conditions from bias originating from the printing process. The results obtained for the detection of 10 9 M oligonucleotide targets by the double hybridization scheme as revealed by solid state fluorescence (Figure 6) are in relatively good agreement with the obtained electrochemical signals (Figure 7), which suggests that the electrochemical detection correctly reflects the behavior observed by fluorescence. The difference in S/N ratio between Figures 6 and 7 is believed to result from easily oxidized optically inactive residues detached from the aldehyde-functionalized glass surface during the release and fragmentation step. The ability of SWSV to concentrate and detect trace amounts of materials makes it highly sensitive to impurities in the sample matrix. Since our microarray scanner limits the detection of fluorescence signals to concentrations higher than 10 11 M, it has not been possible to compare the fluorescence and electrochemical responses over the full range of detection of our system. As expected, a good correlation between the Fc/Fc+ peak intensity and the single-strand DNA target concentration is observed (Figure 8). The intensity was logarithmically related to the target concentrations up to 10 12 M where it starts to plateau. The presence of a saturation regime suggests passivation of the working electrode by adsorbed species at high concentration. The specificity was evaluated with two negative controls, one containing a nonspecific ss-DNA target and another one exempt 8090
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’ ACKNOWLEDGMENT This work was supported by the Natural Sciences and Engineering Research Council of Canada and the Canada Research Chair Program. P. Harding Lepage thanks the GeorgesElie-Amyot Foundation for a Ph.D. scholarship. The authors . Madore, Dr. L. Bissonnette, acknowledge Dr. A. Najari, Dr. E and J.-L. Simard for technical assistance and helpful discussions. ’ REFERENCES
Figure 8. Double hybridization of targets: differential current intensity at the Fc/Fc+ oxidation peak measured by SWSV (after subtraction of the negative control) versus the logarithm of DNA target concentration. (a) Complementary aggregate and (b) nonspecific aggregate. The current change (a) is the subtraction of waveforms (Figure 7a,c) for all concentrations while series (b) is the subtraction of waveforms (Figure 7b,c). Error bars represent variability of the mean of n > 3 different slides. Considering a probed volume of 10 μL and detection limit of 4 10 16 M, an electrochemical detection of about 4 zeptomoles or about 2500 copies of oligonucleotides is therefore afforded.
of the target (Figure 7b,c). In these conditions, lower current is observed in comparison to the perfect match (Figure 7a). Nevertheless, these results demonstrate that the double hybridization method is sensitive and can discriminate a specific target from others.
’ CONCLUSION The sensitive and specific electrochemical detection of as few as zeptomoles of single-strand DNA (2500 copies in 10 μL) was attained with a new amplification strategy based on the use of electroactive aggregates and the superior sensitivity of absorptive stripping square wave voltammetry. This approach exploits the unique combination of the large number of electroactive moieties reporting each hybridization event indebted to the polymeric nature of our transducer and the excellent DNA recognition capabilities of cationic polythiophenes in the aggregate form. It therefore combines the ease of electrochemical detection methods to the greater hybridization properties of PNA toward DNA. This opens interesting possibilities for the future development of multiparametric, integrated, and portable electrochemical devices for diagnostics at point of care. ’ ASSOCIATED CONTENT
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Supporting Information. Details of materials, probe immobilization strategies, aggregates formation, hybridization of species, electrochemical measurements, microarray fabrication, and fluorescence measurements. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected].
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