Direct Electrocatalytic mRNA Detection Using PNA-Nanowire Sensors

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Anal. Chem. 2009, 81, 612–617

Direct Electrocatalytic mRNA Detection Using PNA-Nanowire Sensors Zhichao Fang and Shana O. Kelley* Leslie Dan Faculty of Pharmacy, Department of Pharmaceutical Sciences, and Faculty of Medicine, Department of Biochemistry, University of Toronto, Ontario M5S 3M2, Canada We report an electrochemical nucleic acids sensing system that exhibits high sensitivity and specificity when challenged with heterogeneous samples of RNA. The platform directly detects specific RNA sequences in cellular and clinical samples without any sample labeling or PCR amplification. The sensor features an electrode platform consisting of three-dimensional gold nanowires, and DNA or RNA hybridization is detected using an electrocatalytic reporter system. In this study, probes made of peptide nucleic acid (PNA) are used to detect a newly identified cancer biomarkersa gene fusion recently associated with prostate cancer. The system is able to detect the fusion sequence with 100 fM sensitivity, and retains high sensitivity even in the presence of a large excess of non-complementary sequences. Moreover, the sensor is able to detect the fusion sequence in as little as 10 ng of mRNA isolated from cell lines or 100 ng total RNA from patient tissue samples. The PNA-nanowire nucleic acids sensor described is one of the first electrochemical sensors to directly detect specific mRNAs in unamplified patient samples. Because of the growing demand for tools for bioanalysis, many different types of sensors have been developed for the detection of biomolecular targets. Biosensors convert biological reactions into physicochemical readouts and are important tools for clinical diagnostics and the biological sciences.1 As nucleic acids are important biomarkers for disease diagnosis and prognostic profiling, their detection has been targeted as a critical direction of biosensing development.2 DNA or RNA detection via complexation of a target sequence to solid support modified with a complementary sequence is widely used in various types of biosensors, including those providing optical,3-9 gravimetric,10 piezoelectric,11 and electrical or electrochemical12-22 readouts. Electrochemical sensors have long been viewed as particularly attractive for bioanalysis because of their high sensitivity, high selectivity, high speed, low detection limit, low cost, and ease of use.17 While several techniques exist for the quantitative electrochemical detection of DNA,23-30 most of them rely on multistep sandwich assays that measure a signal change due to the hybridization of an unmodified target to a probe sequence * To whom correspondence should be addressed. E-mail: shana.kelley@ utoronto.ca. (1) Scheller, F. W.; Wollenberger, U.; Warsinke, A.; Lisdat, F. Curr. Opin. Biotechnol. 2001, 12, 35–40. (2) Baker, M. Nat. Biotechnol. 2006, 24, 931–937.

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immobilized on the electrode surface. In addition, most electrochemical biosensors can only detect short synthetic DNA sequences with detection limits in the nanomolar range. Moreover, some of these biosensing devices rely on a “signal-off” approaches that increase the risk of false positives. The sensitivity of the “signal-on” devices that do exist can be limited by high background signals. These deficiencies are highlighted by the lack of systems that can detect nucleic acids sequences in heterogeneous samples, where very high sensitivity and the ability to discriminate closely related sequences is essential. (3) Jin, R.; Cao, Y. C.; Thaxton, C. S.; Mirkin, C. A. Small 2006, 2, 375–380. (4) Fabris, L.; Dante, M.; Braun, G.; Lee, S. J.; Reich, N. O.; Moskovits, M.; Nguyen, T.-Q.; Bazan, G. C. J. Am. Chem. Soc. 2007, 129, 6086–6087. (5) Feng, C. L.; Zhong, X. H.; Steinhart, M.; Caminade, A.-M.; Majoral, J. P.; Knoll, W. Small 2008, 4, 566–571. (6) Goodrich, T. T.; Lee, H. J.; Corn, R. M. J. Am. Chem. Soc. 2004, 126, 4086–4087. (7) Erickson, D.; Liu, X.; Krull, U.; Li, D. Anal. Chem. 2004, 76, 7269–7277. (8) Steemers, F. J.; Ferguson, J. A.; Walt, D. R. Nat. Biotechnol. 2000, 18, 91–94. (9) Willner, I.; Cheglakov, Z.; Weizmann, Y.; Sharon, E. Analyst 2008, 133, 923–927. (10) Mannelli, I.; Minunni, M.; Tombelli, S.; Wang, R.; Spiriti, M. M.; Mascini, M. Bioelectrochemistry 2005, 66, 129–138. (11) Wu, V. C. H.; Chen, S. H.; Lin, C. S. Biosens. Bioelectron. 2007, 22, 2967– 2975. (12) Zeglis, B. M.; Barton, J. K. Nat. Protoc. 2007, 2, 357–371. (13) Xiao, Y.; Qu, X.; Plaxco, K. W.; Heeger, A. J. J. Am. Chem. Soc. 2007, 129, 11896–11897. (14) Wang, J.; Kawde, A.-N.; Jan, M. R. Biosens. Bioelectron. 2004, 20, 995– 1000. (15) Zhang, Y.; Kim, H.-H.; Heller, A. Anal. Chem. 2003, 75, 3267–3269. (16) Lapierre, M. A.; O’Keefe, M.; Taft, B. J.; Kelley, S. O. Anal. Chem. 2003, 75, 6327–6333. (17) Drummond, T. G.; Hill, M. G.; Barton, J. K. Nat. Biotechnol. 2003, 21, 1192–1199. (18) Katz, E.; Willner, I. Electroanalysis 2003, 15, 913–947. (19) Park, S. J.; Taton, T. A.; Mirkin, C. A. Science 2001, 295, 1503–1506. (20) Lapierre-Devlin, M. A.; Asher, C. L.; Taft, B. J.; Gasparac, R.; Roberts, M. A.; Kelley, S. O. Nano Lett. 2005, 5, 1051–1055. (21) Gasparac, R.; Taft, B. J.; Lapierre-Devlin, M. A.; Lazareck, A. D.; Xu, J. M.; Kelley, S. O. J. Am. Chem. Soc. 2004, 126, 12270–12271. (22) Hahm, J.-I.; Lieber, C. M. Nano Lett. 2004, 4, 51–54. (23) Kerman, K.; Saito, M.; Tamiya, E. Anal. Bioanal. Chem. 2008, 379, 1–7. (24) Won, B. Y.; Yoon, H. C.; Park, H. G. Analyst. 2008, 133, 100–104. (25) Aoki, H.; Tao, H. Analyst 2007, 132, 784–791. (26) Kerman, K.; Vestergaard, M.; Nagatani, N.; Takamura, Y.; Tamiya, E. Anal. Chem. 2006, 78, 2182–2189. (27) Liu, J.; Tiefenauer, L.; Tian, S.; Nielsen, P. E.; Knoll, W. Anal. Chem. 2006, 78, 470–476. (28) Raymond, F. R.; Ho, H. A.; Peytavi, R.; Bissonnette, L.; Boissinot, M.; Picard, F. J.; Leclerc, M.; Bergeron, M. G. BMC Biotechnol. 2005, 5, 10. (29) Macanovic, A.; Marquette, C.; Polychronakos, C.; Lawrence, M. F. Nucleic Acids Res. 2004, 32, e20. (30) Ozkan, D.; Kara, P.; Kerman, K.; Meric, B.; Erdem, A.; Jelen, F.; Nielsen, P. E.; Ozsoz, M. Bioelectrochemistry 2002, 58, 119–126. 10.1021/ac801890f CCC: $40.75  2009 American Chemical Society Published on Web 12/16/2008

Scheme 1. (A) Schematic Illustration of Electrocatalytic Detection of Nucleic Acids Using Nanowire Sensors, (B) Representative Scanning Electron Micrograph (SEM) of Gold Nanowires Used in This Studya, and (C) Sequences of Probes Complementary to TMPRSS2 (P2), ERG (P3), or the TMPRSS2:ERG Fusion (P1)

a

Scale Bar ) 100 nm.

Here, we report an electrochemical “signal-on” approach that permits the detection of medically relevant RNAs in complex biological samples (Scheme 1). The approach uses peptide nucleic acids (PNAs) coupled with an electrochemical assay and a nanoscale electrode platform. This system uses two redox reporter groups for readout, Ru(NH3)63+, a DNA-binding and cationic electron acceptor, and Fe(CN)63-, an anionic electron acceptor. Ru(NH3)63+ is attracted by the DNA film at the electrode surface through electrostatic interactions with the negatively charged phosphate backbone. During a negative potential sweep, Ru(III) is reduced and regenerated by the Fe(III) oxidant for multiple turnovers. The multiple redox cycles of Ru(III) amplifies the reduction signal, thus reporting on the amount of hybridization at the electrode surface. Previously, we showed that ultrasensitive nucleic acids detection with pM sensitivity could be achieved when this electrocatalytic reporter system was used in conjunction with DNA probes displayed on nanowire surfaces.16,20,21 However, this system proved unable to assay complex mixtures of sequences and therefore was only useful in a model environment. We therefore sought to improve this method, and to do so, investigated PNA probes, which are uncharged and generate low background signals. Indeed, introducing this material into the assay yields even higher levels of sensitivity and does so even in the presence of a high background of non-complementary sequences. EXPERIMENTAL SECTION Chemical and Materials. DNA sequences were obtained from the Centre for Applied Genomics in the Hospital for Sick Children (Toronto, Canada). PNA was obtained from Biosynthesis Inc. (U.S.A.). 1,1′-Carbonyldiimidizole, 1,6-hexamethylenediamine, 99.8% anhydrous 1,4-dioxane, N-succinimidyl 3-(2-pyridyldithio)propionate,dithiothreitol,6-mercapto-1-hexanol(97%MCH),hexaamineru-

thenium chloride, potassium ferricyanide, and potassium ferrocyanide trihydrate were purchased from Sigma-Aldrich Canada Ltd. Electrochemical Measurements. All cyclic voltammetry (CV) measurements were conducted at room temperature with a Bioanalytical Systems Epsilon potentiostat at a scan rate of 100 mV/s. Differential pulse voltammetry (DPV) signals were measured using a potential step of 5 mV, pulse width of 25 ms, pulse period of 100 ms, pulse amplitude of 50 mV; these conditions are equivalent to a scan rate of 50 mV/s. A three-electrode configuration was used consisting of a modified gold working nanowire electrode, a platinum wire auxiliary electrode, and an Ag/AgCl reference electrode. Electrocatalytic currents were measured in solutions of 10 µM Ru(NH3)63+ and 1 mM Fe(CN)63- in 25 mM sodium phosphate (pH 7), and 25 mM NaCl (any deviations from these conditions are described in individual figure captions.) Stable signals were obtained after 2 scans, and thus the second scan was used for quantitation in all experiments. Cathodic charge (Q) was quantitated by integrating background-subtracted voltammograms. Signal changes corresponding to hybridization were calculated as follows ∆Q (%) ) [((Qfinal - Qinitial)/Qinitial) × 100]. Error bars shown on individual figures correspond to standard error collected from multiple independent trials of each experiment. Preparation of Nanowire Electrodes. Electrodes composed of three-dimensional gold nanowires were fabricated by templated electroless deposition within a polycarbonate membrane and were characterized and optimzed as described previously.21,31 Au nanowires about 200 nm in length and 10 nm in diameter were exposed from the membrane surface after oxygen plasma etching (31) Menon, V. P.; Martin, C. R. Anal. Chem. 1995, 67, 1920–1928.

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for 150 s. The scanning electron microscope (SEM) image (Scheme 1B) shows a representative field of the nanowires used in these experiments The nanowire electrodes generated for different experiments were scanned in a solution containing of 3 mM Ru(NH3)63+ in 25 mM sodium phosphate (pH 7), and 25 mM NaCl by CV to confirm that the working electrode area remained constant for different electrodes. Preparation and Purification of Oligonucleotides. DNA oligonucleotides modified on the 5′-terminus with a hexanediamine-based linker were prepared and purified as described previously.32 All oligonucleotides were stringently purified using reversed-phase HPLC. The following probe and target sequences were used in experiments employing synthetic oligonucleotides. DP1 (DNA probe): SH-5′ ATA AGG CTT CCT GCC GCG 3′ CT . P1 (PNA probe-1): NH2-Cys-Gly- ATA AGG CTT CCT GCC GCG CT -CONH2. P2 (PNA probe-2): NH2-Cys-Gly- TCA ATA TGA CCT GCC GCG CT -CONH2. P3 (PNA probe-3): NH2-Cys-Gly- ATA AGG CTT CCT TGA TAT GA -CONH2. T1 (TMPRSS2:ERG target): 5′ AGC GCG GCA GGA AGC CTT AT3′. T2 (TMPRSS2 target): 5′ AGC GCG GCA GGT CAT ATT GA3′. T3 (ERG target): 5′ TCA TAT CAA GGA AGC CTT AT3′. T4 (non-complementary target): 5′ TTT TTT TTT TTT TTT TTT TT3′. Oligonucleotides were quantitated by measuring 260 nm absorbance using extinction coefficients calculated from the Integrated DNA technologies Web site (http://www.idtdna.com/ analyzer/Applications/OligoAnalyzer/). Preparation of mRNA and Total RNA Targets. mRNA containing the TMPRSS2:ERG fusion was extracted from the VCaP cell line with the Dynabeads mRNA Direct Kit (Invitrogen, U.S.A.). mRNA from MRC-5 and HeLa cell lines was extracted with the same kit and used as negative controls. Approximately 200 ng mRNA was obtained from 3 million cells, and 10 ng was applied to an electrode for analysis. Total RNA from prostate tumor frozen tissue was provided by Dr. Jeremy A. Squire of the Princess Margaret Hospital (Toronto, Ontario), which was extracted using the Trizol (Invitrogen, U.S.A.) protocol followed by DNase treatment. Approximately 10 ug RNA was obtained from ∼50 mg tumor tissue, and 100 ng was applied to an electrode for analysis. Modification of Nanowires with Probe Sequences for Electrochemical Measurements. Single-stranded thiolated DNA probes were immobilized on nanowire electrodes in solution containing 5 µM SH-DNA, 500 nM MCH, 25 mM sodium phosphate (pH 7), 25 mM NaCl, and 50 mM MgCl2 in a dark humidity chamber at room temperature for 15 min. To denature the single-stranded thiolated PNA probes before immobilization, they were heated at 55 °C for 10 min. They were then applied onto nanowire electrodes in solution containing 10 µM SH-PNA, 1 µM MCH, 25 mM sodium phosphate (pH 7), and 25 mM NaCl in a dark humidity chamber at room temperature overnight. Following deposition, electrodes were rinsed in 25 (32) Taft, B. J.; O’Keefe, M. O.; Fourkas, J. T.; Kelley, S. O. Anal. Chim. Acta 2003, 496, 81–91.

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mM sodium phosphate (pH 7), 25 mM NaCl buffer. MCH is added in the immobilization solution to (i) achieve an optimal probe film density and (ii) reduce non-specific adsorption. The adsorption of DNA on the nanowires was confirmed by monitoring the attenuation of the electrochemical signal obtained from a solution of 2 mM ferrocyanide in 25 mM sodium phosphate (pH 7) and 25 mM NaCl solution (data not shown). Electrochemical Detection of Target Hybridization. The electrocatalytic current obtained from nanowire electrodes modified with thiolated probe was measured as described above, the rinsed nanowire electrodes were then exposed to target sequences, and then hybridization was detected through enhancement of the electrocatalytic signal. Hybridization solutions typically contained target sequences in 25 mM sodium phosphate (pH 7), and 25 mM NaCl. 100 mM MgCl2 was added during hybridization when DNA probes were used. Electrodes were incubated at 37 °C in a thermostatted humidity chamber in the dark and were washed extensively with buffer before electrochemical analysis. The conditions used for individual experiments varied depending on the size and source of the target nucleic acid; details of different hybridization trials are provided in the figure captions. RESULTS AND DISCUSSION Investigation of the Effect of PNA Probes on Electrocatalytic Nucleic Acid Detection Sensitivity. We chose a target sequence for these studies with significant medical relevance: a prostate cancer-related gene fusion, recently discovered to be present in 50% of prostate cancer tumors and also thought to have promise as a prognostic factor.33,34 While conventional biomarkers for prostate cancer testing, like the prostate-specific antigen, have been successfully used to diagnose and monitor patients, they lack the ability to provide prognostic information quickly.35 The recently discovered gene fusions are highly significant biomarkers, therefore, as they not only serve as markers of prostate cancer, they can also aid in its classification and could someday guide treatment solutions. Typically, direct sequencing or fluorescent in situ hybridization (FISH) is used for gene fusion detection, and these techniques are difficult to use with high throughput. We endeavored to develop an assay that would allow fast, ultrasensitive detection of these sequences to further the study of gene fusions as medical indicators. To generate an assay with high sensitivity and specificity, we looked at a panel of probe sequences and types. We compared two different probe structures: one made of DNA which features a phosphodiester backbone, and one made of peptide nucleic acid (PNA), which has a pseudopeptide backbone.36-38 Unlike DNA, (33) Tomlins, S. A.; Rhodes, D. R.; Perner, S.; Dhanasekaran, S. M.; Mehra, R.; Sun, X.; Varambally, S.; Cao, X.; Tchinda, J.; Kuefer, R.; Lee, C.; Monite, J. E.; Shah, R. B.; Pienta, K. J.; Rubin, M. A.; Chinnaiyan, A. M. Science 2005, 310, 644–648. (34) Nam, R. K.; Sugar, L.; Yang, W.; Sricastava, S.; Klotz, L. H.; Yang, L.-Y.; Stanimirovic, A.; Encioiu, E.; Neill, M.; Loblaw, D. A.; Trachtenberg, J.; Narod, S. A.; Seth, A. Br. J. Cancer 2007, 97, 1690–1695. (35) Stephan, C.; Jung, K.; Lein, M.; Diamandis, E. P. Eur. J. Cancer 2007, 43, 1918. (36) Brandt, O.; Hoheisel, J. D. Trends Biotech. 2004, 22, 617–622. (37) Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.; Freier, S. M.; Driver, D. A.; Berg, R. H.; Kim, S. K.; Norden, B.; Nielsen, P. E. Nature 1993, 365, 566–568.

Figure 1. Electrocatalytic detection of synthetic DNA targets using (A) DNA-nanowire sensors and (B) PNA-nanowire sensors. Cyclic voltammetry (CV) was used to quantitate charge at electrodes exposed to different target/probe sequence pairs. (insets) Cyclic voltammograms illustrating electrocatalytic signals before (dotted line) and after (solid line) hybridization with 1 µM cDNA target (T1) under optimal conditions.21 When DNA probes were used, hybridization with T1 was induced by introducing a solution containing 100 mM MgCl2, 25 mM sodium phosphate (pH 7), and 25 mM NaCl at 37 °C for 30 min for DNA probes. When PNA probes were used, hybridization with T1 was conducted in 25 mM sodium phosphate (pH 7), and 25 mM NaCl buffer at 37 °C for 30 min. Error bars shown represent standard errors calculated from multiple trials.

PNA is a charge-neutral compound, but just like DNA and RNA, PNA can hybridize with complementary strands, forming righthanded, double-helical complexes according to Watson-Crick rules. PNA-DNA duplexes are more thermally stable than DNADNA duplexes, which improves the efficiency of capture of an oligonucleotide target. The fact that PNA is charge neutral has been used to great benefit in several electrochemical or electronic sensing assays that use changes in substrate electrostatics to read out the presence of a target sequence.23,27,28 We therefore hypothesized that this type of probe could make nanowire-based electrocatalytic nucleic acids detection even more sensitive and robust. Both DNA (DP1) and PNA (P1) probes featuring the sequence of the prostate cancer related fusion site were made (see Scheme 1); both contained thiols to facilitate immobilization on gold nanowires. In addition, a series of synthetic targets were made that permitted not only the sensitivity but also the specificity of the system to be analyzed. In addition to generating a target 100% complementary to the TMPRSS2:ERG splice site (T1), we also made 50% complementary targets corresponding to the sequences of the unspliced wild-type (wt) genes. The 20-nucleotide targets containing portions of the wt sequences of TMPRSS2 (T2) and ERG (T3) were made to allow the binding of half-complementary targets to be assessed, and in addition, a 20-mer of thymine nucleotides (T4) was made as a completely non-complementary target. When the DNA probe was immobilized on Au nanowires and challenged with the 100% complementary fusion target (T1) and the 50% complementary wt targets (T2 and T3), significant differences were observed in the magnitudes of electrocatalytic signals observed after hybridization (Figure 1A). T1 yielded a much larger signal change than T2 or T3; the half-complementary probe-target pairs yielded distinguishable but small signals compared to the T4 target that has 0% complementarity. Nonethe(38) Ratilaimen, T.; Holmen, A.; Tuite, E.; Nielsen, P. E.; Norden, B. Biochemistry 2000, 39, 7781–7791.

less, the assay exhibits good specificity and high levels of discrimination against sequences that are not completely complementary. When the PNA probe was substituted for the DNA probe, a number of changes were observed. First, the background signals observed at probe-only electrodes were significantly decreased (Figure 1B). This reflects the fact that the PNA probe is uncharged and therefore unable to facilitate electrocatalysis until a negatively charged target is bound. This background attenuation has a strong beneficial effect on the performance of the assay, as the suppression of the background signal amplifies the signal changes that are observed in the presence of the target. Here, instead of monitoring 50% signal changes, changes over 2000% are observed, which minimizes the risk of false negatives. The fact that the experimental error is similar for trials run with both DNA and PNA probes is similar, but the magnitude of the signal changes corresponding to hybridization increase by 40-fold, indicates that the signal-to-noise ratio improves for this device configuration. Moreover, the signals obtained with the half-complementary targets remained low, and the control target T4 did not produce any signal increase. Interestingly, the amount of hybridization observed between the PNA probe and T3 was suppressed relative to that observed with the analogous DNA probe. The fact that T3 gives a lower signal than T2 even though both targets have the same degree of complementarity with the probe indicates that another factor must be affecting the results. PNA probes typically form more dense monolayers than DNA probes (PNAs to form monolayers with 3-fold higher densities under the conditions used here16,25); this could account for the trend observed, as it would be more difficult for the target to penetrate the denser PNA monolayer to reach the buried complementary portion of P1. Detection Limit of PNA-Nanowire Sensors and Performance with Cellular RNA. Detection limits for electrochemical nucleic acids assays can vary over a wide range. While the majority are not able to detect targets at concentrations lower than nanomolar, there are several examples of methods that exhibit Analytical Chemistry, Vol. 81, No. 2, January 15, 2009

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Figure 2. Detection limit of PNA-nanowire sensors. (A) Detection limit in solutions containing only the target sequence. CV was used to quantitate the detection limit for PNA-nanowire sensors when the target sequence T1 was introduced in solution. Using a noncomplementary target (T4) to assess background levels, a positive signal change can be obtained down to 100 fM fully complementary target (T1). (B) Detection limit in solutions containing the target sequence and 0.05 mg/mL HeLa mRNA. Values shown represent currents measured over the background observed with HeLa RNA only. Hybridization performed under the same conditions as those described in Figure 1. Error bars shown represent standard errors calculated from multiple trials.

sensitivities to picomolar and femtomolar concentrations of targets.15,21,25 These are rare, and these levels of sensitivity have not been confirmed in heterogeneous samples with high background levels of non-complementary sequences. The electrocatalytic assay used here was originally found to have nanomolar sensitivity when used with bulk gold electrodes and DNA probes.16 When DNA-modified gold nanowires were used as an electrode platform instead, the sensitivity of the assay was significantly improved, with picomolar concentrations of synthetic targets detectable.21 However, attempts to use this system for detection in biological samples were not successful (Kelley and Fang, unpublished work). In the system reported here, modified only by changing probes from DNA to PNA molecules, we were able to obtain a detection limit for electrocatalytic detection of a synthetic target probe of 100 fM (Figure 2), a significant improvement over previous results.21 We then challenged the robustness of this detection limit by introducing a high background of non-complementary RNA isolated from the HeLa cell line, a cervical cancer cell line that does not contain the fused gene complementary to the probe. In the presence of 0.05 mg/mL of HeLa RNA, we were still able to reliabily detect 1 pM (7 pg/mL) concentrations of the complementary target. While the detection limit of our system did increase, a very high level of sensitivity was preserved. This level of sensitivity, coupled with outstanding specificity, indicates that this assay is appropriate for biological testing. Detection of RNA Targets from Cell Lines and Tissue Samples. The next step in testing the robustness of the assay was to determine if the gene fusion could be directly detected in RNA samples from cultured human cell lines. We acquired VCaP cells, a prostate cancer derived cell line that contains the gene fusion,33 and isolated mRNA. As a negative control, we used mRNA extracted from the HeLa cervical cancer cell line that lacks the gene fusion. As an additional negative control, we also tested mRNA from the lung fibroblast MRC-5 cell line (Figure 3). 616

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Figure 3. Electrocatalytic detection of sequence targets in cellular RNA. VCaP is the TMPRSS2:ERG gene fusion positive cell line; MRC-5 and HeLa are used as negative controls. Hybridization of the RNA samples of the RNA samples with the fusion probe (P1) was monitored, as was the hybridization of VCaP mRNA with the WT probes P2 and P3. Target solutions containing 10 ng mRNA, 25 mM sodium phosphate (pH 7), and 25 mM NaCl. Preparation of PNA films and hybridization were performed as described in Figure 1.

Comparison of the electrocatalytic signal changes for the three cell lines clearly showed a differential response for the fusionpositive and fusion-negative cells. The VCaP cell line, which bears the gene fusion, showed a much larger electrochemical response than either of the fusion-negative cell lines. As little as 10 ng of mRNA at a concentration of 1 ng/µL produced a positive result, again indicating that this sensing system has a high level of sensitivity even when presented with a complex mixture of sequences. We confirmed that the response we monitored was indeed specific by examining additional probes (Figure 3). Probes complementary to the TMPRSS2 gene (P2) and complementary to the ERG gene (P3) were monitored alongside the fusion probe. Both probes showed significantly smaller signal changes when challenged with VCaP mRNA, even though the fusion positive RNA contains a sequence half-complementary to both probes. Just as was observed with synthetic targets, the amount of hybridization with P3 was lower than with P2, further confirming the specificity of the gene-fusion specific signal readout with the corresponding probe (Figure 3). These results also demonstrate a need for a panel of probes for the correct identification of the fusion, as otherwise, overexpression of wild-type genes could confound the analysis. The ultimate application of the type of method described here is the analysis of patient samples to determine whether a tumor tissue sample contains the gene fusion. To explore this application, we tested total RNA samples isolated from prostate tumor tissue. PCR and sequencing of both samples revealed that the fusion gene was present in only one of the samples (data not shown). We again challenged our PNA-nanowire devices to analyze for the presence of the prostate cancer gene fusion and were able to detect a significant difference in the response of the sensor to the two samples (Figure 4). While the fusion negative RNA produced a small signal change corresponding to the levels obtained with the negative controls shown in Figure 3, the fusion positive RNA produced a signal change more than 6 times larger. These experiments, using a small amount of total RNA (100 ng), indicate that this system provides a means to detect specific RNA sequences in small, clinically relevant samplessa long-standing

Figure 4. Electrocatalytic detection of gene fusion in total RNA from tumor tissue samples. DPVs illustrating electrocatalytic signals before (dotted line) and after (solid line) hybridization with total RNA from tumor tissue samples. ∆Q was quantitated from CVs. Target solutions containing 100 ng total RNA, 25 mM sodium phosphate (pH 7), and 25 mM NaCl. Preparation of PNA films and hybridization were performed as described in Figure 1. Delta Q values listed on plots represent averages collected from multiple trials. Values in parentheses are standard errors.

challenge in the area of electrochemical biosensor development. The methodology here is competitive both in sensitivity and specificity with conventional FISH and sequencing approaches to fusion identification, but the analysis is much faster and amenable to automation. CONCLUSIONS The implementation of low-cost electrochemical sensors in clinical laboratories requires the generation of devices that are sensitive and selective. Here, we demonstrate that the use of (i) PNA probes, (ii) an electrocatalytic reporter system, and (iii) a nanowire electrode platform produce such qualities by suppressing background signals and effectively capturing low levels of analyte molecules. This approach is effective even in highly heterogeneous samples, which differentiates it from most other electrochemical approaches. Moreover, the assay was able to detect a specific

biomarker in tumor RNA, indicating that the selectivity is sufficient for use with clinical samples. ACKNOWLEDGMENT Financial support of this project was provided by Genome Canada, the Prostate Cancer Research Foundation, the Ontario Centres of Excellence, and NSERC. We thank Stuart Rae for preparation of the RNA samples from cell lines and Dr. Jeremy A. Squire of the Princess Margaret Hospital for providing the RNA samples from tumour tissue and useful information on prostate cancer related gene fusions.

Received for review November 20, 2008.

September

7,

2008.

Accepted

AC801890F

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