Rapid, Sensitive, and Label-Free Impedimetric Detection of a Single

Apr 5, 2010 - Rapid, Sensitive, and Label-Free Impedimetric Detection of a Single-Nucleotide Polymorphism Correlated to Kidney Disease. Alessandra ...
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Anal. Chem. 2010, 82, 3772–3779

Rapid, Sensitive, and Label-Free Impedimetric Detection of a Single-Nucleotide Polymorphism Correlated to Kidney Disease Alessandra Bonanni, Martin Pumera, and Yuji Miyahara* International Center for Materials Nanoarchitectonics (MANA)/Biomaterials Center, National Institute for Material Science (NIMS), Ibaraki, Japan We present a protocol for the very rapid and sensitive detection of a specific mutation of the COL4A5 gene (exon 29, A-C mismatch) which was found in people affected by Alport syndrome (AS) and their families. Disposable electrochemically printed electrodes were used to immobilize a single-stranded oligonucleotide probe that was complementary to the AS-correlated gene. The detection principle is based on changes in the impedance spectra of the redox probe ferro/ferricyanide after hybridization with synthetic target DNA. Detection was performed either for mutated or for healthy (wild-type) gene copies. The high sensitivity obtained with this protocol (LOD in the picomolar range) was additionally enhanced to the femtomolar range by performing the detection in the presence of Ca2+. In fact, the specific binding of the metal ions in the presence of an A-C nucleotide mismatch induced a further impedance change, thus improving the discrimination between the mutated and healthy gene, as the signal amplification is achieved only for the former. Electrochemical impedance spectroscopy (EIS) is a rapidly developing technique for the transduction of biosensing events at the surface of an electrode.1 EIS has become a very attractive tool for numerous applications in genosensing2-8 due to its effective direct probing of the interface properties (capacitance and electron transfer resistance) of modified electrodes.9 One advantage of this technique is that oligonucleotide labeling is not required for DNA detection. The success of biosensors based on * To whom correspondence should be addressed. Fax: (+81)29-860-4714. E-mail: [email protected]. (1) Berggren, C.; Stalhandske, P.; Brundell, J.; Johansson, G. Electroanalysis 1999, 11, 156–160. (2) Bonanni, A.; Pividori, M. I.; Campoy, S.; Barbe, J.; del Valle, M. Analyst 2009, 134, 602–608. (3) Peng, H.; Soeller, C.; Cannell, M. B.; Bowmaker, G. A.; Cooney, R. P.; Travas-Sejdic, J. Biosens. Bioelectron. 2006, 21, 1727–1736. (4) Keighley, S. D.; Li, P.; Estrela, P.; Migliorato, P. Biosens. Bioelectron. 2008, 23, 1291–1297. (5) Lisdat, F.; Schafer, D. Anal. Bioanal. Chem. 2008, 391, 1555–1567. (6) Davis, F.; Hughes, M. A.; Cossins, A. R.; Higson, S. P. J. Anal. Chem. 2007, 79, 1153–1157. (7) Kjallman, T. H. M.; Peng, H.; Soeller, C.; Travas-Sejdic, J. Anal. Chem. 2008, 80, 9460–9466. (8) Caliskan, A.; Erdem, A.; Karadeniz, H. Electroanalysis 2009, 21, 2116– 2124. (9) Katz, E.; Willner, I. Electroanalysis 2003, 15, 913–947.

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“label-free” impedance sensing has been widely demonstrated.10-17 EIS has been used as a tool for studying DNA hybridization3,11,18-22 and single-nucleotide polymorphisms (SNPs).23-28 An SNP is a DNA sequence variation occurring when a single nucleotide in the genome differs between members of the same species. Most of the sequence variation in human DNA is attributed to SNPs caused by environmental factors,29 and SNPs occur every 100-300 base pairs.30 These variations can affect whether humans develop diseases and respond to pathogens, chemicals, drugs, vaccines, and other agents.31 Multiple SNPs have been correlated to the development of cystic fibrosis, (10) Berggren, C.; Bjarnason, B.; Johansson, G. Electroanalysis 2001, 13, 173– 180. (11) Bonanni, A.; Esplandiu, M. J.; Pividori, M. I.; Alegret, S.; del Valle, M. Anal. Bioanal. Chem. 2006, 385, 1195–1201. (12) Peng, H.; Soeller, C.; Vigar, N. A.; Caprio, V.; Travas-Sejdic, J. Biosens. Bioelectron. 2007, 22, 1868–1873. (13) Kafka, J.; Pa¨nke, O.; Abendroth, B.; Lisdat, F. Electrochim. Acta 2008, 53, 7467–7474. (14) Bonanni, A.; Calvo, D.; del Valle, M. Electroanalysis 2008, 20, 941–948. (15) Daniels, J. S.; Pourmand, N. Electroanalysis 2007, 19, 1239–1257. (16) Chen, C. P.; Ganguly, A.; Wang, C. H.; Hsu, C. W.; Chattopadhyay, S.; Hsu, Y. K.; Chang, Y. C.; Chen, K. H.; Chen, L. C. Anal. Chem. 2009, 81, 36– 42. (17) Weng, J.; Zhang, J. F.; Li, H.; Sun, L. P.; Lin, C. H.; Zhang, Q. Q. Anal. Chem. 2008, 80, 7075–7083. (18) Piro, B.; Haccoun, J.; Pham, M. C.; Tran, L. D.; Rubin, A.; Perrot, H.; Gabrielli, C. J. Electroanal. Chem. 2005, 577, 155–165. (19) Ma, K.-S.; Zhou, H.; Zoval, J.; Madou, M. Sens. Actuators, B 2006, 114, 58–64. (20) Moreno-Hagelsieb, L.; Foultier, B.; Laurent, G.; Pampin, R.; Remacle, J.; Raskin, J. P.; Flandre, D. Biosens. Bioelectron. 2007, 22, 2199–2207. (21) Gheorghe, M.; Guiseppi-Elie, A. Biosens. Bioelectron. 2003, 19, 95–102. (22) Lee, T. Y.; Shim, Y. B. Anal. Chem. 2001, 73, 5629–5632. (23) Bardea, A.; Patolsky, F.; Dagan, A.; Willner, I. Chem. Commun. 1999, 21– 22. (24) Ito, T.; Hosokawa, K.; Maeda, M. Biosens. Bioelectron. 2007, 22, 1816– 1819. (25) Patolsky, F.; Lichtenstein, A.; Willner, I. Nat. Biotechnol. 2001, 19, 253– 257. (26) Wang, Y.; Li, C. J.; Li, X. H.; Li, Y. F.; Kraatz, H. B. Anal. Chem. 2008, 80, 2255–2260. (27) Akagi, Y.; Makimura, M.; Yokoyama, Y.; Fukazawa, M.; Fujiki, S.; Kadosaki, M.; Tanino, K. Electrochim. Acta 2006, 51, 6367–6372. (28) Gong, H.; Zhong, T. Y.; Gao, L.; Li, X. H.; Bi, L. J.; Kraatz, H. B. Anal. Chem. 2009, 81, 8639–8643. (29) Kong, A.; Gudbjartsson, D. F.; Sainz, J.; Jonsdottir, G. M.; Gudjonsson, S. A.; Richardsson, B.; Sigurdardottir, S.; Barnard, J.; Hallbeck, B.; Masson, G.; Shlien, A.; Palsson, S. T.; Frigge, M. L.; Thorgeirsson, T. E.; Gulcher, J. R.; Stefansson, K. Nat. Genet. 2002, 31, 241–247. (30) Wang, W. Y. S.; Barratt, B. J.; Clayton, D. G.; Todd, J. A. Nat. Rev. Genet. 2005, 6, 109–118. (31) Yue, P.; Moult, J. J. Mol. Biol. 2006, 356, 1263–1274. 10.1021/ac100165q  2010 American Chemical Society Published on Web 04/05/2010

Table 1. Summary of Exon 29 and DNA Sequences Used in This Work

Alzheimer’s disease, Parkinson’s disease, diabetes, kidney disease, and various cancers.31,32 The need for a sensitive and rapid detection of SNPs has become an important issue. The early identification of these polymorphisms provides an opportunity for either the diagnosis or the treatment of the disease. Compared with more classical methods,33,34 a genosensor can meet the requirements of low cost, simplicity, and rapidity of analysis while generating a highly sensitive response. In this work, we used an impedimetric genosensor in a labelfree protocol for the detection of a single-base mismatch correlated to the development of Alport syndrome (AS). AS is one of the most common inherited causes of kidney failure in the world. AS is a progressive renal disease with cochlear and ocular involvement which is caused by a mutation in the COL4A5 gene.35 Early diagnosis is very important to initiate proper treatment at the initial stage of the illness. At present, the detection of AS involves invasive procedures and time-consuming analysis, such as singlestrand conformation polymorphism (SSCP) analysis.32 In this study, we rapidly and sensitively detected the A-C mutation in exon 29 (2499G > A)35 of the COL4A5 gene, which was found to be present in the genome of patients affected by AS.32 With the aim of optimizing the protocol, synthetic sequences corresponding to the COL4A5 gene were used as the DNA probe and target. A single-stranded DNA oligonucleotide probe (ssDNA), complementary to a nonmutated region of exon 29, was immobilized onto the electrode surface by physical adsorption. Hybridization was then performed both with mutated and healthy gene copies, obtaining respectively a mismatched duplex and a fully complementary duplex. Amplification of the impedimetric signal was obtained using Ca2+ ions. Metal cations are known for their (32) Hertz, J. M.; Juncker, I.; Persson, U.; Matthijs, G.; Schmidtke, J.; Petersen, M. B.; Kjeldsen, M.; Gregersen, N. Hum. Mutat. 2001, 18, 141–148. (33) Rapley, R.; Harbron, S. Molecular Analysis and Genome Discovery; Wiley: New York, 2004. (34) Oefner, P. J.; Underhill, P. A. Am. J. Hum. Genet. 1995, 57, 1547–1547. (35) Zhou, J.; Leinonen, A.; Tryggvason, K. J. Biol. Chem. 1994, 269, 6608– 6614.

ability to interact with double-stranded DNA (dsDNA).36,37 In several studies, specific interactions of nucleobases, nucleosides, and nucleotides with metal cations have been described and it has been suggested that some metal ions show an affinity toward the sugar-phosphate backbone while others bind preferentially to nucleobases.38 In the present study, Ca2+ ions were found to specifically bind to the mismatched duplex, thus improving the limit of detection of the SNP correlated to Alport syndrome. EXPERIMENTAL SECTION Materials. ssDNA oligonucleotides associated with Alport syndrome were prepared by Tsukuba Oligo Service Co. (Tsukuba, Japan). Their sequences, as well as the sequence of exon 29 of the COL4A5 gene, are listed in Table 1. Stock solutions of the oligonucleotides were diluted with sterilized Milli-Q water (18.2 Ω cm resistivity), separated into fractions, and stored at -20 °C. When required, a single fraction was defrosted. Potassium ferricyanide (K3[Fe(CN)6]), potassium ferrocyanide (K4[Fe(CN)6]), and calcium perchlorate tetrahydrate (Ca(ClO4)2 · 4H2O) were purchased from Sigma-Aldrich (St. Louis, MO). Tris (tris(hydroxymethyl)aminomethane) was supplied by Wako (Osaka, Japan). Propidium iodide cell stain solution (P378) was purchased from Dojindo (Kumamoto, Japan). The following buffer solutions were employed: 0.1 M PBS (0.1 M NaCl, 10 mM sodium phosphate buffer, pH 7.0), TSC1 (0.75 M NaCl, 75 mM trisodium citrate, pH 7.0), TSC2 (0.30 M NaCl, 30 mM trisodium citrate, pH 7.0), and Tris-ClO4 (20 mM Tris, adjusted to pH 8.7 with HClO4). All solutions were made up using Milli-Q water. Disposable-type screen-printed carbon electrodes (DEP chips, EE-PP model) were provided by Biodevice Technology (Nomi, (36) Ono, A.; Cao, S.; Togashi, H.; Tashiro, M.; Fujimoto, T.; Machinami, T.; Oda, S.; Miyake, Y.; Okamoto, I.; Tanaka, Y. Chem. Commun. 2008, 4825– 4827. (37) Bin, X. M.; Kraatz, H. B. Analyst 2009, 134, 1309–1313. (38) Luk, K. F. S.; Maki, A. H.; Hoover, R. J. J. Am. Chem. Soc. 1975, 97, 1241– 1242.

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Scheme 1. Schematic of the Experimental Protocol

Japan). They consisted of a three-electrode system including a carbon-based working electrode, a Ag/AgCl reference electrode, and a carbon-based counter electrode. The working electrode area was 2.64 mm2. Immobilization of the ssDNA Probe. The probe oligonucleotide was immobilized onto the DEP chip surface by dry physical adsorption. A 3 µL volume of ssDNA probe in TSC1 buffer solution at the optimized concentration (see the Optimization of the DNA Probe Concentration section) was deposited onto the electrode surface for 20 min at 60 °C. The electrode was washed twice in TSC2 buffer with gentle stirring at room temperature to remove excess, nonadsorbed material. Hybridization with the ssDNA Target. DEP chips modified with ssDNA probes were incubated in an Eppendorf tube with the hybridization solution (TSC1 buffer) containing the desired concentration of DNA target (total volume 100 µL). The incubation was performed at 42 °C for 30 min, with gentle stirring. Two brief washing steps were then performed in TSC2 buffer at 42 °C.11 Three different ssDNA target sequences were used in this step: the wild type, which resulted in the formation of a fully complementary duplex, a sequence with a single-base mismatch (mutant), which resulted in the formation of a mismatched duplex, and a noncomplementary sequence (nc). Addition of Ca2+ Ions. DEP chips modified with dsDNA were incubated in a solution of Ca(ClO4)2 · 4H2O (0.4 mM in Tris-ClO4 buffer) for 2 h at room temperature under gentle stirring.26 This was followed by two washing steps in Tris-ClO4 buffer. DEP chips modified with the three different DNA targets (wild type, mutant, and nc) were employed in this experiment. Intercalation of Propidium Iodide (PI). In a different experiment, DEP chips modified with dsDNA were incubated in a solution of PI (100 µM in PBS buffer) for 30 min at room temperature under gentle stirring. The chips were then washed twice in PBS buffer. The electrode surface was then observed with a fluorescence microscope to confirm the intercalation of the DNA binder into the dsDNA. A Nikon Eclipse-Ti microscope (Nikon, Tokyo, Japan) with a Semrock LF561-A-000 filter (IDEX, Washington) was used. 3774

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Impedimetric Detection. Impedance measurements were recorded between 0.1 MHz and 0.1 Hz at a sinusoidal voltage perturbation of 10 mV amplitude. The experiments were carried out at an applied potential of 0.18 V (vs a Ag/AgCl reference electrode) in a 0.1 M PBS buffer solution containing 10 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1 molar ratio) as a redox probe. A Randles equivalent circuit was used to fit the obtained impedance spectra, represented as Nyquist plots in the complex plane. The χ2 goodness of fit was calculated for each fitting by the Autolab Frequency Response Analyzer (FRA) software (Eco Chemie, The Netherlands). The impedimetric spectra were recorded in the following order: (1) bare electrode, (2) ssDNA probe modified electrode, (3) dsDNA modified electrode, (4) dsDNA/Ca2+ modified electrode.

RESULTS AND DISCUSSION Scheme 1 shows the protocol for ssDNA probe immobilization and hybridization using three different ssDNA targets: fully complementary (wild type), single-base mismatch (mutant), noncomplementary (nc). The protocol comprised two main parts: (A) the impedimetric detection of the hybridization event and (B) the signal amplification step to improve the impedimetric response while detecting a single-nucleotide mismatch. Impedimetric Detection of the Hybridization Event. Figure 1 shows Nyquist plots obtained in a whole biosensing experiment and the Randles equivalent circuit used to fit the experimental data (right-hand corner). In the circuit, the parameter R1 corresponds to the resistance of the solution, R2 (also called Rct) represents the resistance to the charge transfer between the solution and the electrode surface, W is the Warburg impedance due to the contribution of the diffusion, and CPE (constant phase element) is associated with the capacitance of the double layer (due to the interface between the polarized electrode and the electrolytic solution comprising the PBS buffer and the redox marker). The use of a CPE instead of a capacitor results in better fitting of the experimental data, and this is generally

Figure 1. Nyquist plots, -Zi vs Zr, of the bare DEP chip surface (b), probe modified DEP chip surface (O), fully complementary duplex modified DEP chip surface (0), mismatched duplex modified DEP chip surface (2), and negative control with a noncomplementary target (9) (concentration of the DNA probe, 3 × 10-8 M; concentration of the DNA target, 3 × 10-11 M. All measurements were performed in 0.1 M PBS buffer solution containing 10 mM K3[Fe(CN)6]/K4[Fe(CN)6]. Right-hand corner: Randles equivalent circuit used for data fitting.

due to the nonhomogeneous nature of the electrode surface.39,40 Among these electrical parameters, we focused on the change of the charge transfer resistance (Rct) value recorded after any further step of the biosensing protocol. In fact, the charge transfer process, due to the redox reaction of the couple K3[Fe(CN)6]/K4[Fe(CN)6] at the applied potential, is strongly influenced by any electrode surface modification. For this reason it is possible to follow the biosensing event by simply monitoring the variation of Rct. In the Nyquist plot, the Rct value corresponds to the diameter of the semicircle. The time constant of the semicircles was also monitored after any further DEP chip surface modification, and no significant changes were observed. The related difference of frequencies at the apex of the semicircle was within ±1 experimental step of the scanned frequency. In addition, the χ2 goodness-of-fit test was performed for every fitting to validate the calculations. In all cases, the calculated values for each circuit remained in the range of 0.001-0.2, much lower than the tabulated value for 50 degrees of freedom (67.505 at the 95% confidence level). Figure 1 shows that the Rct of the bare electrode (filled circles) significantly increased after ssDNA probe immobilization (empty circles) onto the DEP chip surface. This is due to hindrance of the electron transfer process of [Fe(CN)6]3-/4to the electrode surface after modification.9 The negative charges on the phosphate backbone of the immobilized ssDNA probe repelled the negatively charged redox couple, thus increasing the Rct value. The steric hindrance introduced with the formation of an ssDNA probe film on the electrode surface also contributed to the increment of Rct. After hybridization with the wild type, a fully complementary duplex should be formed and a decrease in charge transfer resistance value was observed (empty squares). A similar drop in Rct after the duplex formation was reported by other authors (39) Macdonald, J. R. Impedance Spectroscopy; Wiley: New York, 1987. (40) Gabrielli, C. Use and Application of Electrochemical Impedance Techniques; Solartron Analytical: Farnborough, U.K., 1990.

for DNA adsorbed onto carbon41 or carbon nanotube modified electrodes42 or covalently immobilized onto a gold surface.43 To understand this behavior, the mechanism of ssDNA immobilization onto the electrode surface and its hybridization with the complementary target solution should be clarified. Several interactions are involved in the physical adsorption of DNA oligonucleotides onto a carbon surface.44 Even though hydrophobic interactions between the electrode surface and oligonucleotide nitrogenous bases represent the main adsorption mechanism, other effects, such as electrostatic and van der Waals interactions, contribute to the adsorption process. Previous studies on this topic demonstrated that ssDNA molecules, compared with dsDNA, interacted and adsorbed quite strongly onto carbon material because nitrogenous bases are more exposed and are free to undergo hydrophobic interactions with the electrode surface.45 In addition, atomic force microscopy (AFM) studies demonstrated that single-stranded molecular films had larger heights than the double-stranded oligonucleotides, which suggests that ssDNA oligonucleotides tend to fold back on themselves.45 When the electrode surface is incubated in a solution containing the complementary target oligonucleotide at the proper concentration and ionic strength, the formation of a double-helical structure is thermodynamically favored, thus reducing the interaction of the ssDNA oligonucleotide molecule with the electrode surface as the bases are involved in the formation of hydrogen bonds inside the helix. These conformational changes could explain the reduced Rct observed after the hybridization step, due to the increased availability of the electrode surface for the redox marker. Moreover, the folded structure of ssDNA would become more open after hybridization, resulting again in a “lower impedance” structure, due to the reduced steric hindrance onto the electrode surface. When a DEP chip modified with an ssDNA probe was incubated in a solution containing the noncomplementary target (nc), the Rct variation during the hybridization step was negligible (filled squares), thus indicating that a DNA duplex was not formed and additional adsorption of nc ssDNA did not take place. Finally, when hybridization was performed with the sequence containing the SNP (mutant), the Rct (filled triangles) value dropped by less than that observed with the wild-type sequence, thus allowing discrimination between the two targets. The smaller decrease in Rct was due to less efficient hybridization, caused by the presence of a single mismatch in the sequence. However, the Rct value was also significantly different from that provided by the noncomplementary target, thus indicating that a non-negligible affinity interaction took place, despite the presence of the mismatch. Optimization of the DNA Probe Concentration. EIS measurements with different amounts of probe oligonucleotide were (41) Davis, F.; Nabok, A. V.; Higson, S. P. J. Biosens. Bioelectron. 2005, 20, 1531–1538. (42) Cai, H.; Xu, Y.; He, P. G.; Fang, Y. Z. Electroanalysis 2003, 15, 1864–1870. (43) Gooding, J. J.; Chou, A.; Mearns, F. J.; Wong, E.; Jericho, K. L. Chem. Commun. 2003, 1938–1939. (44) Pividori, M. I.; Alegret, S. DNA Adsorption on Carbonaceous Materials; Springer: Berlin, Heidelberg, 2005. (45) Paquim, A. M. C.; Oretskaya, T. S.; Brett, A. M. O. Biophys. Chem. 2006, 121, 131–141.

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Figure 2. Curve representing experiments for optimization of the ssDNA probe concentration (∆p ) Rct(probe) - Rct(blank)). Error bars correspond to triplicate experiments.

carried out to optimize the ssDNA probe concentration. The optimized concentration should ensure a full coverage of the electrode surface, avoiding nonspecific adsorption of the ssDNA target. Figure 2 shows the variation (∆p) of the charge transfer resistance between the blank (bare) and probe-modified electrode plotted versus the ssDNA probe concentration. The increment of the ssDNA probe concentration was associated with an enhancement of the Rct value until a plateau was reached. At that point, the electrode surface was considered to be completely covered with the immobilized oligonucleotide. Any additional increment in the ssDNA probe concentration did not result in a further increment of the charge transfer resistance. For that reason, an ssDNA probe concentration of 3 × 10-8 M was chosen for all the experiments. Impedimetric Response toward the ssDNA Target Concentration. The impedimetric response after the hybridization step was recorded for ssDNA target concentrations from 3 × 10-14 to 3 × 10-8 M. The concentration of the ssDNA probe was kept constant at the optimized value of 3 × 10-8 M. The experiment was carried out to calculate the limit of detection (LOD) of the genosensor, either in the detection of the wild-type sequence or in the differentiation between the wild type and a single-nucleotide mismatch (mutant). In Figure 3, the results are expressed as the relative Rct variation between the values obtained in the different experiments (i.e., DNA immobilization or hybridization) and the Rct value due to the bare electrode. This relative variation is represented as a ratio of ∆ increments (see ∆ratio ) ∆s/∆p; see the caption of Figure 3). The elaboration required for the comparison of data from different electrodes has already been used and extensively explained in previous works.11 In this case, the ∆s/∆p value should be