Anal. Chem. 2008, 80, 1336-1340
Label-Free Detection of DNA Hybridization at a Liquid|Liquid Interface Mikhail Yu. Vagin,*,† Stanislav A. Trashin,† Arkady A. Karyakin,† and Marco Mascini‡
Faculty of Chemistry, M.V. Lomonosov Moscow State University, Lenin’s Hills, GSP-3, 119991, Moscow, Russia, and Department of Chemistry, University of Florence, via della Lastruccia 3, 50019, Sesto F.no, Florence, Italy
A novel electrochemical approach for label-free detection of DNA primary sequence has been proposed. The flow of nonelectroactive ions across a liquid|liquid interface was used as an electrochemical probe for detection of DNA hybridization. Disposable graphite screen-printed electrodes shielded with a thin layer of inert polymer plasticized with water-immiscible polar organic solvent were modified by probe oligonucleotide and used as a DNA sensor. The specific DNA coupling has been detected with impedance spectroscopy by decrease of iontransfer resistance. The detection limit was of 10-8 M of target oligonucleotide. The reported sensor was suitable for discrimination of a single mismatch oligonucleotide from the full complementary one. The reported DNA sensor was advantageous over known physicochemical approaches, providing the most significant changes in the measured parameters. DNA recognition, based on its hybridization with immobilized oligonucleotide probes, has a particular interest during the recent years, with a potential for a variety of applications, including drug discovery,1 study of gene expression,2 screening of genetic material for mutations,3 investigation of the molecular basis of infectious diseases,4 and sequencing of particular genes of interest among complex DNA samples.5 These technologies commonly relied on immobilization of a single-stranded DNA probe onto optical,6-8 electrochemical,6,9-16 piezoelectric6,17-20 transducers for * To whom correspondence should be addressed. E-mail:
[email protected]. msu.ru. Tel.: (+7-495) 939-2804. Fax: (+7-495) 939-4675. † M.V. Lomonosov Moscow State University. ‡ University of Florence. (1) Debouck, C.; Goodfellow, P. N. Nat. Genet. 1999, 21, 48-50. (2) Bertucci, F.; Houlgatte, R.; Nguyen, C.; Viens, P.; Jordan, B. R.; Birnbaum, D. Lancet Oncol. 2001, 2, 674-682. (3) Pusztai, L.; Ayers, M.; Stec, J.; Hortobagyi, G. N. Oncologist 2003, 8, 252258. (4) Bryant, P. A.; Venter, D.; Robins-Browne, R.; Curtis, N. Lancet Infect. Dis. 2004, 4, 100-111. (5) Chee, M.; Yang, R.; Hubbell, E.; Berno, A.; Huang, X. C.; Stern, D.; Winkler, J.; Lockhart, D. J.; Morris, M. S.; Fodor, S. P. A. Science 1996, 274. (6) Christopoulos, T. K. Anal. Chem. 1999, 71, 425R-438R. (7) Piunno, P. A. E.; Krull, U. J.; Hudson, R. H. E.; Damha, M. J.; Cohen, H. Anal. Chim. Acta 1994, 288, 205-214. (8) Hanafi-Bagby, D.; Piunno, P. A. E.; Wust, C. C.; Krull, U. J. Anal. Chim. Acta 2000, 411, 19-30. (9) Wang, J.; Jiang, M.; Fortes, A.; Mukherjee, B. Anal. Chim. Acta 1999, 402, 7-12. (10) Wang, J.; Jiang, M.; Mukherjee, B. Anal. Chem. 1999, 71, 4095-4099. (11) Hashimoto, K.; Ito, K.; Ishimori, Y. Sens. Actuators, B 1998, 46, 220-225.
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subsequent recognition of complementary DNA (target) strand in sample solution. Direct (label-free) electrochemical detection of a hybridization reaction represents a very attractive approach for detecting bioaffinity interactions.9,21-23 Such a route can be greatly accomplished by monitoring changes in electronic or interfacial properties accompanying the binding event. This approach greatly simplifies the sensing protocol, because it eliminates the need for the indicator addition/association step. Moreover, the reagentless manner offers an instantaneous detection of bioaffinity complex formation and therefore the real-time monitoring of binding. After the pioneer publication of Koryta et al.,24 electrochemistry of the interface between two immiscible electrolyte solutions became an important tool of electroanalytical chemistry.25 Transfer of ions through the interface results in an increase of current similar to that of the faradic processes at the metal-solution interface. This allows amperometric detection of nonelectroactive ions, which is of great importance in analytical chemistry. The structure of the water|organic interface, which is considered as a model of half of a biological membrane, is highly important for the life sciences. Thus, in recent years, growing (12) Garnier, F.; Korri-Youssoufi, H.; Srivastava, P.; Mandrand, B.; Delair, T. Synth. Met. 1999, 100, 89-94. (13) Brett, C. M. A.; Brett, A. M. O.; Serrano, S. H. P. Electrochim. Acta 1999, 44, 4233-4239. (14) Berggren, C.; Stlhandske, P.; Brundell, J.; Johansson, G. Electroanalysis 1999, 11, 156-160. (15) Bardea, A.; Patolsky, F.; Dagan, A.; Willner, I. Chem. Commun. 1999, 1, 21-22. (16) Maruyama, K.; Motonaka, J.; Mishima, Y.; Matsuzaki, Y.; Nakabayashi, I.; Nakabayashi, Y. Sens. Actuators, B 2001, 3756, 1-5. (17) Rickert, J.; Gopel, W.; Beck, W.; Jung, G.; Heiduschka, P. Biosens. Bioelectron. 1996, 11, 757-768. (18) Tombelli, S.; Minunni, M.; Mascini, M. Methods 2005, 37, 48-56. (19) Tombelli, S.; Minunni, M.; Santucci, A.; Spiriti, M. M.; Mascini, M. Talanta 2006, 68, 806-812. (20) Minunni, M.; Tombelli, S.; Fonti, J.; Spiriti, M. M.; Mascini, M.; Bogani, P.; Buiatti, M. J. Am. Chem. Soc. 2005, 127, 7966-7967. (21) Hianik, T.; Passechnik, V. I.; Sokolikova, L.; Snejdarkova, M.; Sivak, B.; Fajkus, M.; vanov, S. A.; Franek, M. Bioelectrochem. Bioenerg. 1998, 47, 47-55. (22) Hianik, T.; Snejdarkova, M.; Sokolikova, L.; Meszar, E.; Krivanek, R.; Tvarozek, V.; Novotny, I.; Wang, J. Sens. Actuators, B 1999, 57, 201-212. (23) Corbisier, P.; Van Der Lelie, D.; Borremans, B.; Provoost, A.; De Lorenzo, V.; Brown, N. L.; Lloyd, J. R.; Hobman, J. L.; Csoregi, E.; Johansson, G.; Mattiasson, B. Anal. Chim. Acta 1999, 387, 235-244. (24) Koryta, J.; Vanysek, P.; Brezina, M. J. Electroanal. Chem 1976, 67, 263266. (25) Reymond, F.; Fermin, D.; Lee, H. J.; Girault, H. H. Electrochim. Acta 2000, 45, 2647-2662. 10.1021/ac701923c CCC: $40.75
© 2008 American Chemical Society Published on Web 01/19/2008
interest has been given to studies of biomolecules at the liquid|liquid interface.26-33 Organic cations were chosen as electrochemical probes for ion transfer across a 1,2-dichloroethane (DCE)|water microinterface affected by the presence of oligonucleotides in water phase.26 Recently, Osakai et al. observed adsorption of long-chain DNA onto a DCE|water interface assisting surfactant dimethyldistearylammonium tetraphenylborate transfer from the organic phase.32 Long-chain DNA samples with high concentration were used in both works (widespread oligonucleotide masses;26 4500 and 5200 kDa).32 However, in both studies, the hybridization event has not been studied. The difficulties associated with the instability of a liquid|liquid interface, which restricts its employment in a wide variety of applications, can be overcome by solidifying of the organic phase by adjunction of polymers.34-37 Alternatively, the mechanic stability of a liquid|liquid interface can be achieved on solid electrodes covered with thin liquid films38-41 and microdroplets.42 The overall electrode process at a solid electrode shielded with a thin layer of redox mediator solution in water-immiscible organic solvent consists of electron transfer (ET) at a solid electrode|organic layer interface and ion transfer (IT) across an organic layer|water interface to compensate a charge of the oxidized mediator in organic solvent. A similar system with a redox polymer as a mediator was elaborated for investigation of thermodynamics of ion transfer across a liquid|liquid interface.43 In the present work, we report on the observation of DNA hybridization on a supported liquid|liquid interface. The flow of transferring ions was used as an electrochemical probe for direct detection of DNA hybridization. Simple procedures for fabrication of shielded electrode and its subsequent modification with probe DNA give promise for further development of the proposed sensors. The reported DNA sensor is advantageous over known physicochemical approaches providing the most significant changes in the measured parameters. EXPERIMENTAL SECTION Materials. All experiments were carried out with Millipore (MilliQ Plus) water. All inorganic salts were purchased from Sigma. (26) Horrocks, B. R.; Mirkin, M. V. Anal. Chem. 1998, 70, 4653-4660. (27) Lillie, G. C.; Holmes, S. M.; Dryfe, R. A. J. Phys. Chem. B. 2002, 106, 1210112103. (28) Vagin, M. Y.; Malyh, E. V.; Larionova, N. y. I.; Karyakin, A. A. Electrochem. Commun. 2003, 5, 329-333. (29) Amemiya, S.; Yang, X.; Wazenegger, T. L. J. Am. Chem. Soc. 2003, 125, 11832. (30) Yuan, Y.; Amemiya, S. Anal. Chem. 2004, 76, 6877-6886. (31) Vagin, M. Y.; Trashin, S. A.; Ozkan, S. Z.; Karpachova, G. P.; Karyakin, A. A. J. Electroanal. Chem 2005, 584, 110-116. (32) Osakai, T.; Komatsu, H.; Goto, M. J. Phys. Condens. Matter 2007, 19, 375103. (33) Shinshi, M.; Sugihara, T.; Osakai, T.; Goto, M. Langmuir 2006, 22, 59375944. (34) Kakutani, T.; Ohkouchi, T.; Kakiuchi, T.; Senda, M. Anal. Sci. 1985, 1, 219. (35) Osakai, T.; Kakutani, T.; Senda, M. Anal. Sci. 1985, 3, 512. (36) Marecek, V.; Janchenova, H.; Brezina, M.; Betti, M. Anal. Chim. Acta 1991, 244, 15. (37) Borchardt, M.; Dumschat, C.; Cammann, K.; Knoll, M. Sens. Actuators, B 1995, 25, 721-723. (38) Cheng, Y.; Murtomaki, L.; Corn, R. M. J. Electroanal. Chem. 2000, 483, 88-94. (39) Shi, C.; Anson, F. C. J. Phys. Chem. 1998, 102, 9850-9854.
Long-chain poly(vinyl chloride) (PVC), o-nitrophenyl octyl ether (NPOE), tetrahydrofuran (THF), and tetrabutylammonium tetrakis(4-chlorophenyl)borate (TBATClPB) were Selectophore grade and purchased from Fluka. Aqueous supporting electrolyte was 0.1 M NaCl, 10 mM Na2HPO4, pH7.4. Redox polymer polyphenotiazine (PPTA) was synthesized as described elsewhere.43 The degree of polymerization was ∼10. The obtained polymer was completely insoluble in water but soluble in various organic solvents. The estimated degree of oxidation (the quantity of units containing quinoimine groups) was not more than 0.2. The polymer possesses a high degree of crystallinity (∼70%). Two pairs of Probes and Targets were used: (A) probe, C18H35, 5′-CTC AGG TTG CTG ACA TTT-3′; target, 5′-A AAA TGT CAG CAA CCT GA-3′ (18-mer oligonucleotide modified at 5′-end by hydrophobic alkyl linker C18H35 against 18-mer complement oligonucleotide with sequence from Salmonella genome, MW 6.5 kDa). (B) probe, C12H25, 5′-GCG CGC GAA CGG-3′; complement target, 5′-CCG CCA ATA AAG TTC ACA AAA CGC CGT TCG CGC GC-3′; single mismatch target, 5′-CCG CCA ATA AAG TTC ACA AAA CGC CGT TCA CGC GC-3′ (12-mer oligonucleotide modified at 5′-end by hydrophobic alkyl linker C12H25 against 35mer single mismatch oligonucleotide). Electrodes. Graphite screen-printed electrodes (SPE) were made with Gwent Scientific graphite paste. Working electrode diameter was 2 mm. Counter electrode has a sufficiently higher surface. The outer Ag|AgCl|1 M KCl electrode was used as a reference. The gold and glassy carbon disk electrodes were made by pressing a gold wire i.d. 1 mm (99.99% purity, Good Fellow) or glassy carbon rod i.d. 1 mm in a Teflon tube. The working electrode was pretreated by polishing with 0.05-mm Al2O3 (Aldrich) followed by washing in concentrated sulfuric acid for several minutes. The following procedure was used to modify the surface of SPE with a shielding layer of organic membrane of PVC plasticized by NPOE solvent. First, PVC solution in THF was added in TBATClPB and PPTA solution in NPOE. Then the electrode surface was covered with 5 µL of solution and THF was allowed to evaporate. Finally, electrode covered with dried membrane was used as a working electrode. The PVC/NPOE ratio used for electrode surface shielding was 1 or 5%. In order to immobilize oligonucleotides modified by alkyl chains, 7 µL of aqueous buffer solution of an appropriate DNA probe was placed onto a membrane-modified electrode. The electrode was left for a 2 h to carry out the adsorption. Then an aqueous drop was shaken off. Further, in order to conduct the hybridization, 7 µL of aqueous buffer solution of an appropriate DNA target was placed onto the membrane electrode. And the electrode was left for 45 min to carry out the hybridization. Finally, impedance measurements were carried out with the membrane electrode. (40) Shi, C.; Anson, F. C. Anal. Chem. 1998, 70, 3114-3118. (41) Shi, C.; Anson, F. C. J. Phys. Chem. 1999, 103, 6283-6289. (42) Scholz, F.; Komorsky-Lovric, S.; Lovric, M. Electochem. Commun. 2000, 2, 112-118. (43) Karyakin, A. A.; Vagin, M. Y.; Ozkan, S. Z.; Karpachova, G. P. J. Phys. Chem. B 2004, 108, 11591-11595.
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Instrumentation. Electrochemical measurements on SPE were made in a common glass beaker with an Autolab instrument (Eco-Chemie, Utrecht, The Netherlands) with impedance unit. Impedance measurements on disk electrodes were done with a Solartron 1255 frequency response analyzer with a homemade lownoise potentiostat in a two-compartment electrochemical cell. The working and counter electrodes were placed in the same compartment. The counter electrode in impedance measurements with disk electrodes was a platinum disk encircling the working disk electrode. All measurements and procedures were made at room temperature. RESULTS AND DISCUSSION The electrochemical system based on a solid electrode shielded with liquid gel of water-immiscible organic solvent was proposed for direct electrochemical detection of DNA hybridization. Disposable graphite SPEs modified by PVC gel,, which was plasticized with a solution of hydrophobic redox polymer PPTA in waterimmiscible polar organic solvent NPOE, were used as working shielded electrodes in aqueous solution of background electrolyte. The electrode reaction coupled the ET due to PPTA redox reaction at the electrode|NPOE interface with anion transfer across the NPOE|water IT to keep an electroneutrality in the organic shielding layer.39,40 This made the electrode performance sensitive to the IT process. Oligonucleotides are extremely hydrophilic molecules. Thus, even an addition of ethanol to aqueous solutions of oligonucleotides leads to their precipitation. Therefore, oligonucleotides modified by hydrophobic alkyl linkers were used as hybridization probes immobilized by adsorption onto a liquid|liquid interface. Simple incubation of shielded electrodes in an aqueous solution of probe was used for surface modification with oligonucleotides. Such shielded electrodes with immobilized probe were used as disposable ones. Adsorption of DNA onto shielded electrodes was investigated by means of impedance spectroscopy. Shielded electrodes incubated with either pure buffer or with 1 × 10-7 M solutions of DNA without hydrophobic anchor showed rather similar spectra displayed in Figure 1. Incubation with 5 × 10-8M solution of probe A oligonucleotide resulted in increase of semicircle diameter (Figure 1). Treatment of a shielded electrode with immobilized probe in pure buffer did not cause changes in impedance spectra. On the contrary, incubation of probe A electrodes with solutions containing target A starting from 1 × 10-8 M concentration resulted in significant admittance increase (Figure 1). Shielded electrodes with immobilized probe were reproducible: spectra for five electrodes at each probe concentration possesses less than 15% deviation in admittance at a certain frequency. Measurements with the same electrode had even lower deviation of 2%. Analytical performance characteristics of the system were investigated by impedance measurements after incubation of probe-modified electrodes in an aqueous solution of target oligonucleotides (target). Figure 1 presents impedance spectra in admittance plots of shielded electrodes with adsorbed probe A after incubation with different concentrations of target A oligonucleotide. Measurements with pure buffer were as control experiments. It is seen, that the impedance spectrum changes 1338 Analytical Chemistry, Vol. 80, No. 4, February 15, 2008
Figure 1. Impedance spectra in admittance plots of shielded electrodes (1% PVC-NPOE membrane (10 mM TBATClPB and 0.3 mg/mL PPTA) formed onto the SPE surface). (+) After incubation in buffer solution; (9); after adsorption of DNA probe A (5 × 10-8 M); (O, 0, 4, 3, ]) responses of the shielded electrodes with immobilized probe A to incubation with different concentrations of target A: (O) 1 × 10-8, (0) 5 × 10-8, (4) 1 × 10-7, (3) 5 × 10-7, and (]) 1 × 10-6 M. Solid lines illustrate theoretical curves fit to the proposed equivalent circuit (inset). Coincided spectra are omitted for clarity.
after hybridization. The observed spectrum transformation is recognized in admittance plots at high-frequency domain. The response of the probe-modified shielded electrodes to complement target was highly reproducible: the deviation of spectra for electrode series with the same target concentration was in the frame of 5%. Probe-modified shielded electrodes with noncomplement oligonucleotide did not cause any changes in spectra with respect to pure buffer. Probe-modified electrodes were tested with different target concentrations. It is seen from Figure 1 that the increase of target concentration caused greater changes in spectra. Thus, the developed electrochemical sensor can be used for quantitative detection of single-strained DNA. Impedance spectra are always investigated relative to suitable equivalent circuits. It is seen, that all impedance spectra in Figure 1 can be separated in two parts: at high frequencies (>3 kHz) and at low frequencies (
