Anal. Chem. 2006, 78, 2447-2449
Magnetic Nanoparticles Applied in Electrochemical Detection of Controllable DNA Hybridization Xiaoli Zhu, Kun Han, and Genxi Li*
Department of Biochemistry and National Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, 210093 Nanjing, P. R. China
Electrochemical detection of hybridized DNA strands was achieved with a magnetic nanoparticle modified electrode and the commonly used electrochemical couple K3[Fe(CN)6]/K4[Fe(CN)6]. The detection proved to be fast and very simple. Furthermore, magnetic nanoparticles could be employed to control the DNA hybridization process. An inhibited or an enhanced degree of hybridizing could be produced. Electrochemical hybridization assays for DNA sequence analysis and diagnostics have become popular in recent years.1 Compared with the conventional optical methods, the electrochemical method can potentially utilize smaller microlocations (or nanolocations) and reduce the cost and complexity of array technology. Electrochemical detection usually involves the immobilization of a single-stranded probe nucleic acid to a solid substrate and the production of an electrochemical signal when the desired target hybridizes to the probe. Considerable improvements to the development of chemically modified electrodes and more sensitive electrochemical indicators have been made in the past few years;1-5 however, there still exist many problems; for example, low sensitivity, complexity, and the destruction of the DNA. Today, nanomaterials, due to their quantum-scale effect, smallscale effect, surface effect, etc., may partly resolve the problems of traditional chemically modified electrodes. Gold nanoparticles (NPs) are the most widely used nanomaterial.6 In addition, * To whom correspondence should be addressed, E-mail address: genxili@ nju.edu.cn (1) (a) Steel, A. B.; Herne, T. M.; Tarlov, M. J. Anal. Chem. 1998, 70, 46704677. (b) Thorp, H. H. Trends. Biotechnol. 1998, 16, 117-121. (c) Boon, E. M.; Ceres, D. M.; Drummond, T. G.; Hill, M. G.; Barton, J. K. Nat. Biotechnol. 2000, 18, 1096-1100. (d) Patolsky, F.; Lichtenstein, A.; Willner, I. J. Am. Chem. Soc. 2001, 123, 5194-5205. (e) Palecek, E.; Fojta, M. Anal. Chem. 2001, 73, 74A-83A. (f) Wang, J.; Cai, X. H.; Rivas, G.; Shiraishi, H.; Dontha, N. Biosens. Bioelectron. 1997, 12, 587-599. (2) Umek, R. M.; Lin, S. W.; Vielmetter, J.; Terbrueggen, R. H.; Irvine, B.; Yu, C. J.; Kayyem, J. F.; Yowanto, H.; Blackburn, G. F.; Farkas, D. H.; Chen, Y. P. J. Mol. Diagn. 2001, 3, 74-84. (3) Boon, E. M.; Ceres, D. M.; Drummond, T. G.; Hill, M. G.; Barton, J. K. Nat. Biotechnol. 2000, 18, 1096-1100. (4) Park, S. J.; Taton, T. A.; Mirkin, C. A. Science 2002, 295, 1503-1506. (5) Oleinikov, A. V.; Gray, M. D.; Zhao, J.; Montgomery, D. D.; Ghindills, A. L.; Dill, K. J. Proteome Res. 2003, 2, 313-319. (6) Shiigi, H.; Tokonami, S.; Yakabe, H.; Nagaoka, T. J. Am. Chem. Soc. 2005, 127, 3280-3281. 10.1021/ac051962x CCC: $33.50 Published on Web 03/07/2006
© 2006 American Chemical Society
titanium dioxide (TiO2) NPs,7,8 magnetic nanoparticles,9-11 ZnSe/ CdSe nanowire,12 etc., have also been applied in DNA adsorption, hybridization assays, controllable DNA switch, and so on. In this paper, we report a very simple procedure to detect DNA hybridization. The oligonucleotides need not be modified, and the very commonly used electrochemical redox couple K 3[Fe(CN)6]/ K4[Fe(CN)6] can be employed to produce the electrochemical signal.13,14 Since the electrochemical redox couple does not bind to the DNA, the DNA retains its natural state and can be recycled. Furthermore, a controllable hybridizing process for DNA can be achieved by employing magnetic nanoparticles. EXPERIMENTAL SECTION Chemicals and Apparatus. (Aminopropyl)-triethoxysilane (APTS)-coated ferriferrous oxide nanoparticles (APTS-Fe3O4 NPs) were prepared according to the literature.10,15,16 The transmission electron micrograph of APTS-Fe3O4 NPs revealed that the NPs were not aggregated and had an average diameter of ∼18 nm (data not shown). Oleic acid-coated ferriferrous oxide nanoparticles (OA-Fe3O4 NPs) and oligonucleotides (Guaranteed Oligos, HPLC-purified) were purchased from Anhui Jinke Magnetic liquids Co., Ltd. and Shanghai Shenergy Biocolor BioScience & Technology Co., Ltd., respectively. Other chemicals were all of analytical grade. All solutions were prepared with doubly distilled water, which was purified with a Milli-Q purification system (Branstead, USA) to a specific resistance of >16 MΩ cm-1. Electrochemical experiments were carried out with a VMP Potentiostat (PerkinElmer, USA) and a three-electrode system. A one-compartment glass cell with a modified pyrolytic graphite (7) Rajh, T.; Saponjic, Z.; Liu, J. Q.; Dimitrijevic, N. M.; Scherer, N. F.; VegaArroyo, M.; Zapol, P.; Curtiss, L. A.; Thurnauer, M. C. Nano Lett. 2004, 4, 1017-1023. (8) Meier, K. R.; Gratzel, M. ChemPhysChem. 2002, 3, 371. (9) Katz, E.; Weizmann, Y.; Willner, I. J. Am. Chem. Soc. 2005, 127, 91919200. (10) Bruce, I. J.; Sen, T. Langmuir 2005, 21, 7029-7035. (11) Kinsella, J. M.; Ivanisevic, A. J. Am. Chem. Soc. 2005, 127, 3276-3277. (12) Ramanathan, K.; Bangar, M. A.; Yun, M.; Chen, W.; Myung, N. V.; Mulchandani, A. J. Am. Chem. Soc. 2005, 127, 496-497. (13) Maruyama, K.; Mishima, Y.; Minagawa, K.; Motonaka, J. J. Eletroanal. Chem. 2001, 510, 96-102. (14) Radi, A. E.; Sanchez, J. L. A.; Baldrich, E.; O’Sullivan, C. K. Anal. Chem. 2005, 77, 6320-6323. (15) Bruce, I. J.; Taylor, J.; Todd, M.; Davies, M. J.; Borioni, E.; Sangregorio, C.; Sen, T. J. Magn. Magn. Mater. 2004, 284, 145-160. (16) Philipse, A. P.; Bruggen, M. P. B.; Pathmamanoharan, C. Langmuir 1994, 10, 92-99.
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(PG) working electrode, a saturated calomel reference electrode (SCE), and a platinum wire auxiliary electrode was used for the measurements. Transmission electron micrographs were obtained using a JEM-200CX transmission electron microscope (JEOL, Japan). Atomic force microscopy was performed using an SPI3800N scanning probe microscope (Seiko, Japan). UV-vis spectroscopy was performed using a UV-2201 spectrometry (Shimadzu, Japan). Preparation of Single-Stranded Oligonucleotide-Modified Electrode. The substrate PG electrode was prepared by inserting a PG rod into a glass tube and fixing it with epoxy resin. Electrical contact was made by adhering a copper wire to the rod with the help of Woods Alloy. The PG electrode was first polished on rough and fine sandpapers, then its surface was polished to mirror smoothness with an alumina (particle size ∼0.05 µm)/water slurry on silk. Eventually, the electrode was thoroughly washed by ultrasonicating in both doubly distilled water and ethanol for ∼5 min. A 1-mg portion of APTS-Fe3O4 NPs was dissolved in 1 mL of doubly distilled water and treated by ultrasonicating for ∼10 min. A 20 µL portion of the suspension was evenly spread onto the surface of the PG electrode. The electrode surface was covered with an Eppendorf tube for the first 2 h to prepare uniform films and dried overnight in the air. Then the modified electrode was thoroughly rinsed with purified water and dried again. The APTS-Fe3O4 NPs-modified electrode was immersed in a 400-µL solution containing 10 µM of a single-stranded target oligonucleotide (ssDNA I, sequence: CATCGTTGCTAC) for 4 h at room temperature ∼25 °C, then rinsed with purified water and dried. Hybridization on the ssDNA I-Modified Electrode. The ssDNA I-modified electrode was immersed in a 400-µL solution containing 2.5 mM MgCl2 and 10 µM of either the complementary single-stranded oligonucleotide (ssDNA II, sequence: GTAGCAACGATG) or noncomplementary single-stranded oligonucleotide (ssDNA III, sequence: TCCAGTTGACCT). This solution had been preheated to 80 °C. During the attempted hybridization, the solution was cooled slowly to room temperature over a period of 2 h. Afterward, the electrode was taken out of the solution, rinsed with purified water, and dried. The hybridized electrode was reproduced by washing the electrode with preheated 95 °C water for 1 min. We expected that through this process, the duplex pair would be denaturized and the ssDNA II would be washed off, leaving ssDNA I still immobilized on the APTS-Fe3O4 NPs-modified electrode surface. Magnetoswichable Controlled DNA Hybridization. The ssDNA I-modified electrode was immersed in a 400-µL solution containing 2.5 mM MgCl2, 10 µM ssDNA II, and 1 mg mL-1 OAFe3O4 NPs. Over the whole period of hybridizing, a magnetic field over or below the electrode was applied which would either repel or attract the OA-Fe3O4 NPs from or to the surface of the electrode (see Figure S1 in Supporting Information). The solution had been preheated to 80 °C, and the reaction was run as described above. As above, the electrode was taken out of the solution, rinsed with purified water, and dried. Electrochemical Measurements. The K3[Fe(CN)6]/K4[Fe(CN)6] couple was used as the electrochemical indicator. The test solution, which contained 5 mM NaCl and 5 mM K3[Fe(CN)6]/ 2448 Analytical Chemistry, Vol. 78, No. 7, April 1, 2006
Figure 1. Cyclic voltammograms of 5 mM K3[Fe(CN)6]/K4[Fe(CN)6] obtained at a APTS-Fe3O4 NPs-modified electrode (solid line), a ssDNA I-modified electrode (dotted line), and a ssDNA II hybridized electrode (dashed line). Scan rate: 200 mV s-1.
K4[Fe(CN)6], was first bubbled thoroughly with high-purity nitrogen for 5 min. Then a stream of nitrogen was blown gently across the surface of the solution to maintain the solution anaerobic throughout the experiment. Cyclic voltammetry was carried out in a potential range from -200 to 600 mV. All experiments were performed at room temperature of ∼25 °C. RESULTS AND DISCUSSION Amino-silane has been proven to bind oligonucleotide probes through the phosphate moiety, and these probes retain a capacity for base pair-specific hybridization with a solution-state DNA target strand to form the duplex pair.17 Here, we employed APTS-Fe3O4 NPs to immobilize an oligonucleotide probe and electrochemically detect the pair. Characterization of the APTS-Fe3O4 NPs-modified electrode was by atomic force microscopy (see Figure S2 in the Supporting Information). Cyclic voltammetric detection of the hybridization is shown in Figure 1. A decline in the peaks currents for K3[Fe(CN)6]/K4[Fe(CN)6] is observed after the ssDNA Imodified electrode is immersed in a hybridization solution of ssDNA II, and no change of the peaks can be observed when a solution of ssDNA III is applied instead of ssDNA II (data is not shown), implying that ssDNA II has hybridized with ssDNA I on the APTS-Fe3O4 NPs-modified electrode. Figure 1 also shows that the peaks currents of the K3[Fe(CN)6]/K4[Fe(CN)6] couple decreased after the APTS-Fe3O4 NPs modified electrode was immobilized with the ssDNA I probe. Nevertheless, further obvious decline of the peaks currents can be observed after the probe has hybridized with the target DNA, and no change occurs if a noncomplementary ssDNA is applied. UV-vis spectroscopy was employed to quantify the amount of ssDNA adsorbed on the electrode surface. The difference in the absorbance at 260 nm for the solution of ssDNA I before and after adsorption on the APTS-Fe3O4 NPs modified electrode is 0.118, and the difference of the absorbance at 260 nm for the solution of ssDNA II and ssDNA III before and after hybridization is 0.102 and 0.008, respectively. Through calculating, the total (17) Lemeshko, S. V.; Powdrill, T.; Belosludtsev, Y. Y.; Hogan, M. Nucleic Acids Res. 2001, 29, 3051-3058.
amount of ssDNA I adsorbed onto the surface of APTS-Fe3O4 NPs is 4.35 × 10-4 µmo, and the surface density of the DNA is 2.2 nM cm-2 (for the 0.196 cm2 surface area of the PG electrode), indicating that a large amount of ssDNA I is adsorbed onto the surface of the APTS-Fe3O4 NPs-modified electrode, which will benefit from high sensitivity. The hybridizing fraction of ssDNA II on the APTS-Fe3O4 NPs-modified electrode is ∼86%. We have evaluated the reproducibility of DNA immobilization on an APTS-Fe3O4 NPs-modified electrode. After reproduction, the redox peak currents of the electrode can return to the level for the ssDNA I-modified electrode, and the reproduced electrode can still capture ssDNA II. After having reproduced the modified electrode five times, the electrode is still sensitive to ssDNA II, with only a little passivation, indicating good reproducibility. Further studies reveal that this modified electrode can also be used for lower concentrations of ssDNA II and that there is a linear relationship between the concentration of ssDNA II and the peak current of the K3[Fe(CN)6]/K4[Fe(CN)6] couple (see Figures S3 and S4 in the Supporting Information). The oxidative peak current is linear to the ssDNA II concentrations in the range from 5 to 300 nM (the linear regression equation is y ) 20.24147 - 0.00816x; R ) 0.999). The detection limit is 2 nM. Magnetism of Fe3O4 NPs has also been found to be able to control the DNA hybridization process. Figure 2 is the cyclic voltammograms for a magnetism-controlled hybridization. It is observed that the peak current for K3[Fe(CN)6]/K4[Fe(CN)6] remains approximately unchanged, although a magnetic field that attracts the previously added OA-Fe3O4 NPs in the solution of ssDNA II to the electrode surface is applied. However, if the magnetic field which will repel the OA-Fe3O4 NPs from the electrode surface is applied, a measurable decline in the peak currents for K3[Fe(CN)6]/K4[Fe(CN)6] can be observed, indicating that hybridizing has occurred. UV-vis spectral experiments have also confirmed the above results. In the case that the magnetic field attracts the OA-Fe3O4 NPs, the hybridizing fraction is 3.8% for 2 h, 6.2% for 6 h, 7.9% for 24 h, and remains unchanged later. However, if the magnetic field is applied in a reversed position, the hybridizing fraction is
Figure 2. Cyclic voltammograms of 5 mM K3[Fe(CN)6]/K4[Fe(CN)6] obtained at ssDNA I-modified electrode (solid line). Ma and Mr lines are separately the curves after hybridization with ssDNA II in a magnetic field which has attracted for 12 h or repelled for 2 h. Scan rate: 200 mV s-1.
calculated to be 131.1% for 2 h, and remains unchanged later. Therefore, the OA-Fe3O4 NPs are working as a magnetoswich here, either improving or blocking DNA hybridization. ACKNOWLEDGMENT This work has been supported by the National Natural Science Foundation of China (Grant No. 90406005) and the Program for New Century Excellent Talents in University, the Chinese Ministry of Education. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review November 3, 2005. Accepted February 8, 2006. AC051962X
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