Peptide−Nucleic Acid-Modified Ion-Sensitive Field-Effect Transistor

VT, and a decrease in the saturated drain current, ID. These results demonstrate that direct detection of DNA hybridization should be possible using a...
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Anal. Chem. 2007, 79, 52-59

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Peptide-Nucleic Acid-Modified Ion-Sensitive Field-Effect Transistor-Based Biosensor for Direct Detection of DNA Hybridization Takeshi Uno,* Hitoshi Tabata, and Tomoji Kawai

The Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan

Here, we report the development of a peptide-nucleic acid (PNA)-modified ion-sensitive field-effect transistor (IS-FET)-based biosensor that takes advantage of the change in the surface potential upon hybridization of a negatively charged DNA. PNA was immobilized on a silicon nitride gate insulator by an addition reaction between a maleimide group introduced on the gate surface, the succinimide group of N-(6-maleimidocaproyloxy) succinimide, and the thiol group of the terminal cysteine in PNA. The surface was characterized after each step of the reaction by X-ray photoelectron spectroscopy analysis, and the kinetic analysis of the hybridization events was assessed by surface plasmon resonance. In addition, we measured the ζ-potential before and after PNA-DNA hybridization in the presence of counterions to investigate the change in surface charge density at the surface-solution interface within the order of the Debye length. On the basis of the ζ-potential, the surface charge density, ∆Q, calculated using the Grahame equation was approximately 4.0 × 10-3 C/m2 and the estimated number of hybridized molecules was at least 1.7 × 1011/cm2. The I-V characteristics revealed that the PNA-DNA duplexes induce a positive shift in the threshold voltage, VT, and a decrease in the saturated drain current, ID. These results demonstrate that direct detection of DNA hybridization should be possible using a PNA-modified IS-FET-based biosensor. PNA is particularly advantageous for this system because it enables highly specific and selective binding at low ionic strength. Microarray-based technology for analysis of gene expression and detection of gene mutations has become indispensable for both clinical and basic research. Recently, analysis of genomic mutations for drug-metabolizing enzymes (pharmacogenomics), including detection of single nucleotide polymorphisms (SNP), has received increasing attention for the prediction of the efficacy and side effects of therapeutic drugs. Differences in the cytochrome P450 (CYP) superfamily are of interest because they are important mediators of drug metabolism.1 CYP2C9 and CYP2C19, in particular, metabolize a large number of therapeutically * Corresponding author. Phone: +81-6-6879-8424-5131. Fax: +81-6-6875-2440. E-mail: [email protected].

52 Analytical Chemistry, Vol. 79, No. 1, January 1, 2007

important drugs. For example, CYP2C9 is responsible for the metabolism of the isomer of warfarin that is principally responsible for the drug’s anticoagulant effect2-5 and CYP2C19 metabolizes the prototype anticonvulsant drug S-mephenytoin via 4′-hydroxylation.6,7 SNP typing of these drug-metabolizing enzymes is an important target in tailor-made medical therapy. It is expected that the combination of genomic and pharmacologic information along with improved silicon-based technology and data processing will allow generation of new highly specific, sensitive, and reliable biosensors. Biosensors based on biologically modified ion-sensitive field-effect transistors (IS-FETs) are very attractive because of their small size and weight, high reliability, fast response, portability, and low cost of mass production. They can be subdivided into four FETs according to the biorecognition element that is used for detection: enzyme-modified FET, immunologically modified FET, DNA-modified FET, and cell-based FET. Basic mechanisms of potential generation for biologically modified IS-FETs include potential changes caused by (i) a catalytic reaction product (e.g., H+ generation or between an enzyme and its substrate), (ii) surface polarization effects or changes in the dipole moment (e.g., antigen-antibody binding or DNA hybridization), and (iii) potential changes that are coming from living biological systems.8 IS-FET-based pH and enzymemodified FET sensors are already commercially available and commonly used.9-12 Recently, FET-based DNA sensors have received considerable attention for use in both clinical and (1) Wilson, J. F.; Weale, M. E.; Smith, A. C.; Gratrix, F.; Fletcher, B.; Thomas, M. G.; Bradman, N.; Goldstein, D. B. Nat. Genet. 2001, 29, 265-269. (2) Wen, S. Y.; Wang, H.; Sun, O. J.; Wang, S. Q. World J. Gastroenterol. 2003, 9 (6), 1342-1346. (3) Takahashi, H.; Ieiri, I.; Wilkinson, G. R.; Mayo, G.; Kashima, T.; Kimura, S.; Otsubo, K.; Echizen, H. Blood 2004, 103 (8), 3055-3057. (4) Pickering, J. W.; McMillin, G. A.; Gedge, F.; Hill, H. R.; Lyon, E. Am. J. PharmacoGenomics 2004, 4 (3), 199-207. (5) Sandberg, M.; Johansson, I.; Christensen, M.; Rane, A.; Eliasson, E. Drug Metab. Dispos. 2004, 32, 484-489. (6) Kim, W. J.; Sato, Y.; Akaike, T.; Maruyama, A. Nat. Mater. 2003, 2, 815820. (7) Morais, S. M. F.; Wilkinson, G. R.; Blaisdell, J.; Nakamura, K.; Meyer, U. A.; Goldstein, J. A. J. Biol. Chem. 1994, 269 (22), 15419-15422. (8) Schoning, M. J.; Poghossian, A. Analyst 2002, 127, 1137-1151. (9) Yuqing, M.; Jianguo, G.; Jianrong, C. Biotechol. Adv. 2003, 21, 527-534. (10) Ito, Y. Sens. Actuators, B 2000, 64, 152-155. (11) Dzyadevich, S. V.; Korpan, Y. I.; Arkhipova, V. N.; Alesina, M. Y.; Martelet, C.; El’Skaya, A. V.; Soleatkin, A. P. Biosens. Bioelectron. 1999, 14, 283287. 10.1021/ac060273y CCC: $37.00

© 2007 American Chemical Society Published on Web 11/17/2006

research applications. However, whether IS-FET-based biosensors will be sufficiently sensitive for detecting hybridization events has not been clear because the charge of bound nucleotides is neutralized by counterions. In addition, only surface charge density changes within the order of the Debye length can be detected.8,13-16 In a previous paper, we pointed out that the hybridization of an immobilized peptide-nucleic acid (PNA) with a complementary DNA induces a decrease in saturation current and a positive shift in threshold voltage.17,18 Several groups also have recently reported that an IS-FET can detect surface potential changes resulting from the surface adsorption of charged molecules in an aqueous environment.19-26 We have particularly focused on PNA as a probe and have attempted the direct detection of DNA hybridization. PNA is a structural DNA analogue with a charge-neutral N-(2-aminoethyl) glycine backbone replacing the negatively charged phosphate backbone of DNA. PNA highly discriminates mismatch DNA and has stronger binding affinity for complementary DNA than its DNA counterpart at lower ionic strength.27-39 To better interpret the results obtained from IS-FET, we carried out surface analysis, including as X-ray photoelectron spectroscopy (XPS) and surface plasmon resonance (SPR). We also measured (12) Hara, M.; Yasuda, Y.; Toyotama, H.; Ohkawa, H.; Nozawa, T.; Miyake, J. Biosens. Bioelectron. 2002, 17, 173-179. (13) Bergveld, P. Biosens. Bioelectron. 1991, 6, 55-72. (14) Bergveld, P. Sens. Actuators, A 1996, 56, 65-73. (15) Schasfoort, R. B. M.; Kooyman, R. P. H.; Bergveld, P.; Greve, J. Biosens. Bioelectron. 1990, 5, 103-124. (16) Schasfoort, R. B. M.; Bergveld, P.; Kooyman, R. P. H.; Greve, J. Anal. Chim. Acta 1990, 238, 323-329. (17) Uno, T.; Ohtake, T.; Tabata, H.; Kawai, T. Jpn. J. Appl. Phys. 2004, 43 (12B), L1584-L1587. (18) Ohtake, T.; Hamai, C.; Uno, T.; Tabata, H.; Kawai, T. Jpn. J. Appl. Phys. 2004, 43 (9A/B), L1137-L1139. (19) Kim, D. S.; Jeong, Y. T.; Park, H. J.; Choi, P.; Lee, J. H.; Lim, G. Biosens. Bioelectron. 2004, 20, 69-74. (20) Kim, D. S.; Jeong, Y. T.; Lyu, H. K.; Park, H. J.; Kim, H. S.; Shin, J. K.; Choi, P.; Lee, J. H.; Lim, G.; Ishida, M. Jpn. J. Appl. Phys. 2003, 42, 4111-4115. (21) Sakata, T.; Miyahara, Y. ChemBioChem 2005, 6, 703-710. (22) Souteyrand, E.; Cloarec, J. P.; Martin, J. R.; Wilson, C.; Lawrence, C.; Mikkelsen, S.; Lawrence, M. F. J. Phys. Chem. B 1997, 101, 2980-2985. (23) Frits, J.; Cooper, E. B.; Gaudet, S.; Sorger, P. K.; Manalils, S. R. Proc. Natl. Acad. Sci. U.S.A. 2002, 99 (29), 14142-14146. (24) Hahm, J.; Lieber, C. M. Nano Lett. 2004, 4 (1), 51-54. (25) Li, Z.; Chen, Y.; Li, X.; Kamins, T. I.; Nauka, K.; Williams, R. S. Nano Lett. 2004, 4 (2), 245-247. (26) Berney, H.; West, J.; Haefele, E.; Alderman, J.; Lane, W.; Collins, J. K. Sens. Actuators, B 2000, 68, 100-108. (27) Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.; Ferler, S. M.; Driver, D. A.; Berg, R. H.; Kim, S. K.; Norden, B.; Nielsen, P. E. Nature 1993, 365, 566-568. (28) Nielsen, P. E.; Egholm, M.; Buchardt, O. Bioconjugate Chem. 1994, 5, 3-7. (29) Nielsen, P. E.; Haaima, G. Chem. Soc. Rev. 1997, 73-78. (30) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science 1991, 254, 1497-1500. (31) Weiler, J.; Gausepohl, H.; Hauser, N.; Jensen, O. N.; Hoheisel, J. D. Nucleic Acids Res. 1997, 25 (14), 2792-2799. (32) Macanovic, A.; Marquette, C.; Polychronakos, C.; Lawrence, M. F. Nucleic Acids Res. 2004, 32 (2), e20. (33) Aoki, H.; Umezawa, Y. Analyst 2003, 128, 681-685. (34) Aoki, H.; Umezawa, Y. Electroanalysis 2002, 14 (19-20), 1405-1410. (35) Aoki, H.; Buhlmann, P.; Umezawa, Y. Electroanalysis 2000, 12 (16), 12721276. (36) Ozkan, D.; Kara, P.; Kerman, K.; Meric, B.; Erdem, A.; Jelen, F.; Nielsen, P. E.; Ozsoz, M. Bioelectrochemistry 2002, 58, 119-126. (37) Hook, F.; Ray, A.; Norden, B.; Kasemo, B. Langmuir 2001, 17, 8305-8312. (38) Kambhampati, D.; Nielsen, P. E.; Knoll, W. Biosens. Bioelectron. 2001, 16, 1109-1118. (39) Cherny, D. I.; Fourcade, A.; Svinarchuk, F.; Nielsen, P. E.; Malvy, C.; Delain, E. Biophys. J. 1998, 74, 1015-1023.

Figure 1. Schematic representation of the hybridization of PNAmodified IS-FET by DNA in solution. The IS-FET consists of a p-type silicon substrate with two n-doped regions (source and drain), separated by a short channel that is covered by a silicon nitride gate insulator.

the ζ-potential, which is a well-established analytical method for characterizing the electrochemical surface properties. A change in the ζ-potential upon hybridization implies that the surface charge density changes within the order of the Debye length of the IS-FET. To date, very few studies have examined the change in the ζ-potential upon hybridization. Finally, we sought to demonstrate the direct detection of DNA hybridization by a PNAmodified IS-FET-based biosensor that responds to the change in the surface potential. In the current studies, we characterized the surface physical and electrochemical properties as well as the kinetics of PNADNA duplex formation. We also discuss the possible utility of the PNA-modified IS-FET-based biosensor. EXPERIMENTAL SECTION CYP2C9 Gene and Probe Design. The CYP2C9 gene encodes a member of the CYP superfamily of enzymes. The CYP2C9 gene lies on chromosome 10 q22.33 and consists of nine exons. Five alleles of CYP2C9, including the wild-type CYP2C9*1 and the mutants CYP2C9*2 (C430T), CYP2C9*3 (A1075C), CYP2C9*4 (T1076C), and CYP2C9*5 (C1080G), have been found on exons 3 and 7. The CYP2C9 mutation examined in this study was CYP2C9*2 (exon 3). All oligonucleotides were synthesized by Sigma Genosys Japan K.K., and PNAs with ethylene glycol (O) spacers at the cysteine terminus were from Fasmac Co. Ltd. The sequences of the synthesized oligonucleotides were as follows: CYP2C9*1 (wild-type), Cys-OO-TGAGGACCGTGTTCA; CYP2C9*2 (mutant), Cys-OO-TGAGGACTGTGTTCA; negative control, Cys-OO-GGCAGTGCCTCACAA; target DNA1 (complementary to CYP2C9*1), 5′-TGAACACGGTCCTCA-3′; target DNA2 (complementary to CYP2C9*2), 5′-TGAACACAGTCCTCA-3′. Preparation of PNA-Modified IS-FET and DNA Hybridization. An n-channel depletion type IS-FET was fabricated by Hitachi Ultra LSI systems. A schematic representation of the fabricated IS-FET microarray chip is shown in Figure 1. The IS-FET consists Analytical Chemistry, Vol. 79, No. 1, January 1, 2007

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Scheme 1. Schematic Representation of the Process of Covalently Immobilizing the Probe PNA on the Gate Insulator

Table 1. XPS Elemental Surface Analysis of Silicon Nitride before and after the Sequential Modification Steps surface XPS (at. %)

C1s

N1s

O1s

Si2p

Si3N4 substrate sulfo-EMCS/ATEOS on Si3N4 immobilization of PNA on Si3N4

9.8 28.4 39.4

21.3 15.2 26.0

41.7 42.8 29.1

27.1 13.6 6.5

of a p-type silicon substrate with two n-doped regions (source and drain), which are separated by a short channel covered by the gate insulator. The gate insulator is a double layer of SiO2-Si3N4, and each layer is 100 nm thick. The length of the gate region was between 10 and 300 µm, and the width was fixed at 1000 µm. Scheme 1 shows a schematic representation of the covalent immobilization process of the probe PNA on the Si3N4 gate insulator. The surface modification of the Si3N4 gate region was performed under the following conditions. Prior to aminosilanation, the gate surface was soaked in piranha solution (1:3 H2O2/ H2SO4) for 4 min and then washed with deionized distilled water. The Si3N4 surface was next irradiated with UV light for 30 min. The gate surface was immediately immersed for 1 h at room temperature in a solution of acetone containing 1% 3′-aminopropyltriethoxysilane (APTES; Shin-Etsu Chemical) and then washed with acetone. After drying, the IS-FET was baked at 110 °C for 30 min to silanize the surface with the aminosilane coupler. The silanized gate surface was immersed for 1 h at 37 °C in PBS(-) (0.01 M phosphate-buffered saline without Ca2+ and Mg2+, pH 7.2-7.4) containing 1 mM N-(6-maleimidocaproyloxy) sulfosuccinimide, sodium salt (sulfo-EMCS; Dojindo), to introduce maleimide groups by a condensation reaction between the amino group of the silanized surface and the succinimide group of sulfo-EMCS. The gate surface was washed with PBS(-). Probe PNA in PBS(-) was immobilized on the sulfo-EMCS-modified gate surface by addition reaction for 1 h at 37 °C between the maleimide group 54 Analytical Chemistry, Vol. 79, No. 1, January 1, 2007

on the surface and the thiol group of the terminal cysteine of PNA. The gate surface was then washed with PBS(-) and exposed for 1 h at 37 °C to an aqueous solution of 1 mM 6-hydroxy-1hexanethiol to remove nonspecifically bound DNA. After immobilization of the probe PNA on the gate surface of the IS-FET, the gate surface was hybridized for 1 h at 60 °C with 4 µM of target DNA in 0.2× SSC (3 mM sodium citrate containing 30 mM sodium chloride solution) and then washed with the same buffer. Measurements of I-V characteristics were performed with a Keithley 4200-SCS semiconductor analyzer using a semiconductor characterization system with three terminals: source, drain, and gate. A standard Ag/AgCl electrode was used as the reference, and the electrolyte was 0.2× SSC. All measurements were carried out in the dark to avoid photogeneration of charge carriers in the

Figure 2. XPS spectra obtained for (a) N1s and (b) C1s of the silicon nitride surface before and after the following sequential modifications: (a-1 and b-1) the silicon nitride surface following hydrophilic treatment by irradiation with UV light; (a-2 and b-2) the APTES-silanized and sulfo-EMCS-treated surface; (a-3 and b-3) the PNA-immobilized surface.

Figure 3. XPS high-resolution spectra of the fitted curves for (a) N1s and (b) C1s. The lower part of each panel shows the fitted curve for the APTES-silanized and sulfo-EMCS-treated surface, and the upper part shows the fitted curve for the PNA-immobilized surface.

semiconducting electrode. During the measurement of ID-VD characteristics, the drain-source voltage was swept from 0 to 4 V at a gate voltage (VG) of -3 V. For ID-VG characteristics, the gate-source voltage was swept from -4 to 0 V at a drain voltage (VD) of 2 V. XPS Measurements. Each immobilization step was characterized using XPS. Measurements were made using a JEOL JPS9000MC spectrometer equipped with an Al KR aluminum anode (1486.6 eV). The samples used in these measurements have a double layer of SiO2-Si3N4 on a silicon substrate and a cross section of 1 × 1 cm2. Immobilization of PNA was performed as described in the previous section. All of the binding energy peaks were calibrated in relation to the Si2p peak at 99.2 eV. The calibration showed the presence of a C1s hydrocarbon peak at 284.8 eV. Peaks from all high-resolution core spectra were fitted using the nonlinear least-squares curve fitter of Origin 7.0 scientific graphing and analysis software. SPR Kinetic Measurements. SPR measurements used a BIACORE 3000 apparatus (BIACORE K.K.), and analysis of SPR data was performed using BIAevaluation software. The measurements were carried out using a Au sensor chip modified with 1 mM 6-amino-1-hexanethiol (6-AHT) hydrochloride (Dojindo). The 6-AHT-modified Au sensor chip was then docked into the BIACORE apparatus, and the probe PNA was immobilized on the

sensor chip by injecting PBS(-) containing 1 mM sulfo-EMCS at 37 °C at a constant flow rate of 10 µL/min. This introduced maleimide groups on the 6-AHT-modified Au surface by condensation reaction of amino groups. After washing with PBS(-), PNA was immobilized on the sensor chip by injecting PBS(-) containing 4 µM probe PNA at 37 °C at a constant flow rate of 10 µL/ min. After washing again with PBS(-) at the same flow rate and temperature, the sensor chip was exposed to an aqueous solution of 1 mM 6-hydroxy-1-hexanethiol. Kinetic measurements of the interaction between the probe PNA on the sensor chip and the target DNA were carried out by injecting 150 µL of 0.2× SSC containing the target DNA at a flow rate of 20 µL/min at 40 °C. The dissociation time for the measurements was 900 s. Measurement of the Melting Temperature (Tm). The Tm values were determined in 0.2× SSC by measuring the hypochromicity at 260 nm as a function of the temperature using a Ultrospec 3300 pro PTR UV-vis spectrophotometer (Amersham Biosciences). In these experiments, the temperature was increased from 30 to 90 °C in steps of 2.0 °C/min. The two strands were mixed in equimolar amounts, and annealing was performed by heating the sample to 90 °C, followed by cooling to 30 °C in steps of 1 °C/min. ζ-Potential Measurements. The ζ-potential was measured at Otsuka Electronics Co., Ltd. with an ELS-8000 electrophoretic Analytical Chemistry, Vol. 79, No. 1, January 1, 2007

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Figure 4. SPR responses for PNA-DNA hybridization. (a) CYP2C9*2 was used as a probe PNA, and the target DNA was complementary to CYP2C9*2 (target DNA 2). The target DNA concentrations were (from top to bottom) 5.0, 2.5, 1.0, and 0.1 µM. (b-1) The top sensorgram shows CYP2C9*2 with a target DNA containing a single base mismatch (5.0 µM). (b-2) The bottom sensorgram shows immobilized PNA with a single base mismatch (CYP2C9*1) with the CYP2C9*2 complimentary target DNA (5.0 µM).

light-scattering spectrophotometer in 10 mM NaCl or 0.2× SSC containing hydroxypropyl cellulose-coated particles. The electrophoretic mobility is determined by a frequency shift owing to the Doppler effect, and the ζ-potential is calculated using the Smoluchowski equation obtained from the electrophoretic mobility.40-43 RESULTS AND DISCUSSION Surface Characterization. Table 1 summarizes the results of XPS survey scans of the elemental surface composition of silicon nitride before and after the various steps of modification. An increase in the C1s content and a decrease in the Si2p content were observed during the immobilization process, and the O21s content of the silicon nitride substrate was above 40%. This indicates that a highly oxygenated surface layer was formed over the silicon nitride surface after hydrophilic treatment by irradiation with UV light. This highly oxygenated silicon oxynitride layer was chemically reacted with APTES to immobilize the PNA.44 Figure 2 shows the XPS spectra for N1s and C1s from the surface irradiated with UV light (Figure 2, curves a-1 and b-1), the surface silanized with APTES and treated with sulfo-EMCS (Figure 2, curves a-2 and b-2), and the PNA-immobilized surface (Figure 2, curves a-3 and b-3). Following the condensation reaction between APTES and sulfo-EMCS, the XPS spectra (Figure 2, parts a and b) show two peaks for the N1s curve at binding energies of 397.6 and 400.4 eV, and the peak of the higher binding energy increased markedly after PNA immobilization. A similar behavior was observed for the C1s XPS spectra. We observed two peaks in the C1s spectra at binding energies of 285.2 and 288.6 eV due (40) Koch, S.; Woias, P.; Meixner, L. M.; Drost, S.; Wolf, H. Biosens. Bioelectron. 1999, 14, 413-421. (41) Matsumoto, H.; Koyama, Y.; Tanioka, A. J. Colloid Interface Sci. 2003, 264, 82-88. (42) Horiuchi, K.; Dutta, P. Lab Chip 2006, 6, 714-723. (43) Sze, A.; Erichson, D.; Ren, L.; Li, D. J. Colloid Interface Sci. 2003, 261, 402-410. (44) Wu, P.; Hogrebe, P.; Grainger, D. W. Biosens. Bioelectron. 2006, 21, 12521263.

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to the amide peak generated by the sulfo-EMCS reaction, and the amide peak increased significantly because of the peptide backbone of PNA. In addition, the position of the peak for the aliphatic chain was shifted from 284.8 to 286 eV.44-50 Putative assignments for these peaks were made using a nonlinear least-squares approximation (Figure 3, parts a and b). The fitted curve in the lowest part of Figure 3, parts a and b, corresponds to Figure 2, curves a-2 and b-2, respectively, and the fitted curve in the upper part of Figure 3, parts a and b, corresponds to Figure 2, curves a-3 and b-3, respectively. The dotted lines in Figure 3, parts a and b, show the data obtained, and the solid lines show the fits. The N1s peak was divided into four peaks: (1) SixNy; (2) -N-C- and -NH2; (3) -NCdO; (4) -NH3+. The C1s peak was divided into three peaks: (1) -CC-; (2) -C-N-; (3) -NCdO. These findings are similar to previously reported results from studies of silicon and titanium oxides surfaces silanized with N-(2-aminoethyl)-3-aminopropyltrimethoxysilane and modified with sulfosuccinimidyl 4-[N-maleimidomethyl]-cyclohexane 1-carboxylate.47-50 Collectively, the XPS data confirm that PNA was successfully immobilized onto the silicon nitride substrate. Kinetic Measurements. These results confirmed the immobilization of PNA on the silicon nitride surface using XPS analysis. We next examined the molecular recognition at the surface-solution interface. We used SPR to make kinetic measurements in the heterogeneous system between immobilized PNA and DNA strands in solution (Figure 4, parts a and b). At (45) Strother, T.; Hamers, R. J.; Smith, L. M. Nucleic Acids Res. 2000, 28 (18), 3535-3541. (46) Shlyakhtenko, L. S.; Gall, A. A.; Weimer, J. J.; Hawn, D. D.; Lyubchenko, Y. L. Biophys. J. 1999, 77, 568-576. (47) Choi, W. K.; Koh, S. K.; Jung, H. J. Vac. Sci. Technol. 1999, A17 (6), 33623367. (48) Rezania, A.; Johnson, R.; Lefkow, A. R.; Healy, K. E. Langmuir 1999, 15, 6931-6939. (49) Ganapathy, R.; Manolache, S.; Majid, S.; Ferencz, D. J. Biomater. Sci., Polym. Ed. 2001, 12, 1027-1049. (50) Lin, C. W.; Lin, J. C. J. Biomater. Sci., Polym. Ed. 2001, 12, 543-557.

Table 2. Comparison of the Tm, kd, ka, and KD Values PNA probe CYP2C9*2 CYP2C9*2 CYP2C9*2 CYP2C9*1

target DNA target DNA3(PM) target DNA1(1MM) target DNA4(2MM) target DNA3(1MM)

Tm (°C)

ka (1/sM)

74.8 67.2 42.0 58.1

1.0 × 5.7 × 103 5.4 × 102 2.2 × 103 104

kd (1/s)

KD (M)

10-4

5.6 × 1.2 × 10-3 3.6 × 10-3 2.7 × 10-3

5.4 × 10-8 2.0 × 10-7 6.8 × 10-6 1.2 × 10-6

Table 3. Summary of ζ-Potentials Determined from the Osmosis Plot Spectra values of the ζ-potential (mV) 10 mM NaCl 30 mM NaCl

Figure 5. Osmosis plot spectra: (a) the silicon nitride surface following hydrophilic treatment by irradiation with UV light; (b) the silicon nitride surface following modification with PNA; (c) hybridization of the immobilized PNA with the target DNA in solution.

the same time, we measured the Tm for PNA-DNA duplexes in a homogeneous system (PNA and DNA in solution) to compare with the dissociation equilibrium constants obtained from the heterogeneous system (immobilized PNA and DNA in solution). Figure 4a shows the binding of immobilized CYP2C9*2 PNA to four different concentrations of a complementary DNA. Increasing the target DNA concentration from 0.1 to 5.0 µM increased the association rates. At higher target DNA concentrations (2.5 and 5.0 µM), the increase in the association rate and the decrease in the dissociation rate were faster than at lower DNA concentrations (1 and 0.1 µM). This decrease in the rate of dissociation is

(a) -50.2 (a) -36.0

(b) -26.0 (b) -26.4

(c) -53.9 (c) -36.1

presumably due to the influence of the nonspecifically bound DNA; however, the dissociation rate appeared to reach a minimum at concentrations below 1.0 µM. In Figure 4b, the immobilized PNAs on the sensor chip are CYP2C9*2 (Figure 4b-1) and CYP2C9*1 (Figure 4b-2). The target DNAs have a single base mismatch for CYP2C9*2 (Figure 4b-1) and a single mismatch for CYP2C9*1 (Figure 4b-2), respectively. The association rates were lower than that measured for the completely matched pair, and the dissociation rates tended to decrease more quickly. Table 2 summarizes the Tm values, the dissociation and association rate constants (kd and ka, respectively), and the dissociation equilibrium constants (KD’s). The KD is 5.4 × 10-8 M and the Tm is 74.8 °C for a perfect match. The most thermally stable duplexes exhibited the lowest kd and highest ka values. In addition, for the PNA-DNA duplexes, there was a good correlation between the calculated KD in the heterogeneous system and the measured Tm in the homogeneous system.51-54 Surface Potential Measurements. After characterizing the surface of the PNA-modified silicon nitride and the kinetics for PNA-DNA hybridization, we examined the change in the surface potential before and after hybridization by measuring the ζ-potential with an electrophoretic light-scattering spectrophotometer. The osmosis plot spectra are shown in Figure 5, and the ζ-potential values determined from them are listed in Table 3. Figure 5a shows the spectrum for silicon nitride subjected to hydrophilic treatment by irradiation with UV light. Figure 5, parts b and c, shows the spectra of the PNA-modified silicon nitride and the hybridization of DNA strands by the immobilized PNA, respectively. The negatively charged silicon nitride substrate was shifted toward a zero ζ-potential (from -50.2 to -26.0 mV) due to the immobilization of the uncharged PNA; however, the ζ-potential of the immobilized PNA did not reach zero. This is probably due to the coverage of the immobilized PNA. Thereafter, hybridization of the negatively charged DNA induced a negative shift of the ζ-potential from -26.0 to -53.9 mV. It is an interesting point that the ζ-potential after DNA hybridization shows a similar value of (51) Jensen, K. K.; Orum, H.; Nielsen, P. E.; Norden, B. Biochemistry 1997, 36, 5072-5077. (52) Burgener, M.; Sanger, M.; Candrian, U. Bioconjugate Chem. 2000, 11, 749754. (53) Peterson, A. W.; Wolf, L. K.; Genorgiadis, R. M. J. Am. Chem. Soc. 2002, 124, 14601-14607. (54) Persson, B.; Stenhag, K.; Nilsson, P.; Larsson, A.; Uhlen, M. Anal. Biochem. 1997, 246, 34-44.

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Figure 6. Measurements of I-V characteristics: (a) The ID-VD characteristics for the change in ID before and after hybridization. The solid line indicates the ID-VD curve for the PNA-immobilized IS-FET, and the dotted line indicates the ID-VD curve for hybridization of the PNAmodified IS-FET by the target DNA. (b) Comparison of the change ratios in the ID before and after hybridization of the PNA-modified IS-FET with the complementary and noncomplementary target DNAs. (c) The ID-VG characteristics for the change in the VT before and after hybridization. The solid line indicates the PNA-immobilized IS-FET, and the dotted line indicates hybridization of the PNA-modified IS-FET with DNA. (d) The local area of (c).

the silicon nitride (SixNy) surface. In general, there is no chemical similarity between the PNA-terminated silicon nitride surface and that of the bare SixNy subjected to hydrophilic treatment. Accordingly, this may imply that the degree of dissociation of the hydroxy group on the silicon oxynitride layer is equivalent to that of the hybridized DNA phosphate backbone in the same electrolyte. In addition, the change in the ζ-potential tended to increase with decreasing ionic strength of the solution. Therefore, the ζ-potential measurements showed that the hybridization event causes a change in the surface potential and that a low ionic strength leads to an increase in the ζ-potential. These results indicate that the change in the surface potential is detectable in the presence of counterions and that a change in the surface charge density occurs within the order of the Debye length. The structure of a DNA molecule can be described as a circular cylinder with negative charges evenly distributed over its surface. Following the formation of PNA-DNA duplexes, some region of the cylinder may overlap on the order of the Debye length. Therefore, these results support the idea that the IS-FET-based biosensor can be used for detecting hybridization events. Moreover, we expect that the IS-FET based on PNA will be more 58 Analytical Chemistry, Vol. 79, No. 1, January 1, 2007

effective than the one based on DNA for detecting hybridization because PNA-DNA binding is unaffected by the salt concentration. Our next step, therefore, was to determine whether an IS-FET based on PNA can be used as a DNA sensor. This device was based on an electrolyte-insulator-semiconductor capacitor structure. When a positive voltage, VG, is applied to the gate (0 < VG < VT, where VT is the threshold voltage), the majority carrier is pushed away and a depleted region (space-charge region) is formed. With further increases in the VG (VT < VG), mobile electrons are induced at the surface, and the surface is inversed so that it becomes an n-type channel. For the PNA-based IS-FET, a negative charge is induced at the uncharged PNA-modified gate surface due to DNA hybridization, and the mobile electrons are reduced at the same gate voltage. Therefore, it is expected that a decrease in the drain current (ID) and a positive shift in the VT due to the decrease of the ID will be observed. To investigate this principle, we carried out measurements of the I-V characteristics. Figure 6, parts a and c, shows representative results for ID-VD and ID-VG characteristics, respectively. The change in ID before and after hybridization at a given source-

drain voltage (VD) is shown in Figure 6a. The black line indicates the I-V curve of the PNA-modified IS-FET, and the red line indicates the I-V curve after DNA hybridization. As we expected, the saturated ID decreased by 5.5 µA. To evaluate this change in more detail, we compared the change in the ID in the presence of complementary and noncomplementary DNA (positive and negative controls, respectively) (Figure 6b). The change ratio (∆I) is expressed as ∆I (%) ) (I1 - I0)/I1 × 100, where I1 is the saturated ID before hybridization and I0 is the saturated ID after hybridization. The values for the positive control were larger than those for the negative control, but the differences varied from 5% to 14.8%. Although these changes support the possibility of developing an IS-FET-based biosensor, it will be necessary to employ a system for measuring differential arrangement. The change in the square root of the ID before and after hybridization at a given VG is shown in Figure 6c, and the local area is shown in Figure 6d. A linear extrapolation of the I-V curve to x ) 0 was performed to obtain the VT value. The black line indicates the I-V curve of the PNA-modified IS-FET, and the red line indicates the I-V curve after DNA hybridization. The VT values were -3.23 and -3.17 V, respectively. A positive shift in the VT of 60 mV was observed after hybridization, and the negative control was also shifted in the positive direction by 20 mV (data not shown). These positive shifts are due to negatively charged DNA at the gate surface. Therefore, the changes in the ID and the VT upon PNA-DNA hybridization support the idea that a change in the charge density at the interface induces a change in the surface potential. Recently Macanovic and co-workers reported that an impedance-based detection of single-strand DNA sequences was possible by using PNA-modified Si chips.32 A flatband potential shift due to hybridization of DNA was found to be -375 mV by electrochemical impedance measurements. This method provides a more sensitive approach for detection of hybridization events compared to our system. However, it is expected that the PNA-modified IS-FET-based biosensor would have better sensitivity compared to the presented results by employing an optimized gate structure and a system for measuring differential arrangement. This will be the subject of further study. Finally, we calculated the surface charge density (∆Q) from the change in the surface potential by setting the stern potential (σ0) in the Grahame equation (eq 1) equal to the ζ-potential. We

then estimated the number of hybridized molecules from the calculated ∆Q. The Grahame equation is

σ0 ) x8w0kTc0sinh

( ) eψ0 2kT

(1)

where σ0 is surface charge density, ψ0 is surface potential, k is the Boltzmann constant, T is absolute temperature, e is elementary charge, 0 is the permittivity of free space, w is the dielectric constant of water, and c0 is the buffer ionic strength. We assumed an upper value of 15 charges per 15-mer DNA molecule. On the basis of the ζ-potential, the ∆Q is approximately 4.0 × 10-3 C/m2, and the estimated number of hybridized molecules is at least 1.7 × 1011 molecules/cm2. The ideal value for the amount of immobilization is approximately 4 × 1012 molecules/cm2, which is higher than the estimated value; however, taking the degree of dissociation and the hybridization efficiency into consideration, the estimated value is reasonable. These results confirm that hybridization causes a negative shift in the ζ-potential and a positive shift in the VT. CONCLUSIONS In the current studies, we demonstrated the immobilization of PNA on a silicon nitride substrate and the formation of doublestranded PNA-DNA at the surface-solution interface. We also showed that there is a good correlation between the calculated KD and the measured Tm from the PNA-DNA duplexes. The ζ-potential measurements revealed that the change in the surface potential is detectable in the presence of counterions and that a change in the surface charge density occurs within the order of the Debye length. Collectively, these results suggested that DNA hybridization can be directly detected by the PNA-modified ISFET-based biosensor. ACKNOWLEDGMENT This work was supported by a Grant-in-Aid for Core Research, Evolutional Science and Technology of the Japan Science and Technology Agency. Received for review February 13, 2006. Accepted October 2, 2006. AC060273Y

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