Electrochemical Imaging of Localized Sandwich DNA Hybridization

conductivity were sensitively detected by the SECM tip. The proposed method allows imaging of a ... regions on a DNA microarray glass slide, through t...
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Anal. Chem. 2007, 79, 7206-7213

Correspondence

Electrochemical Imaging of Localized Sandwich DNA Hybridization Using Scanning Electrochemical Microscopy Ilaria Palchetti,* Serena Laschi, Giovanna Marrazza, and Marco Mascini

Dipartimento di Chimica, Universita` degli Studi di Firenze, Via della Lastruccia 3, 50019 Sesto Fiorentino (Fi), Italy

Imaging of localized hybridization of nucleic acids immobilized on gold-DNA chip was performed by means of the feedback mode of scanning electrochemical microscopy (SECM). Thiol-tethered oligodeoxynucleotide (HS-ODN) probes, spotted on a gold surface, were hybridized with unmodified target sequence via sandwich hybridization with a biotinylated signaling probe. Spots where sequence-specific hybridization had occurred were developed by adding a streptavidin-alkaline phosphatase conjugate and biocatalyzed precipitation of an insoluble and insulating product. As a consequence, the surface conductivity of the spotted region of the chip where hybridization had taken place changed. These changes in conductivity were sensitively detected by the SECM tip. The proposed method allows imaging of a DNA array in a straightforward way. Analysis of real samples was also performed coupling this method with polymerase chain reaction. The imaging of 60 nM PCR amplicon (255 bp) was demonstrated. Electrochemical detection schemes of DNA hybridization are generally considered cost-effective alternatives to conventional optical assays, as electrochemistry offers high sensitivity in combination with simplicity of instrumentation, as well as the possibility of miniaturization.1-6 In this context, the scanning electrochemical microscope (SECM), has demonstrated a high level of performance for studying immobilized biomolecules and biological processes,7 and some approaches in the use of SECM as readouts of hybridization * To whom correspondence should be addressed. E-mail: [email protected]. Tel.+390554573323. Fax +390554573397. (1) Lucarelli, F.; Marrazza, G.; Turner, A. P. F.; Mascini, M. Biosens. Bioelectron. 2004, 19, 515-530. (2) Kerman, K.; Kobayashi, M.; Tamiya, E. Meas. Sci. Technol. 2004, 15, R1R11. (3) Del Giallo, M. L.; Lucarelli, F.; Cosulich, E.; Pistarino, E.; Santamaria, B.; Marrazza, G.; Mascini, M. Anal. Chem. 2005, 77, 6324-6330. (4) Farabullini, F.; Lucarelli, F.; Palchetti, I.; Marrazza, G.; Mascini, M. Biosens. Bioelectron. 2007, 22, 1544-1549. (5) Lucarelli, F.; Marrazza, G.; Mascini, M. Biosens. Bioelectron. 2005, 20, 2001-2009. (6) Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 8916-8920. (7) Bard, A. J.; Mirkin, M.V. Scanning Electrochemical Microscopy; Marcel Dekker: New York, 2001.

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in DNA (micro)array were reported in the literature.8-14 Toth et al.8 reported preliminary studies for detecting DNA hybridization using glucose oxidase; the imaging of surface-confined DNA molecules and hybridization through guanine oxidation induced by the tip generated Ru(bpy)3+ has been shown by Wang and Zhou,9 with probe immobilized on classical microarray glass slides. Zhou et al. proposed the use of SECM to image DNA hybridization with silver enhancement:10 oligodeoxynucleotide (ODN) probes, immobilized on glass slides, were hybridized with biotinylated target, and hybridization was developed by a silver staining process (adsorption of streptavidin-gold nanoparticles followed by silver particle deposition) with consequent increase of surface conductivity. Komatsu et al. used SECM to visualize DNA duplex regions on a DNA microarray glass slide, through the use of ferrocenylnaphthalene diimide as an electrochemically active DNA hybrid indicator.13 Schuhmann and co-workers reported a labelfree electrochemical detection scheme:11,12 synthetic ODN were spotted on conducting surfaces and investigated by SECM in electrolytes containing [Fe(CN)6]3-/4- as a negatively charged redox mediator. Under these conditions, significant decreases in positive feedback currents were observed above DNA-modified regions due to local appearance of repulsion between deprotonated phosphate groups of the immobilized DNA strands and the [Fe(CN)6]3-/4- anions. Recently Fortin et al.14 described a patterning process achieved using the direct mode of SECM, where the electrical field established between the SECM tip and the gold interface was used to drive the local deposition of micrometersized polypyrrole spots to which an ODN strand was linked covalently. The detection of the hybridization reaction with biotinylated complementary strands was possible after subsequent reactions with streptavidin and biotinylated horseradish peroxidase, which, biocatalyzing the oxidation of 4-chloro-1-naphthol in (8) Gyurcsanyi, R. E.; Jagerszki, G.; Kiss, G.; Toth, K. Bioelectrochemistry 2004, 63, 207-215. (9) Wang, J.; Zhou, F. J. Electroanal. Chem. 2002, 537, 95-102. (10) Wang, J.; Song, F.; Zhou, F. Langmuir 2002, 18, 6653-6658. (11) Turcu, F.; Schulte, A.; Hartwich, G.; Schuhmann, W. Biosens. Bioelectron. 2004, 20, 925-932. (12) Turcu, F.; Schulte, A.; Hartwich, G.; Schuhmann W. Angew. Chem., Int. Ed. 2004, 43, 3482-3485. (13) Komatsu, M.; Yamashita, K.; Uchida, K.; Kondo, H.; Takenaka, S. Electrochim. Acta 2006, 51, 2023-2029. (14) Fortin, E.; Mailley, P.; Lacroix, L.; Szunerits, S. Analyst 2006, 131, 186193. 10.1021/ac070474h CCC: $37.00

© 2007 American Chemical Society Published on Web 08/15/2007

the presence of H2O2, and the precipitation of the insoluble product 4-chloro-1-naphthon on the hybridized areas on the gold film, caused a local alteration of conductivity.14 In this paper, we propose a SECM-based DNA array using a sandwich hybridization assay scheme with biotinylated signaling probe. The analytical method relied on the use of a gold conducting surface on which thiol-tethered ODN (HS-ODN) probes were spotted. Chemisorption of thiol-derivatized oligonucleotides onto gold substrates was extensively characterized using a number of methods, including XPS, ellipsometry, 32Pradiolabeling, neutron reflectivity, and electrochemical methods.1,6,15,16 As well reported in literature, precise control over surface coverage and probe availability can be achieved by creating mixed monolayers of the thiol-tethered DNA probe and a spacer thiol, such as 6-mercapto-1-hexanol (MCH). MCH largely displaced nonspecifically adsorbed oligonucleotides that interacted with the gold surface also or only through nitrogen-containing bases, thus dramatically increasing the availability of the surfacebound probes for specific hybridization. Neutron reflectivity measurements16,6 confirmed that, after treatment with MCH, probe molecules “stand up” consistently with the primary attachment through the thiol group. Moreover, assembly of MCH created a compact layer, which efficiently prevented the nonspecific adsorption of the complementary or noncomplementary sequences on the gold surface. The authors6,16 also observed that hybridization of the surface-bound probe was highly dependent on surface coverage. A combination of steric and electrostatic hindrance, arising from too tightly packed thiol-tethered probes, was found to severely inhibit the hybridization of the complementary sequence. In contrast, a nearly 100% hybridization efficiency was estimated for the optimized thiolated probe/MCH mixed monolayer. Thus, in order to achieve optimal surface coverage and probe availability, a mixed monolayer of the thiol-modified ODN probe and MCH was created, by a two-step method, where first the gold substrate was exposed to a micromolar solution of HS-ODN, followed by exposure to a millimolar solution of MCH. Then, unmodified ODN targets as well as PCR products were captured at the sensor interface via sandwich hybridization with surfacetethered probes and biotinylated signaling probe.3-5 The resulting biotinylated hybrids were coupled with an enzyme conjugate and then exposed to a proper substrate solution. SECM investigation of the hybridization process was performed in feedback mode. As reported in the literature,7 different modes of SECM operation are possible working with an enzymatic label, i.e., the collection mode, where the tip is moved over the immobilized enzymes and the product of the enzymatic reaction is detected. Wittstock et al.17 reported that compared to collector experiments much higher lateral resolution has been demonstrated with the enzyme-generated feedback mode. In this mode, a quasi reversible redox mediator enables communication between an amperometric tip electrode and the enzyme-modified surface. The mediator is converted at the tip, and the product of this reaction represents (15) Levicky, R.; Herne, T. M.; Tarlov, M. J.; Saija, S. K. J. Am. Chem. Soc. 1998, 120, 9787-9792. (16) Steel, A. B.; Herne, T. M.; Tarlov, M. J. Anal. Chem. 1998, 70, 46704677. (17) Wittstock, G.; Yu, K.; Haisali, H. B.; Ridgway, T. H.; Heineman, W. R. Anal. Chem 1995, 67, 3578-3582.

a cofactor for the enzymatic reaction, by which in return the educt is generated for the electrochemical reaction at the tip electrode. This leads to an increase of the tip current iT over the tip current iT∞ in the bulk solution. At an unmodified insulating surface, the tip current falls below the reference value iT∞ because the surface blocks the diffusion of the mediator toward the tip surface. In the present work, alkaline phosphatase (AP), a quite common enzyme label, was used. AP does not require a redox-active cosubstrate and, thus, cannot be imaged with the SECM positive feedback mode.17 In the literature5,18-20 was reported the development of biosensors based on the enzymatic biocatalyzed precipitation of an insoluble and insulating product onto the sensing interface. A similar strategy was adopted in this paper. Since chemisorption of thiolated probe was achieved on a gold surface and since gold is conductive, the enzyme-mediated selective precipitation of an insoluble and insulating product was studied by means of SECM in the feedback mode as detection system of the hybridization reaction. Localized change in conductivity was sensitively detected by the SECM tip and allowed high-quality imaging of DNA arrays. The proposed probe immobilization and hybrid labeling procedures were never used for hybridization detection using SECM; thus, each step of the molecular assembly was characterized by the feedback mode of SECM using a ferrocene derivative as neutral mediator. In respect to the approach reported in ref 14, a different DNA immobilization procedure and a completely different enzymatic label were tested. Furthermore, the effects of target concentration and of nonspecific interactions were also deeply studied. Long PCR amplicons (255 bp) at very low concentration (60 nM) were analyzed; imaging results demonstrated the possibility to analyze real samples by using this new strategy. EXPERIMENTAL SECTION Materials. NaH2PO4, Na2HPO4‚2H2O, NaCl, Tris, HClO4, NaClO4, K4[Fe(CN)6], ferrocenemonocarboxylic acid (FMCA), MCH, dodecylthiol, 2-propanol, ethanol, streptavidin-alkaline phosphatase (1000 U/mg), 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium (NBT) mixture, bovine serum albumin (BSA), magnesium chloride, and diethanolamine were obtained from Sigma-Aldrich-Fluka (Milan, Italy). MilliQ water was used throughout this work. Synthetic oligonucleotides were obtained from MWG Biotech AG. The capture probe (CP) sequence was as follows: 5′-HS(CH2)6-GCG CGC GAA CGG-3′. The target was an unmodified oligonucleotide with the following complementary sequence: 5′CCG CCA ATA AAG TTC ACA AAACG CCG TTC GCG CGC-3′. The signaling probe was 5′-TTT GTG AAC TTT ATT GGC GGTEG-biotin-3′. All oligonucleotide stock solutions were prepared in 0.5 M sodium phosphate buffer (pH 7.0) and stored frozen. Genomic DNA from Lysteria (ATCC 19115D) was purchased from LGC Promochem. Taq polymerase, dNTP mixture, and PCR buffer II were obtained from Takara. SECM analysis of PCR samples were performed with 5′-HS-(CH2)6-CCT AGC AGG TCT-3′ as CP* and 5′-CGC TTG CGA ATT TAA CCA AT-TEG-biotin-3′ as signaling probe and were obtained from MWG Biotech. (18) Willner, I.; Patolsky, F.; Lichtenstein, A. Anal. Sci. 2001, 17, i351-353. (19) Patolsky, F.; Lichtenstein, A.; Willner, I. Nat. Biotechnol. 2001, 19, 253257. (20) Alfonta, L.; Singh, A. K.; Willner, I. Anal. Chem. 2001, 73, 91-102.

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The gold chip was a gold electrode (6.5-mm diameter), with gold evaporated on a quartz disk (thickness 165 µm) obtained by International Crystal Manufacturing. Before use, each gold electrode was immersed in a boiling solution of 5 mL of H2O, 1 mL of H2O2 30%, and 1 mL of NH3 28% for 10 min. Caution! This mixture reacts violently. The solution has to be handled with extreme care to avoid personnel injury. Then, the electrode was immersed in ethanol for at least 1h. SECM Setup and Measurement. SECM apparatus was an Uniscan instrument SECM 270 (Uniscan). Pt disk microelectrodes with 10- and 25-µm diameters (CH Instruments) were used as amperometric SECM tip. SECM experiments were performed in the feedback mode at room temperature. A transparent Plexiglas one-compartment electrochemical cell was used throughout the work. When the tip is far from the substrate and a potential is applied, the steady-state current, iT∞ is recorded. In the positive feedback results, a higher tip current is observed (iT > iT∞) when the tip is closer to the substrate, meaning that the substrate is conductive, whereas in the negative feedback results, a lower tip current is observed (iT < iT∞) when the tip is closer to the substrate.7,21,22 Approach curves are presented in the dimensionless form of IT(L) versus (L), where IT(L) ) iT/iT∞ and L ) d/a, where d is the distance between the tip and the substrate and a the tip radius.7,21,22 Theoretical fits of experimental values were obtained from equations reported in refs 7 and 23 and the theoretical curve for pure negative feedback was obtained from refs 7 and 24. Each result was confirmed repeating the approach on different parts (at least three) of the substrate surface. The area scan and 3D figures were obtained by the Isoplot software (Uniscan) and converted in gray scale by the Corel PhotoPaint 11 Graphic suite. 1. Approach Curve Experiments. Approach curve experiments at an HS-ODN-modified surface were performed in a threeelectrode configuration, where the tip potentials (Etip), tip currents, and substrate potentials (Es) were measured against a saturated Hg/Hg2SO4 reference electrode and a stainless steel counter electrode. The tip was a disk Pt microelectrode with a diameter of 25 µm. HS-ODN-modified surfaces were obtained by incubating 100 µL of 30 µM thiolated CP in 0.5 M phosphate buffer, overnight at room temperature. Experiments were carried out in a 2 mM solution of FMCA or 1 mM K4Fe(CN)6 in 20 mM Tris-ClO4 buffer, 50 mM NaClO4, pH 8.6. After molecular assembly (with ODN or amplicon target), the approach curves were performed using a 10-µm disk Pt tip and Ag/AgCl wire pseudoreference electrode. Measurements were carried out in 2 mM FMCA in 0.1 M phosphate buffer, pH 7.4, 0.1 M NaCl with Etip ) +0.600 V and Es ) -0.100 V versus Ag/ AgCl wire. 2. Cyclic Voltammetry (CV) Experiments. CV experiments were performed with a 10-µm disk Pt tip in 2 mM FMCA in 0.1 M phosphate buffer, pH 7.4, 0.1 M NaCl, applying a Es ) -0.100 (21) Kwak, J.; Bard, A. J. Anal. Chem. 1989, 61, 1221-1227. (22) Mirkin, M. V.; Horrocks, B. R. Anal. Chim. Acta 2000, 406, 119-146. (23) Tsionsky, M.; Bard, A. J.; Mirkin, M. J. Phys. Chem. 1996, 100, 1788117888. (24) Bard, A. J.; Fan, F.; Mirkin, M. V. The handbook of surface imaging and visualization; Hubbard, A. T., Ed.; CRC Press, Inc.: Boca Raton, FL, 1995; pp 667-679.

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V versus Ag/AgCl. Dodecylthiol- or MCH-modified surfaces were obtained by dipping, overnight and at room temperature, the gold electrodes into a solution of 100 mL of 2-propanol/10 µL of dodecylthiol and into a 1 mM solution of MCH in deionized water, respectively. 3. Imaging Measurement. Imaging measurements were carried out using 2 mM FMCA in 0.1 M phosphate buffer, pH 7.4, 0.1 M NaCl as mediator, Etip ) +0.600 V, Es ) -0.100 V versus Ag/AgCl wire. The tip was a disk Pt microelectrode with a diameter of 10 µm. Prior to recording the imaging, an approach curve was performed to place the SECM tip at a working distance within the regime of feedback. PCR Amplification. The PCR conditions for the amplification of a 255-bp sequence was as follows: 10 µg of lyophilyzed genomic DNA was dissolved in 100 µL of sterilized and deionized water and the resultant solution stored overnight at +4 °C, prior to further treatments. The 100 µL of PCR reaction mixture contains 1× PCR buffer II, 200 µM dNTPs, 800 nM top primer, 800 nM bottom primer, 2.5 × 10-2 U/µL Taq polymerase, and 100 ng of genomic DNA. The amplification was performed with a MJ Research PTC-150 thermocycler (MJ Research Inc.). After the initial denaturation of the template (5 min at 95 °C), the PCR consisted of 35 cycles (95 °C/90 s, 58 °C/80 s, 72 °C/120 s). A final extension at 72 °C for 7 min concluded the process. The concentration of the amplicons was finally determined by fluorescence measurements using the Picogreen dye and a TD-700 fluorometer (Analytical Control), and λ-DNA standard solution as recommended by the TD-700 operating manual. Biological Assembly for the Detection of Hybridization. The thiolated CP solution (1 µM in 0.5 M sodium phosphate buffer, pH 7.0) was deposited as a drop of 40 µL on a bare gold surface (approach curve experiment) or spotted by means of a nanodispenser (BJQ3000 Biodot) (imaging experiments). The CPs were arrayed in spots in three rows of 10 nL at a distance of 1 mm center to center. Chemisorption was allowed to proceed overnight (16 h) with the chip stored in a Petri dish to protect the solutions from evaporation. The bottom of a large closed Petri dish (8.5-cm diameter) was covered with a plastic dish (7-cm diameter); the chips were set down on the upper side of the plastic dish, whereas under the other side there was a water-soaked paper pad to maintain high humidity. The Petri dish was wrapped with Parafilm and kept at room temperature. DNA chips were postassembled with MCH (1 mM in deionized water, 30 min, and at room temperature). A volume of 40 µL was deposited onto the surface. The chips were then washed twice with 40 µL of 0.5 M phosphate buffer, pH 7.0. Hybridization was performed in a Petri dish at room temperature by exposing the CP spots for 30 min to 40 µL of a 0.5 M sodium phosphate buffer containing 0.15 µM of a biotinylated signaling probe and a proper concentration of target sequence. After hybridization, the chips were washed twice with 40 µL of DEA buffer, to remove nonspecifically adsorbed sequences. The composition of DEA buffer was 0.1 M diethanolamine, 1 mM magnesium chloride, and 100 mM KCl, pH 9.6. After the hybridization step, the chip was reacted with 40 µL of a solution containing 0.8 U/mL streptavidin-AP conjugate and 10 mg/mL BSA (blocking agent) in DEA buffer. After 20 min,

Figure 1. Scheme of the assay: (A) probe immobilization; (B) detection scheme.

the chips were washed twice with 40 µL of DEA buffer. The enzyme-modified chips were then incubated with 40 µL of BCIP/ NBT for 20 min. After precipitation of the product, the chips were washed with 40 µL of 0.1 M phosphate buffer, 0.1 M NaCl pH 7.0. RESULTS AND DISCUSSION Evaluation and Characterization of HS-ODN/MCH-Modified Gold Surface. The detection scheme is reported in Figure 1. The first step consists of the immobilization of thiol-tethered CPs by chemisorption; then the DNA chip is postassembled with MCH to increase hybridization efficiency and to avoid unspecific adsorption of target strands (Figure 1A). When a gold surface is exposed to thiol-containing compounds (HS-R), by varying the terminal group of R, surface properties, including chemical and electrochemical reactivity, are different.24-28 Thus, an evaluation of the behavior of the HS-ODN/MCH mixed adsorbate was performed. As first, the CP-modified surface was evaluated. Approach curves obtained in 2 mM FMCA solution showed a positive feedback (Figure 2A, open circles), indicating that the redox (25) Liu, B.; Bard, A. J.; Li, C.-Z.; Kraatz, H.-B. J. Phys. Chem. B 2005, 109, 5193-5198. (26) Liu, B.; Bard, A. J.; Mirkin, M. V.; Creager, S. E. J. Am. Chem. Soc. 2004, 126, 1485-1492. (27) Wittstock, G.; Schuhmann, W. Anal. Chem. 1997, 69, 5059-5066. (28) Wittstock, G.; Hesse, R.; Schuhmann, W. Electroanalysis 1997, 9, 746750.

reaction of this mediator at the thiol-tethered ODN-modified gold electrode proceeds virtually unimpeded. This experimental evidence is in accordance to what was reported in ref 25, where different neutral or positively charged mediators were tested, demonstrating that uncharged and cationic mediators are not blocked by the DNA monolayer. Since, during experimental conditions, the total charge of FMCA is null, considering that at neutral or alkaline pH the carboxylic function of FMCA is dissociated29 and, on the contrary, that the ferrocene moiety (reduced form) is converted to the positively charged oxidized ferricinium species, a similar conclusion can be extended also to this mediator. The tip-generated [Fe(CN)6]3- was used as redox probe to confirm that the CP was really immobilized on the gold surface (Figure 2A open squares). As reported in refs 11, 12, and 25, above a DNA layer, the anionic phosphate groups repel the tip-generated [Fe(CN)6]3-/4- molecules. This process hinders their diffusion toward the gold electrode, reduces the rate of recycling, and leads to a drop in the observed tip current. A decrease of the steadystate current was experimentally observed (for Es ) -0.250 V versus Hg/Hg2SO4), implying that the substrate blocks diffusion of species from the tip. A measurable positive feedback (data not shown) was observed as the substrate potential became more negative. Similar results were reported in ref 25 and indicate that (29) Benkeser, R. A.; Goggin, D.; Schroll G. J. Am. Chem. Soc. 1954, 40254026.

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Figure 2. Approach curve experiments on a HS-ODN-modified (A) and on a thiol-modified/unmodified (B) gold substrate. In (A) are reported the approach curves obtained in (O) 2 mM FMCA and (0) 1 mM K4Fe(CN)6 solution in 20 mM Tris-ClO4 buffer + 50 mM NaClO4, pH 8.6, over a CP-modified chip, Etip ) +0.100 V, Es ) -0.250 V vs Hg/Hg2SO4. For all the experiments, a tip with a diameter of 25 µm was used. The solid lines are the SECM theoretical curves. In (B) are reported the CVs of 2 mM FMCA at a Pt electrode (diameter 10 µm), scan rate 50 mV/s; Es ) -0.100 V vs Ag/AgCl. Curve 1 at ∞; curve 2 on a bare gold electrode; curve 3 on a MCH-modified gold electrode; curve 4 on a dodecanethiolmodified gold electrode.

Figure 3. Approach curve obtained on different interfaces after molecular assembly (A) and varying target concentration (B). (A) CP-modified substrate after hybridization with 40 nM target and molecular assembly (0) and after enzyme-catalyzed precipitate of insulating solid (O). The dashed line is the theoretical curve for pure negative feedback according to ref 23. (B) Approach curves on substrates after precipitate of insulating solid, varying the target concentration: (3) 40 nM; (4) 4 nM; (O) 400 pM; (0) 40 pM. The solid lines are the SECM theoretical curves. The approaching speed was 1 µm/s.

the rate of the regeneration of [Fe(CN)6]4- at the HS-ODNmodified gold was faster at higher substrate overpotential. Following ref 25, the standard rate constant for electron transfer (k°) between Fe[(CN)6]3- and the gold substrate modified and unmodified with ODN was calculated, and the results are (2.8 ( 0.8) × 10-6 and (7.5 ( 0.5) × 10-4 cm s-1, respectively. These values are in line to that reported in ref 25, and the almost 3 orders of magnitude of difference confirm that the regeneration of the mediator is slowed down at the ODN-modified surface. Then, the MCH-modified gold substrate was evaluated and compared with results obtained at a bare and long-chain alkanethiol-modified gold surface; in the last case, a negative feedback was expected, due to the blocking properties of longchain alkanethiol.26,28 Figure 2B shows different cyclic voltammograms recorded with the same tip electrode in a solution of 2 mM FMCA over the bare gold substrate (curve 2) and after treatment with MCH (curve 3) or dodecylthiol (curve 4). Curve 1 corresponds to a batch experiment with the tip located far away from any surface. At the bare gold surface (curve 2), the re-reduction of the mediator (FMCA) occurred (positive feedback). Curve 3 corresponds to the experiment over the MCH-modified gold substrate. After approach of the tip to near-field distance to such a surface, consumed mediator molecules can be reconverted into their initial oxidation state, leading to an increase in the tip current (positive feedback). As reported in ref 26, regeneration of mediator 7210

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over short-chain thiol layers can be explained in term of direct electron tunneling or through electron transfer at the pinhole. As expected, the tip current is decreased at the dodecylthiol-modified gold surface (curve 4), meaning that the re-reduction is impeded at this modified substrate. As expected, the mixed adsorbate (HS-ODN/MCH) on the gold substrate allows the electrochemical recycling of FMCA, establishing a regime of positive feedback. Evaluation of DNA Hybridization Using the Feedback Mode of SECM. The scheme of the assay is reported in Figure 1B. The selectivity is guaranteed by the CP; whereas, as reported in refs 3 and 30, the use of a signaling probe, preannealed to target DNA to form partially duplex target molecules, allows the labeling of the target and the prevention of secondary structure that can interfere with hybridization to the surface-tethered CP. In the proposed scheme, the biotinylated hybrid was labeled with the streptavidin-AP conjugate. After this molecular assembly, the chip was incubated with the enzymatic substrate. The electrochemical transduction of the hybridization process was performed by means of feedback mode of SECM, taking advantage of the biocatalyzed precipitation of an insulating indigo derivative5,18,19 that blocks regeneration of the mediator at the substrate surface. (30) Maldonado-Rodriguez, R.; Espinosa-Lara, M.; Loyola-Abitia, P.; Beattie, W. G.; Beattie, K. L. Mol. Biotechnol. 1999, 11, 13-25.

Figure 4. SECM images of DNA spots. (A) Area scan of array of spots of 2.5 × 2.0 mm, with the CPs arrayed in spots in three rows at a distance of 1 mm center to center. (B) Cross-section line scan reporting the current values vs relative horizontal distance of a part of the area described by the white line depicted in (A). The concentration of target was 40 nM. Tip scanning speed 10 µm/s.

Figure 5. 3D SECM image of a DNA spot. The concentration of target was 40 nM. Tip scanning speed 25 µm/s.

In Figure 3A are reported the approach curves over the hybridized ODN gold surface after molecular assembly. Curve 1 (open squares) was obtained after the incubation of the biotinylated hybrid with the streptavidin-AP conjugate. The redox reaction of FMCA proceeds virtually unimpeded. Curve 2 (open circles) was recorded after the molecular assembly and the following incubation of the BCIP/NBT solution. In this case, the insulating solid blocks the regeneration of the mediator at the electrode surface. The change in feedback current is thus linked to the successful ODN hybridization and molecular assembly. Incubation with a noncomplementary target was also carried out in order to evaluate nonspecific interactions; approach curves show a positive feedback behavior not significantly different from the CP-modified gold surface (data not shown). The effect of target concentration was examined in the range 40 pM-400 nM (Figure 3B). When the target concentration is high (g40 nM), the approach curve becomes congruent with the theoretical curve over an insulating substrate. Thus, SECM will not detect amounts greater than 40 nM because the deposition of more insulating substrate will not further decrease SECM current. On the other hand, target concentrations lower than 4 nM alter the SECM approach curves from the behavior over insulating surfaces to that over surfaces with behaviors between completely conductive and insulating situations (curve c, open circles) or to that over conductive surfaces (curve d, open

Figure 6. Approach curve obtained on a PCR amplicon hybridmodified gold substrate after precipitattion of insulating solid. The approaching speed was 1 µm/s.

squares). To have the best resolution, further experiments were performed using a 40 nM target. Imaging of DNA Hybridization Using the Feedback Mode of SECM. In imaging experiments, the SECM tip is scanned in constant height above the gold substrate after ODN hybridization and molecular assembly, while simultaneously monitoring the tip current as a function of the x/y tip position. The dependence of the microelectrode response on the tip to substrate distance and on the nature of the substrate allows revealing spatially resolved information about chemical properties and the morphology of the surface, as reported in Figure 4, with the imaging of an array of spots of 2.5 × 2.0 mm of the same probe after hybridization with target oligonucleotides. From the analysis of the signal height obtained from horizontal line scans across the spots,17 the reproducibility was evaluated and a current mean value of 6 × 10-10 ( 2 × 10-10 A (n ) 9) was found. High-quality 3D SECM images of CP-target hybridized spots were reported in Figure 5. The current values to the left and right of the spot are indicative for the positive feedback from the MCHmodified gold surface. Drastically lowered current values above the spots signify reduced rates of redox recycling. The insulating precipitate is formed with high precision around the spot, confirming the blocking of the gold surface to nonspecific binding of biomolecules during hybridization and enzyme labeling from the MCH layer. Moreover, to further prevent the nonspecific adsorption on the gold surface, 1% BSA was added to the solution of streptavidin-conjugated enzyme.3,4 Analytical Chemistry, Vol. 79, No. 18, September 15, 2007

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Figure 7. Array of ODN after hybridization of PCR amplicon and precipitation of insulating product. SECM images of DNA spots (A, C) and cross-section line scans (B, D), along the white line of (A) and (C), respectively. CP* spotted in two locations at a distance 1 mm center to center (A, B); CP* spotted in one location (B, D). Tip scanning speed 25 µm/s.

Detection of PCR-Amplified Sequences. PCR products (255 bp) yielded by the procedure described in the Experimental Section were diluted to 60 nM using a 0.5 M sodium phosphate buffer containing 0.15 µM biotinylated signaling probe. The double-stranded DNAs were then thermally denatured by using a boiling water bath (5 min at 100 °C); amplicon strand reannealing was retarded by cooling the sample in an ice-water bath for 1 min. A 40-µL aliquot of this solution was placed directly onto the probe-modified chip for 30 min. After hybridization, the surface was washed with 40 µL of DEA buffer, to remove nonspecifically adsorbed sequences. Then the biomolecular assembly was performed as reported above. In Figure 6 is shown an approach curve over the hybridized PCR amplicons on the CP*-modified gold surface after molecular assembly and incubation with BCPI/NBT solution. As can be seen, a negative feedback was obtained as expected. However, for the same concentration value and the same hybridization time, the magnitude of the feedback effect obtained with the amplicon is less important than that obtained with ODN. This could be probably overcome by increasing the hybridization time as well as the enzyme incubation time.3 Figure 7A shows the imaging of PCR amplicon hybridized spots, whereas in Figure 7B is reported the horizontal line scan across the same spots; this was obtained by sampling the current nearly in the center. The distance between the spots is 1 mm 7212

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center to center, and in each position, a negative feedback effect is demonstrated. Panels C and D in Figure 7 show the imaging and the crosssection line scan, respectively, of a different portion of the same chip where the CP* was spotted only in one position; in this case, it was clearly evident that specific hybridization took place only on the CP* spot. CONCLUSIONS Imaging of localized DNA hybridization has been demonstrated. The method relies on the inherent sensitivity of SECM for the detection of the small variation of surface conductivities. Target concentrations at the nanomolar level can be easily imaged. This concentration level is sufficient for most applications involving gene expression and sequence analysis. Moreover, the reliability of the method is also demonstrated, with the low nonspecific signal. Thus, a highly sequence specific (single base mismatch) DNA analysis can be accomplished. Although the concept of using this SECM-based method to image a DNA array is demonstrated only with a low-density chip, miniaturizing the assay by increasing the performance of the spotting system, visualization of a medium- to high-density DNA array should be possible. Moreover, immobilization of different CPs should be possible. and thus. multiplex analysis should be carried out.

Ongoing research is devoted to investigate the possibility of using other stable enzyme conjugates as a label of the hybridization reaction in order to image DNA spots through an enzymegenerated positive feedback mode, thus detecting an increase of the signal instead of a decrease. An important advantage of this format will be the use of less expensive surface material, i.e., glass, for probe immobilization.

ACKNOWLEDGMENT Support from the Italian Government in the framework of the PRIN 2003 and PRIN 2005 projects. Dr. F. Lucarelli is thanked for helpful discussions. Received for review March 8, 2007. Accepted July 15, 2007. AC070474H

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