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Langmuir 2006, 22, 1932-1936
Differential Labeling of Closely Spaced Biosensor Electrodes via Electrochemical Lithography Rebecca Y. Lai,†,‡,§ Sang-ho Lee,⊥ H. T. Soh,⊥,| Kevin W. Plaxco,*,†,‡,| and Alan J. Heeger†,‡,§,# Center for Polymers and Organic Solids, Department of Mechanical Engineering, Biomolecular Science and Engineering Program, Department of Chemistry and Biochemistry, Department of Physics, and Materials Department, UniVersity of California, Santa Barbara, Santa Barbara, California 93106 ReceiVed August 4, 2005. In Final Form: NoVember 22, 2005 Electrochemical biosensors offer the promise of exceptional scalability and parallelizability. To achieve this promise, however, will require the development of new methods for the differential labeling of closely spaced electrodes with specific biomolecules such as DNA or proteins. Here we report a simple, highly selective method for passivating and differentially labeling closely separated gold electrodes with oligonucleotides or other biomolecules. Analogous to photolithography, where a light-sensitive resist is selectively removed to expose specific surfaces to further modification, we passivate gold electrodes with a self-assembled alkanethiol monolayer that protects them from modification. The monolayer is then electrochemically desorbed at relatively low potentials, allowing for the subsequent labeling of the now exposed array element with a specific sensing biomolecule. The observed passivation is highly efficient: using a C11-OH monolayer as the passivating agent, we do not observe any detectable cross-contamination of adjacent electrodes (95 µm separation) upon labeling with a stem-loop DNA probe. Critically, the conditions employed are sufficiently gentle that depassivation reduces the DNA load on adjacent electrodes by only ∼1%, allowing for the sequential labeling of multiple, closely spaced electrodes. This technology paves the way for labeling multiple array elements sequentially without observable cross-contamination in a fast and controlled manner.
Introduction The site-specific functionalization of densely packed electrode arrays with specific biological molecules, such as DNA or polypeptides, is an active area of research.1,2 Methods for immobilization of biomolecules on electrode surfaces fabricated from a wide range of materials, including gold, carbon, and glass, have recently become available.3,4 Most of these techniques, however, do not simultaneously meet the requirements of resolution, speed, and the ability to differentially label each electrode in complex, densely packed, and often inaccessible sensor arrays. For example, while several groups have used precisely positioned microdrop dispensing systems5 to achieve spatial resolutions of several micrometers, this technique requires microscopic alignment to achieve uniformity of the deposit as well as to prevent cross-contamination between probes. Microcontact printing,6 in contrast, offers sub-micrometer spatial resolution but cannot be employed when multiple coating layers are required. Still higher spatial resolution can be achieved using nanografting,7 but this technique is time-consuming and requires complex and expensive infrastructure. Dip-pen nanolithography,8-10 a technique that uses a scanning probe coated with * Correspondence should be addressed to KWP. Phone: (805) 893-5558. Fax: (805) 893-4120. † Center for Polymers and Organic Solids. ‡ Department of Chemistry and Biochemistry. § Department of Physics. ⊥ Department of Mechanical Engineering. | Biomolecular Science and Engineering Program. # Materials Department. (1) Drummond, T. G.; Hill, M. G.; Barton, J. K. Nat. Biotechnol. 2003, 21, 1192. (2) Ramsay, G. Nat. Biotechnol. 1998, 16, 40. (3) Love, J. C.; Estroff, L. A.; Kriebel, J. K.; Nuzzo, R. G.; Whitesides, G. A. Chem. ReV. 2005, 105, 1103. (4) Wang, J. Electroanalysis 2005, 17, 7. (5) Yershov, G.; Barsky, V.; Belgovskiy, A.; Kirillov, E.; Kreindlin, E.; Ivanov, I.; Parinov, S.; Guschin, D.; Drobishev, A.; Dubiley, S.; Mirzabekov, A. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 4913. (6) Kumar, A.; Whitesides, G. M. Appl. Phys. Lett. 1993, 63, 2002. (7) Xu, S.; Liu, G. Y. Langmuir 1997, 13, 127.
molecules as a pen to pattern the surface, has also been described. Despite the nanometer-scale resolution of this patterning technique, this approach is limited by requirements for a high level of stability and solubility of the coating molecules. In general, any technique that uses a mechanical probe to selectively modify a surface will require high resolution and ready access to the sensor surface, rendering it inconvenient for the fabrication of complicated or inaccessible sensor arrays. In contrast to microcontact, photolithographic, and dip-pen lithographic techniques, several approaches to the in situ fabrication of DNA arrays have been reported to date. For example, potential-assisted immobilization of biotin-modified DNA onto agarose-avidin-coated platinum microelectrodes has been reported.11,12 Similarly, the in situ synthesis of DNA on array electrodes has recently been achieved using electrochemically generated acid to site-specifically deprotect the growing oligonucletide chain.13 Here, we describe a complimentary electrochemical lithographic technique for the rapid, convenient, and selective labeling of closely packed electrodes with specific thiol-containing biomolecules. Inspired by photolithography, in which a light-sensitive resist that covers a surface is selectively removed to expose specific regions to further modification, the electrodes are first passivated with a molecular monolayer which is subsequently removed via electrochemistry in order to immobilize the sensing biomolecule. Materials and Methods Reagent grade chemicals, including 1-mercaptohexanol (C6OH), 11-hydroxy-1-undecanethiol (C11-OH), ethanol, sulfuric acid, and potassium ferricyanide, (all from Aldrich, St. Louis, MO), (8) Piner, R. D.; Zhu, J.; Xu, F.; Hong, S. H.; Mirkin, C. A. Science 1999, 283, 661. (9) Hong, S.; Mirkin, C. A. Science 2000, 288, 1808. (10) Schwartz, P. V. Langmuir 2002, 18, 4041. (11) Huang, Y.; Ewalt, K. L.; Tirado, M.; Haigis, R.; Forster, A.; Ackley, D.; Heller, M. J.; O’Connell, J. P.; Krihak, M. Anal. Chem. 2003, 73, 1549. (12) Sosnowski, R. G.; Tu, E.; Butler, W. F.; O’Connell, J. P.; Heller, M. J. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 1119. (13) Egeland, R. D.; Southern, E. M. Nucleic Acids Res. 2005, 33, e125.
10.1021/la052132h CCC: $33.50 © 2006 American Chemical Society Published on Web 01/13/2006
Labeling of Closely Spaced Biosensor Electrodes
Figure 1. Optical image of the microfabricated gold electrode pairs employed in this study. The electrode pair was fabricated on a glass wafer using standard photolithographic techniques. potassium phosphate monobasic, dibasic and sodium chloride (Fisher, Fairlawn, NJ) were used without further purification. Thiol- and amine-modified oligonucleotides were obtained from Synthegen (Houston, TX) and Biosource (Foster City, CA). Methylene blue (MB) was conjugated to the 3′ end of the amino- and thiol-modified stem-loop probe oligonucleotide (MB-DNA: 5′-HS-(CH2)6-GCGAGGTAAAACGACGGCCAGTCTCGC-(CH2)7-NH2-3′) via succinimide ester coupling (MB-NHS, EMP Biotech, Germany). The sequence of the full complement target for this probe was 5′-ACTGGCCGTCGTTTTAC-3′. The sequence of the thiol-modified linear probe oligonucleotide (LP-DNA) was 5′-HS-(CH2)6CTGGCCGGCGATATA-3′. The target sequence for this element was 5′-TATATCGCCGGCCAG-3′ with MB conjugated to its 3′ end. Electrochemistry was performed using a CH Instruments Electrochemical Work Station (Austin, TX). The electrolyte in which the voltammograms were collected contains 10 mM phosphate buffer (pH 7) and 1 M NaCl unless specified in the figure captions. The gold working electrode pairs used in this study were fabricated on a glass plate using standard microfabrication techniques (Figure 1). Details of the fabrication processes are included in the Supporting Information (Figure S.I.1). The patterned electrodes were cleaned by immersing in piranha (3:1 H2SO4:H2O2) for 5 min and then thoroughly rinsed in deionized water. A platinum wire was used as the counter electrode. All electrochemical potentials are reported versus a Ag/AgCl reference electrode.
Langmuir, Vol. 22, No. 4, 2006 1933 In most cases, the gold electrode pair was passivated with a monolayer of C11-OH formed by immersing the electrodes in a 2 mM ethanolic solution of 11-hydroxy-1-undecanethiol for 1 h. In some experiments, however, both electrodes were modified with LP-DNA/C6-OH. All electrochemical oxidative desorptions were performed in 1 mM H2SO4 (pH ∼ 3) using a standard three-electrode system at a scan rate of 500 mV/s. To obtain complete desorption of the C11-OH monolayer from the lower electrode, four cycles of potential scan from -0.5 to +1.8 V were applied to the electrode while keeping the upper electrode at open circuit (Figure S.I.2 of the Supporting Information). After this treatment, cyclic voltammograms of the gold electrode were recorded in 1 mM potassium ferricyanide to examine the surface of the freshly regenerated electrode. MB-DNA was dissolved in 10 mM phosphate buffer (pH 7), 5 mM MgCl2, and 100 mM NaCl solution to a final oligonucleotide concentration of 1 µM. The electrode pair, with the C11-OH passivating layer now covering only the upper electrode, was then immersed in this aqueous solution for 1 h to allow the oligonucleotides to chemisorb to the lower electrode where the C11-OH has been removed via oxidative desorption. The electrodes were then immersed in a 1 mM C6-OH for ∼2 h to displace nonspecifically bound oligonucleotides. The C11-OH passivating layer in the upper electrode could subsequently be removed for immobilization of another oligonucleotide probe sequence utilizing the same method. Prior to interrogation with the complement target oligonucleotides, the electrodes were incubated in 10 mM phosphate buffer (pH 7) and 1 M NaCl for ∼1 h.
Results Oxidative desorption is a relatively gentle means of removing the alkanethiol passivating layer and does not produce any measurable degradation of the DNA films adsorbed to adjacent electrodes. We initially labeled both upper and lower electrodes with a mixed monolayer of thiol-modified linear-probe DNA (LP-DNA) and C6-OH. Cyclic voltammograms of 1 mM ferricyanide obtained with these electrodes indicate that both were successfully modified with the self-assemble monolayer (SAM) and are effectively indistinguishable (Figure 2). After stripping the lower electrode via oxidative desorption, we recover the diffusion controlled voltammogram typical of a SAM-free electrode with peak separations ∼ 68 mV and currents comparable to voltammograms obtained from an unmodified gold electrode of the same geometry (data not shown). This suggests that the
Figure 2. Cyclic voltammograms of 1 mM ferricyanide demonstrates that the stripping of the first electrode does not significantly alter the passivation of the adjacent electrode. Prior to the stripping of the lower electrode, both electrodes were modified with LP-DNA and C6-OH. Upon stripping, the redox peaks of the lower electrode increase significantly, as expected for a bare gold electrode. In contrast, after the stripping of the lower electrode, the passivation of the upper electrode remains almost entirely unchanged.
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Figure 3. Background subtracted AC voltammograms obtained with the differentially labeled electrodes using C1l-OH as a passivating layer. The voltammogram of the upper electrode is essentially flat, indicating the absence of cross-contamination. In the absence and presence of complementary DNA (at 200 nM) the strong peak reduction observed indicates the MB-DNA retains its ability to detect hybridization. The hybridization time was 35 min.
oxidative desorption causes effectively complete stripping of the LP-DNA/C6-OH monolayer. Voltammograms obtained with the upper electrode, which was not stripped, were not measurably affected (despite our ability to monitor changes of current of as little as ∼1%) by the oxidative desorption of the closely packed neighboring electrode (Figure 2), indicating that the stripping procedure is sufficiently benign that it does not adversely affect electrodes (labeled with biomolecules) only 95 µm from the stripped electrode. Long-chain alkanethiol layers, but not those comprised of shorter, C-6 alkanethiols, are highly resistant to crosscontamination and thus prevent the mislabeling of adjacent electrode elements. Following stripping of the lower electrode, we labeled it with E-DNA,14 a molecular beaconlike stem-loop DNA containing the electroactive reporter group methylene blue (MB-DNA). Upon hybridization, the distance between the label and the electrode is significantly increased, leading to a significant reduction in electron transfer. This allows us to detect the chemisorbed oligonucleotides via both cyclic voltammetry (CV) and alternating current voltammetry (ACV) and to show that they retain their selective recognition properties. AC voltammograms obtained with the MB-DNA modified electrode before and after exposure to a complementary oligonucleotide target demonstrate a large reduction in the current arising from the MB-DNA, indicating that the MB-DNA is acting as a sensitive and selective electronic signature for DNA hybridization (Figure 3). In contrast to previous reports,15 however, we find that C6OH is a poor passivating layer as we also observe substantial MB-DNA type signals arising from the upper, supposedly passivated electrode (Figure 4). This cross-contamination presumably arises as a result of the formation of pinholes and other defect sites in the C6-OH monolayer. Alternatively the thiolcontaining DNA may have C6-OH molecules in the passivating layer via thiol-thiol exchange.16,17 To circumvent the poor passivating properties of C6-OH, we conducted a parallel experiment using C11-OH as the passivating layer. With this (14) Fan, C.; Plaxco, K. W.; Heeger, A. J. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 9134. (15) Wallti, C.; Wirtz, R.; Germishuizen, W. A.; Bailey, D. M. D.; Pepper, M.; Middelberg, A. P. J.; Davies, A. G. Langmuir 2003, 19, 981. (16) Yang, G.; Amro, N. A.; Starkewolfe, Z. B.; Liu, G. Langmuir 2004, 20, 3995. (17) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. Soc. 1987, 109, 3559.
passivation chemistry, no measurable cross-contamination is observed (Figure 5). Furthermore, this technique enables the ability to differentially label multiple electrodes by using existing oligonucleotide-SAM monolayers as protection against the deposition of subsequent oligonucleotides as additional array elements are in turn modified. To monitor the level of cross-contamination that occurs during such sequential labeling, we modified both elements of a twoelectrode array with an unlabeled, linear-probe thiolated oligonucleotide (LP-DNA) and then incubated the electrodes in 1 mM C6-OH to anneal the passivating oligonucleotide-labeled monolayers. We then stripped the LP-DNA/C6-OH mixed monolayer off of the lower electrode which we subsequently modified with MB-DNA. Tests with ferricyanide suggest that this step does not significantly affect the existing LP-DNA/ C6-OH monolayer (data not shown). Finally, we incubated the electrode pair in 1 mM C6-OH to expel any physically adsorbed probe molecules. The absence of MB reduction signal observed in the upper electrode coated with LP-DNA/C6-OH implies the absence of cross-contamination (
