© Copyright 2007 American Chemical Society
JULY 31, 2007 VOLUME 23, NUMBER 16
Letters Selective Immobilization of DNA and Antibody Probes on Electrode Arrays: Simultaneous Electrochemical Detection of DNA and Protein on a Single Platform Jason C. Harper, Ronen Polsky, David R. Wheeler, Shawn M. Dirk, and Susan M. Brozik* Biosensors & Nanomaterials, Sandia National Laboratories, PO Box 5800, MS-0892, Albuquerque, New Mexico 87185 ReceiVed June 14, 2007 A proof of concept procedure for the electroaddressable covalent immobilization of DNA and protein on arrayed electrodes along with simultaneous detection of multiple bioagents in the same sample solution is described. Carboxyphenyldiazonium was selectively deposited onto five of nine individually addressable electrodes in an array via bias assisted assembly. Amine functionalized DNA probes were covalently coupled to the carboxyl surface via carbodiimide chemistry. This was followed by the covalent immobilization of diazonium-antibody conjugates into the remaining four electrodes via cyclic voltammetry. Simultaneous electrochemical detection of a DNA sequence related to the breast cancer BRCA1 gene and the human cytokine protein interleukin-12, which is a substantial component in the immune system response and attack of tumor cells, is reported. These results demonstrate the possibility of selective patterning of diverse biomolecules on a single device and may have significant implications for future development of microarrays and biosensors.
Microarray technology has become an important tool for high throughput analysis of biological systems.1 Currently, DNA and antibody microarrays are used independently to obtain genomic and proteomic information. However, it is becoming increasingly evident that, for meaningful gene expression data to be obtained, DNA and protein microarray data must be obtained for the same sample.2 Thus, a single microarray that can provide high throughput genomic, proteomic, and whole cell signatures for several bioagent targets would be a significant advancement, providing more complete and reliable information while sim* Corresponding author. E-mail:
[email protected]. (1) (a) Kozarova, A.; Petrinac, S.; Ali, A.; Hudson, J. W. J. Proteome Res. 2006, 5, 1051-1059. (b) Sobek, J.; Bartscherer, K.; Jacob, A.; Hoheisel, J. D.; Angenendt, P. Comb. Chem. High Throughput Screening 2006, 9, 365-380. (2) (a) Gygi, S. P.; Rochon, Y.; Franza, B. R.; Aebersold, R. Mol. Cell. Biol. 1999, 19, 1720-1730. (b) Ideker, T.; Thorsson, V.; Ranish, J. A.; Christmas, R.; Buhler, J.; Eng, J. K.; Bumgarner, R.; Goodlett, D. R.; Aebersold, R.; Hood, L. Science 2001, 292, 929-934.
plifying the overall assay protocol. Additionally, orthogonal methods of biological detection would facilitate the development of multianalyte biosensors capable of discriminating between similar bioagents. An important advancement that is necessary for the development of such integrated sensors is the realization of a “universal toolset”3 that is capable of selectively manipulating diverse biomolecules such as DNA, proteins, and peptides onto the same array format. We believe one potential answer lies in the utilization of aryl diazonium salts. Diazonium-functionalized electrodes, introduced by Delamar et al.,4 may be assembled onto various conducting and semiconducting substrates, and can allow for the selective functionalization of closely spaced microelectrode surfaces via bias assisted assembly. Modification of gold surfaces with diazonium (3) Medintz, I. Nat. Mater. 2006, 5, 842. (4) Delamar, M.; Hitmi, R.; Pinson, J.; Save´ant, J.-M. J. Am. Chem. Soc. 1992, 114, 5883-5884.
10.1021/la701775g CCC: $37.00 © 2007 American Chemical Society Published on Web 06/30/2007
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Scheme 1. DNA and Antibody Probe Electrode Array Selectively Functionalized via Electroaddressable Deposition of Diazonium Salts
Figure 1. Optimization of carboxyphenyldiazonium electrodeposition conditions for maximum current response from DNA sandwich assay with 10 µM target DNA (2). Error bars are the standard deviation of measurements obtained from three or more electrodes.
salts has shown certain advantages over the more common assembly of thiol onto gold chemistry, including a strong covalent bond, higher stability, and a wider potential window for electrochemical detection methods.5 Diazonium chemistry has been used to immobilize a variety of biomolecules including DNA,6 proteins,7 and peptides.5 The direct electrically addressable immobilization of diazonium-modified proteins onto electrodes was used to obtain direct electron transfer to horseradish peroxidase (HRP),8 and to construct a reagentless9 and a multianalyte electrochemical immunosensor.10 Herein, we describe a proof of concept procedure for the electroaddressable covalent immobilization of DNA and protein on the same array along with simultaneous detection of multiple bioagents in the same sample solution. For this demonstration we have chosen targets for the array that include a 60-mer DNA sequence, 2, related to the breast cancer BRCA1 gene, and the human cytokine protein interleukin-12 (IL-12), which is a substantial component in the immune system response and attack of tumor cells. Scheme 1 depicts the preparation of the DNA and antibody probe microarray. Carboxyphenyldiazonium was selectively deposited onto electrodes 1, 3, 5, 7, and 9 via bias assisted assembly (step A). The array was then treated with a 5′-aminefunctionalized DNA probe sequence, 1, in the presence of 1-ethyl3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), covalently linking the singlestranded DNA to the carboxyl-modified electrodes (step B). The array was exposed to a solution of diazonium-modified antibodies, which were covalently immobilized onto electrodes 2, 4, 6, and 8 via cyclic voltammetry (CV, step C).8 Electrically assembled diazonium films can range from a thin submonolayer formation to highly stacked multilayer structures (5) (a) Liu, G.; Bo¨cking, T.; Gooding, J. J. J. Electroanal. Chem. 2007, 600, 335-344. (b) Laforgue, A.; Addou, T.; Be´langer, D. Langmuir 2005, 21, 68556865. (6) Lee, C.-S.; Baker, S. E.; Marcus, M. S.; Yang, W.; Eriksson, M. A.; Hamers, R. J. Nano Lett. 2004, 4, 1713-1716. (7) (a) Corgier, B. P.; Marquette, C. A.; Blum, L. J. J. Am. Chem. Soc. 2005, 127, 18328-18332. (b) Liu, G.; Gooding, J. J. Langmuir 2006, 22, 7421-7430. (8) Polsky, R.; Harper, J. C.; Dirk, S. M.; Arango, D. C.; Wheeler, D. R.; Brozik, S. M. Langmuir 2007, 23, 364-366. (9) Polsky, R.; Harper, J. C.; Wheeler, D. R.; Dirk, S. M.; Rawlings, J. A.; Brozik, S. M. Chem. Commun. 2007, 2741-2743. (10) Polsky, R.; Harper, J. C.; Wheeler, D. R.; Dirk, S. M.; Arango, D. C.; Brozik, S. M. Biosens. Bioelectron. 2007, accepted.
Figure 2. Simultaneous electrochemical detection of DNA and protein on a single individually addressable electrode array treated with target and nontarget interferants: (1) 10 µM complementary target DNA sequence 2 with 400 ppb cytokine IL-1β control, (2) 10 µM random DNA control sequence 4 with 400 ppb target cytokine IL-12, and (3) 10 µM complimentary target DNA sequence 2 with 400 ppb target cytokine IL-12. Current response was normalized to account for differing probe density obtained from DNA or Ab functionalized electrodes. Error bars are the standard deviation from five (DNA) or four (Ab) electrodes on the array. Inset: Photograph of the gold array used in this work. The chip is comprised of nine individually addressable Au disk electrodes (500 µm diameter), a common counter/pseudoreference electrode (Au bar), with 10 square contact pads on the perimeter.
depending on the deposition conditions.11 Previous work has shown that there is an inherent tradeoff between film thickness and efficient electron-transfer kinetics.12 Electrodeposition (11) (a) Anariba, F.; DuVall, S. H.; McCreery, R. L. Anal. Chem. 2003, 75, 3837-3844. (b) Kariuki, J. K.; McDermott, M. T. Langmuir 2001, 17, 59475951. (12) (a) Harper, J. C.; Polsky, R.; Dirk, S. M.; Wheeler, D. R.; Brozik, S. M. Electroanalysis 2007, 19, 1268-1274. (b) Uetsuka, H.; Shin, D.; Tokuda, N.; Saeki, K.; Nebel, C. E. Langmuir 2007, 23, 3466-3472.
Letters
conditions were optimized to allow for effective DNA immobilization and hybridization. Optimization of diazoniummodified antibodies was reported previously.10 Five different electrochemical immobilization protocols were compared for DNA detection: (1) a 30 s duration chronoamperometric (CA) step to -1 V; (2) a 1 min CA step to -1 V; (3) a 2 min CA step to -1 V; (4) a CV from 0 to -1 to 0 V at 100 mV/s; and (5) 2 CVs from 0 to -1 to 0 V at 100 mV/s. For diazonium depositions it has been reported that potential sweep methods lead to thicker and less ordered films than fixed potential depositions.12 This higher film thickness from sweep depositions has been attributed to rearomatization of the phenyl rings on oxidative sweeps, increasing film conductivity.13 While potential sweeps can result in a higher density of active sites on the surface, the increased film thickness can significantly impair electron-transfer kinetics. This phenomenon is clearly demonstrated in Figure 1 in which the results of sandwich DNA hybridization assays for each deposition protocol are presented. HRP was used as an electrochemical label for these experiments (see Supporting Information). All five electrodeposition methods resulted in favorable signals when compared to a control sample (DNA probe incubated with a noncomplementary random DNA sequence, 4). The significantly lower signal of a 2 CV deposition compared to a 1 CV deposition reflects the increased film thickness and electrode blocking when using increasing numbers of potential sweeps. The CA deposition protocols exhibited increased signals with increasing deposition time, with a signal maximum obtained from a 2 min CA deposition. Figure 2 shows the electrochemical detection of target DNA sequence 2, and target protein, IL-12, in the presence of control interferants. Control interferants are a random DNA sequence, (13) (a) Adenier, A.; Combellas, C.; Kanoufi, F.; Pinson, J.; Podvorica, F. I. Chem. Mater. 2006, 18, 2021-2029. (b) Pinson, J.; Podvorica, F. I. Chem. Soc. ReV. 2005, 34, 429-439.
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4, or the similar human cytokine, IL-1β. After treatment with the biological sample solution, the array is washed and incubated with a solution containing biotinylated detection DNA probe, sequence 3, and biotinylated secondary detection antibodies. This treatment is again followed by a wash and a brief incubation in avidin-modified HRP. After washing, the CA response at each electrode is monitored in a solution containing HRP substrate and tetramethylbenzidine (TMB) mediator. The average current response from the five DNA- and four antibody-functionalized electrodes show that the array successfully distinguished between target and interferant DNA and protein. Additionally, the device was capable of simultaneous detection of target DNA and protein using a single protocol. Assay optimization, including the introduction of blocking groups and reducing the concentration of detection and label molecules, is expected to improve the overall response of the electrodes. In conclusion, our results demonstrate the possibility of selective patterning of diverse biomolecules on a single device, with subsequent multianalyte detection in the presence of interferants, and have significant implications for future development of microarrays and biosensors. Acknowledgment. Sandia is a multiprogram laboratory operated by Sandia Corporation, a Lockheed Martin Company, for the United States Department of Energy under contract DEAC04-94AL85000. Supporting Information Available: DNA sequences 1, 2, 3, 4, and DNA detection calibration curve, as well as related instrumentation, reagents, and procedures. This material is available free of charge via the Internet at http://pubs.acs.org. LA701775G
