DNA-Directed Protein Immobilization on Mixed Self-Assembled

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DNA-Directed Protein Immobilization on Mixed Self-Assembled Monolayers via a Streptavidin Bridge Jon Ladd,† Christina Boozer,† Qiuming Yu,† Shengfu Chen,† Jiri Homola,†,‡ and Shaoyi Jiang*,† Department of Chemical Engineering, University of Washington, Seattle, Washington 98195, and Institute of Radio Engineering and Electronics, Academy of Sciences, Chaberska 57, 18251 Prague, Czech Republic Received January 14, 2004. In Final Form: June 10, 2004 The simultaneous detection of multiple analytes is an important consideration for the advancement of biosensor technology. Currently, few sensor systems possess the capability to accurately and precisely detect multiple antigens. This work presents a simple approach for the functionalization of sensor surfaces suitable for multichannel detection. This approach utilizes self-assembled monolayer (SAM) chemistry to create a nonfouling, functional sensor platform based on biotinylated single-stranded DNA immobilized via a streptavidin bridge to a mixed SAM of biotinylated alkanethiol and oligo(ethylene glycol). Nonspecific binding is minimized with the nonfouling background of the sensor surface. A usable protein chip is generated by applying protein-DNA conjugates which are directed to specific sites on the sensor chip surface by utilizing the specificity of DNA hybridization. The described platform is demonstrated in a custom-built surface plasmon resonance biosensor. The detection capabilities of a sensor using this protein array have been characterized using human chorionic gonadotropin (hCG). The platform shows a higher sensitivity in detection of hCG than that observed using biotinylated antibodies. Results also show excellent specificity in protein immobilization to the proper locations in the array. The vast number of possible DNA sequences combine with the selectivity of base-pairing makes this platform an excellent candidate for a sensor capable of multichannel protein detection.

1. Introduction The ability to create a protein array capable of fast, reliable parallel detections would greatly improve screening capabilities in medical diagnostics,1-3 environmental monitoring, as well as other fields. Although many novel techniques for the application of proteins to surfaces have recently been developed,4-7 protein arrays are routinely generated using spotting techniques,8,9 whereby a protein of interest is dropped onto the surface and allowed to adsorb in the contact area, often times irreversibly bound to the surface via a chemical linkage.6,10,11 This method of random immobilization generates an array of specific proteins with no designated orientation and unknown * To whom correspondence may be addressed. E-mail: sjiang@ u.washington.edu. † Department of Chemical Engineering, University of Washington. ‡ Institute of Radio Engineering and Electronics, Academy of Sciences. (1) Rhodes, D. R.; Chinnaiyan, A. M. J. Invest. Surg. 2002, 15, 275279. (2) Young, R. A. Cell 2000, 102, 9-15. (3) Epstein, C. B.; Butow, R. A. Curr. Opin. Biotechnol. 2000, 11, 36-41. (4) Mooney, J. F.; Hunt, A. J.; McIntosh, J. R.; Liberko, C. A.; Walba, D. M.; Rogers, C. T. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 1228712291. (5) Lee, K. B.; Park, S. J.; Mirkin, C. A.; Smith, J. C.; Mrksich, M. Science 2002, 295, 1702-1705. (6) Martin, B. D.; Gaber, B. P.; Patterson, C. H.; Turner, D. C. Langmuir 1998, 14, 3971-3975. (7) Ringeisen, B. R.; Callahan, J.; Wu, P. K.; Pique, A.; Spargo, B.; McGill, R. A.; Bucaro, M.; Kim, H.; Bubb, D. M.; Chrisey, D. B. Langmuir 2001, 17, 3472-3479. (8) MacBeath, G.; Schreiber, S. L. Science 2000, 289, 1760-1763. (9) Templin, M. F.; Stoll, D.; Schrenk, M.; Traub, P. C.; Vohringer, C. F.; Joos, T. O. Trends Biotechnol. 2002, 20, 160-166. (10) Disley, D. M.; Cullen, D. C.; You, H. X.; Lowe, C. R. Biosens. Bioelectron. 1998, 13, 1213-1225. (11) Collioud, A.; Clemence, J. F.; Sanger, M.; Sigrist, H. Bioconjugate Chem. 1993, 4, 528-536.

conformation,12 limiting the reactivity and usefulness of the protein on the surface. Immunological-based detection is possibly the only method shown to successfully detect analytes ranging from viruses to proteins to whole bacteria.13 This method relies on immobilized antibodies, or antibody fragments, on a surface, which can then detect the presence or absence of a target analyte.14,15 The random orientation inherent in most protein immobilization techniques limits the lower detection limits of these platforms.16 The linking of these proteins to the surface makes the chips difficult to store due to the presence of antibodies on the surface. The type and number of detectable analytes are limited by the antibodies linked on the surface. The linked antibodies dictate the chip’s end function and not the other way around. In addition to the random orientation of the detecting element present on the protein chip, the generation of false positives is a serious issue faced by many protein arrays. This is due to a surface that fouls in the presence of other proteins. Because of the complex nature of proteins, many modified substrates will nonspecifically adsorb proteins. The use of a nonfouling background, then, becomes more important. One method to create a nonfouling background is the use of oligo(ethylene glycol) (OEG), which has been shown to render surfaces protein resistant.17-19 Self-assembly is a convenient means for (12) Figeys, D. Proteomics 2002, 2, 373-382. (13) Iqbal, S. S.; Mayo, M. W.; Bruno, J. G.; Bronk, B. V.; Batt, C. A.; Chambers, J. P. Biosens. Bioelectron. 2000, 15, 549-578. (14) de Wildt, R. M. T.; Mundy, C. R.; Gorick, B. D.; Tomlinson, I. M. Nat. Biotechnol. 2000, 18, 989-994. (15) Lal, S. P.; Christopherson, R. I.; dos Remedios, C. G. Drug Discovery Today 2002, 7, S143-S149. (16) Chen, S. F.; Liu, L. Y.; Zhou, J.; Jiang, S. Y. Langmuir 2003, 19, 2859-2864. (17) Palegrosdemange, C.; Simon, E. S.; Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1991, 113, 12-20.

10.1021/la049867r CCC: $27.50 © 2004 American Chemical Society Published on Web 08/18/2004

Protein Immobilization

functionalizing metal surfaces with well-ordered monolayers. OEG thiols have been shown to passivate goldcoated surfaces.18,20,21 The specificity that single-stranded DNA (ssDNA) shows for hybridizing with its complementary strand22 provides a basis for the site-directed immobilization of proteins for use in a multichannel sensor. An array of unique sequences of immobilized ssDNA can be created on the surface of a sensor chip by a variety of methods.23-26 Different proteins linked to DNA strands complementary to those immobilized on the chip surface can be directed to the specific functionalized areas through the specificity of DNA hybridization. DNA hybridization has also been used to immobilize molecular components such as nanoparticles and polypeptides.27,28 Niemeyer et al. have previously conjugated streptavidin to ssDNA. These conjugates were then incubated with biotinylated antibodies. In their work, biotinylated complementary ssDNA was bound to streptavidin adsorbed onto microtiter plate wells.29 The conjugates were immobilized on the surface via DNA hybridization. Although the end result is the conversion of a DNA surface into a protein surface, their process requires extra incubation steps, the biotinylation of antibodies, and the direct adsorption of proteins on the chip surface. Niemeyer et al. focused on demonstrating the feasibility of using DNA-directed immobilization but did not investigate detections using the resulting platform. In this work, detections were performed using a surface based on self-assembled monolayers (SAMs) to better control protein orientation and conformation. A simplified method of conjugate preparation linking DNA directly to the protein of interest was used. Biotinylated ssDNA was patterned using a flow cell onto a nonfouling mixed SAM background via streptavidin. Antibody conjugated directly to a complementary ssDNA was then used to quickly and specifically convert the DNA chip into a protein chip. The functionalization process is illustrated in Figure 1. The resulting protein chip was then used for detection of human chorionic gonadotropin (hCG) in a custom-built surface plasmon resonance (SPR) biosensor and compared to previous work done with biotinylated antibodies.30 Results demonstrate the feasibility of using this simple platform to achieve high sensitivity and specificity, which are important in biosensor technology, but have not been fully addressed. SPR (18) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714-10721. (19) Nelson, K. E.; Gamble, L.; Jung, L. S.; Boeckl, M. S.; Naeemi, E.; Golledge, S. L.; Sasaki, T.; Castner, D. G.; Campbell, C. T.; Stayton, P. S. Langmuir 2001, 17, 2807-2816. (20) Mrksich, M.; Whitesides, G. M. Using Self-Assembled Monolayers That Present Oligo(ethylene glycol) Groups To Control the Interactions of Proteins with Surfaces. In Poly(Ethylene Glycol): Chemistry and Biological Applications; Harris, J. M., Zalipsky, S., Eds.; ACS Symposium Series 680; American Chemical Society: Washington DC, 1997; pp 361-373. (21) Ostuni, E.; Yan, L.; Whitesides, G. M. Colloids Surf., B 1999, 15, 3-30. (22) Neumann, T.; Knoll, W. Isr. J. Chem. 2001, 41, 69-78. (23) Blanchard, A. P.; Kaiser, R. J.; Hood, L. E. Biosens. Bioelectron. 1996, 11, 687-690. (24) Goldmann, T.; Gonzalez, J. S. J. Biochem. Biophys. Methods 2000, 42, 105-110. (25) Boncheva, M.; Scheibler, L.; Lincoln, P.; Vogel, H.; Akerman, B. Langmuir 1999, 15, 4317-4320. (26) Schena, M.; Heller, R. A.; Theriault, T. P.; Konrad, K.; Lachenmeier, E.; Davis, R. W. Trends Biotechnol. 1998, 16, 301-306. (27) Niemeyer, C. M. Trends Biotechnol. 2002, 20, 395-401. (28) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382, 607-609. (29) Niemeyer, C. M.; Sano, T.; Smith, C. L.; Cantor, C. R. Nucleic Acids Res. 1994, 22, 5530-5539. (30) Boozer, C.; Yu, Q. M.; Chen, S. F.; Lee, C. Y.; Homola, J.; Yee, S. S.; Jiang, S. Y. Sens. Actuators, B 2003, 90, 22-30.

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Figure 1. Illustration of the general construct for the DNAimmobilized sensor platform. Streptavidin is immobilized on a mixed BAT/OEG SAM. Biotinylated ssDNA is bound to the streptavidin. Antibody conjugated to a complementary ssDNA (A) is immobilized on the surface through hybridization to the proper location. Antibody conjugated to a noncomplementary ssDNA (B) does not bind. The conjugated antibody is then used for detection.

is a relatively new technique for real-time, quantitative monitoring of biomolecular interactions. One of the key advantages of SPR technology is the ability to carry out detections without the need of labeling proteins, e.g. with fluorescent tags, which has significant consequences and problems.15,31-33 Recently, SPR sensors have been developed to support multichannel capacity,30,34 indicating that SPR-based biosensors could be used to conduct arraybased detections. 2. Experimental Section 2.1. Surface Plasmon Resonance Sensor. SPR is a technique used to measure binding events to a surface in real time. For the custom-built SPR sensor used in this work, a parallel polychromatic light beam passes through an optical prism coated with a thin metal layer and excites surface plasmons at the interface between a metal layer on an optically transparent substrate and sample, Figure 2a. The excitation of surface plasmons is accompanied by the transfer of optical energy into the surface plasmon and the dissipation of optical energy in the metal layer, resulting in a narrow dip in the spectrum of reflected light, Figure 2b. The wavelength at which resonant excitation occurs depends on the refractive index of the analyte in the proximity of the SPR surface. Thus the amount of captured analyte can be quantified by measuring the refractive index change-induced shift in resonant wavelength.35,36 2.2. Preparation of Self-Assembled Monolayer. A mixture of oligo(ethylene glycol) alkanethiol (OEG) and biotinylated alkanethiol (BAT) was dissolved in absolute ethanol. The BAT, a C15 alkanethiol chain linked to a biotin headgroup by three ethylene glycol groups, was used as the specific binding element of the SAM, targeting streptavidin, while the OEG, a C10 alkanethiol chain linked to a hydroxyl headgroup by four ethylene glycol groups, created a nonfouling background. The mixture had a total thiol concentration of 100 nM and consists of a 1:4 ratio of BAT:OEG. The ratio has been shown to maximize avidin binding to the resulting mixed SAM.19,37 SPR sensor chips, BK7 glass chips 32 mm × 15 mm × 1.5 mm (Schott), were coated with 2 nm of Cr and 48 nm of Au by electron beam evaporation. These chips were exhaustively rinsed with absolute ethanol and water. Chips were then dried by blowing with dry N2. The chips were then cleaned by UV-ozone for 20 min. Following UV cleaning, these chips were again exhaustively (31) Kodadek, T. Chem. Biol. 2001, 8, 105-115. (32) Elia, G.; Silacci, M.; Scheurer, S.; Scheuermann, J.; Neri, D. Trends Biotechnol. 2002, 20, S19-S22. (33) Zhu, H.; Snyder, M. Curr. Opin. Chem. Biol. 2003, 7, 55-63. (34) Nikitin, P. I.; Beloglazov, A. A.; Kochergin, V. E.; Valeiko, M. V.; Ksenevich, T. I. Sens. Actuators, B 1999, 54, 43-50. (35) Homola, J. Sens. Actuators, B 1997, 41, 207-211. (36) Homola, J.; Dostalek, J.; Chen, S. F.; Rasooly, A.; Jiang, S. Y.; Yee, S. S. Int. J. Food Microbiol. 2002, 75, 61-69. (37) Jung, L. S.; Nelson, K. E.; Stayton, P. S.; Campbell, C. T. Langmuir 2000, 16, 9421-9432.

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Figure 2. (a) Schematics of the prism-based SPR sensor with wavelength interrogation. The sensor consists of a prism coupler and a thin SPR-active metal layer. (b) Typical spectra calculated for two different refractive indices for the SPR sensor consisting of a BK-7 glass prism and a thin gold layer. rinsed with water followed by absolute ethanol. The cleaned chips were immersed in the OEG/BAT solution described above overnight. 2.3. Patterning Oligonucleotides on the Chip Surface. A sensor chip was mounted into a custom-built SPR sensor. An acrylic flow cell was separated into two separate flow channels using a 50 µm deep Mylar gasket. This flow cell was mounted onto the sensor chip functionalized with the OEG/BAT monolayer. Streptavidin (Sigma-Aldrich) in a Tris-EDTA (TE) (10 mM Tris, 1 mM EDTA, pH 7.4) with 1 mg/mL bovine serum albumin (BSA) solution was flowed in both chambers at a concentration of 0.05 mg/mL. All flow events were carried out at a flow rate of 50 µL/min. While flow was used to functionalize the sensor surface with DNA, inkjet printing or conventional spotting techniques could also have been used. Biotinylated single-stranded oligonucleotides (bDNA) were immobilized on the layer of streptavidin. Custom oligonucleotides (Synthegen) were modified with a 5′ end attachment of a C6 linker and a biotin molecule. Two different sequences of DNA were used

Sequence A: 5′-TCCTGTGTGAAATTGTTATCCGCT-3′ (MW 7760.2) Sequence B: 5′-GTAATCATGGTCATAGCTGTT-3′ (MW 6886.7) Sequence A or B was flowed into one of the flow channels at a concentration of 100 nM in TE buffer with 1 mg/mL BSA for 10 min. This created a distinct DNA spot on the sensor surface. 2.4. Synthesis of Antibody-DNA Conjugate. AntibodyDNA conjugates were synthesized using a modification of the procedure used by Niemeyer et al.29 An amine group on the surface of the antibody was linked to the sulfhydryl group on a modified ssDNA. The oligonucleotide was modified with a C6 spacer between the sulfhydryl group and the 5′ end of the DNA sequence. The thiol oligonucleotide was chosen to be complementary to one of the biotinylated sequences immobilized on the streptavidin surface. The antibody-DNA linkage was accomplished using a sulfo-SMPB (Pierce) cross-linker. Monoclonal to β-hCG rabbit anti-hCG (Scripps Laboratories) or polyclonal anti-lysozyme (Research Diagnostics, Inc.) was reacted with the sulfo-SMPB cross-linker at a 10× molar excess in phosphate buffered saline (PBS) (100 mM phosphate, 150 mM NaCl, pH 7.4) for 30 min at room temperature. Unreacted cross-linker was removed using a microconcentrator (30k MW cutoff; Millipore). Solution buffer was switched to PBS with EDTA (PBE) (100 mM phosphate, 150 mM NaCl, 5 mM EDTA, pH 7.4). Purified products were reacted with one sequence of thiol-modified oligonucleotide in a 1:1 molar equivalent for 30 min at room temperature. Antibody-DNA conjugates were purified using a microconcentrator (100k MW cutoff; Millipore). This purification step was performed twice to remove all free, unreacted DNA. Solution buffer was switched to PBS and products are stored at 4 °C. Product yields were

quantified using a UV spectrophotometer at wavelengths of 260 and 280 nm. 2.5. Antibody Immobilization. Antibody-DNA conjugates were immobilized onto the sensor surface by making use of the specificity of DNA hybridization. Antibody-DNA conjugates were flowed in the SPR flow channels at a concentration of 0.02 mg/ mL in a TE buffer (10 mM Tris, 1 mM EDTA, pH 7.4) with 1 mg/mL BSA for 30 min. 2.6. Detection. hCG (MW 37 kDa) (Scripps) or lysozyme (MW 14 kDa) (Sigma) was flowed across the sensor surface and bound to the immobilized antibody. A sandwich assay was used in the detection of analyte. A direct detection response was first observed when analyte was passed over the sensor surface for 20 min, followed by a buffer wash for 10 min. A polyclonal secondary antibody was then flowed across the sensor surface for 20 min. This secondary antibody bound to unbound epitopes on the immobilized analyte, producing an amplification response. Polyclonal goat anti-rabbit hCG (Scripps) or polyclonal antilysozyme (Sigma-Aldrich) was used in this amplification step.

3. Results and Discussion 3.1. Nonfouling Surface. The OEG/BAT mixed SAM provides a nonfouling background for the binding of streptavidin onto the surface.19,37 This is due to the OEG portion of the BAT behind the biotin headgroup and the rest of the OEG background. Once the streptavidin and biotinylated oligonucleotide are on the surface, the nonfouling properties of this surface may change considerably. A nonfouling surface is a necessity for sensor chips. If the surface used for sensing fouls, or nonspecifically adsorbs large amounts of protein, the detection response cannot be validated. This will result in false detections. The sensor surface used in this work maintains its nonfouling characteristics following the immobilization of streptavidin and the biotinylated oligonucleotide. Figure 3 shows the specificity of the DNA surface to the complementary antibody-DNA conjugates. An antibody conjugated to a noncomplementary oligonucleotide is flowed across the surface. A minimal (∼0.1 nm) binding response is observed. This binding is less than 1% of the binding seen when an antibody conjugated to a complementary oligonucleotide is passed across the surface. Figure 4 shows the nonspecific adsorption of the mouse monoclonal anti-hCG antibody used in the conjugation procedure. Again, minimal nonspecific binding (