Decoration of Discretely Immobilized Cowpea Mosaic Virus with

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Decoration of Discretely Immobilized Cowpea Mosaic Virus with Luminescent Quantum Dots Igor L. Medintz,*,† Kim E. Sapsford,†,‡ John H. Konnert,§ Anju Chatterji,| Tianwei Lin,| John E. Johnson,| and Hedi Mattoussi*,⊥ Center for Bio/Molecular Science and Engineering Code 6900, Laboratory for the Structure of Matter Code 6812, and Division of Optical Sciences Code 5611, U.S. Naval Research Laboratory, Washington, D.C. 20375, George Mason University, 10910 University Boulevard, MS 4E3, Manassas, Virginia 20110, and Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037 Received December 21, 2004. In Final Form: March 31, 2005 This report describes two related methods for decorating cowpea mosaic virus (CPMV) with luminescent semiconductor nanocrystals (quantum dots, QDs). Variants of CPMV are immobilized on a substrate functionalized with NeutrAvidin using modifications of biotin-avidin binding chemistry in combination with metal affinity coordination. For example, using CPMV mutants expressing available 6-histidine sequences inserted at loops on the viral coat protein, we show that these virus particles can be specifically immobilized on NeutrAvidin functionalized substrates in a controlled fashion via metal-affinity coordination. To accomplish this, a hetero-bifunctional biotin-NTA moiety, activated with nickel, is used as the linker for surface immobilization of CPMV (bridging the CPMVs’ histidines to the NeutrAvidin). Two linking chemistries are then employed to achieve CPMV decoration with hydrophilic CdSe-ZnS core-shell QDs; they target the histidine or lysine residues on the exterior virus surface and utilize biotin-avidin interactions. In the first scheme, QDs are immobilized on the surface-tethered CPMV via electrostatic attachment to avidin previously bound to the virus particle. In the second strategy, the lysine residues common to each viral surface asymmetric unit are chemically functionalized with biotin groups and the biotinylated CPMV is discretely immobilized onto the substrate via NeutrAvidin-biotin interactions. The biotin units on the upper exposed surface of the immobilized CPMV then serve as capture sites for QDs conjugated with a mixture of avidin and a second protein, maltose binding protein, which is also used for QD-protein conjugate purification. Characterization of the assembled CPMV and QD structures is presented, and the potential uses for protein-coated QDs functionalized onto this symmetrical virion nanoscaffold are discussed.

Introduction Hybrid nanostructures that combine inorganic materials, such as nanoparticles or nanocrystals and nanorods, with the biological functions provided by proteins or DNA constitute a promising area of nanotechnology where materials interface with biology. For example, proteins can contribute structural and catalytic functions to the hybrid construct while nanoparticles contribute electronic, luminescent, or magnetic properties. These properties could be utilized for driving the signal transduction necessary for creating molecular scale diagnostic or sensing devices. One of the goals in designing these hybrids is to create new types of functional nanoscale devices, such as sensors.1-4 Rather than using lengthy schemes for synthesizing and purifying these hybrid materials, which can be further complicated by the labile nature of * Corresponding authors. E-mail: (H.M.) Hedimat@ ccs.nrl.navy.mil, (I.M.) [email protected]. † Center for Bio/Molecular Science and Engineering Code 6900, U.S. Naval Research Laboratory. ‡ George Mason University. § Laboratory for the Structure of Matter Code 6812, U.S. Naval Research Laboratory. | Department of Molecular Biology, The Scripps Research Institute. ⊥ Division of Optical Sciences Code 5611, U.S. Naval Research Laboratory. (1) Niemeyer, C. M. Angew. Chem., Int. Ed. 2001, 40, 4128-4158. (2) Seeman, N. C.; Belcher, A. M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 6451-6455. (3) Katz, E.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 6042-6108. (4) Oakley, B. A.; Hanna, D. M. IEEE Trans. Nanobiosci. 2004, 3, 74-84.

biomolecules, it would be simpler to exploit the programmable properties of the biological systems along with their ability to self-assemble to form these new nanostructures. Due to its base-pairing complementarily, DNA has long been used in creating a variety of multifunctional hybrid organic/inorganic nanostructures, such as hybrid nanowires and transistors.3,5,6 However, DNA has only four naturally occurring bases, which limits the number of possible combinations and types of available chemical interactions. Proteins on the other hand have 20 commonly occurring amino acids as their monomers, with more rare variants and many more synthetic analogues available. Furthermore, these amino acids carry different functionalities (e.g., amines, thiols, carboxyls, hydroxyls). This greatly expands the number of combinations and types of chemistries available. Since this endeavor seeks to create and control structures on the nanometer scale, protein building blocks of the appropriate size with well-ordered structures and chemically programmable functionalities are of particular interest.7,8 Icosahedral virus and cowpea mosaic virus (CPMV) particles, in particular, constitute an excellent example of ordered protein-based building blocks and nanoscaffolds.9-11 The coat protein shell of these 30 nm (5) Daniel, M. C.; Astruc, D. Chem. Rev. 2004, 104, 293-346. (6) Lee, S. H.; Mao, C. D. Biotechniques 2004, 37, 517-519. (7) Storhoff, J. J.; Mucic, R. C.; Mirkin, C. A. J. Cluster Sci. 1997, 8, 179-216. (8) Jones, A. H.; Lvov, Y. M. Cell Biochem. Biophys. 2003, 39, 23-43. (9) Wang, Q.; Kaltgard, E.; Lin, T. W.; Johnson, J. E.; Finn, M. G. Chem. Bio. 2002, 9, 805-811. (10) Wang, Q.; Lin, T. W.; Johnson, J. E.; Finn, M. G. Chem. Bio. 2002, 9, 813-819.

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Figure 1. (A) A ribbon diagram showing the asymmetric repeat protein unit which constitutes the CPMV capsid and the three insertion sites used to generate the HIS mutants in this study. (B) Model of the CPMV structure. Coordinates were obtained from entry 1ny7.pdb in the Protein Data Bank. Locations of particular 6-histidine mutation insertion sites are indicated with different colors: CPMV-C′-C′′ HIS, residues 144-45 of the small (s) subunit, blue; CPMV-EF HIS, residues 298-99 of the large (l) subunit, purple; CPMV-C-ter HIS, before stop codon in small (s) subunit, teal. (C) Chemical structure of biotin-X-NTA.

diameter particles consists of 60 copies of two different protein subunits: the small (s) subunit, consisting of the A domain and the large (l) subunit made up of the B and C domains. See parts A and B of Figure 1. In addition, the virus capsid accumulates to high levels in plants (facilitating large scale expression and purification), is stable over wide pH and temperature ranges, and is amenable to genetic alterations/modifications.11-14 The structure of the virus has been resolved to 2.8 Å resolution allowing rational design and incorporation of differing peptides (11) Wang, Q.; Lin, T.; Tan, L.; Johnson, J. E.; Finn, M. G. Angew. Chem., Int. Ed. 2002, 41, 459-462.

that bear desired properties and functionalities on the virus surface.11-14 Both wildtype and mutant CPMVs have been decorated or functionalized with a variety of species including poly(ethylene glycol), fluorescent dyes, DNA, proteins, and gold nanoparticles; CPMVs have also been successfully immobilized onto surfaces.11,15-21 Colloidal semiconductor nanocrystals or quantum dots (QDs) exhibit unique electronic properties as well as (12) Lomonossoff, G. P.; Johnson, J. E. Curr. Opin. Struct. Biol. 1996, 6, 176-182. (13) Wellink, J. Plant Virology Protocols; Foster, G., Taylor, S., Eds.; Human Press: Totowa, NJ, 1998; p 205. (14) Douglas, T.; Young, M. Adv. Mater. 1999, 11, 679-681.

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controllable size-dependent optical and spectroscopic properties along with a strong resistance to chemical and photodegradation. This makes them useful in a variety of bio-oriented applications, as well as potential building blocks for nanoscale electronic devices.22-28 Studies have used QDs as fluorophores in a variety of bio-oriented investigations, ranging from live cell imaging to bioassays.22-28 We have demonstrated their utility in numerous immunoassays and shown them to be excellent energy donors in fluorescence resonance energy transfer (FRET) investigations.26-28 QDs are also effective scaffolds for assembling FRET-based solution-phase biosensing nanostructures.27 We have explored protein-based strategies for modulating QD photoluminescence (PL) via FRET as well as surface tethering protein-functionalized QDs.28a,b To further develop and broaden the use of these materials, flexible and reproducible methods for patterning QDs onto surfaces need to be developed; this could pave the way for subsequent development of optoelectronic and sensing devices.21,29-32 Exploiting protein-based self-assembly to attach or tether QDs onto surfaces is particularly appealing, due to the simplicity and the potential for highorder design that can be intrinsically imparted onto the assembled nanostructures. Herein, we explore strategies for discretely immobilizing various CPMV mutants onto modified surfaces in a controlled fashion and then functionalizing the immobilized virus particles with luminescent QDs. CPMV with its unique three-dimensional (3-D) symmetry presents a nanoscaffold with an available protein surface that can be functionalized with a variety of species. We utilize step-by-step surface self-assembly to form surfaceimmobilized CPMV nanostructures functionalized with luminescent QDs. This approach permits one to circum(15) Raja, K. S.; Wang, Q.; Gonzalez, M. J.; Manchester, M.; Johnson, J. E.; Finn, M. G. Biomacromolecules 2003, 4, 472-476. (16) Wang, Q.; Raja, K. S.; Janda, K. D.; Lin, T.; Finn, M. G. Bioconjugate Chem. 2003, 14, 38-43. (17) Blum, A. S.; Soto, C. M.; Wilson, C. D.; Cole, J. D.; Kim, M.; Gnade, B.; Chatterji, A.; Ochoa, W. F.; Lin, T.; Johnson, J. E.; Ratna, B. R. Nano Lett. 2004, 4, 867-870. (18) Chatterji, A.; Ochoa, W. F.; Paine, M.; Ratna, B. R.; Johnson, J. E.; Lin, T. Chem. Biol. 2004, 11, 855-863. (19) Strable, E.; Johnson, J. E.; Finn, M. G. Nano Lett. 2004, 4, 13851389. (20) Chatterji, A.; Ochoa, W.; Shamieh, L.; Salakian, S. P.; Wong, S. M.; Clinton, G.; Ghosh, P.; Lin, T.; Johnson, J. E. Bioconjugate Chem. 2004, 15, 807-813. (21) Smith, J. C.; Lee, K.-B.; Wang, Q.; Finn, M. G.; Johnson, J. E.; Mrksich, M.; Mirkin, C. A. Nano Lett. 2003, 3, 883-886. (22) Dubertret, B.; Skourides, P.; Norris, D. J.; Noireaux, V.; Brivanlou, A. H.; Libchaber, A. Science 2002, 298, 1759-1762. (23) Jaiswal, J. K.; Mattoussi, H.; Mauro, J. M.; Simon, S. M. Nature Biotech. 2003, 21, 47-51. (24) Mattoussi, H.; Mauro, J. M.; Goldman, E. R.; Anderson, G. P.; Sundar, V. C.; Mikulec, F. V.; Bawendi, M. G. J. Am. Chem. Soc. 2000, 122, 12142-12150. (25) Goldman, E. R.; Balighian, E. D.; Mattoussi, H.; Kuno, M. K.; Mauro, J, M.; Tran, P. T.; Anderson, G. P. J. Am. Chem. Soc. 2002, 124, 6378-6382. (26) Clapp, A. R.; Medintz, I. L.; Mauro, J. M.; Fisher, B.; Bawendi, M. G.; Mattoussi, H. J. Am. Chem. Soc. 2004, 126, 301-310. (27) Medintz, I. L.; Clapp, A. R.; Mattoussi, H.; Goldman, E. R.; Fisher, B.; Mauro, J. M. Nat. Mater. 2003, 2, 630-638. (28) (a) Medintz, I. L.; Trammell, S. A.; Mattoussi, H.; Mauro, J. M. J. Am. Chem. Soc. 2004, 126, 30-31. (b) Sapsford, K. E.; Medintz, I. L.; Golden, J. P.; Deschamps, J. R.; Uyeda, H. T..; Mattoussi, H. Langmuir 2004, 20, 7720-7728. (29) Constantine, C. A.; Gattas-Asfura, K. M.; Mello, S. V.; Crespo, G.; Rastogi, V.; Cheng, T.-C.; DeFrank, J. J.; Leblanc, R. M. Langmuir, 2003, 19, 9863-9867. (30) Lu, N.; Chen, X.; Molenda, D.; Naber, A.; Fuchs, H.; Talapin, D. V.; Weller, H.; Muller, J.; Lupton, J. M.; Feldmann, J.; Rogach, A. L.; Chi, L. Nano Lett. 2004, 4, 885-888. (31) Jaffer, S.; Nam, K. T.; Khademhosseini, A.; Xing, J.; Langer, R. S.; Belcher, A. M. Nano Lett. 2004, 4, 1421-1425. (32) Baumle, M.; Stamou, D.; Segura, J.-M.; Hovius, R.; Vogel, H. Langmuir 2004, 20, 3828-3831.

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vent potential problems, such as solution cross-linking and aggregation, that may arise from the multiple functional sites available on CPMV cross-reacting with multiple QDs in solution when using covalent chemical modification.11,24 Experimental Section Reagents. Unless otherwise stated, chemicals were of reagent grade and used as received. Waveguides used in the surface studies consisted of plain microscope slides (Daigger & Co Inc., Vernon Hills, IL). The (3-mercaptopropyl)triethoxysilane was purchased from Fluka (AG, Buchs). N-(γ-Maleimidobutyryloxy) succinimide ester (GMBS), avidin, and NeutrAvidin where purchased from Pierce (Rockford, IL). Maltose binding protein (MBP) was expressed and purified as described previously.33,34 The biotin linker used in this study, succinimidyl-6-(biotinamido) hexanoate, EZ-Link NHS-LC-Bt, was purchased from Pierce. The biotin-X-NTA (Figure 1C; abbreviated Bt-X-NTA in figures only) was obtained from Biotium, Inc. (Hayward, CA). AlexaFluor 647 used for control experiments was purchased from Molecular Probes (Eugene, OR). Polyoxyethylenesorbitan monolaurate (Tween 20), dimethyl sulfoxide (DMSO), and anhydrous toluene were purchased from Sigma. Absolute ethanol was supplied by Warner-Graham Co (Cockeysville, MD). The poly(dimethylsiloxane) (PDMS), used to prepare the flow cells, was obtained from Nusil Silicone Technology (Carpintera, CA). Preparation of HIS-CPMV Mutants. A total of three histidine (HIS) CPMV mutants were used in this study (Figure 1A,B): the CPMV-C-ter HIS mutant expressing a poly-HIS tag (HIS6) at the C-terminus of the s subunit, the CPMV-EF HIS mutant with HIS6 inserted at residues 298-299 of the l subunit, and the CPMV-C′-C′′ HIS mutant expressing a HIS6 inserted at residues 144-145 of the s subunit. Mutational insertion of the HIS6 residues was accomplished using recombinant DNA techniques, and subsequent purification of mutant CPMV is as described.10,11,13-16 Briefly, oligonucleotides were used to direct PCR amplification of the appropriate CPMV gene, which were then ligated and transformed into E. coli cells to generate a cDNA plasmid. This plasmid was subsequently used to produce the specific HIS mutants using oligonucleotide site-directed mutagenesis. The primary leaves of young cowpea seedlings were mechanically inoculated with the plasmids, and this initial inoculum was extracted 7 days after inoculation from the infected leaves. This plant extract was then used to infect approximately 50 plants which were then harvested 3 weeks later and the resulting CPMV was isolated and purified using standard procedures.13 Preparation of Labeled CPMV. Biotinylation of both the wild-type CPMV and the HIS CPMV mutants was achieved using NHS-LC-Biotin, which targets the epsilon amine of lysine residues on the virus surface, using a 7000:1 molar ratio of NHS-biotin:CPMV, according to the manufacturer’s instructions. Biotin-labeled CPMV was separated from unincorporated biotin using 50 kDa MWCO dialysis membranes and dialyzing against phosphate buffer saline (PBS: 10 mM PB + 150 mM NaCl). CPMV was labeled with NHS-AlexaFluor-647 using the same procedure as described previously for NHS-fluorescein, resulting in an approximate dye/virus ratio of 60:1.18 Preparation and Purification of Avidin-Functionalized QDs. CdSe-ZnS core-shell nanocrystals were prepared stepwise using high-temperature solution chemistry from organometallic precursors. CdSe cores were first prepared by reacting cadmium and selenium precursors at ca. 300-340 °C in a hot coordinating solvent made of trioctylphosphine/trioctylphosphine oxide (TOP/TOPO) mixed with amine ligands.35,36 Purification of the growth solution, via size selective precipitation, to isolate CdSe nanocrystals was followed by ZnS overcoating carried out at lower (33) Medintz, I. L.; Goldman, E. R.; Lassman, M. E.; Mauro, J. M. Bioconjugate Chem. 2003, 14, 909. (34) Medintz, I. L.; Mauro, J. M. Anal. Lett. 2004, 37, 209. (35) Murray, C. B.; Norris, D. J.; Bawendi, M. G. J. Am. Chem. Soc. 1993, 115, 8706-8715. (36) Peng, Z. A.; Peng, X. J. Am. Chem. Soc. 2001, 123, 183-184.

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Figure 2. (A) Spectral properties of 570 nm emitting QDs and AlexaFluor 647 dye. The dashed blue and red lines represent the 488 and 633 nm excitation wavelengths. Black solid lines represent 550 nm long pass and 600 nm short-pass filters used as blocks when illuminating QDs at 488 nm. (B) Schematic representation of the PDMS patterning (1) and assay (2) flow cells used in this study. temperature 150-190 °C.37 The QDs were rendered water soluble by replacing the native TOP/TOPO organic capping ligands with bidentate dihydrolipoic acid (DHLA, dithiol-alkyl-COOH) ligands.24 The spectral characteristics of a 570 nm emitting QD solution are shown in Figure 2A; data shown in this study focused on QD solutions emitting at 570 or 590 nm, but studies with other size nanocrystals showed similar results. The bioconjugate assembly of QDs functionalized with the bacterial periplasmic maltose binding protein (MBP) and avidin were prepared following reported procedures.24,25 Briefly, between 50 and 100 pmol of QD was mixed with MBP and avidin at molar ratios of ∼15 MBP/QD and ∼1 avidin/QD in 250 µL of 10 mM NaTetraborate buffer pH 9.55 (borate buffer). Bioconjugates were allowed to self-assemble for 45 min at room temperature. The MBP conjugates to the surface of the QDs via its unique HIS-tag function.26-28 The reason for co-conjugating the MBP and avidin to the QD surface is that the MBP later aids in the purification of the bioconjugated QDs, by allowing them to bind to an amylose column. This allows removal of excess avidin, which does not stick to the column with washing followed by elution of the conjugate with maltose. Purification of the QD-conjugates was carried out as follows: QDs conjugated to a mixture of avidin and MBP (abbreviated MBP-QD-avidin) were allowed to adhere to amylose resin (New England Biolabs, Beverly, MA) in a gel column. Excess proteins (avidin in this case) were washed off the amylose resin with borate buffer, and MBP-QD-avidin bioconjugates were eluted with 10 mM maltose in borate buffer. Eluted solutions generally contained between 10 and 30 pmol of avidinfunctionalized QDs, and this solution is referred to as the “stock” solution in future sections dealing with the MBP-QD-avidin conjugates. Preparation of NeutrAvidin-Functionalized Waveguides. The microscope slides, used as waveguides for fluorescent characterization, were cleaned by immersion for 30 min in KOH (10% w/v) in isopropyl alcohol, followed by rinsing with Milli-Q water and drying under a nitrogen stream. Silanization of the slides was achieved by immersion for 1 h in anhydrous toluene solution containing 2% (3-mercaptopropyl)triethoxysilane, under an inert atmosphere. Slides were then washed in toluene and nitrogen-dried. NeutrAvidin was attached to the substrate by (37) (a) Hines, M. A.; Guyot-Sionnest, P.; J. Phys. Chem. B 1996, 100, 468-471. (b) Dabbousi, B. O.; RodriguezViejo, J.; Mikulec, F. V.; Heine, J. R.; Mattoussi, H.; Ober, R.; Jensen, K. F.; Bawendi, M. G. J. Phys. Chem. B 1997, 46, 9463.

Medintz et al. immersing the silanized slides first in 1 mM GMBS prepared in absolute ethanol for 30 min at room temperature, followed by washing with water and overnight incubation at 4 °C in a solution containing 25 µg/mL of NeutrAvidin in PBS. The slides were rinsed and either patterned immediately with the biotin-labeled species or stored in PBS at 4 °C. The PDMS flow cells used in this study consist of two types: (1) the patterning flow cell which is typically used to expose the waveguide surface to the biotinlabeled species; (2) the assay PDMS flow cell, whose channels run perpendicular to the patterning PDMS flow cell, which is used for further exposure of the waveguide surface to various reagent test solutions (Figure 2B). The PDMS flow cells were prepared by mixing part A and part B PDMS precursors (MED4011) in a 10:1 ratio. The mixture was then degassed and poured into flow cell molds, machined in poly(methyl methacrylate), PMMA, using a CNC mill. The PDMS-containing molds were then degassed to remove any trapped air incurred from the pouring process and then baked at 60 °C for 1 h. The molds were allowed to cool to room temperature before the PDMS flow cells were released from the PMMA molds and rinsed extensively with PBS prior to use. Patterning of CPMV on Substrates followed by Decoration with Hydrophilic QDs. Two reaction schemes were investigated to facilitate the controlled immobilization of the CPMV particles followed by labeling of the virus with QDs; these are outlined in Figure 3. For Scheme 1 shown in Figure 3A, NeutrAvidin functionalized substrates were assembled with sixchannel patterning PDMS flow cells (see Figure 2B) and exposed to a 10 µg/mL biotin-X-NTA solution in PBS, overnight at 4 °C. Note that in all studies described the biotin-X-NTA solution was precharged with nickel (Ni2+) prior to use. The channels were then rinsed with 1 mL of PBS and filled with a solution containing 75 µg/mL CPMV in PBS plus 1% Tween-20 for 1 h at room temperature. While still in the patterning PDMS flow cell, channels were rinsed and exposed to a solution of biotin-X-NTA in PBS (concentration ) 50 µg/mL) for 1 h at room temperature, followed by further rinsing and exposure to 100 µg/mL avidin solution in PBS containing 0.05% Tween (PBST) for 1 h. The slides were rinsed, disassembled from the patterning PDMS flow cell, and reassembled in the assay PDMS flow cell (Figure 2B). Channels were filled with 50 pM QDs dispersed in borate buffer containing varying amounts of Tween (0-0.5%) and allowed to incubate for 3 h at room temperature. The channels were rinsed and disassembled from the assay PDMS flow cell, and the functionalized substrate was dried and imaged using a total internal reflection imaging system (NRL Array Biosensor), equipped with an argon ion laser (488 nm) or a 635 nm diode laser for excitation and a CCD camera for fluorescence collection using appropriate filters, as previously described in ref 22. For Scheme 2 shown in Figure 3B, the NeutrAvidin slides were assembled with six-channel patterning PDMS flow cells and exposed to either a 20 µg/mL biotinylated-MBP (biotin-MBP) solution (positive control)22 or a 135 µg/mL biotin-CPMV (for wildtype or mutants) solution for 2 h; both solutions were prepared in PBST, and experiments were carried out at room temperature. The channels were then rinsed with 1 mL of PBST and the slides removed from the patterning PDMS flow cell and reassembled in the assay PDMS flow cell where they were exposed to MBP-QD-avidin in borate buffer containing varying amounts of Tween (0-0.1%) for 3 h. The channels were then rinsed with 1 mL of borate buffer, the slide was disassembled from the assay PDMS flow cell, and the functionalized substrate was dried and imaged as described above.22 Modeling the Capacity for QD Capture by Immobilized CPMV. Using the crystallographic structure of CPMV,11-14 a close packed array of CPMV virions was simulated. The QD threedimensional structure was simulated using a sphere of 65 Å. A simulation was run where the QD (sphere of 65 Å) was rolled over the surface of the close packed array of CPMV virions. All areas of contact were highlighted, and this surface area was compared to a “flat” two-dimensional surface of the same area.

Results and Discussion Controlled Surface Immobilization of CPMV. As schematically outlined in Figure 3 two reaction schemes were employed to facilitate the controlled and patterned

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Figure 3. Schemes for patterning CPMV virus on a substrate and decoration with QDs (not to scale). (A) Scheme 1. Glass slides were functionalized with NeutrAvidin and the surface exposed to biotin-X-NTA. Biotin binds NeutrAvidin leaving the Ni-NTA function available to coordinate to HIS6 on CPMV particles. The upper exposed surface of immobilized CPMV is then exposed to biotin-X-NTA, then avidin, and finally to DHLA-capped QDs. (B) Scheme 2. CPMV mutants labeled with NHS-ester-LC-biotin (LC-Bt) were first immobilized on the NeutrAvidin functionalized glass slide. The biotinylated CPMV mutants were then exposed to MBP-QD-avidin conjugates (consisting of ∼1 avidin/QD and ∼20 MBP/QD).25

immobilization of the CPMV on glass substrates, followed by decoration of the virus particles with luminescent QDs. In the first scheme detailed in Figure 3A, immobilization of the HIS CPMV mutants utilized biotin-X-NTA to bridge NeutrAvidin on the substrate with the HIS functions on the virus particles; this scheme takes advantage of the biotin-avidin interaction (KD ∼ 10-15) and the strong affinity of NTA for HIS groups (KD ∼ 10-9 to 10-12).38,39 In the second scheme (Figure 3B) CPMV mutants were labeled with NHS-ester-LC-biotin and the resulting biotin functionality on the virus was then used for both the immobilization of the CPMV on the surface of the waveguide and their subsequent interaction with avidin conjugated nanocrystals. Control experiments using AlexaFluor-647-labeled HIS CPMV mutants (labeled on the lysines residues with a dye/virus ratio of ca. 60:1), limited to Scheme 1, permitted us to optimize the reaction parameters between the biotinX-NTA functionalized surface and the HIS CPMV by allowing us to visualize the resulting patterned substrates using a 633 nm diode laser for excitation of the AlexaFluor647 dye.40 Figure 4A shows the fluorescence image collected with a CCD camera where the columns (X-axis) were exposed to different concentrations of biotin-X-NTA while rows (Y-axis) were exposed to AlexaFluor-labeled wild-type CPMV (CPMV), CPMV-C-ter HIS, or CPMV(38) Hermanson, G. T. Bioconjugate Techniques; Academic Press: New York, 1996. (39) Ueda, E. K. M.; Gout, P. W.; Morganti, L. J. Chromatogr., A 2003, 988, 1-23. (40) Sapsford, K. E.; Liron, Z.; Shubin, Y. S.; Ligler, F. S. Anal. Chem. 2001, 73, 5518-5524.

EF HIS. Data indicate that the wild-type CPMV does not attach to the exposed Ni-NTA functionalities on the biotin-X-NTA, an expected result since the virus does not express any available surface-exposed HIS6 (Figure 3A, rows 1*, 2*). The CPMV-C-ter HIS was found to bind promiscuously to both the NeutrAvidin and biotin-X-NTA regions of the slide, as demonstrated by the continuous signal observed along the entire length of rows 3* and 4* in Figure 4A. There are several potential reasons for the nonspecific binding observed for the CPMV-C-ter HIS mutant; this particular mutant may be more labile and may have denatured during the assay, or mutation at this particular site may have caused the virus to become more surface active, perhaps by rearranging some of the surface charges. However, the CPMV-EF HIS was specifically immobilized in the discrete squares (also called “data containing squares” in the figure legends) that had been exposed to Ni-NTA, which we attribute to the HIS-metal affinity coordination; the progressive decrease in the fluorescence intensity correlates well with the decrease in the biotin-X-NTA concentration (Figure 4A, rows 5*-6*).41,42 The CPMV-EF-HIS was found not to bind to the NeutrAvidin surface alone as demonstrated by the lack of signal generated in column [1], rows 5* and 6*, in Figure 4A in which the slide was exposed to PBS and not the biotin-X-NTA. This clearly demonstrates the required presence of the biotin-X-NTA for the discrete immobilization of the CPMV-EF-HIS via HIS-metal affinity (41) Hainfeld, J. F.; Liu, W.; Halsey, C. M. R.; Freimuth, P.; Powell, R. D. J. Struct. Biol. 1999, 127, 185-198. (42) Xu, C.; Xu, K.; Gu, H.; Zhong, X.; Guo, Z.; Zheng, R.; Zhang, X.; Xu, B. J. Am. Chem. Soc. 2004, 126, 3392-3393.

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Figure 4. Optimization of HIS CPMV immobilization. (A) Fluorescence image (using 633 nm excitation) collected of a slide patterned with decreasing concentrations of biotin-X-NTA (µg/mL) (patterning channels, columns [1]-[6]) then exposed to either AlexaFluor 647-labeled wild-type CPMV (150 µg/mL) or HIS CPMV mutants (75 µg/mL) (assay channels, rows [1*]-[6*]) for 1 h. Various dose-response curves were obtained in characterizing and optimizing the surface chemistry. (B) An expanded plot of the CPMV dye fluorescence net intensity (background subtracted) collected from a data square versus concentration of Biotin-X-NTA (slide image not shown; data average of four squares; curve R2 ) 0.969). The data square is defined as the intersection of the patterning and assay channels. Inset shows an expanded portion of the initial concentration points. (C) Plot of the net intensity versus concentration of AlexaFluor-647 labeled CPMV-EF HIS (slide image not shown; data average of four squares; curve R2 ) 0.995). The star indicates the concentration of CPMV used in some of the subsequent experiments, 75 µg/mL. All data were fit using a sigmoidal Hill four-parameter and in most cases the average standard deviation between data squares on one slide, exposed to the same conditions, was between 25 and 30%, whereas variation between slides was found to be between 10 and 15%.

coordination. A third CPMV HIS mutant, CPMV-C′-C′′ HIS, was also found to specifically bind biotin-X-NTA functionalized regions of the slide (data not shown). To further optimize this surface self-assembly, the effects of the biotin-X-NTA and Alexa-CPMV-EF HIS concentrations on the net fluorescence intensity obtained from the substrate were studied (intensity versus concentration are presented in parts B and C of Figure 4, slide images are not shown). It is apparent from the data shown in parts B and C of Figure 4 that saturation in the CPMV surface coverage (as reflected in the collected net fluorescence intensity) can be reached either by increasing the biotin-X-NTA on the substrate for a given virus concentration, or by increasing the concentration of the virus particle for a given biotin-X-NTA density on the surface. One should keep in mind that the virus is ∼7800 times larger than the biotin-X-NTA in terms of mass. Following these results, experiments were limited to using the HIS mutants CPMV-EF HIS and CPMV-C′-C′′ HIS, and we used solution parameters approaching saturation for the virus particles (75 µg/mL, 13 nM) and above saturation for the biotin-X-NTA (10 µg/mL, 14 µM). Decoration/Labeling of Surface-Immobilized CPMV with Luminescent QDs. Self-Assembly of Hydrophilic QDs onto Avidin-Coated Virus Particles. Immobilization of luminescent QDs on the surface-tethered CPMV particles was carried out by targeting the HIS groups on the exposed top surface of the virus particles (Scheme 1, Figure 3A) for reaction with a second round

of biotin-X-NTA solution, followed by exposure to hen egg avidin. The avidin tetramer binds to the available biotin moieties on the upper surface of CPMV with strong affinity, providing a top layer of exposed avidin, which we targeted for interactions with DHLA-capped QDs.25 We have previously shown that negatively charged DHLA-capped CdSe-ZnS QDs can electrostatically self-assemble with positively charged avidin;25 QD-avidin conjugates formed using this route were employed in immunoassay studies to detect solubilized toxins and explosives.25 This interaction is known to be electrostatic in nature because when the same type of QDs are exposed to NeutrAvidin, a deglycosolated form of avidin that has a much lower isoelectric point and is negatively charged at the pH of the buffer (unlike the avidin which is positively charged), very little interaction is observed.25 Figure 5 shows the resulting CCD image of the CPMV-EF-HIS (not AlexaFluor labeled) functionalized with QDs using this route (as in Scheme 1, Figure 3A). Slides were imaged by collecting the QD PL signal with a CCD camera, using 488 nm laser excitation. Figure 5 clearly shows that using the biotin-X-NTA/avidin combination allows our DHLA-capped QDs to be specifically captured by the CPMV-EF HIS mutant which is discretely immobilized on the substrate since only the squares exposed to the correct order of reagents allow QD decoration (patterning columns 4-6). The patterning columns 1-3 demonstrate (due to the low signal intensity) that the QDs have very little interaction with either the unfunctionalized Neutr-

Immobilized Virus with Luminescent Quantum Dots

Figure 5. Decoration/labeling of avidin-functionalized CPMV with DHLA-capped QDs. The fluorescence image shows patterns of the CPMV labeled with QDs via Scheme 1 and excited at 488 nm. Order of patterning exposure is shown above the image (numbers indicate channels exposed to that reagent) and assay exposure is shown to the right.

Avidin surface (column 1) or the CPMV-EF-HIS either alone or when functionalized with a top layer of biotinX-NTA (columns 2 and 3). Although there does appear to be some nonspecific interaction with these regions, due to the presence of some signal intensity, the overall intensity of the regions of the slide exposed to the correct sequence of reagents that facilitate QD immobilization (patterning columns 4-6) is much higher. The observed nonspecific binding of the QDs, while low, is decreased further by the addition of Tween into the QD solution (rows [2*]-[6*]). Tween is typically added to buffers to reduce nonspecific binding of proteins due to electrostatic interactions. It was also observed that as the concentration of the Tween in the borate buffer of the QD solution was increased (rows [2*]-[6*]), the intensity of the final immobilized QD squares decreases (patterning columns 4-6), suggesting that Tween is also interfering with the interaction between the immobilized avidin and the QDs. This confirms our previous findings on the nature of the electrostatic nature of the self-assembly of avidin onto DHLA-capped QDs in solution and now extends it to “surface-tethered” avidin.25 We also attempted to capture DHLA-capped QDs directly onto the virus particles using the HIS CPMV mutants (CPMV-EF HIS and CPMV-C′-C′′ HIS) that had been successfully immobilized on the substrate using Scheme 1. This attempt relied on the ability of the ZnS surface of DHLA-QDs to recognize and coordinate to polyhistidine (HIS) functionalities (HIS-zinc metal affinity coordination).27,43 However, collected images showed little to no interactions of these QDs with the HIS mutated CPMVs (data not shown). Possible reasons for this observation include the fact that the HIS functions are not well exposed (not protruding) from the virus surface and thus not available for effective coupling directly to the QD surface or virus surface charges may prevent the QDs from approaching the HIS residues. (43) Medintz, I. L.; Konnert, J. H.; Clapp, A. R.; Stanish, I.; Twigg, M. E.; Mattoussi, H.; Mauro, J. M.; Deschamps, J. R. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 9612-9617.

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Binding of Avidin Conjugated QDs onto Biotin-Functionalized CPMV. While successful in patterning CPMV with QDs, Scheme 1 is relatively lengthy and produces non-negligible background signal (see Figure 5). Therefore, we opted to explore an alternative method, still relying on biotin-avidin interactions, but utilizing preassembled MBP-QD-avidin conjugates, as outlined in Figure 3B (Scheme 2). In addition to preparing self-assembled MBPQD-avidin conjugates in solution, we have also shown that biotin-avidin binding is a robust linking strategy to attach QDs onto surface-tethered proteins.28 Here the virus (both wildtype and mutants) is first labeled with biotin via its surface lysines and then immobilized onto the NeutrAvidin slide. The slide was then exposed to avidin-functionalized QDs (MBP-QD-avidin, prepared as described above).25 The fluorescence images of the QD PL for the biotin-CPMVC′-C′′ HIS and biotin-CPMV-EF HIS mutants are shown in parts A and B of Figure 6, respectively. Biotin-MBP was immobilized in column 1, as a positive control, since our previous studies have shown that this protein can link MBP-QD-avidin to a NeutrAvidin surface.28 Data in parts A and B of Figure 6 clearly show that the biotinfunctionalized CPMV HIS mutants bind MBP-QD-avidin with high specificity, as evident by the discrete bright squares obtained in patterning columns [3] and [4]. However the MBP-QD-avidin was not found to interact with the NeutrAvidin surface alone, as shown in the PBS column [2] of parts A and B of Figure 6, demonstrating the importance of the biotin-functionalized CPMV HIS in the specific immobilization of the MBP-QD-avidin. The effects of the biotin-CPMV-EF HIS and MBP-QD-avidin concentration on the net intensity of the QD PL are shown in parts C and D of Figure 6 (slide images not shown). Data clearly indicate that concentrations as low as 20 µg/mL biotin-CPMV-EF HIS are sufficient to reach the maximum intensity of QD PL signal. Figure 6D also shows that saturation has not been reached when the stock MBPQD-avidin solution (as eluted from the amylose column) was passed over the surface, indicating the ability of CPMV immobilized on the surface to potentially accommodate higher decoration density of MBP-QD-avidin conjugates. Scheme 2 involves fewer steps than Scheme 1, and if one compares the background regions between the squares and in the PBS patterning columns in Figure 5 versus parts A and B of Figure 6 (channels [3*] or [4*]), Scheme 2 also produces lower nonspecific binding of the QD, as observed by lower QD PL intensity in these regions. This second scheme for functionalizing the virus particle with LC-biotin eliminates the need to use a HIS CPMV mutant, since both wild-type and mutant viruses contain lysines that can be targeted in the biotinylation reaction. That said however, it does leave the HIS functions on the mutant CPMV available should one wish to use them for further immobilization or functionalization via HIS-metal interactions. The ability to use either the biotin or the HIS functionalities of the biotin-CPMV-EF HIS to surface immobilize the virus followed by decoration with MBP-QD-avidin conjugates (via biotin-avidin interactions) is demonstrated by the data shown in Figure 7. Here, CPMV immobilization used either Scheme 2 or a combination of Scheme 1 and Scheme 2; see Scheme 3 Figure 7, on the same slide. Biotin-CPMV-EF HIS particles were immobilized either via interactions of the biotin functions with NeutrAvidin on the surface (Figure 7, column [2]) or via interactions of the HIS functions with NTA (in the biotin-X-NTA), previously attached to NeutrAvidin on the substrates (Figure 7, column [4]). Again, the MBP-QD-avidin was not found to interact with the NeutrAvidin surface alone, as shown in the PBS column

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Figure 6. Labeling of biotin-functionalized CPMV with MBP-QD-avidin. Portion of the fluorescence image of (A) biotin-CPMVC′-C′′ HIS and (B) biotin-CPMV-EF HIS decorated with QDs via Scheme 2, using 488 nm excitation. (C) A plot of the QD PL net intensity (background subtracted) collected as a function of the biotin-CPMV-EF HIS solution concentration (slide image not shown; data average of six squares; Curve R2 ) 0.989). (D) A plot of the QD PL net intensity as a function of the QD concentration (reported as % of the avidin-QD stock solution eluted from the amylose column); slide image is not shown; data average of six squares; curve R2 ) 0.998. 75 µg/mL biotinylated CPMV concentration was used.

Figure 7. Decoration/labeling of biotin-functionalized CPMV with MBP-QD-avidin. Virus particles were immobilized on the substrate via either their biotinylated lysine residues (Scheme 2, Figure 3) or HIS functionalities, as depicted in Scheme 3. The 488 nm line was used to excite the waveguide. Conditions for reagent parameters used are shown on top and to the right of the image.

[1] Figure 7, nor was it found to interact with the biotinX-NTA-Ni2+ functionalized region of the slide, column [3].

As a further control CPMV-EF HIS with no biotin was also immobilized onto a biotin-X-NTA functionalized surface (Figure 7, columns [5] and [6]). As can be seen, only columns [2] and [4] coated with biotin-CPMV-EF HIS, immobilized by either biotin-NeutrAvidin [2] or HIS-NTA [4] interaction, produced discrete and specific decoration with MBP-QD-avidin conjugates. Absence of any biotin sites on the immobilized virus in columns 5 and 6 prevented decoration with MBP-QD-avidin, which confirms that it is the avidin-biotin interactions that permits capture of QD conjugates on the virus particle. While increasing concentrations of Tween were found to affect the electrostatic interaction between the surface immobilized avidin and the solution QDs in Scheme 1, Figure 5, the same was not true (as expected) for Schemes 2 and 3 investigated in Figures 6 and 7 (where the interaction is biotin-avidin), as observed by little variation in the net intensity reached in the MBP-QD-avidin functionalized regions. It is worth noting that utilizing either of these residues (lysines/histidines) on the CPMV for subsequent linking or modification highlights the programmable nature of using these virus particles as a nanoscale scaffold for loading QD and other nanoparticle assemblies. These benefits will become more important as CPMV particles derivatized with multiple distinct functionalities are made available, thus providing soluble and controllable platforms for building nanoscale devices. Evaluation of CPMV Capacity to Capture QDs and QD Assemblies. Due to their large surface areas, CPMVs offer a few potential advantages as nanoscacle scaffolds, since a single virus particle should be able to accommodate several QDs and QD conjugates. To test this premise, we carried out a side by side comparison of the fluorescence signals measured from surface patterns formed with either

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Figure 8. Evaluation of the capacity of a close-packed CPMV layer to capture QDs. (A) Sketch of the immobilization scheme used to monitor CPMV ability to capture QDs. (B) Fluorescence image collected using 488 nm excitation. (C) Scale image of a close-packed array of CPMV particles. A single CPMV is circled in black. (D) Scale image obtained by rolling spheres of 65 Å (light green-bottom left corner) in diameter (approximate QD size) over the close-packed layer of virus particles. A single CPMV is circled in black. All areas of contact are highlighted in light green. There is about 40% increase in the QD density expected when a close-packed layer of CPMV is used to capture the QDs compared to a flat surface.

QDs immobilized directly on the surface or QDs captured onto CPMV particles already immobilized on the surface (Figure 8). For this, avidin-functionalized slides were prepared in the same manner as for NeutrAvidin above. The choice of avidin instead of NeutrAvidin for surface capture in these experiments was motivated by the low electrostatic affinity of DHLA-QDs to NeutrAvidin.25 An avidin-functionalized slide (made of only two rows and four columns) was first assembled in the patterning PDMS flow cell format, and half the channels (two of the four columns) were functionalized with biotin-CPMV-C′-C′′ HIS. The slide was then assembled in the assay PDMS flow cell, and the channels (both rows) were exposed to avidin followed by a solution of QDs (590 nm emitting QDs capped with DHLA were used). The resulting fluorescence image, taken using 488 nm excitation, is shown in Figure 8B. The collected image clearly shows a sizable increase in the fluorescence intensity collected from the columns where biotin-CPMV-C′-C′′ was immobilized prior to decoration with QDs, with ∼75% increase in the measured signal (compare columns [4] and [5] to columns [2] and [3]). Preliminary modeling experiments were performed to evaluate the QD packing on CPMV surfaces (described in Methods, see Figure 8C,D). We assume a close-packed arrangement for both CPMV immobilized on a flat surface and QDs immobilized either on a flat substrate or on the arrayed CPMV particles. Results from this modeling indicate that we could expect to derive a minimum increase in the QD density on the CPMV array of ∼40% compared to a monolayer of QDs on a flat surface. Although the 75% fluorescent increase from the above experiment cannot be directly correlated to a quantitative packing value, it clearly indicates the increased packing capacity. We used virus particles of 30 nm and core-shell QDs of ∼6.5 nm in diameter; however, this treatment did not take into account the avidin contribution to the virus dimensions. The addition of an avidin layer, extending

outward from the CPMV structure, could further increase the capture capacity of the CPMV particles. This preliminary result demonstrates the potential utility of such surface-functionalized virus particles as scaffolds for increasing the loading capacity of “2-D” substrates compared to a flat surface. This also has the potential of providing tethered or even soluble nanoscaffolds for loading large numbers of molecular assemblies, such as QD-bioreceptor conjugates. This can substantially improve the signal-to-noise ratio compared to individual assemblies.26,27 Conclusions We have demonstrated that variants of cowpea mosaic virus (CPMV) with specifically designed surface functionalities can be discretely immobilized and patterned onto NeutrAvidin-functionalized substrates via either biotin-avidin or HIS-metal affinity coordination interactions. Once immobilized these virus particles can be further functionalized and efficiently decorated with luminescent QDs, using biotin-avidin interaction or a combination of avidin-biotin and metal-affinity coordination (Figure 3). Although the process described here is innately a 2-D process, assembly using CPMV as a platform does offer advantages over strictly 2-D assemblies consisting of a QD monolayer. Not only does a CPMV covered surface have more area than a corresponding “flat” surface, but CPMV offers prearranged, symmetric, and fixed chemical functionalities for modification, something not easily achieved when creating 2-D assemblies. We have demonstrated that immobilizing virus particles on a surface followed by decoration with luminescent QDs produces a larger fluorescence signal than that generated by QDs immobilized on a similar “flat” surface area. These results could have implications in designing hybrid protein assemblies. For example, the QDs on the CPMV scaffold can in turn act as a scaffold for other proteins.

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The large surface area available on each CPMV particle could provide a scaffold for immobilizing an array of molecular assemblies with different functionalities and properties, so as to allow for a system with multiplexing capabilities. The different programmable chemistries of residues available on the CPMV surface lend themselves to these types of experiments. Inherent virus properties such as organized assembly, extensive exterior surface, enclosure of a large internal space and propensity to form arrays, combined with the unique QD properties, make these hybrids suitable templates for creating and inves-

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tigating new nanocomposite materials with biomedical/ bioelectronic applications. Acknowledgment. I.L.M. was partially supported by a National Research Council Fellowship through NRL. H.M. acknowledges A. Ervin and L. Chrisey at the Office of Naval Research, Grant N001404WX20270, and A. Krishnan at DARPA. NIH (R01 EBB00432-02 to J.E.J.), and NRL grants (N00014-00-1-0671 to J.E.J. and N00014-03-1-0632 to T.L.) are acknowledged. LA0468287