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An immunoassay based on gliding microtubules (MTs) is described for the detection of staphylococcal enterotoxin B. Detection is performed in a sandwic...
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Anal. Chem. 2008, 80, 5433–5440

Toward Single Molecule Detection of Staphylococcal Enterotoxin B: Mobile Sandwich Immunoassay on Gliding Microtubules Carissa M. Soto,*,† Brett D. Martin,† Kim E. Sapsford,‡,§ Amy Szuchmacher Blum,† and Banahalli R. Ratna† Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, D.C. 20375, and George Mason University, 10910 University Boulevard, MS 4E3, Manassas, Virginia 20110 An immunoassay based on gliding microtubules (MTs) is described for the detection of staphylococcal enterotoxin B. Detection is performed in a sandwich immunoassay format. Gliding microtubules carry the antigen-specific “capture” antibody, and bound analyte is detected using a fluorescent viral scaffold as the tracer. A detailed modification scheme for the MTs postpolymerization is described along with corresponding quantification by fluorescence spectroscopy. The resultant antibody-MTs maintain their morphology and gliding capabilities. We report a limit of detection down to 0.5 ng/mL during active transport in a 30 min assay time and down to 1 ng/mL on static surfaces. This study demonstrates the kinesin/MT-mediated capture, transport, and detection of the biowarfare agent SEB in a microfluidic format. Kinesin is an ATP-dependent motor protein that is involved in the intracellular transport of organelles, protein complexes, and mRNA.1 In eukaryotic cells, microtubule-based motility is also known to be involved in many cellular processes including mitosis, meiosis, and cell shape changes.2 Kinesin travels along microtubules (MTs), which are hollow tubular assemblies having a diameter of 25 nm with 8 nm structural periodicity. MTs are made of repeating Rβ-tubulin heterodimers that bind head to tail into protofilaments.3 They can be assembled in vitro from their constituent tubulin protein in the presence of GTP4 and stabilized by the action of taxol.5 Since the discovery of kinesin in 1985,6 the scientific community has worked extensively to determine its structure, mode * To whom correspondence should be addressed. Phone: 202-404-6006. Fax: 202-767-9594. E-mail: [email protected]. † Naval Research Laboratory. ‡ George Mason University. § Current address: U.S. Food and Drug Administration, CDRH/OSEL/DB, Building 64 HFZ-110, 10903 New Hampshire Ave., Silver Spring, MD 20993. (1) Kinbara, K.; Aida, T. Chem. Rev. 2005, 105, 1377–1400. (2) Stock, M. F.; Guerrero, J.; Cobb, B.; Eggers, C. T.; Huang, T. G.; Li, X.; Hackney, D. D. J. Biol. Chem. 1999, 274, 14617–14623. (3) Lowe, J.; Li, H.; Downing, K. H.; Nogales, E. J. Mol. Biol. 2001, 313, 1045– 1057. (4) Cote, R. H.; Borisy, G. G. J. Mol. Biol. 1981, 150, 577–602. (5) Amos, L. A.; Lowe, J. Chem. Biol. 1999, 6, R65-R69. (6) Vale, R. D.; Reese, T. S.; Sheetz, M. P. Cell 1985, 42, 39–50. 10.1021/ac800541x CCC: $40.75  2008 American Chemical Society Published on Web 06/11/2008

of action, and physical properties.7–18 Currently, researchers are investigating the motors in a more device-oriented context.19–28 One can imagine using such nanomachines to construct devices for laboratory-on-a-chip applications for sorting,29 concentration, and detection. Recent reviews describe the current status of the technology toward the utilization of the kinesin-MT system in actual nanodevices.1,30–34 The field still faces great challenges to produce devices for high-throughput data acquisition. Some of (7) Howard, J.; Hudspeth, A. J.; Vale, R. D. Nature 1989, 342, 154–158. (8) Correia, J. J.; Gilbert, S. P.; Moyer, M. L.; Johnson, K. A. Biochemistry 1995, 34, 4898–4907. (9) Vale, R.; Funatsu, T.; Pierce, D. W.; Romberg, L.; Harada, Y.; Yanagida, T. Nature 1996, 380, 451–453. (10) Hua, W.; Young, E. C.; Fleming, M. L.; Gelles, J. Nature 1997, 388, 390– 393. (11) Jiang, W.; Hackney, D. D. J. Biol. Chem. 1997, 272, 5616–5621. (12) Schnitzer, M. J.; Block, S. M. Nature 1997, 388, 386–390. (13) Vugmeyster, Y.; Berliner, E.; Gelles, J. Biochemistry 1998, 37, 747–757. (14) Rice, S.; Lin, A. W.; Safer, D.; Hart, C. L.; Naber, N.; Carragher, B. O.; Cain, S. M.; Pechatnikova, E.; Wilson-Kubalek, E. M.; Whittaker, M.; Pate, E.; Cooke, R.; Taylor, E. W.; Milligan, R. A.; Vale, R. D. Nature 1999, 402, 778–784. (15) Vale, R. D.; Milligan, R. Science 2000, 288, 88–93. (16) Hua, W.; Chung, J.; Gelles, J. Science 2002, 295, 844–847. (17) Yildiz, A.; Selvin, P. R. Acc. Chem. Res. 2005, 38, 574–582. (18) Leduc, C.; Ruhnow, F.; Howard, J.; Diez, S. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 10847–10852. (19) Hess, H.; Matzke, C. M.; Doot, R. K.; Clemmens, J.; Bachand, G. D.; Bunker, B. C.; Vogel, V. Nano Lett. 2003, 3, 1651–1655. (20) Hess, H.; Clemmens, J.; Qin, D.; Howard, J.; Vogel, V. Nano Lett. 2001, 1, 235–239. (21) Hess, H.; Clemmens, J.; Howard, J.; Vogel, V. Nano Lett. 2002, 2, 113– 116. (22) Brown, T. B.; Hancock, W. O. Nano Lett. 2002, 2, 1131–1135. (23) Clemmens, J.; Hess, H.; Lipscomb, R.; Hanein, Y.; Bo ¨hringer, K. F.; Matzke, C. M.; Bachand, G. D.; Bunker, B. C.; Vogel, V. Langmuir 2003, 19, 10967– 10974. (24) Ionov, L.; Stamm, M.; Diez, S. Nano Lett. 2005, 5, 1910–1914. (25) Mukhopadhyay, R. Anal. Chem. 2005, 249A–252A (26) Bachand, G. D. Small 2006, 2, 381–385. (27) Yokokawa, R.; Yoshida, Y.; Takeuchi, S.; Kon, T.; Fujita, H. Nanotechnology 2006, 17, 289–294. (28) Yoshida, Y.; Yokokawa, R.; Suzuki, H.; Atsuta, K.; Fujita, H.; Takeuchi, S. J. Micromech. Microeng. 2006, 16, 1550–1554. (29) Jia, L.; Moorjani, S. G.; Jackson, T. M.; Hancock, W. O. Biomed. Microdev. 2004, 6, 67–74. (30) Bakewell, D. J. G.; Nicolau, D. V. Aust. J. Chem. 2007, 60, 314–332. (31) Hess, H.; Vogel, V. Rev. Mol. Biotechnol. 2001, 82, 67–85. (32) Wang, Z. Phys. Rev. E 2004, 70, 031903. (33) Spetzler, D.; York, J.; Dobbin, C.; Martin, J.; Ishmukhametov, R.; Day, L.; Yu, J.; Kang, H.; Porter, K.; Hornung, T.; Frasch, W. D. Lab Chip 2007, 7, 1633–1643. (34) van den Heuvel, M. G. L.; Dekker, C. Science 2007, 317, 333–336.

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those challenges comprise the integration of active biomolecules into inorganic platforms and the combination of electrical, optical, and physical measurements with fluid handling.33 The kinesin-MT system offers the potential for single molecule detection which is expected to produce very sensitive and fast devices. This advantage is critical in applications such as antibioterrorism efforts or very early identification of pandemic species, where speed of detection is indispensable.30 The toxin staphylococcal enterotoxin B (SEB) is a common source of food borne illnesses35 and has the potential for use as a biological warfare agent,36 making its rapid detection of great importance. A number of sensing technologies have been applied to the detection of SEB.37 Immunoassays in particular have played a key role in rapid SEB detection, with transduction mechanisms including technologies based on fluorescence, chemiluminescence, and electrochemistry, as well as surface plasmon resonance (SPR), mass spectrometry, and piezoelectric cantilevers.38–51 With the exception of the piezoelectric cantilever sensors, which report limits of detection (LOD) in the 2.5 fg/mL-50 pg/mL range, the majority of the antibody based sensors achieve LODs in the 0.1-100 ng/mL range depending on the transduction mechanism and the sample matrix. The work presented herein demonstrates the utilization of kinesin-MT nanomachinery for detection of SEB using a sandwich immunoassay-type construction. In our experimental scheme, we used the well-known gliding assay format (which consists of MTs moving on kinesin-functionalized surfaces (Figure 1A)) to move antibody-containing MTs. Motility experiments are done in a flow cell which is comprised of two parallel glass surfaces separated by thin spacers approximately 100 µm in thickness. The motility flow cell is mounted on a microscope which is used to observe the movement of the motile element via fluorescence. Antibody-MTs carry the antigen specific “capture” antibody, while a virus containing a large number of dye molecules serves as the tracer in the sandwich immunoassay format (Figure 1B). Specifically, the tracer consists of a virus scaffold (cowpea mosaic virus, CPMV) to which a recognition element (Sh-anti-SEB) was coupled (35) Walt, D.; Franz, D. R. Anal. Chem. 2000, 72, 738A–746A. (36) Haes, A. J.; Terray, A.; Collins, G. E. Anal. Chem. 2006, 78, 8412–8420. (37) Ler, S.; Lee, F.; Gopalakrishnakone, P. J. Chromatogr., A 2006, 1133 (12), 1–12. (38) Sapsford, K. Appl. Environ. Microbiol. 2005, 71, 5590–5592. (39) Schlosser, G.; Kacer, P.; Kuzma, M.; Szilagyi, Z.; Sorrentino, A.; Manzo, C.; Pizzano, R.; Malorni, L.; Pocsfalvi, G. Appl. Environ. Microbiol. 2007, 73, 6945–6952. (40) Campbell, G.; Medina, M.; Mutharasan, R. Sens. Actuators, B 2007, 126, 354–360. (41) Maraldo, D.; Mutharasan, R. Anal. Chem. 2007, 79, 7636–7643. (42) Chatrathi, M.; Wang, J.; Collins, G. Biosens. Bioelectron. 2007, 22, 2932– 2938. (43) Branen, J.; Hass, M.; Douthit, E.; Maki, W.; Branen, A. J. Food Prot. 2007, 70, 841–850. (44) Yacoub-George, E.; Hell, W.; Meixner, L.; Wenninger, F.; Bock, K.; Lindner, P.; Wolf, H.; Kloth, T.; Feller, K. Biosens. Bioelectron. 2007, 22, 1368– 1375. (45) Medina, M. J. Agric. Food Chem. 2006, 54, 4937–4942. (46) Medina, M. J. Rapid Methods Autom. Microbiol. 2005, 13, 37–55. (47) Anderson, G.; Lingerfelt, B.; Taitt, C. Sens. Lett. 2004, 2, 18–24. (48) Rucker, V.; Havenstrite, K.; Herr, A. Anal. Biochem. 2005, 339, 262–270. (49) Alefantis, T.; Grewal, P.; Ashton, J.; Khan, A.; Valdes, J.; Del Vecchio, V. Mol. Cell. Probe 2004, 18, 379–382. (50) Shriver-Lake, L. C.; Shubin, Y. S.; Ligler, F. S. J. Food Prot. 2003, 66, 1851– 1856. (51) Khan, A.; Cao, C.; Thompson, R.; Valdes, J. Mol. Cell. Probe 2003, 17, 125–126.

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Figure 1. Experimental scheme. (A) Gliding assay: kinesin is immobilized on a glass surface inside a flow chamber. Kinesin heads (shown in pink) bind to the MT and move towards the positive (+) end of the MT in discrete 8 nm steps. The MTs shown have antigen specific “capture” antibodies coupled (light blue). (B) Immunoassay sandwich assay on an gliding MTs: (1) anti-SEB-MTs being moved by kinesin, (2) known amount of SEB is added to the microfluidic chamber where it binds to the corresponding site at the anti-SEBMTs, a wash follows to remove any unbound SEB, and (3) Cy5CPMV-anti-SEB tracer is introduced, and it binds to the SEB-antiSEB-MT complex; the tracer contains a large number of dyes that fluoresce allowing the imaging of the “moving” tracer. Protein structures are not drawn to scale.

along with a large number (60) of reporter entities (Cy5 dye molecules). We have used similar approaches in the past in the detection of pathogenic DNA52 and in immunoassays53 for signal enhancement. We envision that this tracer will make the system even more robust. The primary goal of this study was to build upon our previous success with gliding assays, by not only reducing the number of steps involved but also demonstrating detection in a sandwich immunoassay format. In our original direct gliding assays, biotinylated MTs were functionalized using a multistep incubation approach.54 Biotin-labeled antibodies were exposed to the biotin binding protein streptavidin, and the resulting complex was then exposed to the biotinylated MTs. With the goal of functionalizing the MTs with capture antibodies in a simpler, more controlled and robust fashion while maintaining their gliding properties (active MTs), we investigated the covalent attachment of Sh-antiSEB antibodies directly to the surface of the MTs (Figure 1A). (52) Soto, C. M.; Blum, A. S.; Vora, G. J.; Lebedev, N.; Meador, C. E.; Won, A. P.; Chatterji, A.; Johnson, J. E.; Ratna, B. R. J. Am. Chem. Soc. 2006, 128, 5184–5189. (53) Sapsford, K. E.; Soto, C. M.; Blum, A. S.; Chatterji, A.; Lin, T. W.; Johnson, J. E.; Ligler, F. S.; Ratna, B. R. Biosens. Bioelectron. 2006, 21, 1668–1673. (54) Martin, B. D.; Soto, C. M.; Blum, A. S.; Sapsford, K. E.; Whitley, J. L.; Johnson, J. E.; Chatterji, A.; Ratna, B. R. J. Nanosci. Nanotechnol. 2006, 6, 1–10.

Prior to the gliding assays, immunoassays were performed using the Naval Research Laboratory (NRL) array biosensor for optimization purposes and as a validation technique. The NRL biosensor55 is a well-established technique which allows us to simultaneously test a large numbers of samples and assay conditions.56 The NRL biosensor has been extensively used on sandwich immunoassays for identification of bacterial, viral, and protein analytes.57 EXPERIMENTAL SECTION Materials. Unless otherwise specified, chemicals were of reagent grade and used as received. All materials were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. Taxol used for stabilization of MTs was from Fisher Scientific (Pittsburgh, PA). Hydroxylamine, N-(γ-maleimidobutyryloxy)succinimide ester (GMBS), N-succinimidyl S-acetylthiopropionate (SATP), and NeutrAvidin were obtained from Pierce Biotechnology, Inc. (Rockford, IL). Poly(dimethyl)siloxane (PDMS), used for making the assay flow cells used in the array biosensor analysis, was obtained from Nusil Silicone Technology (Carpinteria, CA). Borosilicate glass slides from Daigger & Co. Inc. (Vernon Hills, IL) were used as waveguides for the array biosensor assays. Fluorescent labeling of the SEB antibodies was achieved using AlexaFluor 647 carboxylic acid succinimidyl ester (AF647), purchased from Invitrogen Corporation (Carlsbad, CA). Cowpea mosaic virus, in particular the double mutant (DM, 228/2102) used in this study, was obtained from John E. Johnson at The Scripps Research Institute. The DM-CPMV was chosen as it contained 120 inserted cysteine residues on the capsid surface, used for antibody functionalization as described later. Fluorescent labeling of the DM-CPMV capsid was achieved using Cy5-Mono-Maleimide, purchased from GE Healthcare Biosciences Corp (Piscataway, NJ). SEB and sheep (Sh-) and rabbit (Rb-) anti-SEB antibodies were obtained from Toxin Technology, Inc. (Sarasota, FL). All tubulin proteins (tubulin, biotinylated-tubulin, and rhodaminetubulin isolated from bovine brain minus glycerol) were purchased lyophilized from Cytoskeleton Inc. (Denver, CO). Preparation of Viral Tracer: Cy5-CPMV-anti-SEB. Sh-antiSEB (250 µL of 1 mg/mL anti-SEB in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.3)) was mixed with 3.5 µL of GMBS (10 µg/µL) and incubated at room temperature (RT) for 1 h. Excess GMBS was removed by using a prepacked desalting column (Hi-Trap, GE Healthcare Biosciences Corp.) using 50 mM potassium phosphate buffer (PB) pH 7.0. Recovered anti-SEB-GMBS (λmax ) 280 nm) was mixed with 200 µg of double mutant DM-CPMV (228/2102)53 in a final volume of 2 mL (we will refer to the DM-CPMV as simply CPMV from this point on). The mixture was incubated for 16 h, at RT in the dark. The sample was concentrated down to 500 µL using a 100k molecular weight cut off (MWCO) centrifugal filter (Microsep unit, MWCO ) 100 kDa; from VWR International; West Chester, PA). Unreacted anti-SEB-GMBS was separated from the CPMV-anti-SEB product via size exclusion chromatography using (55) Ligler, F. S.; Breimer, M.; Golden, J. P.; Nivens, D. A.; Dodson, J. P.; Green, T. M.; Haders, D. P.; Sadik, O. A. Anal. Chem. 2002, 74, 713–719. (56) Sapsford, K. E.; Charles, P. T.; Ligler, F. S. Anal. Chem. 2002, 74, 1061– 1068. (57) Ligler, F. S.; Sapsford, K. E.; Golden, J. P.; Shriver-Lake, L. C.; Taitt, C. R.; Dyer, M. A.; Barone, S.; Myatt, C. J. Anal. Sci. 2007, 23, 5–10.

Superose 6 (GE Healthcare Biosciences Corp., 18 cm long, 1 cm diameter, flow rate 0.5 mL/min) using 100 mM PB pH 7.0 as the eluent. Coupling of Cy5 dye to CPMV-anti-SEB virus was performed via maleimide chemistry. Virus containing fractions (CPMV-antiSEB, confirmed by UV-visible spectroscopy; λmax ) 260 nm) were pooled and concentrated down to 250 µL using a 100 kDa centrifugal filter. The virus sample was incubated on ice for 5 min prior to the addition of 10 µg/µL of Cy5-Mono-Maleimide in DMSO to have a 100 molar excess of dye relative to reactive thiol groups on the virus. DMSO was added to 25% followed by gentle mixing. The mixture was incubated at room temperature in the dark for 16 h. The sample was purified 2× using a Superose 6 column, eluent 50 mM (PB) pH 7.0 buffer. Typically, the resultant tracer (Cy5-CPMV-anti-SEB) contains one antibody and 60 dyes per virus capsid. Microtubules Preparation: Biotinylated-Rhodamine-MT (BR-MT). Microtubules were polymerized by mixing tubulin solutions in PMG/GTP buffer (80 mM Pipes, 1 mM EGTA, 1 mM MgCl2, 1 mM in GTP, pH 6.9); 5 µL of 20 µg/µL tubulin, 5 µL of 3 µg/µL biotinylated-tubulin, and 4 µL of 1 µg/µL of rhodaminetubulin along with additional PMG/GTP, resulting in a final concentration of 5 µg/µL tubulin. Corresponding solutions were incubated 15 min at 37 °C, followed by the addition of 0.5 µL of 5 mM taxol in DMSO after which the mixtures were incubated another 15 min at 37 °C. Resultant biotinylated-rhodamine MTs (BR-MT) were 4% in rhodamine. It is important to emphasize that MTs must be in their concentrated form (5 µg/µL) for long-term storage at 4 °C and during polymerization. For short-term storage and coupling reactions (∼2 days at room temperature), the MTs may be diluted up to 1 µg/µL in PMG/taxol buffer (PMG buffer 50 µM in taxol). Artificial sulfhydryl groups were incorporated into MTs at naturally occurring lysine groups via SATP reaction. Polymerized BR-MTs were diluted with 50 µL of PMG/GTP buffer and 1 µL of 5 mM taxol. Freshly prepared SATP, 2.2 µL of 1 mg/mL solution in DMSO was added to BR-MT solutions, corresponding to a 5× molar excess of SATP per tubulin. The reaction was incubated for 1 h in the dark at RT. BR-MT-SH were recovered by centrifuging for 30 min, RT at 13 000 rpm. After the supernatant was removed, the pellets were resuspended in 23 µL of PMG/ GTP buffer followed by the addition of 0.5 µL of 5 mM taxol to each vial. BR-MT-SH were stored overnight at 4 °C, in the dark. Deprotection of thiol groups was accomplished by addition of hydroxylamine; 80 µL of warm (37 °C) PMG/taxol buffer and 5 µL of hydroxylamine (0.5 M in PBS buffer pH 7.2 containing 25 mM EDTA, freshly prepared) were added to BR-MT-SH. Solutions were incubated in the dark at RT for 2 h. The MTs were recovered after reaction by centrifuging for 30 min, RT at 13 000 rpm. The BR-MT-SH isolated pellet was resuspended in 50 µL of PMG/ taxol buffer. Antibody Coupling to BR-MT-SH. Resupended BR-MT-SH were mixed with a corresponding amount of anti-SEB-GMBS (preparation as described previously) to obtain 1:100, 1:75, and 1:60 anti-SEB/tubulin ratios in a total of 100 µg of tubulin (see Supporting Information, Table S-1). MTs and antibody mixtures were incubated overnight in the dark at RT. To cap the free thiols 2 µL of 1 µg/µL maleimide-AlexaFluor 488 (AF488; Invitrogen Analytical Chemistry, Vol. 80, No. 14, July 15, 2008

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Corporation) was added and the mixture incubated at RT for 1 h. MTs were recovered by centrifugation and corresponding pellets were resuspended in PMG/taxol buffer up to a concentration of 1 µg/µL in tubulin. The BR-MT-anti-SEB samples were then visualized under a fluorescent microscope (see Supporting Information and Figure 2) to ensure that the modified MTs were intact prior to sandwich assays. Resultant antibody/tubulin ratios were determined by using controls samples and calibration curves (Figures S-1 and S-2) as explained in detailed in the Supporting Information. Patterning of NA Waveguides with Modified MTs for Array Biosensor Analysis. Glass microscope slides were functionalized with NeutrAvidin (NA) as described previously.56 Patterning of the NeutrAvidin waveguides with BR-MT modified with different ratios of anti-SEB, in PMG buffer, was carried out using a 6-channel patterning PDMS flow cell clamped onto the slide surface. The slides were then incubated overnight at 4 °C. In a typically experiment, certain channels were exposed to control samples consisting of either 10 µg/mL Biotin-Rb-anti-SEB (no MTs) or BR-MT (no antibody). SEB Sandwich Immunoassay and Array Biosensor Analysis. After overnight incubation, the waveguide-patterning flow cell assembly was hooked up to an ISMATEC multichannel pump (Cole-Parmer Instruments Company, Vernon Hills, IL) at one end (outlet) and syringe barrels (1 mL) were then attached at the opposite end (inlet), and the flow cell channels rinsed with 1 mL at 0.4 mL/min. The slide was then removed from the PDMS patterning flow cell and placed in 10 mM phosphate buffer (PB) + 150 mM NaCl + 0.05% Tween 20 (PBST) blocking solution containing 1% BSA. After ∼30 min, the slides were rinsed with Milli-Q water and assembled in a 6-channel assay PDMS flow cell, with the flow channels orientated perpendicular to the stripes of immobilized BR-MT, ready for the SEB sandwich immunoassay. First SEB, ranging from 100 to 0 ng/mL in PBST, was injected into the channels and left to incubate for 30 min at RT. The flow cell was then connected to the pump and the channels rinsed with 1 mL of PBST at 0.4 mL/min. Once washed, the channels were then filled with the tracer species, either 10 µg/mL AF647-antiSEB or 10 µg/mL Cy5-CPMV-anti-SEB in PBST, and the slide incubated for 30 min at RT. The flow cell was then connected to the pump and the channels rinsed with 1 mL of PBST at 0.4 mL/ min, before the PDMS assay flow cell was removed and the slide rinsed in Milli-Q water, dried with nitrogen, and imaged on the NRL array biosensor. The NRL array biosensor has been described in detail previously.55 Briefly, evanescent wave excitation of the surface-bound fluorescent species was achieved using a 635 nm, 12 mW diode laser (Lasermax, Rochester, NY). Light was launched into the end of the slide using a focal length lens equipped with a line generator and the resulting fluorescence emission monitored at right angles to the planar surface. A two-dimensional graded index of refraction (GRIN) lens array (Nippon Sheetglass, Summerset, NJ) was used to image the fluorescent pattern onto the Peltier-cooled CCD camera (Spectra Source, Teleris, Westlake Village, CA).58 Longpass (Schott 0G-0665, Schott Glass, Duryea, PA) and band-pass filters (Corion S40-670-S, Franklin, MA) were mounted on the (58) Golden, J.; Shriver-Lake, L.; Sapsford, K.; Ligler, F. Methods 2005, 37, 65– 72.

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device scaffolding to eliminate excitation and scattered light prior to CCD imaging. Data was acquired in the form of digital image files in flexible image transport system (FITS) format and the images analyzed using a custom software application that was written in LabWindows/CVI (National Instruments). The program creates a mask consisting of data squares (enclosing the areas where the capture antibody is patterned) and background rectangles which are located on either side of each data square. The average background value is subtracted from the average data square value, and the net intensity value is calculated and imported into a Microsoft Excel file for data analysis. Gliding Assays. MTs derivatized with anti-SEB using the molar ratio 1:75 sheep anti-SEB/Rβ tubulin (see above). The motility assays were performed at room temperature (21 °C). BRB80 buffer (80 mM PIPES, 1 mM MgCl2, 1 mM EGTA, pH 6.95 with KOH) was prepared, and motility buffer, which has components to prevent MT depolymerization and fluorophore photobleaching, was formulated to contain 1 mM ATP (adenosine 5′-triphosphate) and 10 µM taxol with oxygen scavenging additives (20 mM D-glucose, 20 µg/mL glucose oxidase, 8 µg/mL catalase, 0.2% 2-mercaptoethanol) in BRB80. SEB and the CPMV tracer (Cy5-CPMV-anti-SEB) were diluted into motility buffer for introduction into the flow cell (final concentrations: SEB, 0.5 ng/mL; Cy5-CPMV-anti-SEB, 6.6 µg/mL). The motility ensemble proteins were introduced into the flow cell using the standard capillaryflow technique.20,59,60 The cell was first filled with 20 µL of diluted casein solution (0.5 mg/mL in BRB80, 5 min deposition time), followed by 20 µL of Drosophila kinesin solution (0.1 mg/mL kinesin with 1 mM ATP and 0.2 mg/mL casein in BRB80, 5 min deposition time), and finally with 20 µL of motility buffer containing the anti-SEB-derivatized MTs. The latter solution was formulated by adding 2 µL of 5 µg/µL MTs in PMG/taxol to 18 µL of motility buffer. A time of 5 min was allowed for MT attachment, and the cell was then rinsed with 20 µL of fresh motility buffer to remove unattached MTs. Next, 20 µL of motility buffer-SEB solution was added to the cell, which was then allowed to incubate 30 min to allow SEB capture by the MT-anti-SEB. The cell was then rinsed with 20 µL of fresh motility buffer to remove unattached SEB. Finally, 20 µL of motility buffer-tracer Cy5-CPMVanti-SEB was added to the cell. MT visualization commenced 5 min afterward. MTs and the attached sandwich construction were visualized using an Olympus Opelco BX51 fluorescence microscope using 100× magnification, and MT speeds were quantified using Olympus MicroSuite image analysis software. Drosophila kinesin was kindly provided by Dr. George Bachand. RESULTS AND DISCUSSION Solution studies were undertaken to fully characterize the resulting Sh-anti-SEB (anti-SEB) modified MTs, and initial immunoassays were performed using the NRL array biosensor, prior to the gliding assays. This first assessment allowed us to find out the minimum amount of anti-SEB required on the antibody-MTs for detection and to ensure that anti-SEB on the MTs kept its specificity toward SEB. (59) Bachand, G. D.; Rivera, S. B.; Boal, A. K.; Gaudiso, J.; Liu, J.; Bunker, B. C. Nano Lett. 2004, 4, 817. (60) Bachand, M.; Trent, A. M.; Bunker, B. C.; Bachand, G. D. J. Nanosci. Nanotechnol. 2005, 5, 718.

Figure 3. Fluorescence emission of BR-MT-anti-SEB-AF647 series. Fluorescence of AF647 (excitation at 650 nm, emission at 667 nm) was normalized relative to rhodamine fluorescence (excitation at 544 nm, emission at 576 nm) for quantification purposes. Representative data of MTs in PMG buffer. Negative controls, BR-MT-AF488 lack antibody and showed no fluorescence at 667 nm. Table 1. Coupling Sh-anti-SEB to BR-MTs Figure 2. Microscope images of BR-MT-anti-SEB-AF647 capped with AF488. Images taken in an Olympus OpelcoBX51 fluorescence microscope using an oil immersion 100× objective (see Supporting Information for sample preparation details). (A) Rhodamine channel, (B) AF488 channel. The clarity of the MTs under the AF488 cube is a result of a large level of incorporation of the dye into the MTs, indicating that a large number of free thiols were still available after coupling of GMBS-anti-SEB-AF647. The scale bar corresponds to 5 µm.

Solution Studies. The main purpose of our solution studies was to find methods by which we can incorporate antibodies directly on the MTs via a covalent bond without affecting the gliding properties of the MTs. We utilized our experience with antibody coupling, particularly with maleimide-containing crosslinkers such as GMBS, which reacts with the thiol contained in cysteine (Cys). The MTs do not have a large number of naturally occurring Cys, and the ones available are involved in disulfide bond formation for structural integrity. Therefore, we designed our modification scheme around naturally occurring lysine (Lys), which contains a primary amine, and tends to be more abundant in proteins. Our scheme involves the incorporation of protected artificial thiols through the Lys using the commercially available linker SATP. We adapted known protocols for SATP incorporation and thiol-deprotection to the MTs system. We found that 5× excess of SATP was sufficient to incorporate thiols while keeping the MTs active in gliding assays. An important finding during our solution-phase studies was that the presence of free thiols after antibody incorporation resulted in inactive MTs. When the free thiols were capped by reacting them with a maleimide-containing dye, active MTs were obtained. We chose the dye AF488 so as not to introduce any spectral interference with any of the other dyes used for quantification or with the tracer. Figure 2 shows characteristic fluorescent images of resultant MTs. The MTs are intact and well dispersed indicating that the reactions have neither affected the structure of the MTs nor created aggregates. This is an important result, since other chemistries that utilize bifunctionalcross-linkers such as glutaraldehyde tend to produce MTs that contain large aggregates which may impede MT gliding in the sandwich assays.

calculated molar molar amount resultant molar ratio anti-SEB/ of anti-SEB/ ratio after reaction name tubulin tubulin added anti-SEB/tubulin % yielda R100 R75 R60 a

1:100 1:75 1:60

1.00:100 1.33:100 1.66:100

0.15:100 0.18:100 0.21:100

15.0 13.5 12.6

% Yield based on amount added vs resultant ratio after the reaction.

During our solution studies, we determined reaction molar ratios of antibody/Rβ-tubulin that resulted in viable MTs that contained enough active antibodies for sandwich assays. We found that reaction molar ratios 1:100, 1:75, and 1:60 of anti-SEB/tubulin were all appropriate for sandwich assays in the NRL biosensor (see next section for more details) while maintaining the MTs gliding capabilities. Once we found the best reaction molar ratios, we determined the actual amount of anti-SEB on the MTs. In order to achieve such quantification we coupled the dye AF647 (resultant 1:1 ratio dye/antibody) via the amines of the antibody using NHS ester chemistry (see Supporting Information). A known amount of rhodamine dye is routinely incorporated into the MTs during polymerization for imaging purposes during gliding assays (typically, 4% of repeat units are derivatized). We used that known amount of rhodamine as an internal standard for tubulin quantification. Fluorescence measurements of the BR-MT-anti-SEBAF647 in PMG were performed (Figure 3) to determine corresponding fluorescence output of AF647 relative to rhodamine. Figure 3 shows that indeed the amount of antibody bound (emission at 667 nm) increases as a function of antibody added as expected, indicating that at those conditions saturation has not been reached. By using corresponding calibration curves (Figures S-1 and S-2), we determined the resultant molar ratio of antibody/ tubulin. Values are an average of three measurements (Table 1). NRL Array Biosensor Analysis. Initial screening assays were carried out using the NRL array biosensor. The format of this system allowed us to quickly evaluate various anti-SEB-MTs modifications and the potential limits-of-detection (LODs) obtainable. Unlike the gliding assays, the MTs for the NRL array biosensor analysis required biotinylation so they could be imAnalytical Chemistry, Vol. 80, No. 14, July 15, 2008

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Figure 4. (A) Schematic of the sandwich immunoassay between immobilized BR-MT-anti-SEB and AF647-anti-SEB antibodies in the presence of SEB. (B) CCD image taken using the NRL array biosensor of a waveguide exposed to the SEB sandwich immunoassay. (C) Histogram plot showing the relative fluorescence intensities measured from the CCD image as a function of the SEB concentration, for the various anti-SEB/ MT ratios studied and also the negative control lanes (MTc).

Figure 5. (A) Schematic of the sandwich immunoassay between immobilized BR-MT-anti-SEB and Cy5-CPMV-anti-SEB viral tracer in the presence of SEB. (B) CCD image taken using the NRL array biosensor of a waveguide exposed to the SEB sandwich immunoassay, also used in the gliding assays. Note the patterning channels labeled with “x” refer to a ×10 dilution of the BR-MT-anti-SEB stock solution prior to exposure to the waveguide surface. (C) Histogram plot showing the relative fluorescence intensities measured from the CCD image as a function of the SEB concentration, for the two anti-SEB/MT ratios R75 and R60, the negative control lanes are also included. (/ denotes the conditions used in gliding assays with successful Cy5-CPMV-anti-SEB tracer, Figures 6 and 7).

mobilized onto the waveguide surface. Figure 4A shows a schematic of the initial optimization sandwich immunoassays, here AF647-anti-SEB was used as the tracer. A NeutrAvidin functionalized waveguide is patterned, using a PDMS flow cell, with MTs modified with varying ratios of anti-SEB. The MT patterned waveguide is then assembled in an assay PDMS flow cell to perform the sandwich immunoassay. The assay was two step, involving first a 30 min exposure to the SEB toxin followed by a 30 min exposure to the tracer, before the waveguide was imaged. An example of the resulting CCD image recorded is shown in Figure 4B. This particular waveguide was patterned with MTs prepared using anti-SEB/MTs ratios of 1:75, 1:50, and 1:25 (R75, R50, and R25, respectively). Buffer and MTs with no anti-SEB were used as negative controls, and biotin-labeled anti-SEB (no MTs) was used as a positive control. The waveguide was then exposed to various SEB concentrations (0-100 ng/mL) followed by the AF647-anti-SEB tracer. The CCD image clearly demonstrates that as the number of anti-SEB antibodies on the MT surface increases (i.e., the anti-SEB/MT ratio decreases) so too does the fluores5438

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cence intensity for a fixed SEB analyte concentration. This confirms the presence of the covalently attached anti-SEB on the surface of the MTs. There is also a clear dose-response dependence of the fluorescent signal as a function of the SEB concentration. Both of the negative controls, the buffer, and MTs with no anti-SEB show no fluorescent intensity in the presence of SEB, demonstrating the specificity of the reaction. The intensities from these images were analyzed using a custom software application as described in the Experimental Section. Results from data analysis are summarized in Figure 4C, as a plot of net fluorescence intensity as a function SEB concentration, for each of the anti-SEB/MT ratios studied and the negative control lanes. As expected, MTs modified with greater numbers of antiSEB on their surface (lower ratios) gave better LODs, with R25 and R50 detecting 0.5 ng/mL SEB, while R75 and R100 measured 1.0 ng/mL SEB. After some initial gliding studies with anti-SEB modified MTs, ratios of 1:75 (R75) and 1:50 (R50) anti-SEB/MTs were found to be optimal for capture of SEB on gliding MTs. NRL array

biosensor assays using these modified MTs coupled with the Cy5CPMV-anti-SEB tracer were performed to evaluate potential LODs. A schematic of the immunoassay is illustrated with the resulting CCD image shown in parts A and B of Figure 5, respectively. As before, the negative control, MTs with no anti-SEB, showed no fluorescent signal upon exposure to SEB toxin and the tracer. Both the R75 and R60 anti-SEB modified MTs demonstrated dose dependent responses to SEB exposure, as plotted in Figure 5C. LODs for both R75 and R60 where determined to be 1 ng/mL SEB. The motility assays were performed as described in the Experimental Section. A schematic of the MT-bound sandwich assay is shown in Figure 1. There are 3-5 sheep anti-SEB copies per CPMV tracer.53 During MT formation, the ratio of sheep antiSEB to Rβ-tubulin was chosen as 1:75. In preliminary motility assays using MTs constructed with ratios of 1:25, 1:50, and 1:75, we found that the ratio of 1:25 caused the MTs to completely lose their ability to attach to the immobilized kinesin, and the ratio of 1:50 resulted in an average gliding speed of 0.50 µm/s with roughly one-half of the MTs attached but immobile. The ratio of 1:75 resulted in an average gliding speed of 0.44 µm/s, with virtually all MTs mobile. Spectroscopic study indicated that a starting ratio of 1:75 anti-SEB to Rβ-tubulin in MT formation resulted in a final ratio of 1:555. The evident rejection of the vast majority of SEB-derivatized Rβ-tubulin may arise from anti-SEB attachment at sites necessary for subunit polymerization. There are 13 Rβ-tubulin repeat units in a circular cross-sectional segment of a MT, and each repeat unit has a length of approximately 8 nm. Thus, at the ratio of 1:555 there is an average of one antiSEB molecule for every 341 nm of MT length. Assuming that a 20 nm MT length is occupied per IgG,61–63 there is a spacing of roughly 17 IgG lengths between every attached antibody. Therefore, is unlikely that more than one SEB molecule will be trapped by a single viral tracer during the sandwich assay. The time-dependent speeds of MTs and CPMV tracer during the course of the gliding assay were determined (Figure S-3). For the SEB capture phase, solution SEB concentration was 0.5 ng/ mL; for the Cy5-CPMV-anti-SEB tracer capture phase, solution Cy5-CPMV-anti-SEB concentration was 6.6 µg/mL (1.2 nM). MT speeds were measured using sets of fluorescent images taken with emission collected at 550 nm, and Cy5-CPMV-anti-SEB speeds were measured using sets of images taken with emission collected at 650 nm. Cy5-CPMV-anti-SEB tracer movement, indicating that a successful sandwich immunoassay had taken place on a moving MT, was seen as early as t ) 15 min. When MT speeds were measured, only those without Cy5-CPMV-anti-SEB cargo were analyzed. As expected, the average speeds of MTs and Cy5CPMV-anti-SEB are nearly identical, with average MT speed at 0.49 µm/s and average tracer Cy5-CPMV-anti-SEB speed at 0.50 µm/s. These average speeds are comparable to those found by us in previous gliding studies using streptavidin-bound CPMV as cargo (0.52 µm/s), with similar solution CPMV (61) Sarma, V. R.; Silverton, E. W.; Davies, D. R.; Terry, W. D. J. Biol. Chem. 1971, 246, 3753–3759. (62) Cser, L.; Gladkih, L. A.; Franek, F.; Ostanevich, Y. M. Colloid Polym. Sci. 1981, 259, 625–640. (63) Saphire, E. O.; Parren, P. W. H. I.; Pantophlet, R.; Zwick, M. B.; Morris, G. M.; Rudd, P. M.; Dwek, R. A.; Stanfeld, R. L.; Burton, D. R.; Wilson, I. A. Science 2001, 293, 1155–1159.

Figure 6. Consecutive fluorescence microscope images showing a Cy5-CPMV-anti-SEB tracer being carried by a moving MT that has been covalently derivatized with anti-SEB. SEB is bound between the MT via the anti-SEB antibody and the tracer Cy5-CPMV-antiSEB in a sandwich geometry. The MT is labeled with rhodamine, and images 1, 3, and 5 were acquired at 550 nm emission wavelength. The Cy5-CPMV-anti-SEB tracer is labeled with Cy5, and images 2, 4, and 6 were acquired at 650 nm wavelength. A time of 3 s elapsed between frames. For the SEB capture phase, solution SEB concentration was 0.5 ng/mL; for the Cy5-CPMV-anti-SEB tracer capture phase, solution Cy5-CPMV-anti-SEB concentration was 6.6 µg/mL (1.2 nM).

concentrations. Typically, nonfunctionalized MT average speeds in our experimental setting ranges between 0.60-0.62 µm/s.54 In Figure 6, consecutive fluorescence microscope images are shown, depicting a Cy5-CPMV-anti-SEB tracer being carried by a moving MT. The MT is labeled with rhodamine, and images 1, 3, and 5 were acquired at 550 nm emission wavelength. The Cy5-CPMV-anti-SEB tracer is labeled with Cy5, and images 2, 4, and 6 were acquired at 650 nm wavelength. A time of 3 s elapsed between frames. The presence of the completed sandwich assay on a given MT evidently did not cause disruption of the normal Analytical Chemistry, Vol. 80, No. 14, July 15, 2008

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Figure 7. Transport of Cy5-CPMV-anti-SEB tracer by a MT with SEB bound as an intermediate in a sandwich geometry. A time of 4 s elapsed between each frame from left to right (first 4 frames). The fifth frame shows the first four images superimposed, with the rectilinear motion of the tracer clearly seen. Images were acquired at 650 nm emission wavelength.

MT gliding behavior. In general, MTs were observed to transport only a single tracer, no cases of multiple tracer binding and transport were observed. This is probably because of the very low SEB concentration used. Figure 7 contains additional images of a moving Cy5-CPMV-anti-SEB tracer acquired at 650 nm emission wavelength. A time of 4 s elapsed between each frame. The final frame shows the first four images superimposed, with the rectilinear motion of the tracer clearly seen. In a separate control experiment, Cy5-CPMV-anti-SEB tracer was added to the cell in the presence of gliding anti-SEBderivatized MTs. In this case, no SEB had been added. When the MTs were observed over long periods (>1 h) at 650 nm emission wavelength, absolutely no Cy5-CPMV-anti-SEB was seen moving. This indicates that Cy5-CPMV-anti-SEB tracer does not bind to the derivatized MTs in a nonspecific manner. The close correspondence of MT and Cy5-CPMV-anti-SEB speeds (Figure S-3), the coinciding tracks of Cy5-CPMV-anti-SEB and carrier MT seen in Figure 6, and the rectilinear motion over 12 s (with speeds closely corresponding to those of Figure S-3) seen in Figure 7 clearly demonstrate that Cy5-CPMV-anti-SEB tracer movement is due to transport by a supporting MT. The tracer movement clearly does not arise from a free Browniantype motion, since such movement is random with no linearity. Furthermore, each transported tracer Cy5-CPMV-anti-SEB corresponds to a single bound molecule of SEB, so in this sense single-molecule detection is being observed. This work is distinguishable from previous studies by other groups which successfully had reported selective binding and subsequent transport of virus particles26 and target proteins.64 They had used bifunctional cross-linkers and multilayer assembly approaches to incorporate antibodies into the MTs. In our study, we performed a classical sandwich immunoassay on gliding anti(64) Ramachandran, S.; Ernst, K.-H.; Bachand, G. D.; Vogel, V.; Hess, H. Small 2006, 2, 330–334.

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SEB MTs which were functionalized in solution prior to the gliding assay. The sandwich assay components, SEB, and Cy5CPMV-anti-SEB tracer were introduced separately after the onset of motility. The mobile sandwich assay proved to be an effective way of detecting SEB at very low concentrations of subnanograms per mL. CONCLUSIONS We have shown that MTs can be modified postpolymerization in a controlled way with anti-SEB. However, the amount of antiSEB has to be limited in order to obtain gliding MTs. We also found out that free thiols on the MTs, which resulted from our modification scheme, must be capped to maintain MT activity. To our knowledge this is the first demonstration in which a gliding MT carrying a capture antibody is used for the detection of SEB in a sandwich assay format down to LODs of 0.5 ng/mL. This system has potential applications in laboratory-on-a-chip type of technologies for detection of target analyte in a complex solution. ACKNOWLEDGMENT B.D.M. and K.E.S. contributed equally to this work. We thank DARPA for financial support, John E. Johnson for CPMV mutants, George Bachand for Drosophila kinesin, and George Anderson and Anthony Malanoski for reviewing the manuscript. SUPPORTING INFORMATION AVAILABLE Detailed procedures for determining the amount of anti-SEB in the MTs along with corresponding calibration curves. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review March 14, 2008. Accepted April 18, 2008. AC800541X