Targeted Particulate Adhesion to Cellulose Surfaces Mediated by

Sep 11, 2007 - Jered B. Haun , Lauren R. Pepper , Eric T. Boder , and Daniel A. Hammer ... Veldhuis , J. Christopher Hall , Jamshid Tanha , Roger MacK...
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Langmuir 2007, 23, 10682-10693

Targeted Particulate Adhesion to Cellulose Surfaces Mediated by Bifunctional Fusion Proteins Gautam Pangu,† Eric Johnston,‡ Jordan Petkov,§ Neil Parry,§ Matthew Leach,§ and Daniel A. Hammer*,†,‡ Department of Chemical and Biomolecular Engineering and Department of Bioengineering, UniVersity of PennsylVania, Philadelphia, PennsylVania, and UnileVer Research and DeVelopment Port Sunlight, Wirral, U.K. ReceiVed March 1, 2007. In Final Form: May 13, 2007 The adhesion of particles to surfaces is an integral element in many commercial and biological applications. In this article, we report on the direct measurements of protein-mediated deposition and binding of particles to model cellulose surfaces. This system involves a family of heterobifunctional fusion proteins that bind specifically to both a red dye and cellulose. Amine-coated particles were labeled with a red dye, and a fusion protein was attached to these particles at various number densities. The strength of adhesion of a single particle to a cellulose fiber was measured using micropipette manipulation as a function of the specificity of the protein and its surface density and contact time. The frequency and force of adhesion were seen to increase with contact time in fiber experiments. The dynamics of adhesion of the functionalized particles to cellulose-coated glass slides under controlled hydrodynamic flow was explored using a flow chamber for two scenarios: detachment of bound particles and attachment of particles in suspension as a function of the shear rate and surface density of protein. Highly specific adhesion was observed. The critical shear rate for particle detachment was an increasing function of cellulose binding domain (CBD) density on particle surface. A rapid irreversible attachment of particles to cellulose was observed under flow. Using a family of proteins that were divalent for binding either the red dye or cellulose, we found that particle detachment occurred because of the failure of the cellulose-CBD bond. A comparison of fiber binding and particle detachment results suggests that forces of adhesion of particles to cellulose of up to 2 nN can be obtained with this molecular system through multiple interactions. This study, along with the adhesion simulations currently under development, forms the basis of particulate design for specific adhesion applications.

Introduction The adhesion of particles to surfaces under conditions of fluid flow is an integral element in many industrial processes including but not limited to detergency, paper manufacture, water purification, bacterial adhesion, cell separation, and chemical catalysis. Several features contribute to these phenomena, including material properties, particle deformability and roughness as well as size and shape, particle-substrate and particle-particle interactions, complex hydrodynamic environments, and dynamically changing solution conditions such as surfactant concentration and temperature. Even though each of these features adds to the overall complexity of adhesion, their individual roles can be sorted out through a hierarchy of interfacial and hydrodynamic flow experiments. Often times, the adhesion of particles to a surface is mediated by specific binding between molecules on a particle surface and complementary ligand molecules on the receiving surface. The motivation for specific binding comes from biological systems, where adhesion is mediated by specific protein binding.1,2 A variety of well-designed and controlled adhesion assays have been used to investigate the interplay of chemistry and hydrodynamics in adhesion processes. The techniques that employ measurements on an individual particle include the peel test in which the critical tension required to peel the adherent surfaces is an index of adhesion energy,3-5 total internal reflection to monitor the separation distance between a colloidal sphere and * Corresponding author. E-mail: [email protected]. † Department of Chemical and Biomolecular Engineering, University of Pennsylvania. ‡ Department of Bioengineering, University of Pennsylvania. § Unilever Research and Development Port Sunlight. (1) Rosen, S. D.; Bertozzi, C. R. Curr. Opin. Cell. Biol. 1994, 6, 663-673. (2) Lawrence, M. B.; Springer, T. A. Cell 1991, 65, 859-873.

a surface combined with radiation pressure to apply a force to the sphere,6 and micropipette manipulation to remove micrometersized particles, biological cells, or liposomes from other particle or fiber surfaces,7-10 among others. In addition, the adhesion of a population of particles under hydrodynamic conditions is studied using flow assays that apply steady laminar flow field, radial flow field, or a centrifugal field either to remove prebound particles from surfaces or to deposit adhesive particles to surfaces under force.11-15 In this case, the index of adhesion is the force required to remove some fraction (typically 50%) of the adherent particles. These adhesion assays recreate the actual adhesion environment to different extents and help in systematically analyzing the role of the above-mentioned features in the adhesion process. In this work, we perform direct experimental measurements on a biomolecular system developed to facilitate the targeted deposition and binding of particulates to model cellulose surfaces (3) Dembo, M; Torney D. C.; Saxman, K.; Hammer, D. A. Proc. R. Soc. London, Ser. B 1988, 234, 55-83. (4) Evans, E. A. Biophys. J. 1985, 48, 185-192. (5) Evans, E.; Berk, D.; Leung, A.; Mohandas, N. Biophys. J. 1991, 59, 849860. (6) Walz, J. Y.; Prieve, D. C. Langmuir 1992, 8, 3073-3082. (7) Evans, E.; Berk, D.; Leung, A. Biophys. J. 1991, 59, 838-848. (8) Berk, D.; Evans, E. Biophys. J. 1991, 59, 861-872. (9) Tees, D. F. J.; Waugh, R.; Hammer D. A. Biophys. J. 2001, 80, 668-682. (10) Lin, J.; Silas, J. A.; Bermudez, H.; Milam, V. T.; Bates, F.; Hammer, D. A. Langmuir 2004, 20, 5493-5500. (11) Tempelman, L. A.; Hammer, D. A. Biophys. J. 1994, 66, 1-13. (12) Zhang, Y.; Eniola, A. O.; Graves, D. J.; Hammer, D. A. Langmuir 2003, 19, 6905-6911. (13) Cozen-Roberts, C.; Quinn, J. A.; Lauffenburger, D. A. Biophys. J. 1990, 58, 107-125. (14) Chu, L.; Tempelman, L. A.; Miller, C.; Hammer, D. A. Am. Inst. Chem. Eng. J. 1994, 40, 692-703. (15) Lotz, M. M. B.; Carol, A.; Erickson, H. P.; Mcclay, D. R. J. Cell Biol. 1989, 1795-1805.

10.1021/la700603u CCC: $37.00 © 2007 American Chemical Society Published on Web 09/11/2007

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Figure 1. Schematic diagram of the biomolecular family with binding domains for RR6 and cellulose.

using two adhesion assays. Cellulose (C6H10-O5)n is a longchain polysaccharide carbohydrate of β-glucose. It forms the primary structural component of the green plant cell walls, and it occurs naturally in its most pure form in cotton fiber.16 The experimental biomolecular system involves a family of heterobifunctional fusion proteins. These proteins were prepared by fusing a variable heavy-chain fragment from a heavy-chain llama antibody (VHH) against optically absorbing reactive red dye azo RR6, henceforth designated as VhhRR6, with a cellulose binding domain (CBD) that is typically a structurally and functionally independent noncatalytic module found in many polysaccharide degrading enzymes. Thus, these proteins are bifunctional in nature with one domain binding to RR6 and the other domain binding to cellulose in various affinities. The construction and verification of the binding affinity of these fusion molecules to RR6 and cellulose is described in detail elsewhere.17 Four proteins in this family were used in this study: single-chain protein VhhRR6CBD that binds to both RR6 and cellulose, proteins bivalent in either VhhRR6 or CBD ((VhhRR6)2CBD and VhhRR6(CBD)2, respectively), and single-chain protein VhhRR6 that binds to RR6 alone (Figure 1). Amine-coated micrometer-sized polystyrene particles are decorated with RR6, and single/multiple fusion proteins from the family are attached to these particles in various number densities. Glass fibers and glass slides coated with cellulose were used as the model surfaces to study the adhesion of these particles to cellulose. We employed LangmuirBlodgett deposition18-22 and spin coating,23-26 which are the well-studied and established techniques to produce smooth, ultrathin cellulose films on glass fibers and slides, respectively. (16) Kontturi, E. Surface Chemistry of Cellulose: From Natural Fibers to Model Surfaces. Ph.D. Thesis, Eindhoven University of Technology, The Netherlands, 2005. (17) Lewis, W.; Keshavarz-Moore, E; Windust, J; Bushell, D.; Parry, N. Biotechnol. Bioeng. 2006, 94, 625-632. (18) Schaub, M.; Wenz, G.; Wegner, G.; Stein, A.; Klemm, D. AdV. Mater. 1993, 5, 919-922. (19) Buchholz, V.; Wegner, G.; Stemme, S.; O ¨ dberg, L. AdV. Mater. 1996, 8, 399-402. (20) Holmberg, M.; Berg, J.; Stemme, S.; O ¨ dberg, L.; Rasmusson, J.; Claesson, P. J. Colloid Interface Sci. 1997, 186, 369-381. (21) Holmberg, M.; Wigren, R.; Erlandsson, R.; Claesson, P. M. Colloids Surf., A 1997, 129-130, 175-183. (22) Buchholz, V.; Adler, P.; Ba¨cker, M.; Ho¨lle, W.; Simon, A.; Wegner, G. Langmuir 1997, 13, 3206-3209. (23) Neuman, R. D.; Berg, J. M.; Claesson, P. M. Nordic Pulp Paper Res. J. 1993, 8, 96-104. (24) Geffroy, C.; Labeau, M. P.; Wong, K.; Cabane, B.; Cohen Stuart, M. A. Colloids Surf. 2000, 172, 47-56. (25) Gunnars, S.; Wågberg, L.; Cohen Stuart, M. A. Cellulose 2002, 9, 239249. (26) Konturri, E.; Thune, P. C.; Niemantsverdriet, J. W. (Hans) Langmuir 2003, 19, 5735-5741.

The forces and frequency of adhesion of a single particle to a cellulose-coated glass fiber are measured by using micropipette manipulation as a function of the adhesive affinity and specificity of the protein and its surface density and contact time between particle and fiber. In addition, the dynamics and strength of adhesion of the functionalized particles to cellulose-coated glass slides under controlled hydrodynamic flow are explored using parallel plate flow chamber assays for two scenarios: detachment of statically bound particles and attachment of particles in suspension under flow as a function of shear rate, in addition to above-mentioned parameters. The results show a highly specific and strong adhesion of the particles to cellulose only when a protein containing a CBD domain is used and a strong dependence of the extent of adhesion on the surface density of CBD domains as well as applied shear force. Furthermore, the magnitudes of average normal and shear forces to detach the particles as measured from the pipet and hydrodynamic assays are seen to complement each other. This study provides some useful insights into the adhesive behavior of the antibody-coated particles, which could pave the way for the systematic design of particulate systems with engineered deposition properties. Experimental Methods Bovine serum albumin (BSA), phosphate-buffered saline (PBS), trimethylsilyl cellulose, sodium borate, sodium chloride, hydrochloric acid, ethanol, chloroform, and hexamethyl disilazane were purchased from Sigma (St. Louis, MO). Tris-Cl buffer (pH 8.0) was purchased from Fisher Scientific (Pittsburgh, PA). RR6, VhhRR6, VhhRR6CBD, (VhhRR6)2CBD, VhhRR6(CBD)2, and rabbit anti-Vhh antibodies were kindly supplied by Unilever R & D, Port Sunlight, U.K. FITC goat anti-rabbit IgG (H+L) antibody was obtained from Zymed Laboratories (San Francisco, CA). Amine-coated polystyrene particles (mean diameter 6.34 µm) were products of Bangs Laboratories (Fishers, IN). Particle Preparation. Amino-functionalized polystyrene particles (mean diameter 6.34 µm) were functionalized with optically absorbing hapten azo dye reactive red (RR6) (molecular formula C35H25N9O22S6, molecular weight 1116.02 g/mol). An aqueous particle suspension (1 mL) was mixed with 500 µL of a 0.1% solution of RR6 in borate buffer (100 mM Na2B4O7, 10 H2O, 50 mM NaCl, pH 9.5) overnight at room temperature on a rotary mixer. The particles were washed several times with PBS by centrifugation to remove excess dye. The particles were then resuspended in 2 mL of blocking buffer (100 mM Tris-Cl, pH 8.0) and mixed as before for 2 h. They were then washed five times with PBS and stored at 4 °C. The antibodies were individually dissolved in appropriate volume of PBS to prepare a solution of 2 mg/mL concentration. To functionalize the particles for an adhesion experiment, 30 µL of the particle suspension was washed twice with 0.5 mL of PBS. These particles were then mixed

10684 Langmuir, Vol. 23, No. 21, 2007 with a 50 µL aliquot of a 2 mg/mL protein solution and 920 µL of PBS on a rotary mixer for 4 h. To vary the number of cellulose binding sites on the particle surface, mixtures of specific and non-specific antibody solutions in different ratios were used while preparing the particles for an experiment. They were then washed four times with PBS and stored at 4 °C. The particle suspension was sonicated for 2 min in a Branson 3510 water bath sonicator (Branson Ultrasonic Corp., Danbury, CT) before using it in an adhesion experiment. Fiber Preparation. Glass fibers (6 to 7 µm diameter, MO-SCI Corp., Rolla, MO) were inserted into the tips of a micropipette and secured with UV curing adhesive (NOA 71, Norland Products, Inc, New Brunswick, NJ) under a low power inspection microscope. The adhesive was cured overnight under 365 nm UV illumination via a Spectroline E-Series (Spectronics Corp, Westbury, NY). The pipettes that hold the fibers were inserted into modified pipet holders (MPH-1, E.W. Wright, Guilford, CT) with a protective 2 mm outside diameter glass sleeve inserted into one end. The back end of the micropipette was used to pull the fiber end into the protective tube. The fiber was washed with ethanol by injecting it into the side port of the holder. It was then rinsed with DI water through the same port. The remaining water was wicked out of the tube with lint-free tissue. Hexamethyl disilazane was introduced into the side port until the holder was filled. Then a 1.5 cm3 siliconized polypropylene microcentrifuge tube (Marsh Biomedical Products, Inc, Rochester, NY) was partially filled with hexamethyl disilazane. The holder was then placed into the tube, and the pipet was slid into the tube such that the fiber was immersed in the liquid. Following overnight incubation to hydrophobicize the fibers, fibers were dip coated with trimethylsilyl cellulose (TMSC) in a Langmuir trough (MicroTrough X, Kibron Inc.). A 0.5 mg/mL solution of TMSC in chloroform was added to the water surface, and the surface tension was set to 20 dyne/cm. The glass fibers were coated using a MicroTrough dipping accessory that allows manual dipping in the trough, through five cycles of lowering and raising the fiber. The fibers were then exposed to 10% HCl vapor for 2 min to regenerate TMSC on their surface back to cellulose. To image the fibers using scanning electron microscopy, samples were mounted on an aluminum specimen stub using double-sided carbon tape. They were then plated for 25 s using a Cressington Sputter Coater 108 with a gold-palladium target (Au 60, Pd 40) at 30 mA and 0.08 mb of argon. The samples were imaged using an FEI Strata DB235 SEM at an accelerating voltage of 5 kV. Figure 2a,b shows the scanning electron microscopy images of TMSC-coated glass fibers with and without pretreatment with hexamethyl disilazane, respectively. Peeling of the cellulose layer can be discerned to some extent for fibers that were not hydrophobicized. However, a uniform TMSC coating can be observed on fibers pretreated with hexamethyl disilazane. Slide Preparation. Microscope glass slides (75 mm × 25 mm × 1 mm, Corning Life Sciences) for flow chamber experiments were cleaned with 95% ethanol, dried, and hydrophobicized by incubating overnight with 1 mL of hexamethyl disilazane in a closed glass box. Furthermore, each slide was spread with 2 mL of a TMSC in chloroform solution (20 mg/mL concentration) and was immediately spun up to 1000 rpm for ∼30 s using a spin coater. It was further exposed to vapors of 10% HCl for 2 min to regenerate cellulose. For details about this setup, we refer to Kontturi et al.26 RR6 and Antibody Density Determination on a Particle Surface. The density of RR6 molecules on the particle surface was determined by absorbance measurements on an ELx 800 96 well microplate reader (Biotek Instruments, Winooski, VT). A calibration curve was generated from absorbance measurements of five RR6 solutions of 10-fold increasing dilutions starting from 0.1% concentration in borate buffer at 550 nm. The amount of RR6 per particle in a suspension of particles functionalized with RR6 was then determined from its absorbance measurement using the calibration curve. The amount of protein on the particle surface was quantified with flow cytometry. A suspension of particles (100 µL) functionalized with VhhRR6 protein (107 particles/mL) as described in the particle preparation section was incubated with 100 µL of a solution of rabbit anti-VHH antibody (diluted 1:1000 with PBS+ (1% denatured BSA solution in PBS)) for 1 h on ice. The suspension

Pangu et al. was washed three times with PBS+ and incubated with 100 µL of 10 µg/mL secondary fluorescent FITC goat antirabbit IgG (H+L) solution for 1 h on ice. The particles were washed three times with PBS+, resuspended in 500 µL of PBS+, and stored at 4 °C. Particles coated with secondary antibody alone were used as a negative control, and the fluorescence shifts were converted to site densities using a calibration curve relating the mean peak florescence of quantum FITC calibration particles (medium level) to their molecules of an equivalent soluble fluorochrome (MESF). It should be noted that we have used a polyclonal antibody to detect the RR6 fragment. Therefore, in the detection of molecules at the surface and of available binding sites, we sometimes observe some nonlinearity in translating the number of fluorescent events to molecular density at the surface. Micropipette Manipulation Assay. Micropipette manipulation is described in detail in many papers.7-10, 27-30 Briefly, micropipettes constructed of 0.8 mm borosilicate glass tubing (Friedrich and Dimmock, Milville, NJ) were made using a needle/pipet puller (model 730, David Kopf Instruments, Tujunga, CA) and microforged using a glass particle to give the tip a smooth, flat edge. Micropipettes were filled with DI water and mounted on a micromanipulator (model MHW-12, Narishige, Tokyo), and the pipet was connected via tubing to a manometer. A negative pressure, produced through the manometer using a syringe, was created in the pipet to pick up an antibody-coated particle. Fiber-touching experiments were performed inside a chamber designed and manufactured in house from 1/4 in. thick polycarbonate. The top and bottom were 22 mm × 22 mm no. 1.5 microscope cover glasses (Fisher Scientific, Pittsburgh, PA) and were attached to the chamber using an inert tube sealing compound (no. 510, Chase Instruments, Rockwood, TN). A clean chamber was assembled prior to each experiment, and the volume of the chamber was ∼2 cm3. The pipet was inserted into the side of the chamber. The fiber was introduced into the front of the chamber, at 90° with respect to the pipet, and the particle was brought into contact with the fiber using the micromanipulator. The pipet was repeatedly retracted from and brought back into contact with the fiber using a piezoelectric controller (model 17PAZ007, Melles Griot, Boulder, CO), and the maximum deflection of the fiber was recorded. The actuator was controlled using a GP-IB interface driven with LabVIEW 5.1 (National Instruments, Austin, TX). Experiments were imaged using a video camera (model 4915, Cohu Inc, San Diego, CA) and recorded using a Sony SVO-9500MD VTR (Sony Medical Systems, Montvale, NJ). Recorded images were digitized with a frame grabber board (PCI-1408, National Instruments) and analyzed using LabVIEW and IMAQ Vision software (National Instruments). This procedure was repeated at least 20 times for each particle. The location on the fiber was changed each time in case any residue from the contact remained on the fiber. The contact time between the particle and the fiber was varied, and the frequency and the force of adhesion were measured as a function of contact time. Flow Adhesion Assay. Flow adhesion experiments were performed in a parallel-plate chamber employing a tapered flow channel, the design of which has been described elsewhere.31,32 The schematic of the design is shown in Figure 3. By allowing the sides of the channel to follow the streamlines formed by 2D stagnation point flow, the shear rate (γ) varies along the length of the channel as γ)

6Q z 12 L h w1

(

)

where Q is the volumetric flow rate, h is the channel height, w1 is the channel entrance width, z is the axial position from the inlet, and (27) Kim, D. H.; Klibanov, A. L.; Needham, D. Langmuir 2000, 16, 28082817. (28) Evans, E.; Klingenberg, D. J.; Rawicz, W.; Szoka, F. Langmuir 1996, 12, 3031-3037. (29) Bermudez, H.; Brannan, A. K.; Hammer, D. A.; Bates, F. S.; Discher, D. E. Macromolecules 2002, 35, 8203-8208. (30) Bermudez, H.; Hammer, D. A.; Discher, D. E. Langmuir 2004, 20, 540543. (31) Usami, S.; Chen H.-H.; Zhao Y.; Shien S.; Skalak R. Ann. Biomed. Eng. 1993, 21, 77-83. (32) Brunk, D. K.; Hammer, D. A. Biophys. J. 1997, 72, 2820-2833.

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Figure 2. Scanning electron microscope images of glass fibers coated with TMSC (a) after and (b) without using hexamethyl disilazane to make the fibers hydrophobic. L is the channel length. This design allows the observation of particlesurface adhesion at various shear rates during one experiment. Templates with a tapered flow channel were cut from 0.01 in. thick Duralastic sheeting (Allied Biomedical, Goose Creek, SC) and placed over a cellulose-coated glass slide. The template and slide were placed in the bottom well of the flow chamber and secured with the top of the flow chamber. The dimensions of the flow channel thus formed were h ) 0.023 cm, w1 ) 0.1 cm, and L ) 6.6 cm. The flow chamber was assembled in deionized water to keep air from getting into the channel. The whole assembly was mounted on the stage of a Nikon Diaphot inverted microscope with phase-contrast optics (Nikon, Tokyo, Japan) connected to a Cohu black and white CCD camera (Cohu Inc., San Diego, CA) and a Sony SVO-9500MD S-VHS recorder (Sony Medical Systems, Montvale, NJ). The buffer and particle suspensions were drawn through the chamber with an infusion/withdrawal syringe pump (Harvard Apparatus, South Natick,

MA). The chamber was gently perfused with PBS for at least 10 min before introducing the particle suspension into it. For particle detachment experiments, 200 µL of the particle suspension (∼ 5 × 107/mL) was injected into the chamber, and the particles were allowed to settle on the slide surface for 45 min. The chamber was stepped down from inlet to outlet in steps of 0.2 cm, and images of the particles that settled on the slide surface in the field of vision of the microscope were recorded. Flow was initiated with the syringe pump at a desired flow rate, after which the nonadherent particles were detached from the slide and cleared from the chamber. The images of the slide surface at the same locations as before were recorded. The particle numbers at a slide location before and after the flow was initiated were obtained through digital image analysis of these images using LabView software (National Instrument, Austin, TX). In general, two flow rates were used for two separate particle suspensions for every ratio of specific to non-

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Figure 3. (a) Side view of the parallel-plate flow chamber. (b) Top view of the tapered channel. specific antibody in incubating solution, and each flow rate was run in duplicate or triplicate. For attachment experiments, a particle suspension (106 particles/ mL) was injected into the chamber at 0.4 mL/min. The number of particles adherent to different locations on the slide under flow was measured by stepping the chamber down from inlet to outlet in steps of 0.4 cm and manually counting the number of adherent particles in the field of view. A particle was considered to be adherent if it remained stationary for >10 s. The entire chamber was traversed at least three times during one experiment (∼26 min). Every ratio of specific to non-specific antibody was run in duplicate or triplicate.

Results RR6 and Protein Site Density on the Particle Surface. The average density of RR6 molecules on the particle surface from absorbance measurements of two different dilutions of 1 mL of an aqueous particle suspension mixed with 500 µL of a 0.1% solution of RR6 in borate buffer was found to be 7.89 × 106 ((3.56 × 106) molecules/µm2. To quantify the amount of protein on the particle surface, four particle samples functionalized with proteins VhhRR6, VhhRR6CBD, (VhhRR6)2CBD, and VhhRR6(CBD)2, respectively, were treated with primary and secondary antibodies as described in Experimental Methods. For each sample, a particle sample functionalized with the corresponding protein and secondary antibodies (no primary antibody) was used as a negative control. The results show that the secondary antibody does not nonspecifically bind with protein, and a large rightward shift in fluorescence was observed in the presence of primary antibody, as shown in Figure 4. This shift was converted to site densities using a calibration curve relating the mean peak florescence of quantum FITC calibration particles to their molecules of equivalent soluble fluorochrome (MESF). The results for the number of secondary antibody units per square micrometer of particle surface, which is directly related to the site density of protein, for all four proteins are shown in Figure 5. Because each protein is of a different size and valency, the surface densities of each protein are different. Micropipette Manipulation Results. In micropipette manipulation experiments, the frequency of adhesion was recorded as a function of contact time between particle and fiber for different particle preparations. Furthermore, the force of adhesion was measured for the fraction of positive adhesive events. A video micrograph of a typical experiment is shown in Figure 6. In panel A, the particle-pipet combination is brought into contact with the fiber. In panel B, the particle that is adherent to the fiber is retracted, and the fiber deflection is used to measure the force

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of adhesion. Figure 7 shows the results of the experiments where particles coated with 100% VhhRR6CBD were used to test the adhesion and particles coated with VhhRR6 only were used as a negative control. The frequency of adhesion is between 80 and 90% for the positive antibody whereas it is between 10 and 65% for the negative antibody as the contact time is varied from 2 to 60 s, as seen in Figure 7a. Thus, reducing the contact time seems to reduce nonspecific binding. The force of adhesion for particles coated with positive antibody ranges from 1 to 2 nN over the range of contact times tested whereas that for particles coated with negative antibody reaches 1 nN at 60 s of contact time, as seen in Figure 7b. Thus, the force of adhesion for specific adhesion is significantly greater than that for nonspecific adhesion for all contact times. Flow Chamber Detachment Results. In detachment experiments, we measured change in the number of bound particles at the same location on the slide before and after the flow was applied. Shear rates were derived from locations of different spots on the slide relative to the flow inlet and the volumetric flow rate, and particle adhesion was calculated as a function of shear rate. To determine the time required to reach steady state for detachment, the number of adherent particles coated with VhhRR6CBD antibody was measured as a function of time at different shear rates (Figure 8). It was observed that the number remained unchanged for a wide range of shear rates after 25 min. Indeed, it is remarkable that most unbinding events take place within the first 5 min. In actual experiments, the slides were washed for 35 min to ensure steady state. Effect of Shear Rate and Specific Antibody Concentration. The use of a tapered gasket plus two flow rates for each antibody concentration in detachment experiments allowed us to vary the shear rate over a wide range. Representative images of particles attached at spots on the slide before and after washing with buffer are shown in Figure 9. To vary the number of binding sites on the particle surface, we varied the ratio of bifunctional to monofunctional (negative control) protein in the incubating solution. The plot of the percentage of particles remaining adherent at steady state as a function of both the shear rate and the number of binding sites is shown in Figure 10a. Detachment with shear rate was observed in all cases. Particles experience greater hydrodynamic forces at higher shear rates, and hence the percentage of detatched particles increases with increasing shear rates. Also, adhesion was greater for particles with a larger percentage of CBD on the particle surface. The negative control (particles incubated with 100% VhhRR6) showed the weakest adhesion. For each of the curves in Figure 10a, the critical shear rate was identified, which is the shear rate at which 50% of the statically bound particles are detached from the slide at steady state. The critical shear rate is an increasing function of the relative percentage of CBD in incubating protein solution (Figure 10b). Effect of Protein Valency on Microsphere Detachment. To study the effect of protein valency on the extent of particle adhesion, detachment experiments were performed with particles functionalized with 100% VhhRR6(CBD)2san antibody with two copies of the CBDsand 100% (VhhRR6)2CBDsan antibody that is bivalent in VhhRR6. The results for these experiments are plotted in Figure 11a with the results for the detachment of particles coated with 100% VhhRR6CBD, as shown in Figure 10a. The basic shape of the detachment curve is the same in all cases. However, it can be seen that particles functionalized with (VhhRR6)2CBD show marginally weaker adhesion than that mediated by the other antibodies, whereas the extent of adhesion for particles functionalized with the remaining two antibodies

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Figure 4. Fluorescence histograms for particle samples. The four negative controls are the particles coated with each of the fusion proteins, followed by the secondary FITC antibody. The positive samples are particles coated with (a) VhhRR6, (b) VhhRR6CBD, (c) (VhhRR6)2CBD, and (d) VhhRR6(CBD)2, followed by primary rabbit anti-VHH antibody and secondary FITC goat anti-rabbit IgG (H+L) antibody. MESF are calculated from the fit of the calibration curve for quantum FITC calibration particles, and site densities are derived from site density ) MESF/(particle surface area).

Figure 6. Video micrograph of a typical fiber-touching experiment: (a) at the point of contact of the particle and the fiber and (b) in the deflected position. ∆x shows the fiber deflection.

Figure 5. Number of molecules of secondary FITC goat antirabbit antibody per unit area of particle for particles functionalized with each of the four proteins.

is almost indistinguishable, especially at higher shear rates. This could have been due to the high density of protein on the particle surface and the shielding of heavy VhhRR6 or CBD domains by their counterparts when bivalent antibodies were used to

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Figure 7. (a) Adhesion frequency and (b) adhesion strength with a cellulose fiber for positive events for VhhRR6-CBD- and VhhRR6-coated particles as a function of contact time.

Figure 8. Determination of the time required to reach steady state in detachment experiments for particles functionalized with 100% VhhRR6-CBD as a function of the shear rate. Detachment attains steady state after ∼25 min.

functionalize the particles. A set of detachment experiments was performed at a flow rate of 600 µL/min (shear rates ranging from 200 to 950 s-1 across the length of the chamber) with particles

functionalized with VhhRR6 solution and either of the bivalent antibody solutions mixed in a 1:1 proportion in an attempt to reduce possible shielding and achieve a more pronounced difference in the performance of bivalent antibodies. The results for these experiments are plotted in Figure 11b with the results for particles functionalized with a 50:50 VhhRR6/VhhRR6CBD solution mixture. These results indicate the strongest adhesion for VhhRR6(CBD)2, followed by VhhRR6CBD and the weakest adhesion again seen for (VhhRR6)2CBD. This conclusion is in good agreement with data presented for biacore studies of the affinity of CBD-cellulose and VhhRR6-RR6 binding.17 These results can be viewed in light of the flow cytometry results for the density of VhhRR6 domains on the particle surface for different antibodies, as shown in Figure 5. Particles coated with (VhhRR6)2CBD have a high density of VhhRR6 domains on their surface but a comparatively lower density of CBD domains because of their bivalency in VhhRR6, and they show the weakest adhesion. Particles coated with VhhRR6(CBD)2 have a low VhhRR6 density but a high CBD density, and they show the strongest adhesion. This indicates that the connection between cellulose and CBD is weaker than that between RR6 and VhhRR6, thus CBD-cellulose binding is the weak link in securing the firm adhesion of particles with the surface. This is in good agreement with published data that demonstrates that antibodies bind with dissociation constant values of 10-7-10-10 M33-35 compared with CBDs reported between 10-4 and 10-6 M.36-38

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Figure 9. Steady-state detachment after 35 min of flow for particles coated with (a) 100% VhhRR6-CBD at 1000 s-1, (b) 50/50 VhhRR6/ VhhRR6-CBD at 1000 s-1, and (c) 100% VhhRR6 at 500 s-1.

Flow Attachment Experiments. In attachment experiments, the number of particles adhering to a cellulose slide under flow was measured as a function of shear rate and time. Particle attachment was observed, even at relatively high shear rates. Figure 12 shows the plots of the number of particles bound to a cellulose slide after 26 min of flow as a function of shear rate for three different particle preparations: particles coated with 100% VhhRR6CBD, 50/50 VhhRR6CBD-VhhRR6, and 100% VhhRR6. The extent of adhesion decreases with increasing shear rate, and the adhesion is greater for the particles that have more CBD. As the CBD surface density increases or the shear rate decreases, the probability of an encounter between a CBD molecule on a particle and a cellulose molecule on a surface increases. Depending upon their starting position across the flow inlet, a very small fraction (∼1 to 2%) of particles can actually come into contact with the cellulose slide as they flow through the chamber. The particles were rarely seen to detach from the (33) van der Linden, R. Unique Characteristics of Llama Heavy Chain Antibodies. Ph.D. Thesis, Utrecht University, The Netherlands, 1999. (34) Davis, P. J.; Parry, N. J. Detergent Composition Comprising Benefits. Patent No. WO0146357, 2001. (35) van der Linden, R.; Frenken, L. G. J; de Geus, B.; Harmsen, M. M.;Ruuls, R. C.; Stock, W.; de Ron, L.; Wilson, S.; Davis, P.; Verrips, C. T. Biochim. Biophys. Acta 1999, 1431, 37-46. (36) Reinikainen, T.; Teleman, O.; Teeri, T. T. Proteins: Struct., Funct., Genet. 1995, 22, 392-403. (37) Tomme, P.; Boraston, A.; McLean, B.; Kormos, J.; Creagh, A.L.; Sturch, K.; Gilkes, N. R.; Haynes, C.; Warren, R. A.; Kilburn, D. G. J. Chromatogr., B: Biomed Sci. Appl. 1998, 715, 283-296. (38) Nigmatullin, R.; Lovitt, R.; Wright, C.; Linder, M.; Nakari-Setala, T.; Gama, M. Colloids Surf., B 2004, 35, 125-135.

slide once attached. Figure 12b shows the particle count at the same spot on the slide as a function of time. The count increases linearly with time except at larger times. Attachment experiments were also performed as a function of particle concentration in suspension. When the concentration of suspension was halved, the bound particle count was seen to go down by approximately 50% (data not shown). The attachment experiments indicate that the fusion protein can react sufficiently strongly and quickly with cellulose to secure particle adhesion even under flow. Even when detached, the fusion protein clearly has the ability to rebind. Effect of Protein Valency on Attachment. To study the effect of protein valency on the dynamics of particle adhesion, attachment experiments were performed with particles functionalized with 100% VhhRR6(CBD)2 and 100% (VhhRR6)2CBD. The results for these experiments are plotted in Figure 13 with the results for the attachment of particles coated with 100% VhhRR6CBD. The VhhRR6(CBD)2 antibody shows the strongest adhesion because of the increased probability of cellulose-CBD binding under flow as a result of high CBD density on the particle surface, whereas the (VhhRR6)2CBD antibody shows the weakest adhesion. However, once again it was observed that the difference in the performance of these antibodies was not substantial and that all bifunctional antibodies could support binding.

Discussion In this study, we have performed direct measurements on molecular systems that will allow the targeted binding of particles to model cellulose surfaces. Polystyrene particles functionalized

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Figure 10. (a) Adherent particles as a function of shear rate for different incubating mixtures. (b) Critical shear rate as a function of the relative amount of CBD in the incubating mixture. Note that these data are at constant total protein coverage.

with red dye are coated with fusion molecules that bind to both the red dye and cellulose in various affinities. The frequency and force of adhesion of these particles to cellulose-coated fibers were measured using micropipette manipulation, and the strength and dynamics of adhesion of these particles to cellulose-coated glass slides under hydrodynamic flow were measured using flow chamber assays. In both cases, highly specific adhesion of particles to cellulose was observed. The frequency and force of nonspecific adhesion were seen to increase with contact time in micropipette manipulation experiments; however, they were always substantially lower than those for specific adhesion. In flow chamber experiments, the controlled flow is the source of hydrodynamic force on the particles. A tapered flow channel was used to alter the shear rate (and shear stress) on identically attached particles in detachment experiments. The strength of adhesion was

quantified in terms of the percentage of particles remaining adherent at a particular spot on the slide after 30 min of washing. At high shear rates, the forces on the particles are strong enough to detach the particles from the slide. The percentage of adherent particles decreases as a strong function of shear rate. The critical shear rate, which is the index of adhesion, is an increasing function of the density of CBD domains on the particle surface. The detachment curves for a 75:25 mixture of positive and negative antibodies and 100% positive antibody overlap each other, especially at higher shear rates, suggesting that a 75:25 mixture might be the optimal antibody surface density for particles. In attachment experiments, particles were seen to bind with cellulose under flow even at shear rates of ∼550 s-1, suggesting quick, strong bond formation between cellulose and CBD. The extent of binding was a strong function of CBD density, shear rate, and

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Figure 11. Detachment curves for particles (a) fully functionalized with either of the bivalent antibodies or VhhRR6-CBD and (b) functionalized with a 50/50 mixture of these antibodies with VhhRR6.

suspension concentration. Kuo and Lauffenburger39 studied the relationship between receptor/ligand binding affinity and adhesion strength of polystyrene particles functionalized with different animal immunoglobin G (IgG) antibodies to the protein A (SpA) surface. The affinities of goat and sheep IgGs to SPA reported in their work are similar to the CBD-cellulose affinities reported in the literature. To compare the results of this work with those reported by Kuo and Lauffenburger,39 the critical shear stress was plotted as a function of receptor number using the data in Figures 5 and 10. The slope of that plot was 2 × 10-5 ( 0.8 × 10-5 (dynes/cm2)/(no. of receptors/bead), which compares reasonably well with that observed for sheep and goat IgGs in ref 39. Detailed simulations of these experiments, using the adhesive dynamics algorithm, that aim to relate the properties of single molecules to observed experimental results are underway. The broken connections in these experiments may either be the RR6-VhhRR6 bond or the cellulose-CBD bond. However, (39) Kuo, S. C.; Lauffenburger, D. A. Biophys. J. 1993, 65, 2191-2200.

the adhesion performance of the particles shows a strong sensitivity toward the density of CBD domains on the particle surface as indicated in Figure 10. If the cellulose-CBD bond were the stronger connection, then only a few of those bonds would be sufficient to secure particle adhesion with the surface. Decreasing the proportion of CBD protein in the incubating solution actually increases the density of VhhRR6 domains on the particle surface. If the RR6-VhhRR6 connection were the weaker link, then the presence of more RR6-VhhRR6 bonds would have made the particle-surface adhesion stronger, but the opposite observation is made experimentally. Furthermore, the antibody bivalent in CBD showed stronger adhesion than the antibody bivalent in VhhRR6 in flow chamber experiments. The strength of adhesion was directly related to the density of CBD domains on particles as indicated in Figures 12 and 13. In both detachment and attachment experiments, it was observed that the bivalent antibodies do not show a great difference in adhesion performance from that of the monovalent antibody. This could be because the incubating solution had the same mass concentra-

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Figure 12. (a) Number of attached particles as a function of shear rate and CBD density after 26 min of flow. (b) Time accumulation of particles on a spot on the slide corresponding to a shear rate of 206 s-1 for fully functionalized particles.

tion (100 µg/mL) of antibody, regardless of the antibody used. Hence, the density of VhhRR6 or CBD domains on the particle surface does not become two times the density for VhhRR6CBD when the antibody bivalent in either VhhRR6 or CBD is used, respectively, as shown in Figure 5. However, the experimental results also suggest that the cellulose-CBD end of the chain is mechanically weaker and hence its engagement is critical in securing particle-surface adhesion. The force of adhesion for particles coated with 100% VhhRR6CBD was ∼2 nN at large contact times in micropipette manipulation experiments. In flow chamber detachment experiments, the critical shear rate for these particles was ∼900 s-1. The force on a 6.34 µm particle at this shear rate is 171 pN from the Stokes’ law.40 Chang and Hammer40 found out that the force (40) Chang, K. C.; Hammer, D. A. Langmuir 1996, 19, 6905-6911.

to break a bond on the particle by shear, F, was related to the tension force on the bond, T, by the relationship T ) 1.369F(a/ 2(L - h))1/2, where a is the particle radius, L is the bond length, and h is the separation between the particle and surface. We can use this expression to compare the results of the two assays. For F ) 171 pN, a ) 3.17 µm, and (L - h) ) 25 nm, the tension T according to the above expression is 1.87 nN, which compares reasonably well with the forces of adhesion observed in micropipette manipulation studies. The above-mentioned force balance applies to a particle bound to a surface by a single bond, whereas multiple bonds are expected to be present in the particlesurface contact zone in both of the adhesion assays used in this work. It has been shown in the literature that the average force required to break multiple bonds generally does not scale linearly with the number of bonds,41-43 so our analysis may be prone to some error. However, we can still use this force balance to make

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Figure 13. Attachment as a function of shear rate for particles functionalized with either of the bivalent antibodies or VhhRR6-CBD.

a general order of magnitude comparison between the results of two assays considering that the bonds in the contact zone are not uniformly stressed in any mode of detachment and most of the load is shared by a small number of bonds at the rear end of the contact zone. To our knowledge, these are the first measurements to directly relate the forces of adhesion between the pipet and flow chamber measurements for a bifunctional antibody. A huge increase in the number of attached particles was observed below a shear rate of 200 s-1 in attachment experiments. This indicates that the average number of CBD-cellulose bonds forming between a particle and cellulose under flow is generally sufficient enough to withstand forces on a particle corresponding to a shear rate e200 s-1. At this shear rate, the adhesion rate constant for particles is estimated to be ∼0.004 s-1 at a Peclet number based on a particle radius of ∼104.

In this article, an experimental system is developed that aims to relate the strength and dynamics of adhesion of an individual particle to cellulose mediated by bifunctional fusion proteins, measured using micropipette manipulation, to that observed for a population of particles under hydrodynamic flow. The experiments represent a reproducible means for relating interfacial force measurements to the adhesiveness of particles and serve as an important step toward the broad goal of developing general physical science principles for designing particulate systems for specific applications. Our future plans involve the assessment of any role that surfactants and electrolytes may play in the binding of particles mediated by antibodies and the development of predictive adhesive dynamics simulations complementary to these experiments that can serve as a tool for particulate system design.

(41) Tees, D. F. J.; Woodward, J. T.; Hammer, D. A. J. Chem. Phys. 2001, 114, 7483-7496. (42) Williams, P. W. Anal. Chim. Acta 2003, 479, 107-115. (43) Seifert, U. Phys. ReV. Lett. 2000, 84, 2750-2753.

Acknowledgment. G.P., E.J., and D.A.H. are grateful for support from Unilever Research at Port Sunlight for this work. LA700603U