Specific Adhesion of Micron-Sized Colloids to Surfaces Mediated by

Gregory P. Robbins , Dooyoung Lee , Joshua S. Katz , Paul R. Frail , Mike J. Therien , John C. Crocker , Daniel A. Hammer. Soft Matter 2011 7 (2), 769...
0 downloads 0 Views 182KB Size
Download PDF
Langmuir 2003, 19, 6905-6911

6905

Specific Adhesion of Micron-Sized Colloids to Surfaces Mediated by Hybridizing DNA Chains Ying Zhang,†,‡ A. Omolola Eniola,†,‡ David J. Graves,† and Daniel A. Hammer*,†,‡,§ Department of Chemical and Biomolecular Engineering, Department of Bioengineering, and Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Received December 13, 2002. In Final Form: April 14, 2003 The hybridization of complementary DNA chains can be used to identify unknown strands. Substantial work has been done to link nanoparticles in solution or on surfaces via DNA hybridization. However, few studies have been done on micron-sized particles that can be visually identified using optical microscopy. In this paper, we used 9.95 µm oligonucleotide coated polystyrene microspheres as probes to identify complementary oligonucleotide sequences immobilized on polystyrene substrate surfaces. We showed that particle adhesion is specific and only seen when beads and surfaces are complementary; nonspecific binding was negligible. Using a flow chamber, we studied the effects of shear rate on microsphere detachment as a function of oligonucleotide site density on the substrate. We show that the critical shear rate at which the beads are detached is an increasing function of oligonucleotide substrate site density. Analysis of the microspheres detached from the slide by flow cytometry demonstrated that the bonds cohesively fail due to the physical desorption of the Neutravidin linkages to the polystyrene slide and not due to the breaking of DNA-DNA bonds. With optimization, the attachment and detachment of micron-sized DNA coated particles on arrays hold promise for the specific, selective identification of DNA sequences.

Introduction Microarrays are two-dimensional patterns involving immobilization of well-defined functional molecules on surfaces, such as proteins and single DNA strands, used for the detection of specific molecular binding. In the past decade, microarrays have been used to detect DNA through oligonucleotide hybridization, in which single strands of DNA with complementary base-pair sequences bind to form a double stranded DNA chain through hydrogen bonding.1,2 This technology is in widespread use in medical, pharmaceutical, and forensic applications.3 In general, DNA hybridization microarrays are created by immobilizing single stranded oligonucleotides on glass, silicon, or plastic substrates.4 Hybridization analysis on microarrays typically involves detecting signals generated by the binding of fluorescently labeled complementary strands to the targeted DNA sequence. Hybridization efficiency can be determined by measuring the intensity of the fluorescent signal obtained by fluorescence scanning, fluorescent microscopic imaging, or mass spectroscopy and comparing this signal to that of a reference sample. The method can allow for the determination of the over- or underexpression of specific genes, when coupled with reverse transcriptase to make DNA from expressed RNA, or for detecting the presence of an unknown oligonucleotide strand from a foreign cell (i.e. pathogen detection). Though powerful, this technology has some limitations, some of * To whom correspondence should be addressed. Current address: Department of Bioengineering, University of Pennsylvania, 120 Hayden Hall, 3320 Smith Walk, Philadelphia, PA 19104. Phone: 215-573-6761. Fax: 215-573-2071. E-mail: hammer@ seas.upenn.edu. † Department of Chemical and Biomolecular Engineering. ‡ Institute for Medicine and Engineering. § Department of Bioengineering. (1) Southern, E. M. Trends Genet. 1996, 12 (3), 110. (2) Southern, E. M. Methods Mol. Biol. 2001, 170, 1. (3) Reichert, J.; Csaki, A.; Kohler, J. M.; Fritzsche, W. Anal. Chem. 2000, 72 (24), 6025. (4) Heller, M. J. Annu. Rev. Biomed. Eng. 2002, 4, 129.

which include its sample volume requirements, lack of sensitivity to point mutations, low fluorescence intensity, and dye photobleaching.5,6 Due to these limitations, methods to increase the selectivity, sensitivity, and stability of microarray-based analysis are currently being investigated. Furtado and co-workers reported that an on-line acoustic wave sensor with better selectivity can be used to detect the hybridization of complementary strands and distinguish them from single-base mutated oligonucleotides. Their studies indicate this method is sensitive to the nature of the mismatch in base pairing and to the location of mutation.7 Attempts to use alternative labels have been made by various groups.5,6,8-10 For example, gold nanoparticles, which are labeled with oligonucleotides and Raman-active dyes, have been used as probes and hybridized to the target sequence on the chip. After silver enhancement, the system has an extended detection limit to 100 attomolar.10 Gold spheres are particularly interesting because they can interact with light through simple fluorescence of molecules attached to them as well as surface plasmon resonance and surface-enhanced Raman scattering (SERS).11-14 The latter can provide sensitivity increases of 6 to 20 orders of magnitude relative to ordinary Raman scattering.15,16 Our goal in this work is to develop a novel detection (5) Bao, P.; et al. Anal. Chem. 2002, 74 (8), 1792. (6) Taton, T. A.; Mirkin, C. A.; Letsinger, R. L. Science 2000, 289 (5485), 1757. (7) Furtado, L. M.; Thompson, M. Analyst 1998, 123 (10), 1937. (8) Csaki, A.; Moller, R.; Straube, W.; Kohler, J. M.; Fritzsche, W. Nucleic Acids Res. 2001, 29 (16), E81. (9) Taton, T. A.; Lu, G.; Mirkin, C. A. J. Am. Chem. Soc. 2001, 123 (21), 5164. (10) Cao, Y. C.; Jin, R.; Mirkin, C. A. Science 2002, 297 (5586), 1536. (11) Gillis, E. H.; Gosling, J. P.; Sreenan, J. M.; Kane, M. J. Immunol. Methods 2002, 267 (2), 131. (12) Vo-Dinh, T.; Allain, L. R.; Stokes, D. L. J. Raman Spectrosc. 2002, 33 (7), 511. (13) Vo-Dinh, T.; Houck, K.; Stokes, D. L. Anal. Chem. 1994, 66 (20), 3379. (14) Otto, A. J. Raman Spectrosc. 2002, 33 (8), 593.

10.1021/la026999g CCC: $25.00 © 2003 American Chemical Society Published on Web 07/03/2003

6906

Langmuir, Vol. 19, No. 17, 2003

system based on the adhesion of optical microspheres through DNA hybridization. When the particle size is several micrometers rather than in the nanometer range, additional factors can influence the selectivity of binding and detachment. The large size of such spheres potentially can provide advantages, such as still greater amplification of an optical signal and even the possibility of producing an instrument-free (naked eye) detection system. Such large spheres can interact with bound chains through a large number of chain/chain interactions, creating an adhesion “patch” on each bead surface. Multiple chain interactions have been shown to result in sharpened dependence on variables that influence adhesion, such as temperature in the case of nanospheres.6,17 The large size of microspheres also provides the possibility of modulating adhesion by applying hydrodynamic flow forces, a characteristic not possible with nanospheres. At large enough Peclet numbers, we hypothesize that imperfectly matched hybridizing chains will separate. This may ultimately provide a better method of distinguishing perfectly complementary nucleotide chains from those where there is a single-base mismatch. We can gain insight into the adhesive behavior of these beads through previous work on the adhesion of cells. The receptor-mediated adhesion of cells to surfaces is involved in many normal physiological processes, including the inflammatory response and lymphocyte homing. The adhesion of cells under flow is a result of the balance between receptor-mediated and hydrodynamic forces. The interactions between the cells and the surfaces are macromolecular, specific bonding forces. The net adhesiveness of the particle is the cumulative result of the number of specific, macromolecular bonds between the cell and the surface and their strength, which are related to the properties of the individual receptors.18 In turn, it is possible to study the interactions of specific binding molecules through controlling the hydrodynamic force on the cell.19,20 The fundamental interactions of leukocyte adhesion receptors have been studied by attachment of adhesion molecules to micron-sized colloidal particles. For example, the dynamics of neutrophil rolling can be recreated with such “cell-free” colloidal mimetics.21-24 By analogy, since single stranded DNA molecules also interact with high-affinity and high-specificity to complementary strands, we would expect that micron-sized colloidal particles labeled with single stranded DNA would bind specifically to DNA surfaces. In a macroscopic adhesion experiment of a micron-sized particle, adhesion molecules will experience forces, and it is useful to have some insight into how DNA chains respond to force. The interactions between single DNA strands have been measured with AFM by Strunz et al.25,26 (15) Kneipp, K.; Kneipp, H.; Itzkan, I.; Dasari, R. R.; Feld, M. S. Chem. Phys. 1999, 247 (1), 155. (16) Kneipp, K.; Kneipp, H.; Itzkan, I.; Dasari, R. R.; Feld, M. S. Chem. Rev. 1999, 99 (10), 2957. (17) Park, S. J.; Taton, T. A.; Mirkin, C. A. Science 2002, 295 (5559), 1503. (18) Swift, D. G.; Posner, R. G.; Hammer, D. A. Biophys. J. 1998, 75 (5), 2597. (19) Alon, R.; Hammer, D. A.; Springer, T. A. Nature 1995, 374 (6522), 539. (20) Park, E. Y.; et al. Biophys. J. 2002, 82 (4), 1835. (21) Eniola, A. O.; Rodgers, S. D.; Hammer, D. A. Biomaterials 2002, 23 (10), 2167. (22) Bhatia, K. S.; Hammer, D. A. Langmuir 2002, 18 (15), 5881. (23) Brunk, D. K.; Hammer, D. A. Biophys. J. 1997, 72 (6), 2820. (24) Rodgers, S. D.; Camphausen, R. T.; Hammer, D. A. Biophys. J. 2001, 81 (4), 2001. (25) Strunz, T.; Oroszlan, K.; Schafer, R.; Guntherodt, H. J. Proc. Natl. Acad. Sci. U.S.A. 1999, 96 (20), 11277. (26) Grange, W.; Strunz, T.; Schumakovitch, I.; Guntherodt, H. J.; Hegner, M. Single Mol. 2001, 2 (2), 75.

Zhang et al.

Figure 1. (A) Schematic diagram of microspheres binding to the target sequence on the microarray. (B) Schematic diagram showing the chemistry used in DNA-mediated adhesion.

They found that the relationship between the force and time for the dissociation of hybridization DNA chains followed the Bell model,27,28

kr ) k°r exp(γF/kbT) where kr is the dissociation rate, k°r is the unstressed dissociation rate constant, γ is the reactive compliance, F is the applied force, kb is the Boltzmann constant, and T is temperature. Both k°r and γ are functions of the degree of chain overlap, N, and they decrease with N. These studies did not explore how single base defects affected the strength of binding between individual molecules, nor did they address the collective behavior of multiple hybridized chains in an adhesive interface, which can ultimately be studied using bead adhesion methods. Compared with AFM, the adhesion of beads will invariably involve multiple hybridizing chains.25,29,30 One of the ultimate goals of our work is to understand how cooperativity among multiple chains manifests itself in bead adhesion experiments and how the features of single chain binding manifest themselves in adhesion experiments. If a clear relationship emerges, it might ultimately be possible to use adhesion measurements to detect molecular features of the chains. In this paper, we allow microspheres coated with single stranded oligonucleotides to contact a surface covered with single stranded oligonucleotides under quiescent conditions, and then we attempt to detach the particles from the surface under an applied flow. A schematic diagram of the experiment is shown in Figure 1. Oligonucleotide (27) Bell, G. I. Nature 1974, 248 (447), 430. (28) Chang, K. C.; Hammer, D. A. Biophys. J. 2000, 79 (4), 1891. (29) Lee, G. U.; Chrisey, L. A.; Colton, R. J. Science 1994, 266 (5186), 771. (30) MacKerell, A. D., Jr.; Lee, G. U. Eur. Biophys. J. 1999, 28 (5), 415.

Adhesion Mediated by Hybridizing DNA Chains

molecules are immobilized on microspheres and polystyrene slide surfaces via biotin-avidin bonds. The oligonucleotides on the microspheres hybridize specifically to spots on the substrate surface containing the complementary sequence. We also study the effects of applied shear force on adherent microspheres to find the critical shear stress necessary to break the bonds between the particles and substrate. Furthermore, we explore the effects of DNA substrate surface density on adhesion strength by incubating the polystyrene slide with DNA solutions of different concentrations. The results show that micron-sized colloidal particles can be engineered to adhere specifically and that detachment force depends on chain density. Experimental Methods Bovine serum albumin (BSA), Dulbecco’s phosphate buffered saline (PBS), lithium chloride, and polyoxyethylene-sorbitan monolaurate (Tween 20) are purchased from Sigma (St. Louis, MO). EDTA solution (0.5 M, pH 8.0) and Tris-HCl buffer (pH 8.0) come from Fisher Scientific (Pittsburgh, PA). Neutravidin is purchased from Pierce Biotechnology, Inc. (Rockford, IL). Oligonucleotides are custom synthesized by Biosource International, Inc. (Camarillo, CA). Superavidin coated microspheres of 9.95 µm diameter are products of Bangs laboratories (Fishers, IN). Fluorescein biotin is supplied by Molecular Probes, Inc. (Eugene, OR). Oligonucleotide sequences used in this study are identical to strands designed by Strunz and co-workers; these chains have been shown to hybridize and have been designed to avoid selfcomplementarity.25 The sequences have 30 bases, with 10 bases used as spacer to improve the hybridization efficiency, and 20 bases available for overlap.31 The oligomer a has the base sequence 5′G-G-C-T-C-C-C-T-T-C-T-A-C-C-A-C-T-G-A-C-A-T-CG-C-A-A-C-G-G-3′. It can bind specifically to the sequence b ) 5′-G-G-C-T-C-C-C-T-T-C-C-C-G-T-T-G-C-G-A-T-G-T-C-A-G-T-GG-T-A-3′. The underlined domains show the regions of base pair overlap. Given the salt concentration of 150 mM in hybridization buffer, the melting temperature of the two sequences is 71 °C.32 In the experiments, both sequences are modified with biotin at their 5′ ends. For verification of specific adhesion of microspheres to the substrate, sequences modified at their 3′ ends with the fluorescent dye Cy5 are used. For flow cytometry, complementary sequences with fluorescein at the 5′ end but missing biotin at the 3′ end were used. Substrate Preparation. Oligonucleotide coated slides are prepared by a method similar to that reported by Rodgers et al.24 In this method, single well, rectangular flexiperm gaskets (Sigma, St. Louis, MO) are placed tightly on polystyrene slides cut from sterile Petri dishes (Fisher, Pittsburgh, PA). The surfaces enclosed by the gaskets are coated with 1600 µL of 100 µg/mL Neutravidin in binding buffer (0.1 M NaHCO3, pH 9.2) overnight. Neutravidin coated surfaces are blocked with a 2 mL solution of 2% denatured BSA in PBS for at least 30 min, after which a self-built microarrayer33 is used to spot samples of 3 nL of oligonucleotide a or b solutions (20 µM) on the slides, or the surfaces enclosed by the flexiperm gaskets are incubated for a period of at least 2 h with 1 mL solutions, which are diluted and have different final concentrations of biotinylated oligonucleotides (2, 5, 10, 15, and 20 nM), to obtain the different surface densities of chains on the slides. Oligonucleotide Site Density Determination on a Slide Surface. To determine the site density on the substrate surface, which is the number of oligonucleotide molecules available for hybridization in each unit area, flexiperm gaskets with eight wells are placed on polystyrene slides. Two wells are used as negative controls: one is incubated with Neutravidin only, and the other is incubated with Neutravidin and Cy5 labeled oligonucleotides. Surfaces enclosed with another five wells are (31) Su, H. J.; Surrey, S.; McKenzie, S. E.; Fortina, P.; Graves, D. J. Electrophoresis 2002, 23 (10), 1551. (32) Breslauer, K. J.; Frank, R.; Blocker, H.; Marky, L. A. Proc. Natl. Acad. Sci. U.S.A. 1986, 83 (11), 3746. (33) Graves, D. J.; Su, H. J.; McKenzie, S. E.; Surrey, S.; Fortina, P. Anal. Chem 1998, 70 (23), 5085.

Langmuir, Vol. 19, No. 17, 2003 6907 coated with oligonucleotide using the method described above, and the concentrations of incubation solutions are 2, 5, 10, 15, and 20 nM, respectively. Oligonucleotide coated surfaces are incubated with a saturating concentration of Cy5 labeled complementary sequences for 30 min at room temperature, followed by washing with PBS five times. The intensities of fluorescence at different positions on the slides are determined quantitatively with ScanArray 5000 (Packard Bioscience, Meriden, CT) and then converted into site densities with a calibration curve, which describes the intensities of fluorescence as a function of known densities of Cy5 molecules on a polystyrene slide.34 Microsphere Preparation. Superavidin coated microspheres are beads made of polystyrene and coated with immunopure Neutroavidin via covalent bonds.35 Oligonucleotide sequences are coated on them using the method recommended by the manufacturer, as follows.36 The beads are rinsed with TTL buffer (100 mM Tris-HCl, pH 8.0; 0.1% Tween 20; and 1 M LiCl) twice and then incubated in biotinylated oligonucleotide solution and TTL buffer mixture for 15 min. The oligonucleotide/microsphere conjugates are washed with 0.15 N NaOH, rinsed with TT buffer (250 mM Tris-HCl, pH 8.0, 0.1% Tween 20) twice, and incubated in TTE buffer (250 mM Tris-HCl, 0.1% Tween 20, 20 mM Na2EDTA, pH 8.0) at 80 °C for 10 min. The conjugates are suspended in a solution of 2% denatured BSA in PBS at 4 °C for at least 30 min before use. All microspheres are coated with oligonucleotide sequence a on the same day that they are used in a flow chamber experiment. The fluorescence intensities on the microsphere surfaces are measured with flow cytometry. The fluorescence shifts are converted to site densities using a calibration curve relating the mean peak fluorescence of Quantum 26 calibration beads to their molecules of equivalent soluble fluorochrome (MESF).23 Flow Adhesion Assay. Adhesion experiments are performed in a parallel-plate flow chamber (Figure 2). The templates with either straight or tapered channel are cut from 0.01 in. thick Duralastic sheeting (Allied Biomedical, Goose Creek, SC) and placed over a slide coated with single stranded oligonucleotides. The template and slide are placed in the bottom well of the flow chamber and secured with the top of the flow chamber. The whole assembly is mounted on the stage of a Nikon Diaphot inverted microscope with phase-contrast optics (Nikon, Tokyo, Japan). Air bubbles are removed by gentle flushing of the chamber with a solution of 2% denatured BSA in PBS with sodium chloride concentration of about 0.15 M. Microspheres suspended in the same solution are connected to the inlet of the flow chamber by a rubber tube and are injected into the chamber. Before a controlled flow is initiated, microspheres are allowed to settle on the slide surface uniformly for 40 min, and the pictures at randomly selected points in the chamber are recorded with a Cohu black and white CCD camera (Cohu Inc., San Diego, CA) and Sony SVO-9500MD S-VHS recorder (Sony Medical Systems, Montvale, NJ). The location of these points was recorded with a digital dimension indicator for reference. After flow is initiated with the infusion/withdrawal syringe pump (Harvard Apparatus, South Natick, MA), nonadherent microspheres are cleared from the chamber. For each experiment, the height of the flow channel is measured by focusing the microscope on top and bottom surfaces. The flow rates generated by the syringe pump are calibrated by timed collection of samples. For a straight channel template, the wall shear rate (γ) is calculated as γ ) 6Q/h2w, where Q is the volumetric flow rate in mL/s, h is the channel height in cm, and w is the channel width in cm. The tapered channel template allows the wall shear stress to vary along the length of the channel. The wall shear rate is defined according to

γ)

6Q z 1L h2w1

(

)

(34) Graves, D. J.; Su, H. J.; Addya, S.; Surrey, S.; Fortina, P. Biotechniques 2002, 32 (2), 346. (35) Material Safety Data Sheet; Bangs Laboratories, I.: Fishers, IN, 2002. (36) Instructions: ProActive Streptavidin Coated Microspheres; Bangs Laboratories, I.: Fishers, IN, 1999.

6908

Langmuir, Vol. 19, No. 17, 2003

Zhang et al.

Figure 3. Fluorescence histograms for the microsphere populations. The three negative samples are as follows: sample A, bare Superavidin coated beads; sample B, bare Superavidin coated beads incubated with fluorescein labeled sequence a; sample C, biotinylated sequence a coated beads incubated with fluorescein labeled sequence a. The positive sample (sample D) is the biotin a coated beads incubated with the fluorescein labeled b (complementary) sequence. Sample E is the Superavidin coated beads incubated with fluorescein biotin. All the beads are suspended in 2% BSA buffers. Site densities for the positive samples are derived from site density ) MESF/(bead surface area × number of dye molecules per DNA), where MESF is calculated from the fit of the calibration curve: MESF ) 1177.8 × fluorescence intensity.

Figure 2. (A) Side view of parallel-plate flow chamber. (B) Top view of tapered channel. (C) Wall shear rate obtained as a function of distance from the inlet using the tapered channel gasket, where h ) 0.023 cm, w1 ) 0.1 cm, and L ) 6.6 cm. where w1 is the width at the channel entrance in cm, z is the axial position from the inlet, and the L is the channel length. Both Q and h have the same definitions as described above.23 The microsphere detachment results are represented as the ratio of the number of microspheres in the visual field of the microscope before and after the controlled flow is applied. The microsphere numbers are obtained through digital image analysis of video records of adhesion experiments, using LabView software (National Instrument, Austin, TX). Error bars are plotted using standard error calculation.

Results Site Density and Verification of Hybridization on the Microsphere Surfaces. Different oligonucleotide sequences are attached to the surfaces of the Superavidin coated microspheres, followed by the suspension of the microspheres in solutions containing fluorescein labeled oligonucleotide sequences. The fluorescence intensities of the microspheres are measured using flow cytometry. Three samples are used as the negative controls: sample A, Superavidin coated microspheres washed with buffers and suspended in 2% BSA; sample B, Superavidin coated microspheres washed with buffer and incubated with fluorescein labeled oligonucleotide sequence a for 30 min and then suspended in 2% BSA; sample C, biotinylated oligonucleotide sequence a is attached to the microspheres, and then the bead population is incubated with fluorescein labeled oligonucleotide molecules with the same base sequences (fluorescein a) and suspended in 2% BSA. The results show that fluorescein a strands do not nonspecifically adsorb to the beads (A and B) and do not bind to biotin a strands (C). In sample D, beads labeled with biotin a strands are incubated with fluorescein b strands (i.e., complementary); the positive shift in fluorescence indi-

cates strands are specifically hybridizing. To measure the amount of avidin on the bead surface, beads are incubated with fluorescein biotin (sample E). Figure 3 shows the fluorescence histograms for the microspheres in these five samples. A large rightward shift of the positive samples from the negative controls confirms the presence of biotinylated oligonucleotides on the surface of the microspheres and the hybridization of complementary strands. The density of oligonucleotides available for the hybridization on the microsphere surfaces for the positive sample D is estimated to be 3250 molecules/µm2. The density of avidin sites on the microsphere surface was measured to be 4588 molecules/µm2. Verification of Specific Adhesion of Microspheres to the Slide Surface. Two kinds of Cy5 labeled oligonucleotide samples are spotted with the microarrayer and immobilized on the slide. One is the sequence biotin b-Cy5, which is complementary to the oligonucleotide biotin a attached on the microsphere surfaces. The other one is the sequence biotin a-Cy5, identical to that on the microsphere surface and therefore not complementary to strand a. These spots in the second case are used as the negative control. The slide is then assembled into the flow chamber and the adhesion experiment is performed. The same slide is checked with a fluorescent scanner to locate the oligonucleotide spots on the slide after the adhesion experiment. We use image analysis software to overlay the images of bead adhesion and the fluorescent scanning pattern (Figure 4). The quantitative measurements of the adhesion of the microspheres to complementary and noncomplementary oligonucleotides and regions devoid of oligonucleotides (devoid of DNA) are shown in Figure 5. These figures confirm that the microspheres bind specifically to the complementary oligonucleotides on the slide surface but do not bind to noncomplementary oligonucleotides or to regions devoid of DNA. Determination of Proper Washing Times. After the microspheres adhere to the slide surface in a flow chamber, a controlled shear stress is used to “wash” the slide. In this process, weak connections between the microspheres

Adhesion Mediated by Hybridizing DNA Chains

Langmuir, Vol. 19, No. 17, 2003 6909

Figure 4. Verification of microsphere adhesion mediated by oligonucleotides. DNA strands are spotted on the surface of an avidin coated polystyrene slide using a microarray. After a short incubation and gentle washing, only microspheres coated with the biotinylated a sequence bind specifically to b, the complementary sequences on the slide: (A) schematic diagram of spot deposition geometry; (B) fluorescent image; (C) fluorescent image of one pair of spots with bound beads, which are visible as small light dots within the larger spotted region.

Figure 5. Quantitative measurements of microsphere adhesion on the regions devoid of oligonucleotides and the areas coated with noncomplementary or complementary oligonucleotides.

and the slide are broken and these unbound microspheres are removed. At the same spot on the slide, changes in the number of bound microspheres after the flow is initiated

Figure 6. Selection of the proper washing time as a function of hydrodynamic shear rates of 666, 944, and 1130 s-1. Detachment reaches steady state after 2 min for all shear rates. DNA densities on substrate surfaces are 18 600 molecules/µm2.

are recorded and analyzed. The extent of microsphere adhesion is measured as a function of the time of exposure to flow with various shear rates (Figure 6). It can be seen that, in all cases, 2 min are enough for the system to reach

6910

Langmuir, Vol. 19, No. 17, 2003

Zhang et al.

Figure 8. Fluorescence histogram of the microspheres after an adhesion experiment performed with fluorescently labeled Neutravidin on the surface. Beads coated with oligonucleotides but not involved in the adhesion experiment are used as negative sample A. Oligonucleotide coated beads involved in the adhesion experiment and interacting with the noncomplementary sequence coated substrate surface are used as negative sample B. Beads bound to the complementary sequence coated surface and then detached from the slide by flow are the positive sample. This experiment indicates Neutravidin is phys-desorbed from the slide surface.

Figure 7. (A) Effects of wall shear rates on microsphere adhesion on slides with different DNA site densities. (B) Effects of concentration of incubation solution on the site density on the substrate slide. Site density in molecules/µm2 is converted from the fluorescence intensity with the equation site density ) exp((fluorescence intensity + 48652)/5714). (C) Effects of chain density on the mean strength of microsphere adhesion. The critical shear rates are obtained from the curves in part A, where “critical” refers to the shear rate that leads to a decrease in adhesion of 50%.

steady state and for the weakly bound microspheres to be removed from the surface. In actual experiments, all the slides are washed for 10 min to ensure steady-state binding is reached. Effect of Shear Rate on Microsphere Detachment. A tapered gasket is used in the flow chamber to regulate the flow with different shear rates at each axial position along the slide. The microsphere adhesions at different spots on the slide are recorded and correlated to the relative locations of the spots on the slide, from which the shear rates are derived. For any given slide, microspheres bind to the slide tightly at the lower shear rates (