Adsorption and Surface Diffusion of DNA Oligonucleotides at Liquid

work, a cooled charge-coupled device camera is substituted for the PMT normally used. Studies of adsorption and surface diffusion of the well-characte...
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Langmuir 1997, 13, 320-329

Adsorption and Surface Diffusion of DNA Oligonucleotides at Liquid/Solid Interfaces Vincent Chan,† David J. Graves,*,† Paolo Fortina,‡ and Steven E. McKenzie†,‡ Department of Chemical Engineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, and Department of Pediatrics, University of Pennsylvania School of Medicine and the Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104 Received July 8, 1996. In Final Form: October 28, 1996X Total internal reflection (TIR)/fluorescence recovery after photobleaching (FRAP), which has been used to study adsorption and surface diffusion of proteins, was modified and applied to study DNA oligonucleotides at liquid/solid interfaces. Conventional TIR/spot FRAP and TIR/pattern FRAP techniques use a photomultiplier tube (PMT) to reveal the adsorption dynamics and surface diffusion rates of biomolecules, respectively. However, they do not provide spatial information on these interfacial processes. In this work, a cooled charge-coupled device camera is substituted for the PMT normally used. Studies of adsorption and surface diffusion of the well-characterized protein bovine serum albumin (BSA) validated the system’s operation. Then, the desorption rate constant for a fluorescently tagged 21-mer DNA oligonucleotide (MW 7140 Da) was determined by spot FRAP. The desorption rate constants for strongly and weakly adsorbed oligonucleotides from (3-aminopropyl)triethoxy silane (APTES) glass were determined to be 0.02 and 0.19 s-1, respectively. These are of the same order of magnitude as those for BSA (MW 67 000 Da) on APTES glass. The surface diffusion coefficients of oligonucleotide are approximately the same as those for BSA and are dependent on the surface concentration of the molecules on APTES-coated glass. Since the molecules differ by a factor of 10 in molecular weight, these results suggest that the shape of a adsorbate molecule and the strength of adsorbate/substrate interactions play a strong role in interfacial adsorption and diffusion. The substitution of a methyl group in APTES for a hydrogen atom increased the desorption rate constants and surface diffusion coefficients significantly.

Introduction The adsorption properties of oligonucleotide and DNA are important in the design of biosensors to detect microbial pathogens and genetic defects and to identify DNA in many other applications. One new method of detection consists of linking small oligonucleotide probes to silanized glass surfaces and exposing them to a solution of the sought DNA molecules which have been fluorescently labeled. Complementary molecules bind, or hybridize, to each other. The understanding of adsorption/ desorption mechanisms is also important in the design of chromatography materials. The work we report here is applicable to these and other areas, but we emphasize hybridization in the following discussion. Hybridization between solution phase DNA and an array of many different types of immobilized DNA probes in microscopic patterns of spots is potentially one of the most promising analytical techniques in molecular biology.1,2 From such an array, in theory, the identity or sequence of an unknown DNA target can be distinguished by the patterns of hybridization. In keeping with the emerging convention for immobilized arrays, the known immobilized DNA molecules are called probes and the unknown DNA molecules in the solution are called targets. Targets are usually labeled either directly with fluorescent or radioactive tags or indirectly with conjugates that then bind fluorescent, chemiluminescent, or radioactive molecules. During the past few years, considerable work has been done on the development of a commercial biosensor based on this principle for disease diagnosis and large scale genomic sequencing.1-13 This new technology, which can be called heterogeneous hybridization, has the po* Author to whom correspondence should be addressed. † Department of Chemical Engineering. ‡ Department of Pediatrics. X Abstract published in Advance ACS Abstracts, January 1, 1997. (1) Chetverin, A. B.; Kramer, F. R. Biotechnol. Adv. 1994, 12, 1093. (2) Jacobs, J. W.; Fodor, S. P. A. Trend Biotechnol. 1994, 12, 19. (3) Boyle, J. S.; Lew, A. M. Trend Genet. 1994, 11, 8.

tential to replace traditional techniques such as Southern blotting because it is less labor intensive, conducts many different tests simultaneously, and uses less of the expensive DNA probe material. However, the behavior of both the target and probe DNAs and their interactions at the liquid/solid biosensor interface are at present poorly understood. In a recent theoretical paper,14 we predicted that under the proper circumstances the rate of hybridization can be substantially enhanced and the required amount of immobilized DNA probe can be minimized. The requirements for such enhancement involve a surface sparsely covered with chemically coupled DNA probes which adsorb targets with high strength and selectivity while the nonprobe-covered regions can also adsorb target DNA nonselectively but with only moderate strength. Under such circumstances, three-dimensional diffusion directly from solution to the probe is relatively slow because of the large number of directional choices for diffusion which a molecule has at each instant.15 In contrast, the target DNA theoretically can diffuse along the surface in a two(4) Eggers, M.; Hogan, M.; Reich, R. K.; Lamture, J.; Ehrlich, D.; Hollis, M.; Kosicki, B.; Powdrill, T.; Beattie, K.; Smith, S.; Varma, R.; Gangadharan, R.; Mallik, A.; Burke, B.; Wallace, D. Biotechniques 1994, 17, 516. (5) Lamture, J. B.; Beattie, K. L.; Burke, B. E.; Eggers, M. D.; Ehrlich, D. J.; Fowler, R.; Hollis, M. A.; Kosicki, B. B.; Reich, R. K.; Smith, S. R.; Varma, R. S.; Hoagon, M. E. Nucleic Acids. Res. 1994, 22, 2121. (6) Maskos, U.; Southern, E. M. Nucleic Acids Res. 1992, 20, 1679. (7) Maskos, U.; Southern, E. M. Nucleic Acids Res. 1993, 21, 4663. (8) Mirzabekov, A. D. Trend Biotechnol. 1994, 12, 27. (9) Pease, A. C.; Solas, D.; Sullivan, E. J.; Cornin, M. T.; Holmes, C. P.; Fodor, S. P. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 5022. (10) Southern, E. M.; Case-Green, S. C.; Elder, J. K.; Johnson, M.; Mir, K. U.; Wang, L.; Williams, J. C. Nucleic Acids Res. 1994, 22, 1368. (11) Kreiner, T. Am. Lab. (Shelton, Conn.) 1996, 28 (5), 39. (12) Livshits, M. A.; Florentiev, V. L.; Mirzabekov, A. D. J. Biomol. Struct. Dyn. 1994, 22, 783. (13) Sanguedolce, L. A.; Graves D. J.; McKenzie, S. E.; Fortina, P. In preparation. (14) Chan, V.; Graves D. J.; McKenzie, S. E. Biophys. J. 1995, 69, 2243.

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dimensional fashion, finding its complementary strand relatively quickly. Such a phenomenon was originally proposed by Adam and Delbruck16-19 for the binding of ligands on cell surfaces. Since then, this relatively rapid 2-D diffusion has led to what has been called reduction of dimensionality enhancement (RD enhancement).15,17 One of the experimental proofs of RD enhancement was provided by Gaspers et al.20 for the hydrolysis of surfaceimmobilized peptides by irreversibly adsorbed collagenase. The work reported here was carried out on both to gain fundamental knowledge and to see if such diffusion enhancement could been seen. Spot and pattern fluorescence recovery after photobleaching (FRAP) using total internally reflected light (TIR) have become important analytical tools for studying the behavior of molecules at solid/liquid interfaces. The process of two-dimensional diffusion is well recognized in biological systems, such as cell membranes, as well as man-made materials.21-24 Several research groups successfully measured the lateral mobility of synthetic lipids on cell membranes25 and proteins on various substrates21-24 by the application of laser TIR fluorescence microscopy and FRAP. We have chosen to apply these methods to measure several physical parameters of DNA targets at the liquid/solid interface. TIR produced by an Ar-ion laser source using spot FRAP and pattern FRAP are two of the most effective experimental techniques for studying coupled diffusion and reaction kinetics at surfaces.26 In this method, biomolecules either are nonspecifically adsorbed to the liquid/ solid interface or they are specifically bound to immobilized complementary molecules. In either case, the fluorescently tagged molecules are illuminated by a laser beam, which is totally internally reflected in a glass plate such as a microscope slide. A thin layer of surface-associated illumination (the evanescent wave) about 0.1 µm in thickness will penetrate into the liquid adjacent to the surfaces.26-28 This evanescent electromagnetic field excites fluorescent molecules at the interface, with minimal interference from fluorescent molecules in solution adjacent to the interface. A low-intensity laser beam with a power of about 5-50 µW permits direct monitoring of the fluorescence intensity at the interface. To photobleach, higher intensity laser beams with a power of about 0.5 W destroy, or bleach, the tagged fluorescence molecules in the same region illuminated by the monitoring beam. As molecules diffuse back into the bleached region either from the surface or solution, the kinetics of the interfacial process can be studied. (15) Berg, O. G.; von Hippel, P. H. Ann. Rev. Biophys. Biophys. Chem. 1985, 14, 131. (16) Adam, G.; Delbruck, M. In Structural Chemistry and Molecular Biology; Rich, A., Davidson, N., Eds.; W. H. Freeman and Co.: San Francisco, 1968; pp 198-215. (17) Axelrod, D.; Wang, M. D. Biophys. J. 1994, 66, 588. (18) Berg, H.; Purcell, E. M. Biophys. J. 1977, 20, 193. (19) Delisi, C. Q. Rev. Biophys. 1980, 13, 201. (20) Gaspers, P. B.; Gast, A. P.; Robertson, C. R. J. Colloid Interface Sci. 1995, 172, 518. (21) Burghardt, T. P.; Axelrod, D. Biophys. J. 1981, 33, 455. (22) Gaspers, P. B.; Robertson, C. R.; Gast, A. P. Langmuir 1994, 10, 2699. (23) Tilton, R. D.; Robertson, C. R.; Gast, A. P. J. Colloid Interface Sci. 1990, 137, 192. (24) Thompson, N. L.; Burghardt, T. P.; Axelrod, D. Biophys. J. 1981, 33, 435. (25) Kubitscheck, U.; Wedekind, P.; Peters, R. Biophys. J. 1994, 67, 948. (26) Thompson, N. L.; Pearce, K. H.; Hsieh, H. V. Eur. Biophys. J. 1993, 22, 367. (27) Rebar, V. A.; Santore, M. M. J. Colloid Interface Sci. 1996, 178, 29. (28) Abney, J. R.; Scalettar, B. A.; Thompson, N. L. Biophys. J. 1992, 61, 542-552.

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Pattern FRAP is an variation of FRAP which can be used to measure surface diffusion coefficients of irreversibly or weakly adsorbed biomolecules that are fluorescently tagged. In this technique, a periodic pattern of photobleaching is created by the interference of two coherent laser beams in a total internally reflected system. This results in a steady-state semisinusoidal concentration distribution of unbleached fluorophore that is 180° out of phase with the illumination pattern. The dissociation rate constants and surface diffusion coefficients for biomolecules on different surfaces have been determined by analyzing such patterns. These include measurements of the diffusion of reversibly adsorbed bovine serum albumin at a quartz surface,21 lipids on supported membranes,29-32 collagenase on 2-furanacryloyl-L-leucylglycyl-L-prolyl-L-alanine peptides attached to a glass surface,22 bovine serum albumin irreversibly adsorbed at polymer surfaces,23,33,34 and the combined diffusion and adsorption of the reversibly adsorbed bovine prothrombin fragment 1 on a supported membrane.35 In traditional TIR/spot FRAP and TIR/pattern FRAP techniques, a photomultiplier tube (PMT) is used for measuring photons generated by the excitation of the fluorescent molecules. While the PMT is extremely sensitive, it provides only temporal information on the biophysical processes occurring during TIR/FRAP in the form of an integrated signal and has a relatively low signal to noise ratio. Quantitative photomicrography also has been combined with pattern FRAP to study the diffusion of fluorescently labeled lipids on supported membranes.31,32 In this paper, we describe a modified TIR/FRAP technique in which a cooled charge-coupled device (CCD) camera is substituted for the PMT. A similar idea was applied recently to fluorescence correlation spectroscopy (FCS) to study the clustering of biomolecules on a planar membrane.36 The biophysical processes that are occurring in the nonprobe-covered region of the liquid/solid interface are shown in Figure 1. In general, the important events which one wishes to understand are diffusion from solution to surface, two-dimensional diffusion on the surface, and adsorption on, and desorption from, the interface. We have measured the surface diffusion coefficients and desorption rates of oligonucleotide and compared them with those of the previously well-studied molecule bovine serum albumin (BSA).21,23,33,34 The adsorption isotherm of our oligonucleotide was also measured. These results suggest that interfacial adsorption and diffusion of biomolecules are strongly influenced by the shape of a molecule (rigid rod in the case of oligonucleotide vs globular for BSA) and the strength of molecule/substrate interactions. They provide important physical parameters for the design and operation of DNA hybridization and separation devices. (29) Kalb, E.; Frey, S.; Tamm, L. K. Biochim. Biophys. Acta 1992, 1103, 307. (30) Tamm, L. K.; Kalb, E. In Molecular Luminescence Spectroscopy; Schulman, S. G., Ed.; John Wiley and Sons, Inc.: 1993; Part III, p 253, and references cited therein. (31) Smith, B. A.; McConnell, H. M. Proc. Natl. Acad. Sci. U.S.A. 1978, 75 (6), 2759. (32) Tamm L. K.; McConnell, H. M. Biophys. J. 1985, 47, 105. (33) Robeson, J. L.; Tilton, R. D. Biophys. J. 1995, 68, 2145. (34) Tilton, R. D.; Gast, A. P.; Robertson, C. R. Biophys. J. 1990, 58, 1321. (35) Huang, Z.; Pearce, K. H.; Thompson, N. L. Biophys. J. 1994, 67, 1754. (36) Huang, Z.; Thompson, N. L. Biophys. J. 1996, 70, 2001.

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Figure 1. Physical model for the behavior of oligonucleotide at a liquid/solid biosensor interface. These DNA molecules can either irreversibly or reversibly adsorb from solution and diffuse on the surface. The -NH2 groups on the surface are the terminal groups of the (3-aminopropyl)triethoxysilane that is chemically bound to the glass.

Materials and Methods DNA and Protein Labeling. Single-stranded DNA (oligonucleotide) with a size of 21 bases was supplied and labeled with fluorescein by Molecular Probes. The sequence of the DNA is 5′-GTC AAG GCT GCC CAA TTT GAG-3′ which encodes a region of the human FcgRIIA gene.37 One fluorescein moeity is present on the 5′ end of each oligonucleotide. The 100 µM stock solution was diluted to lower concentration with phosphate-buffered saline (PBS contains 0.147 M NaCl, 0.008 M Na2HPO4, and 0.002 M NaH2PO4 and has a pH of 7.4.) and lower fluorescein-labeling ratios are prepared by mixing with fluorescein-free oligonucleotide with the same sequence. Bovine serum albumin (BSA, Sigma) was labeled with fluorescein isothiocyanate (FITC) by dissolving the protein and FITC in a buffer of 50 mM sodium bicarbonate and 150 mM NaCl at pH ) 9.0. The solutions were mixed with roughly a 2:1 molar ratio of FITC to BSA. Unreacted FITC was removed by using a Sephadex G-50 column pre-equilibriated in PBS buffer. This procedure yielded 1-1.5 fluorescein groups per protein molecule as determined by a fluorometer (MPF-66, Perkin Elmer). The FITC-BSA was then lyophilized and stored in a -70 °C freezer. FITC-BSA was dissolved in PBS before each experiment. Substrate Preparation. Glass slides (75 × 25 mm, Dow Corning) were cut into three equal pieces roughly 25 × 25 mm before being cleaned. All the cleaning procedures were performed in a semiconductor preparation Class 100 clean room. Twentyfour 25 × 25 mm glass slides were placed in a Teflon wafer carrier and cleaned in 300 mL of Piranha solution (33% by volume of hydrogen peroxide (30% in concentration) and 66% by volume of concentrated sulfuric acid (99% in concentration)) at 130 °C for 10 min.13,38 The slides were rinsed in a deionized water overflow tank for 10 min after cleaning, dried in nitrogen, and heated on a hot plate at 120 °C. The highly acidic conditions applied during the cleaning procedure are expected to generate silica-like surfaces with a net negative charge.27 These cleaned glass slides were used as substrates for TIR/FRAP study or were immediately silanized. The silanization of 24 cleaned glass substrates was carried out in a 300 mL mixture of 1% (3-aminopropyl)triethoxylsilane (APTES) or N-methyl-(3-aminopropyl)trimethoxylsilane (Nmethyl-APTMS) in 95% ethanol (Pharmco) for 20 min at room temperature. The slides were then washed three times with 100% ethanol and dried at 120 °C on a hot plate.13 Total Internal Reflection Microscopy/Fluorescence Recovery after Photobleaching Instrument. The design of this instrument was adapted to an inverted microscope from the basic design of Huang et al.35 Figure 2 shows the overall optical train (37) Cassel, D. L.; Keller M. A.; Surrey, S.; Schwartz E.; Schreiber, A. D.; Rappaport, E. F.; McKenzie, S. E. Mol. Immunol. 1993, 30, 451. (38) Kern, W.; Puotinen, D. A. RCA Rev. 1970, 187.

Chan et al. for the TIR/pattern FRAP apparatus. The basic equipment is a 1W Ar-ion laser (Lexel 85-1, with Etalon), a low-light level cooled CCD camera (KF1400, Princeton Instruments), an inverted microscope (Nikon Diaphot), and a Macintosh Power PC 7100/ 80 with IPLab prism software for image capture and analysis. The CCD chip is a 768 × 512 pixel KAF-0400 chip. All of the equipment and other optical components, except the computer, are placed on an optical table with air-float supports (Newport, San Jose, CA). The laser first passes through a 10/90 mirror type beam splitter (BS1) which produces a weak and a strong beam. A variable neutral density filter (ND) further reduces the power of the weak beam and another 10/90 beam splitter (BS2) again reduces the power of the weak beam to a level of 10-50 µW so that it can be used as a monitoring beam. A shutter (S) is used to control the strong beam which is used to photobleach the fluorescein in 300-550 ms. The photobleach and monitoring beams are then recombined and for pattern photobleaching are directed to a 50/50 cubic beam splitter (CBS) and are then split into two beams of equal intensity. The final separation of these two beams is well-controlled by a set of two convex lenses (L1 with f ) 200 mm and L2 with f ) 50, 75, or 100 mm) that are placed in front of the 50/50 beam splitter. The characteristic distance between interference fringes at the solid/liquid interface is related to this beam separation. The fringe spacing (w) for pattern FRAP is 9 or 10.5 µm. These beams are then positioned by a beam steerer and pass through a 150 mm focal length (L3) convex lens to be focused to a single spot on the surface of the total internal reflection prism. The photons generated by the TIR light are collected by a 10× objective and recorded by the CCD chip of the camera. For spot photobleaching, one of the two beams produced by the cubic beam splitter is blocked. Adsorption and Surface Diffusion Measurements. All of the TIR/FRAP experiments were carried in phosphate buffered saline (PBS) which provides standard physiological conditions and has been used by others in similar experiments.21,33-35 All deionized water used in the experiments was purified in a MilliQ+TOC unit (Millipore). The oligonucleotide concentrations used in this study ranged from 5.6 µM (labeling ratio ) no. of labeled molecules/no. of total molecules ) 0.25-0.5) to 22.6 µM (labeling ratio ) 0.125-0.25). A variation in the labeling ratio is used to control for potential fluorescence quenching due to intermolecular energy transfer. The BSA concentration ranged from 0.1 to 0.4 mg/mL (1.5-6 µM) with LR ) 1, and both bare glass and APTES-coated glass substrates were employed in spot FRAP and pattern FRAP. Usually, 18 µL of a solution containing a given quantity of oligonucleotide or BSA was placed in a probe-clip fluid cell (Grace Bio-Labs) and a silanized or cleaned bare glass slide was put on top to seal the cell. The mixture was equilibrated with the surface for at least 1 h before spot FRAP or pattern FRAP was performed. Then, the probe-clip sample was placed on the stage of the microscope, a drop of immersion oil (Nikon) was put on upper surface of the glass slide as a refractive index matching medium, and a glass prism was put on top to direct the beam at an angle producing the total internal reflection. The angle of incidence to liquid/solid interface was adjusted to about 70° in each case to create a depth of penetration of the evanescent wave of about 0.1 µm. The wavelength of the laser light was set at 488 nm, the objective was 10× (Nikon, plan achromat, 0.25 NA). The monitoring laser beam had a power ranging from 15 to 50 µW, and the photobleaching beam power was set to its maximum value of about 0.25 W. The length scale of the field provided by a 10× objective was calibrated with known scale. The size of the TIR light profile is an ellipse of about 200 × 500 µm. The characteristic fringe spacing in pattern FRAP ranged from 9 to 10.5 µm. This was measured on a fluorescein-coated glass slide by 1-D line profile image analysis and depended on the focal length of L2. The duration of the photobleaching pulse was 300550 ms. The CCD camera was set to take pictures every 0.4-0.8 s before and immediately after the photobleaching. At least three different sets of spot and pattern FRAP were performed in different regions of each sample. An image analysis program (IPLab Spectrum) was used to define a region of interest in the TIR light profile to measure integrated intensity per pixel (at least three different regions are measured per image set), and to obtain spatial information by finding concentration profiles.

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Figure 2. Overall optical train for the TIR/pattern FRAP apparatus. The basic equipment is a 1 W Ar-ion laser (Lexel 85-1, with Etalon), a low-light level cooled CCD camera (Princeton Instruments), an inverted microscope (Nikon Diaphot), and a Macintosh Power PC 7100/80 for image capture and analysis. For spot bleaching studies, one of the two beams from CBS is blocked. The surface densities of adsorbed oligonucleotides were measured separately by using porous glass beads which were coated by APTES (Corning CPG, formerly available from Pierce Chemical). The surface area of the porous glass beads was 34 m2/g and the pore radius was 1350 Å as characterized by the manufacturer. In brief, 0.35 mg of glass beads was weighed, put in an 1.5 mL Ependorf tube, mixed with 15 µL of fluoresceintagged oligonucleotide solution of known concentration in PBS and allowed to sit for 1.5 h. The attainment of equilibrium in this time period was verified by additional studies up to 6 h. The porous glass then was spun down to the bottom of the Eppendorf tube by using a centrifuge at the end of the adsorption period, and fluorescence measurements were made on the separated supernatent. The fluorescence intensity of the solution before, and that of the supernatent after the adsorption process were measured after dilution to a suitable volume by a spectrofluorometer (MDF-66, Perkin Elmer). The excitation and the emission wavelengths of the spectrofluorometer were set at 492 and 514 nm, respectively. The difference between the fluorescence intensities before and after adsorption was then converted to the number of molecules adsorbed from measurement of known fluorescein concentrations. Because the surface area of the porous glass beads is known, the density of the adsorbed molecules per unit area can be determined.

Results and Discussion 1. Mathematical Model for Spot FRAP and Pattern FRAP. We have followed the published theories of both spot24 and pattern FRAP.23,28,39 In Spot FRAP with a large photobleaching area (0.3 mm in the short elliptical TIR light profile), the fluorescence recovery process is dictated only by the adsorption/desorption kinetics of the biomolecules at the liquid/solid interface when diffusion from the bulk is not limiting.24,26,35,39 The fluorescence recovery is often biphasic over time, and has been modelled as biomolecules which can adsorb on the surface at two types of adsorption sites. This behavior has been observed for BSA adsorbed on glass.21 The kinetics of this reversible (39) Hsieh, H. V.; Thompson, N. L. Biophys. J. 1994, 66, 898.

adsorption process are: ka1

A + S1 {\ } C1 k d1

ka2

A + S2 {\ } C2 k d2

(1) (2)

where S1 and S2 are two types of surface binding sites which result in two states of adsorption, C1 and C2 respectively. The mathematical model that describes this process was published elsewhere35,17,24 and the solution for the two component spot FRAP when the bulk normal diffusion rate (DA/(Ceq./Aeq.)2) is much larger than the desorption rate24,35,39-40 is given by

G(t) ) g0 + g1e-kd1t + g2e-kd2t G(t) )

(F(-) - F(t)) (F(-) - F(0))

(3) (4)

where F(t) is the temporal fluorescence signal after photobleaching which is obtained by the image analysis of a series of CCD images in units of signal/pixel, F(-) is the fluorescence intensity before the photobleaching, F(0) is the fluorescence signal immediately after photobleaching and is a free variable,21,24,26,35,39 g0 ) (1 - g1 - g2) is the irreversibly adsorbed fraction, g1 is the reversibly adsorbed fraction with kd1 as its dissociation rate constant (s-1), g2 is the reversibly adsorbed fraction with kd2 as its dissociation rate constant (s-1), DA is the bulk solution diffusion coefficient (cm2/s), Ceq is the total equilibrium concentration of adsorbed molecules (mol/cm2), and Aeq is the total equilibrium concentration of molecules in solution (mol/cm3). A desorption-rate-limited recovery process in (40) Pearce, K. H.; Hof, M.; Lentz, B. R.; Thompson, N. L. J. Biol. Chem. 1993, 268, 22984.

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spot FRAP is implied when the measured desorption rate constants remain the same as the solution concentration changes.21,27,35,40 In our work, all of these parameters are determined by nonlinear least-squares regression (KaleidaGraph) of G(t) vs t. Pattern FRAP can be used to probe simultaneously the adsorption or binding kinetics and surface diffusion rate for weakly bound or adsorbed biomolecules as shown by Huang et al.16 for the surface diffusion of weakly bound bovine prothrombin fragment 1. The recovery rate for biomolecules with two adsorption states can be described by the following expression:28,34,35

G(t) )

)[

(

-π2Dt g1e-kd1t + g2e-kd2t f 1 - (1 - e w2 ) g1 + g2 3

]

(5)

where D is the surface diffusion coefficient (cm2/s), f is the mobile fraction of the biomolecule that can diffuse on the surface, and w (cm) is the characteristic spacing of the fringe pattern. In our case, w is directly measured from a CCD image of a fluorescein-coated glass slide by image analysis. The general approach in this study is to measure the desorption parameters from spot FRAP as described above, substitute these parameters in eq 5, and measure the surface diffusion from pattern FRAP. It must be noted that this correlation assumes that the amplitude33 A or visibility35 of the sinusoidal intensity profile is equal to 1:

(

(πxw))

It ) Ii 1 + A sin

(6)

where It is the local laser intensity, Ii is the incident laser intensity, and A is the visibility or amplitude of the laser fringe pattern. 2. Experimental Verification of the Validity of Spot FRAP with BSA. BSA adsorption and diffusion on fused quartz glass has been characterized using FRAP.21 In this study, BSA was used as a standard for checking the performance of our instrumentation and comparing with the oligonucleotide measurements. A concentration range of 0.1-0.4 mg/mL of BSA with a labeling ratio (labeled molecule:total molecule) of 1 was chosen because it has been shown33 that FITC-BSA has no significant fluorescence quenching at this level that would affect the measured transport parameters. Our experimental method for the measurement of desorption rates by conventional spot FRAP using a temporal signal was similar to that described elsewhere.21 Pattern FRAP was then used for measurement of the surface diffusion coefficient and mobile fraction in the manner of Huang et al.35 The main difference between our study and others is the application of a CCD camera to capture both the temporal and spatial fluorescence signal information during spot or pattern FRAP rather than a PMT and analysis of a 5 × 5 pixel region to obtain the intensity in units of intensity/pixel by image analysis. Such a region is approximately equal to the width of one bright fringe. Figure 3 shows a conventional normalized spot photobleaching recovery curve (G(t) vs time) of 0.1 mg/mL (1.5 µM) BSA in PBS at the bare glass/solution interface. The data were correlated with eq 3, because others have shown that BSA at this concentration reversibly adsorbs in two states and has a surface concentration of approximately 3 × 1012 molecules/cm2 (a closely packed BSA monolayer contains 3.5 × 1012 molecules/cm2).14,21 The slow and fast desorption rates of BSA were 0.0165 and 0.227 s-1, respectively, with a slow desorbed fraction g2 of 0.6 (relative slow desorbed fraction ) g2/(g1 + g2) )

Figure 3. Adsorption and diffusion of BSA. (A) Normalized spot photobleaching recovery (G(t) vs time) of 0.1 mg/mL (1.5 µM) of BSA in PBS buffer at the bare glass/solution interface. By fitting this data with eq 7, the slow and fast desorption rates of BSA are calculated to be 0.0165 and 0.227 s-1, respectively, with a slow desorbed fraction of 0.38 and fast desorbed fraction of 0.6. (B) Normalized pattern FRAP-photobleaching recovery curve. The calculated surface diffusion coefficient of oligonucleotide is about 3.92 × 10-9 cm2/s, and the mobile fraction of adsorbed oligonucleotide is about 0.302 on this recovery curve.

0.61) and a fast desorbed fraction g1 of 0.38 (relative fast desorbed fraction ) g1/(g1 + g2) ) 0.39). The correlation coefficient is 0.9955. Burghardt and Axelrod21 reported that the slow and fast desorption rates of BSA on fused quartz slide were 0.005 s-1 with a desorbed fraction of 0.25 and 0.26 s-1 with a desorbed fraction of 0.15, respectively. This corresponds to a relative slow desorbed fraction g1/(g1 + g2) of 0.64 and a relative fast desorbed fraction g2/(g1 + g2) of 0.36. The discrepancy between our data and theirs21 in the slow desorption rate and the irreversibly adsorbed fracton (1 - g1 - g2) may be due to the fact we used a different and more aggressive cleaning solution for the glass surface than the chromic acid employed in their study. Ours is the standard currently used for silicon wafers. Although their irreversibly adsorbed fraction is higher than ours, the ratios of the number of slow or fast reversibly desorbed molecules to the total number of reversibly adsorbed molecules (relative fast or slow desorbed fraction ) g1,2/(g1 + g2)) are similar in both studies. We examined the interfacial behavior of BSA on (3aminopropyl)triethoxysilane (APTES)-coated glass, which

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Table 1. Adsorption and Transport Parameters of Bovine Serum Albumin (BSA) in PBS Buffer

substrate type bare glass bare glass APTES glass N-methyl-APTES glass bare glass (from ref 8)

BSA solutn concn (µM)

weakly adsorbed fraction g1

fast desorptn rate const kd1 (s-1)

strongly adsorbed fraction g2

slow desorpn rate constant kd2 (s-1)

2-D diffusn coeff (cm2/s)

mobile fraction f

1.5 3 6 6 1.5-150

0.35 ( 0.04 0.23 ( 0.04 0.27 ( 0.07 0.31 ( 0.07 0.25-0.50

0.26 ( 0.03 0.28 ( 0.01 0.28 ( 0.03 0.51 ( 0.12 0.26 ( 0.02

0.65 ( 0.04 0.72 ( 0.08 0.65 ( 0.03 0.50 ( 0.01 0.5-0.75

0.018 ( 0.0014 0.019 ( 0.0006 0.008 ( 0.0005 0.036 ( 0.007 0.005 ( 0.001

(3.3 ( 0.2) × 10-9 (2.7 ( 0.1) × 10-9 (1.8 ( 0.4) × 10-9 (4.9 ( 0.6) × 10-9 (5 ( 1) × 10-9

0.28 ( 0.02 0.20 ( 0.03 0.25 ( 0.03 0.19 ( 0.04

is used as a substrate for DNA biosensors as described earlier.2,5,9 With this surface, we had difficulty in obtaining a reasonable signal to noise ratio in our FRAP measurements when we used BSA concentrations lower than 0.4 mg/mL (6 µM). Table 1 lists the experimental results for spot FRAP with BSA as well as the data of Burghardt and Axelrod.21 About 92.5% (g1 + g2) of the BSA molecules adsorb reversibly on APTES-coated glass. The desorption rate constant from the strongly adsorbed fraction of BSA decreases from 0.018 s-1 for bare glass to 0.0082 s-1 while the weakly adsorbed fractions appear to be similar. The desorption rate constant for the slow fraction on APTEScoated glass is closer to that measured by Burghardt and Axelrod21 on the chromic acid cleaned glass than that measured on bare glass. We have confirmed the high reversibility of BSA adsorption by performing a control experiment in which either the bare or APTES-coated glass with adsorbed BSA was washed with protein-free PBS buffer. Nearly all BSA was removed as indicated by a reduction of TIR fluorescence signal of more than 95%. We also believe that the previously used glass-cleaning method21 (ethanol rinsing after chromic acid washing and storage in ethanol) may have created a very different and less clean surface. We showed this by rinsing the chromic acid cleaned glass slide with laboratory grade ethanol and performing TIR fluorescence signal measurements of BSA before and after a PBS buffer wash. The irreversibly adsorbed fractions of BSA on chromic acid washed glass before and after an additional ethanol rinse were 0 and 15%, respectively. These observations suggest that organic impurities deposited or adsorbed on the chromic acid cleaned glass during the ethanol rinsing and storage steps. A complete explanation for the differences between the two sets of results must await further studies and comparisons with additional protein interfacial data,41-43 but this work validated that our spot FRAP apparatus was comparable in its capabilities to standard PMT-based systems. 3. Spot FRAP of Oligonucleotides on APTESCoated Glass. Figure 4 shows a typical real time fluorescence intensity image of 256 × 171 pixels (captured by a 10× objective and CCD camera with the size bar of 200 µm) during spot FRAP with 11.2 mM oligonucleotide in PBS (labeling ratio ) 0.25). A typical depth of photobleaching is about 55%. Figure 5A shows a conventional analysis of spot FRAP as a normalized integrated signal of fluorescence determined from a 15 × 15 µm region (5 × 5 pixels) of interest against time for a series of images. This is the only type of data available from a PMT-based system. The slow desorption rate constant in this case was found to be 0.024 s-1 with a fraction of 0.561, and the fast desorption rate constant was found to be 0.24 s-1 with a fraction of 0.42 by fitting the normalized fluorescence data with eq 3. The correlation coefficient (R) is (41) Stout, A. L.; Axelrod, D. Photochem. and Photobiol. 1994, 62(2), 239. (42) Adamson, A. W. Physical Chemistry of Surfaces; John Wiley and Sons, Inc.: New York, 1990. (43) Squire, P. G.; Moser, P.; O’Konski, C. T. Biochemistry 1968, 7, 4261.

Figure 4. Typical 170 × 600 µm TIR/spot FRAP light profile showing fluorescence intensity image captured by a 10× objective and CCD camera using spot FRAP with 11.2 µM oligonucleotide in PBS (labeling ratio ) 0.25) on APTES-coated glass substrate.

0.999. Although a two-exponential function fits the data well, the curves actually may be composed of a continuum of desorption rate constants. At present there is no theory for fitting FRAP data with a continuum of rate constants. The two-component model is a convenient way of parameterizing the data which may or may not actually represent two discrete states.21,24 Table 2 lists the adsorption parameters of all the samples that we have studied. The fast desorption rate constants and the slow desorption rate constants for this oligonucleotide are about 40% lower and 100% higher than those of BSA, respectively. It should be noted that an increase in solution concentration of oligonucleotide had no apparent effect on the desorption rate constants and that most of the oligonucleotides adsorb reversibly on APTES-coated glass (i,e., g1 + g2 is close to unity). This reversibility is further supported by results showing a Langmuir adsorption isotherm of oligonucleotide on APTES-coated porous glass as shown in Figure 6A. Since a diffusion-controlled process is concentration dependent, these results provide good evidence that the recovery process is governed only by the kinetics of the desorption.21,35,36,40 For the maximum concentration of oligonucleotide (22.4 µM) that we used in the photobleaching experiments, the number of molecules in solution that are illuminated by a typical evanescent wave with a finite penetration depth of 0.1 µm is 1.35 × 1011 in a 1 cm2 area while the adsorbed density is 1.3 × 1013/ cm2 as shown on Figure 6A. As a result, we are confident

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Figure 5. Normalized photobleaching recovery curves of 11.2 µM oligonucleotide in PBS (labeling ratio ) 0.25) on APTEScoated glass substrate. (A) Normalized spot FRAP-photobleaching recovery curve. By fitting this data with eq 7, the slow and fast desorption rates of the DNA are calculated to be 0.0235 and 0.249 s-1, respectively, with a slow desorbed fraction of 0.424 and fast desorbed fraction of 0.561. (B) Normalized pattern FRAP-photobleaching recovery curve. The calculated surface diffusion coefficient of oligonucleotide is about 1.76 × 10-9 cm2/s, and the mobile fraction of adsorbed oligonucleotide is about 0.288 on this recovery curve.

that the TIR fluorescence signal is almost completely due to fluorescently tagged oligonucleotides that are on the surface. It has been demonstrated that fluorescence quenching caused by a high concentration of fluorescein at the liquid/ solid interface would affect the accuracy of the TIR/spot FRAP experiments.29 The total surface concentration of adsorbed oligonucleotide as determined by a spectrofluorometer is 2.6 × 1012 molecules/cm2 (Figure 6A) at a oligonucleotide solution concentration of 5.6 µM. This density results in a calculated separation between two adjacent DNA molecules (unlabeled or labeled) of about 1.53 × 10-6 cm. This separation between fluorophores of 1.53 × 10-6 cm (when the labeling ratio is equal to 1) becomes even larger for lower fluorescein-labeling ratios. It is known29 that energy transfer between fluorescein molecules occurs to a significant extent only at separation of 8 × 10-7 cm or less. Thus, all of our experiments at 5.6 µM were carried out in the regime of fluorescein surface concentrations without significant quenching. Furthermore, the solution phase aggregation or condensation of DNA which occurs in the presence of both alcohol and cations44,45 should be absent in this study with a PBS (44) Bloomfield, V. A. Biopolymers 1991, 31, 1471.

Chan et al.

buffer. In order to further assure that we determined the correct adsorption and transport parameters of oligonucleotides, we varied the labeling ratio, i.e., the number of fluorescein-labeled oligonucleotides to the total number of oligonucleotides in solution (labeled + unlabeled molecules). Table 2 summarizes the adsorption parameters of oligonucleotide in PBS at different DNA solution concentrations with different labeling ratios. When the labeling ratio was varied from 0.25 to 0.5 in a fixed total concentration of 11.2 µM, no evidence of fluorescence quenching or preferential adsorption of fluorescein-tagged DNA was observed in the experimental parameters for spot FRAP. On the other hand, some apparent trends in the adsorption parameters were seen when the labeling ratio was varied from 0.25 to 0.5 at a lower solution concentration of 5.6 µM (when quenching is even less likely). The strongly adsorbed fraction of oligonucleotide apparently decreases and the weakly adsorbed fraction increases when there is an increase of labeling ratio. However, we believe that the trends either are spurious or perhaps that at low concentration there might have been a slight preferential adsorption of fluorescein-tagged oligonucleotides relative to nonlabeled molecules. The effect of quenching is only observed when the labeling ratio is increased to 1 in the 11.2 µM sample. In this case, pattern FRAP F(∞) is greater than that before photobleaching F(-). This is caused by the shorter distance between adjacent fluorophores of about 7.7 × 10-7 cm (7.6 × 1012 molecules/cm2 as determined from spectrofluorometer measurement) at this high labeling ratio, which is close to the effective quenching distance of 8 × 10-7 cm.33 Stout and Axelrod41 reported on the reversible recovery of TIR fluorescence in deoxygenated medium. We did perform some control FRAP experiments in a deoxygenated medium and detected no apparent difference between deoxygenated and nondeoxygenated medium in our experiments. Although photochemically induced cross-linking may be another cause for the high values for F(∞), our adsorption isotherm measurement (in Figure 6A) strongly suggests that quenching is the main source of high values for F(∞). 4. Experimental Verification of the Validity of Pattern FRAP. A typical normalized pattern recovery curve of G(t) vs time for 0.1 mg/mL (1.5 µM) BSA in PBS buffer on a bare glass surface is given in Figure 3B. The values of g1, g2, kd1, and kd2 for BSA were determined by spot FRAP as described above and were then substituted into eq 5 as fixed parameters to find the two-dimensional surface diffusion coefficient and mobile fraction by a nonlinear least-squares regression procedure. The quantity w is the characteristic size of the interference pattern, which was measured on a fluorescein-coated glass slide before each set of experiments. We have tried to measure A experimentally by fitting the intensity profiles with eq 9, but the intensity profile is not always an ideal sinusoidal intensity profile because spurious reflections result in multiple interacting beams which create nonsinusoidal patterns. From a theoretical calculation of visibility performed by Abney et al.,28 the evanescent wave visibilities of s-polarized and p-polarized light are 0.94 and 0.99, respectively. Without better information, we assume that A is equal to 1 as has been done in other studies.22,23,34,35 Visibility has been measured with an oscillating mirror in one study, and the value was 0.61.29 This is predicted to underestimate the mobile fraction mea(45) Bloomfield, V. A.; Crothers, D. M.; Tinoco, I. The Physical Chemistry of Nucleic Acids; Harper and Row: New York, 1977.

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Table 2. Adsorption and Transport Parameters of Oligonucleotide on APTES Glass in PBS oligoncleotide labeling weakly adsorbed fast desorpn rate strongly adsorbed slow desorpn rate concn (µM) ratio fraction g1 const kd1 (s-1) const kd2 (s-1) fraction g2 5.6 5.6 5.6 11.2 11.2 11.2 22.4

0.25 0.35 0.5 0.25 0.5 1 0.25

0.22 ( 0.04 0.27 ( 0.08 0.42 ( 0.04 0.42 ( 0.04 0.47 ( 0.08 0.34 ( 0.07 0.34 ( 0.06

0.14 ( 0.03 0.19 ( 0.03 0.23 ( 0.05 0.17 ( 0.08 0.17 ( 0.02 0.16 ( 0.09 0.17 ( 0.03

0.69 ( 0.04 0.60 ( 0.07 0.45 ( 0.05 0.50 ( 0.02 0.49 ( 0.11 0.59 ( 0.09 0.43 ( 0.03

0.024 ( 0.005 0.015 ( 0.003 0.033 ( 0.004 0.022 ( 0.005 0.018 ( 0.008 0.018 ( 0.004 0.024 ( 0.006

2-D diffusn coeff (cm2/s)

mobile fraction f

(2.22 ( 0.11) × 10-9 (2.20 ( 0.13) × 10-9 (2.25 ( 0.1) × 10-9 (1.57 ( 0.22) × 10-9 (1.44 ( 0.1) × 10-9 quencha (9.17 ( 0.6) × 10-10

0.29 ( 0.02 0.28 ( 0.03 0.29 ( 0.02 0.27 ( 0.03 0.27 ( 0.01 quencha 0.26 ( 0.02

a Effect of quenching is observed when the labeling ratio is increased to 1 in the 11.2 µM sample. In this case the fluorescence intensity after FRAP F(∞) is greater than that before photobleaching F(-).

Figure 6. (A) Adsorption isotherm of fluorescently tagged oligonucleotide on APTES-coated porous glass. The solid line is a fit to the experimental data by the Langmuir adsorption equation. The maximum concentration of the adsorbed oligonucleotide on the plot corresponds to 33% of a full monolayer of closed-packed molecules. (B) Surface diffusion coefficients of oligonucleotide against the density of adsorbed molecules on APTES-coated glass.

surement, but does not affect the comparison between BSA and DNA. The measured surface diffusion coefficient of BSA was 3.92 × 10-9 cm2/s, and the mobile fraction of adsorbed BSA was 0.302. This surface diffusion coefficient agrees well with the data of Burghardt and Axelrod,21 who reported a value of 5 × 10-9 cm2/s for BSA using spot FRAP with a varying beam width, and is similar to the surface diffusion coefficient of BSA on polystyrene (8 × 10-9 cm2/s).33 Spot FRAP, unlike pattern FRAP, cannot determine the fraction of the adsorbed molecules which can surface diffuse. The similarity between our results and those found by others is good evidence for the utility of a system based on a CCD camera. An increase in the

concentration of BSA in solution did not significantly change the adsorption parameters when the BSA concentration in solution was doubled (3 µM), which agrees with the results of other groups.21 The surface diffusion coefficient of BSA measured by pattern FRAP on APTES glass is about two times smaller than that on bare glass. 5. Pattern FRAP Data for the Oligonucleotide. Figure 5B shows a conventional pattern FRAP recovery curve with 11.2 µM oligonucleotide (labeling ratio ) 0.25) in PBS in units of fluorescence intensity per pixel. The normalized data were regressed with eq 5 after the constants g1, g2, kd1, and kd2 had been determined independently from spot FRAP (Figure 5A). The surface diffusion coefficient was 1.76 × 10-9 cm2/s, and the mobile fraction was 0.288. A summary of diffusion parameters for this oligonucleotide obtained by TIR/pattern FRAP is given on Table 2. These surface diffusion coefficients are of the same order of magnitude as those for BSA. BSA is basically a well-packed globular protein that has molecular weight of about 67 000 and a molecular dimension of about 40 × 40 × 140 Å,38 while the 21 nucleotide long oligonucleotide that we studied has a MW of about 7140 and has the shape of a rigid rod, with molecular dimension of about 5 x 5 × 60 Å.21,39,46 It was determined by using coagulation kinetic measurements47 and hydroxyl radical footprinting48 that small singlestranded DNA molecules with 80 bases or less adsorb on negatively charged latex particles with the long axis parallel rather than perpendicular to the surface. Studies by radioactivity4 and ellipsometry49 confirm such an orientation. However, the orientation of the adsorbed oligonucleotide alone does not explain why it has desorption rate constants and surface diffusion coefficients in the same order of magnitude to those of BSA although it is about ten times smaller in its molecular weight. In comparison with BSA, which has a surface diffusion coefficient of about 8 × 10-9 cm2/s as measured by TIR/ FRAP on polystyrene, fluorescein-labeled RNase A with molecular weight of 13 700 and a molecular dimension of 25 × 25 × 40 Å51 has a much larger surface diffusion coefficient of about 5 × 10-8 cm2/s.33 This example demonstrates that a protein molecule (RNase A) with smaller molecular weight and size diffuses faster than BSA on the same substrate. The relatively low surface diffusion coefficient of oligonucleotides in comparison with that of BSA may indicate a stronger molecule/substrate interaction for nucleic acids than for amino acids. Ly(46) Dwyer, J. D.; Bloomfield, V. A. Biophys. J. 1993, 65, 1810. (47) Walker, H. W.; Grant, S. B. J. Colloid Interface Sci. 1996, 179, 552. (48) Walker, H. W.; Grant, S. B. Langmuir 1995, 11, 3772. (49) Chrisey, L. A.; Roberts, P. M.; Benezra, V. I.; Dressick, W. J.; Dulcey, C. S.; Calvert, J. M. Mater. Res. Soc. Symp. Proc. 1994, 330, 179. (50) Tilton, R. D.; Robertson C. R.; Gast, A. P. Langmuir 1991, 7, 2710. (51) Lyubchenko, Y. L.; Gall, A. A.; Shlyakhtenko, L. S.; Harrington, R. E.; Jacobs, B. L.; Oden, P. I.; Linsay, S. M. J. Biomol. Struct. Dyn. 1992, 10 (3), 589.

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Table 3. Adsorption and Transport Parameters of Oligonucleotide on N-Methyl-APTMS Glass in PBS oligoncleotide labeling weakly adsorbed fast desorpn rate strongly adsorbed slow desorpn rate concn (µM) ratio fractn g1 const kd1 (s-1) const kd2 (s-1) fractn g2 5.6 5.6 11.2 22.4

0.5 1 0.5 0.25

0.31 ( 0.07 0.25 ( 0.08 0.21 ( 0.04 0.51 ( 0.07

0.51 ( 0.15 0.47 ( 0.14 0.43 ( 0.12 0.47 ( 0.1

0.50 ( 0.01 0.51 ( 0.07 0.63 ( 0.02 0.41 ( 0.1

0.033 ( 0.007 0.039 ( 0.012 0.051 ( 0.015 0.057 ( 0.006

2-D diffusn coeff (cm2/s)

mobile fraction

(7.47 ( 0.24) × 10-9 (7.32 ( 0.11) × 10-9 (5.26 ( 0.15) × 10-9 (3.42 ( 0.22) × 10-9

0.22 ( 0.04 0.19 ( 0.03 0.21 ( 0.04 0.21 ( 0.03

ubchenko et al.50 provides experimental evidence that electrostatic interaction is a predominant force holding very large double-stranded DNA molecules (MW > 15 000 000) onto aminopropyl mica surfaces. This is supported by the zwitterionic nature of APTES coated on glass which has superficial concentrations of NH3+ and SiO- on the surface in water at pH 7.52 The positive charge results in strong interactions between the surface and the negatively charged DNA molecules. On the other hand, the so-called walking (desorption/rapid readsorption) mechanism for surface diffusion of weakly bound biomolecules proposed by Broek and Thompson53 generates a surface diffusion coefficient of 1.5 × 10-14 cm2/s, which is a few orders of magnitude smaller than the measured value by pattern FRAP. As a result, the walking mechanism may not be the only factor determining the surface diffusion rate. Lamture et al.5 found that the adsorption of a 36 base long oligonucleotide on APTES-coated glass increased with solution concentration. The surface concentration of adsorbed oligonucleotide increased from 1 × 1010 molecules/ cm2 to 1 × 1011 molecules/cm2 in 0.1 M KOH solution when the solution concentration increased from 5 to 50 µM as detected by radioactivity. At such pH, one would expect more SiO- charges but few NH3+ groups. It is interesting to examine the effect of the surface concentration on the adsorption and surface diffusion process. It is well-known that an increase in the solution or surface concentration of proteins like BSA23 and prothrombin fragment 135 reduces their surface diffusion coefficients, presumably due to steric interactions between adjacent molecules. Table 2 lists the experimental diffusion parameters of oligonucleotide when the solution concentration is increased from 5.6 to 22.4 µM. A measured adsorption isotherm of fluorescently tagged oligonucleotide on model APTES-coated porous glass beads is shown on Figure 6A. Adsorption in the range of solution concentrations that we have studied appears to be a Langmuir isotherm in which the equilibrium surface concentration is proportional to the solution concentration in the lower concentration regime (