J. Phys. Chem. B 2001, 105, 8679-8684
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Single Molecule Study of the Lateral Transport of Four Homooligoncleotides at the Interface of Water and Chemically Modifed Silica† Derrick J. Swinton and Mary J. Wirth* Department of Chemistry and Biochemistry, UniVersity of Delaware, Newark, Delaware 19716 ReceiVed: April 2, 2001; In Final Form: July 14, 2001
The lateral transport of four single-strand homooligonucleotides on a microscopically flat silica surface chemically modified with chlorodimethyloctadecylsilane, in contact with 0.01 M KCl, was investigated using single-molecule spectroscopy. The deoxynucleotides were 20-mers of poly A, poly C, poly T, and poly G, each labeled at the 5′ end with tetramethylrhodamine. Autocorrelation of the burst data, for each homooligonucleotide, indicates a single diffusing species with no significant difference in the diffusion coefficient among the individual oligonucleotides (D ) 3.2 ( 0.3 × 10-6 cm2/s). Examination of burst data for each homooligonucleotide reveals the presence of diffusing molecules occasionally stopping at strong adsorption sites. The frequency of strong adsorption correlates with the number of the amino and amide groups of the base. A histogram of duration times of the strong adsorption events shows that there are two different time scales for strong adsorption events. The autocorrelation decay for the strong adsorption events pooled from all four oligonucleotides fit well to a double-exponential decay with 80% and 20% of the strongly adsorbing molecules having an average desorption time of 16 and 220 ms, respectively.
Introduction The design of new surfaces for chromatographic separation and purification of biomolecules require an understanding of surface defects, which presently limit separation efficiency.1-6 Protein adsorption to surface defects is also a problem in electrophoresis, where zone broadening occurs,7 in medicine, where biocompatibility of materials is an issue,8 in industry, where enzymatic reactors are needed,9 and in proteomics, where methods are being developed to screen of protein function in arrays.10 Genomics and proteomics also employ commercially available biochips containing adsorbed or tethered oligonucleotides to study gene expression through hybridization with complementary strands. Surface defects cause adsorption that interferes with the detection of hybridization. A fundamental understanding of the adsorption of biomolecules to surface defects would have impact on many important technologies. The interaction of oligonucleotides with surfaces of biochips is particularly interesting because the performance relies on the detailed physical chemistry of the surface. A library of oligonucleotides called probes are deposited on a surface as an array of spots, and the sample oligonucleotides, called targets, bind to their complementary probes to form double strands. To detect the double strands, a variety of methods have been devised, including the use of labeled targets. The printed array of probe oligonucleotides often relies on a covalent bond between the surface and the oligonucleotide to prevent desorption of the double strand,11 and the kinetics of hybridization and melting have been studied.12 Because it is possible that hybridization might occur faster if the probes are not covalently bonded to the surface, and this circumvents derivatization of the probes, it is common to print the probe oligonucleotides onto the surface by relying on weak physisorption. In this design, the complementary target oligonucleotides also adsorb, and then the target †
Part of the special issue “Royce W. Murray Festschrift”. * Author to whom correspondence is to be addressed.
and probe oligonucleotides laterally diffuse together, and they subsequently hybridize. The printing process itself relies on the details of lateral transport of the probes as the sizes of the regions are reduced to the nanometer scale.13 For enhancing hybridization speed, Chan et al. have provided a model, as well as adsorption and transport data, showing that fast lateral diffusion and fast reversible adsorption in nonprobe regions enhances the speed of hybridization.14-16 Nonprobe regions are those where no complementary strand is printed. Adsorption into the nonprobe region can be caused either by weak hybridization to a probe having partial complementarity or to adsorption to sites on the substrate. The latter is known to be a substantial problem, and it is the subject of this paper. The investigation of adsorptive defects on engineered surfaces is complicated by the fact that adsorption to defect sites is a rare event, once a surface is sufficiently good for commercial use. Single-molecule spectroscopy has allowed researchers to study events in solution and on surfaces that would otherwise be obscured by ensemble averaging.17 Single-molecule spectroscopy has been used to study the dynamics of DNA and oligonucleotides in solution.18-20 More pertinently, singlemolecule spectroscopy has been used to study strong adsorption of an oligonucleotide, the SP6 promoter primer, to defects on a chemically modified silica surface.21 The results showed the presence of both diffusing and transiently stationary molecules. These strong adsorption events were attributed to adsorption to substrate silanols. The sequence of SP6 contains all four of the bases; therefore, it is not known whether the different bases contribute differently to strong adsorption. The purpose of this paper is to study the lateral transport of single-strand homooligonucleotides and their interaction with strong adsorption sites on a chemically modified silica surface. Experimental Section The experimental apparatus was identical to one previously used,21 where an inverted optical microscope (Zeiss Axiovert
10.1021/jp011234n CCC: $20.00 © 2001 American Chemical Society Published on Web 08/04/2001
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Swinton and Wirth
Figure 1. Plot of photon counts vs time for each homooligonucleotide. The solid bar in each panel denotes an interval where a strong adsorption event occurred.
100) with an XF32 filter set (Omega Optical, Inc.) was used in combination with an external confocal pinhole to allow singlemolecule measurements with a high fluorescence-to-background ratio. A 514.5 nm beam from a mode-locked argon ion laser (Coherent, Inc.) was focused at the interface using a 100× oil immersion objective (NA ) 1.40). The beam size at the focus was evaluated using a CCD camera (Roper Instruments, TEK512B) and surface having a high concentration of oligonucleotide. A ronchi ruling was subsequently placed in the object plane to calibrate the spatial scale of the CCD. The size of the confocal pinhole was 25 µm, chosen to encircle the image of the Gaussian beam over (2s, where s2 is the variance. The pinhole image was refocused onto an actively quenched avalanche photodiode (EG&G Optoelectronics), and its output was time-filtered (Oxford-Tennelec TAC 480) to reduce signal caused by Raman scattering from the solvent. Laser power was varied to ensure that photobleaching and optical trapping were negligible, and the average power used in the experiments was 2 µW. Burst data were collected for 65 s using a 2 ms dwell time and 16384 channels in the multichannel scalar (OxfordTennelec), and each experiment was repeated 10 times. The data were analyzed using Origin and Matlab to autocorrelate burst data and evaluate individual burst characteristics. To prepare a surface, a fused silica microscope slide (ESCO) was derivatized with chlorodimethyloctadecylsilane in dry toluene for 3 h, using butylamine as a catalyst. The microscope slide was then rinsed with toluene and hot MeOH. This procedure gives a sterically limited coverage of 3 µmol/m2 of silane, limited sterically by the methyl side groups of the silicon atom. The rhodamine-labeled homooligonucleotides, obtained from New England Biolabs, were dissolved in 0.01 M KCl, diluted to nanomolar levels and then equilibrated with the surface. The interface was placed in the focal plane of the microscope, and the fluorescence was monitored as the solution was successively diluted until the concentration decreased to one molecule in the beam Γslow(0). However, the number of events is so low that the statistics of small numbers limit determination of the rate constants and relative magnitudes of the decay components. Given the similarities of the autocorrelations of strong adsorption events for the four homooligonucleotides, it is worthwhile to consider that they might all have the same behavior, only a difference in number of events; therefore, pooling the events would provide better statistics. Figure 7 shows an autocorrelation of the pooled events. The data fit well to a double exponential decay, with time constants of 16 and 220 ms. The fit also indicates the concentration ratio of the two types of strong adsorption sites is 4:1. This may seem surprising at first, given the large difference in number of events on the two different time scales. The similar concentration of the two types of sites is a manifestation of the accumulation of adsorbates on the very strong sites, despite rarity of adsorption to these sites. The substantial accumulation to the very strong sites is why they are important to study. Increased accumulation slows hybridization and increases chromatographic tailing. The rarity of the adsorption event present a challenge for their study. New experimental design to study strong adsorption alone, rather than strong adsorption in tandem with diffusion, will be required
Swinton and Wirth
Figure 7. Autocorrelation of 279 pooled strong adsorption events from all four homooligonucleotides (circles). Best fit to an exponential decay (solid line). The results of the regression are shown in the figure.
to obtain the good statistics needed to characterize the rare, very strong adsorption of oligonucloetides. Acknowledgment. This work was supported by the National Science Foundation under Grant CHE-0078847. D.J.S. is grateful to the University of Delaware for a Presidential Fellowship. References and Notes (1) Nawrocki, J. J. Chromatgr. A. 1997, 779, 29-71 (2) Harris, J. M.; Marshall, D. B. J. Microcolumn Sep. 1997, 9, 185191. (3) Cox, G. B. J. Chromatogr. A. 1993, 656, 353-367. (4) Kohler, J.; Kirkland. J. Chromatogr. 1987, 385, 125-150. (5) Bij, K. E.; Horvath, C.; Melander, W. R.; Nahum, A J. Chromatogr. 1981, 203, 65-84. (6) Baba, Y. J. Chromatogr. 1993, 618, 41-55. (7) Figeys, D.; Pinto, D. Electrophoresis 2001, 22, 208-216. (8) Young, B. R.; Pitt, W. G.; Cooper, S. L. J. Colloid Interface Sci. 1988, 124, 28-43. (9) Tsung, E. F.; Tilton, R. D. J. Colloid Interface Sci. 1999, 213, 208217. (10) MacBeath, G.; Schreiber, S. L. Science 2000, 289, 1760-1763. (11) Brockman, J. M.; Nelson, B. P.; Corn, R. M. Annu. ReV. Phys. Chem. 2000, 51, 41-63 (12) Peterlinz, K. A.; Georgiadis, R. M. J. Am. Chem. Soc. 1997, 119, 3401-3402. (13) Piner, D.; Zhu, J.; Xu, F.; Hong, S.; Mirkin, C. A. Science 1999, 283, 661-63. (14) Chan, V.; Graves, D. J.; McKenzie, S. E. Biophys. J. 1995, 69, 2243-55. (15) Chan, V.; McKenzie, S.; Fortina, P.; Graves, D. Langmuir 1997, 13, 320-329. (16) Chan, V.; McKenzie, S.; Surrey, S.; Fortina, P.; Graves, D. J. Colloid Interface Sci. 1998, 203, 197-207. (17) Ambrose, W. P.; Goodwin, P. M.; Jett, J. H.; Van Orden, A.; Werner, J. H.; Keller, R. A. Chem. ReV. 1999, 99, 2929-2956. (18) Nie, S. M.; Chiu, D. T.; Zare, R. N. Anal. Chem. 1995, 67, 28492857. (19) Sauer, M.; Drexhage, K. H.; Lieberwirth, U.; Muller, R.; Nord, S.; Zander, C. Chem. Phys, Lett. 1998, 284, 153-163. (20) Wennmalm, S.; Edman, L.; Rigler, R. Chem. Phys. 1999, 247, 6167. (21) Wirth, M. J.; Swinton, D. J. J. Phys. Chem. B. 2001, 105, 14721477. (22) Wirth, M. J.; Swinton, D. J. Anal. Chem. 1998, 70, 5264-5271. (23) Trautman, J. K.; Macklin, J. J. Chem. Phys. 1996, 205, 221-229. (24) Dickson, R. M.; Cubitt, A. B.; Moerner, W. E. Nature 1997, 388, 355-358. (25) Handbook of Biochemistry, 2nd Ed.; CRC Press: Boca Raton, FL, 1970. (26) Kovaleski, J. M.; Huang, X.; Wirth, M. J. Anal. Chem. 1996, 68, 4119-4123. (27) Mao, Y.; Daniel, L. N.; Whitaker, N.; Saffiotti, U. DNA binding to Crystalline Silica Characterized by Fourier transform Infrared Spectroscopy. EnViron. Health Perspect. 1994, 102, 165-171.
