Kinetics of Porphyrin Adsorption and DNA-Assisted Desorption at the

Jan 27, 2010 - Michael A. O'Connell , Anne de Cuendias , Florence Gayet , Ian M. Shirley , Stuart R. Mackenzie , David M. Haddleton , and Patrick R. U...
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Kinetics of Porphyrin Adsorption and DNA-Assisted Desorption at the Silica-Water Interface Meiqin Zhang,† Hayley V. Powell,† Stuart R. Mackenzie,‡ and Patrick R. Unwin*,† †

Department of Chemistry, University of Warwick, Coventry CV4 7AL, United Kingdom, and ‡Department of Chemistry, University of Oxford, Physical & Theoretical Chemistry Laboratory, Oxford OX1 3QZ, United Kingdom Received September 11, 2009. Revised Manuscript Received November 30, 2009

Evanescent wave cavity ring-down spectroscopy (EW-CRDS) has been used to study in situ the kinetics of the adsorption of 5,10,15,20-tetrakis(4-N-methylpyridiniumyl)porphyrin (TMPyP) from pH 7.4 phosphate buffer solution (PBS) to the silica-water interface and the interaction of calf thymus DNA (CT-DNA) with the resulting TMPyPfunctionalized surface. TMPyP was delivered to the silica surface using an impinging jet technique to allow relatively fast surface kinetics to be accessed. Adsorption was first-order in TMPyP, and the initial adsorption rate constant at the bare surface was found to be k = (4.1 ( 0.6)  10-2 cm s-1. A deceleration in the adsorption kinetics was observed at longer times that could be described semiquantitatively using an Elovich-type kinetic expression. The limiting value of the absorbance corresponded approximately to monolayer coverage (6.2  1013 molecules cm-2). Exposure of the TMPyPmodified silica surface to CT-DNA, achieved by flowing CT-DNA solution over the functionalized surface, resulted in efficient desorption of the TMPyP. The desorption process was driven by the interaction of TMPyP with CT-DNA, which UV-vis spectroscopy indicated involved intercalative binding. The desorption kinetics were also simulated using complementary finite element modeling of convection-diffusion coupled to a surface process.

1. Introduction The ability to probe the region in the vicinity of surfaces and interfaces is important for understanding and optimizing a myriad of interfacial processes, including chemical binding and transformation reactions in environmental molecular science,1,2 heterogeneous catalysis,3,4 adsorption in chromatography,5 and transport in cell membranes.6 Despite the importance of kinetic measurements in understanding the solid/liquid interface, relatively few techniques are available to probe chemical reaction kinetics at dielectric (nonconductive) interfaces.7,8 To reveal the mechanisms involved in these interfacial processes at the molecular level, it is essential to use nondestructive methods that can probe events that occur near the surface. Temperature jump,9 pressure jump,10 and stopped flow methods11 have been applied *Corresponding author: Tel þ44 (0) 2476 523264; Fax þ44 (0) 2476 524112; e-mail [email protected]. (1) Usher, C. R.; Michel, A. E.; Grassian, V. H. Chem. Rev. 2003, 103, 4883– 4940. (2) Al-Abadleh, H. A.; Mifflin, A. L.; Musorrafiti, M. J.; Geiger, F. M. J. Phys. Chem. B 2005, 109, 16852–16859. (3) Duchateau, R. Chem. Rev. 2002, 102, 3525–3542. (4) Ebitani, K.; Fujie, Y.; Kaneda, K. Langmuir 1999, 15, 3557–3562. (5) Wirth, M. J.; Swinton, D. J. Anal. Chem. 1998, 70, 5264–5271. (6) (a) Xu, X.-H.; Yeung, E. S. Science 1998, 281, 1650–1653. (b) Grime, J. M. A.; Edwards, M. A.; Rudd, N. C.; Unwin, P. R. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 14277–14282. (7) Unwin, P. R. J. Chem. Soc., Faraday Trans. 1998, 94, 3183–3195. (8) Hansen, R. L.; Harris, J. M. Anal. Chem. 1998, 70, 4247–4256. (9) (a) Ren, F. Y.; Harris, J. M. Anal. Chem. 1996, 68, 1651–1657. (b) Climent, V.; Coles, B. A.; Compton, R. G. J. Phys. Chem. B 2002, 106, 5988–5996. (10) (a) Hachiya, K.; Sasaki, M.; Ikeda, T.; Mikami, N.; Yasunaga, T. J. Phys. Chem. 1984, 88, 27–31. (b) Wu, C. H.; Lin, C. F.; Lo, S. L.; Yasunaga, T. J. Colloid Interface Sci. 1998, 208, 430–438. (c) Liu, C.; Huang, P. M. Geoderma 2001, 102 1–25. (11) (a) Ikeda, T.; Nakahara, J.; Sasaki, M.; Yasunaga, T. J. Colloid Interface Sci. 1984, 97, 278–283. (b) Taniguchi, M.; Kaneyoshi, M.; Nakamura, Y.; Yamagishi, A.; Iwamoto, T. J. Phys. Chem. 1990, 94, 5896–5900. (c) Strelow, F.; Henglein, A. J. Phys. Chem. 1995, 99, 11834–11838. (d) Bujalski, R.; Cantwell, F. F. Anal. Chem. 2006, 78, 1593–1605. (12) (a) Ludes, M. D.; Wirth, M. J. Anal. Chem. 2002, 74, 386–393. (b) Ludes, M. D.; Anthony, S. R.; Wirth, M. J. Anal. Chem. 2003, 75, 3073–3078.

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to suspensions of solid materials in solution, and fluorescence imaging12 and total internal reflection spectroscopy8,13 have been used to study adsorption/desorption at macroscopic interfaces. Probing adsorption/desorption kinetics can also be accomplished with evanescent wave cavity ring-down spectroscopy (EWCRDS),14,15 which uses the evanescent field formed during total internal reflection at a solid/liquid interface as an optical probe. The exponential decay of the electric field amplitude with distance from the interface, which occurs over a distance of a few hundred nanometers, makes this technique very sensitive to the interfacial region. This, coupled with the inherent sensitivity of cavity ringdown spectroscopy over other optical methods, has made EWCRDS a powerful technique for probing interfacial adsorption and other processes. In particular, EW-CRDS has the capability to provide sensitive kinetic information, and it is this property of which we take advantage in the present study.15-26 (13) McCain, K. S.; Schluesche, P.; Harris, J. M. Anal. Chem. 2004, 76, 930– 938. (14) Van der Sneppen, L.; Ariese, F.; Gooijer, C.; Ubachs, W. Annu. Rev. Anal. Chem. 2009, 2, 13–35. (15) Pipino, A. C. R.; Hudgens, J. W.; Huie, R. E. Rev. Sci. Instrum. 1997, 68, 2978–2989. (16) Pipino, A. C.R.; Hudgens, J. W.; Huie, R. E. Chem. Phys. Lett. 1997, 280, 104–112. (17) Li, F.; Zare, R. N. J. Phys. Chem. B 2005, 109, 3330–3333. (18) Shaw, A. M.; Hannon., T. E.; Li, F.; Zare, R. N. J. Phys. Chem. B 2003, 107, 7070–7075. (19) Everest, M. A.; Black, V. M.; Haehlen, A. S.; Haveman, G. A.; Kliewer, C. J.; Neill, H. A. J. Phys. Chem. B 2006, 110, 19461–19468. (20) Fan, H.-F.; Hung, C.-Y.; Lin, K.-C. Anal. Chem. 2006, 78, 3583–3590. (21) Fan, H.-F.; Li, F.; Zare, R. N.; Lin, K.-C. Anal. Chem. 2007, 79, 3654–3661. (22) Mazurenka, M.; Wilkins, L.; Macpherson, J. V.; Unwin, P. R.; Mackenzie, S. R. Anal. Chem. 2006, 78, 6833–6839. (23) Mazurenka, M.; Hamilton, S. M.; Unwin, P. R.; Mackenzie, S. R. J. Phys. Chem. C 2008, 112, 6462–6468. (24) Schnippering, M.; Powell, H. V.; Zhang, M.; Macpherson, J. V.; Unwin, P. R.; Mazurenka, M.; Mackenzie, S. R. J. Phys. Chem. C 2008, 112, 15274–15280. (25) Powell, H. V.; Schnippering, M.; Mazurenka, M.; Macpherson, J. V.; Mackenzie, S. R.; Unwin, P. R. Langmuir 2009, 25, 248–255. (26) Ruth, A. A.; Lynch, K. T. Phys. Chem. Chem. Phys. 2008, 10, 7098–7108.

Published on Web 01/27/2010

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This paper considers the kinetics of porphyrin adsorption to silica surfaces and the interaction of DNA with the resulting chemically functionalized surface. The silica-aqueous interface is of particular importance in geochemistry2,27 and separation science,5 and understanding how organic molecules, such as porphyrins, interact with this surface provides valuable information on the intrinsic chemical activity of silica. Porphyrins are heterocyclic organic rings containing four pyrrole subunits linked via methine bridges. They have been investigated extensively in various fields, for example, as potent antiviral and antitumor therapeutic agents,28,29 for specific sensing of DNA sequences,30 and as photosensitizers in artificial solar energy systems.31,32 These, and other applications, arise because porphyrins exhibit strong absorption bands in the visible region, possess interesting nonlinear optical properties,33 and are relatively easy to synthesize, and their physicochemical properties can be tuned through careful choice of peripheral substituents and inserted metal ions.34 In the past decade, the interest in supramolecular interactions of water-soluble cationic porphyrins like 5,10,15,20-tetra(Nmethylpyridinium-4-yl)porphyrin (TMPyP, see Figure 1 for chemical structure) with biological molecules has increased steadily, as a consequence of the desire to understand the structure and functions of biomacromolecules.34-37 The binding of a watersoluble porphyrin to nucleic acids has been studied in solution with a variety of spectroscopic methods, including resonance Raman (RR),38 UV-vis spectroscopy, circular dichroism (CD),39 and fluorescence.40 Resonance light scattering (RLS) spectra have also been reported.41 There have been relatively few studies investigating the binding behavior at surfaces. Lei et al.42 determined the rate constant for the binding of FeTMPyP to a surfaceimmobilized salmon testis double-stranded DNA-polymer film using a quartz crystal microbalance. Naue et al.43 studied the binding of a tetrapyridylporphyrin to surface-bound CT-DNA using surface plasmon resonance (SPR). In the present work, we have used EW-CRDS to measure the kinetics of TMPyP adsorption at the silica-water interface under well-defined flow conditions using an impinging jet cell to deliver solution to the interface. Complementary finite element modeling (27) Bancroft, G. M.; Brown Jr., G. E.; Hochella Jr., M. F.; Bunker, B.; Casey, W. H.; Davis, W. H.; Davis, J. A.; Kent, D. B.; Hering, J. G.; Stumm, W.; Hyland, M. M.; Lasaga, A. C.; Nancollas, G.; Zhang, J.-W.; Parks, G. A.; Schindler, P. W.; Sposito, G.; Waite, T. D.; White, A. F. Mineral-Water Interface Geochemistry. In Reviews in Mineralogy; Hochella, M. F., White, A. F., Eds.; Mineralogical Society of America Series 23; Mineralogical Society of America: Washington, DC, 1990. (28) Marzilli, L. G. New J. Chem. 1990, 14, 409–420. (29) Villanueva, A.; Juarranz, A.; Diaz, V.; Gomez, J.; Canete, M. P. AntiCancer Drug Des. 1992, 7, 297–303. (30) Eggleston, M. K.; Crites, D. K.; McMillin, D. R. J. Phys. Chem. A 1998, 102, 5506–5511. (31) Butler, W. L. Acc. Chem. Res. 1973, 6, 177–184. (32) Kay, A.; Gratzel, M. J. Phys. Chem. 1993, 97, 6272–6277. (33) McMillin, D. R.; Shelton, A. H.; Bejune, S. A.; Fanwick, P. E.; Wall, R. K. Coord. Chem. Rev. 2005, 249, 1451–1459. (34) (a) Senge, M. O.; Fazekas, M.; Notaras, E. G. A.; Blau, W. J.; Zawadzka, M.; Locos, O. B.; Ni Mhuircheartaigh, E. M. Adv. Mater. 2007, 19, 2737–2774. (b) de la Torre, G.; Vazquez, P.; Agullo-Lopez, F.; Torres, T. Chem. Rev. 2004, 104, 3723– 3750. (35) Kim, M. Y.; Gleason-Guzman, M.; Izbicka, E.; Nishioka, D.; Hurley, L. H. Cancer Res. 2003, 63, 3247–3256. (36) Wei, C.; Jia, G.; Yuan, J.; Feng, Z.; Li, C. Biochemistry 2006, 45, 6681–6691. (37) Parkinson, G. N.; Ghosh, R.; Neidle, S. Biochemistry 2007, 46, 2390–2397. (38) Blom, N.; Odo, J.; Nakamoto, K.; Strommen, D. P. J. Phys. Chem. 1986, 90, 2847–2852. (39) Sehlstedt, U.; Kim, S. K.; Carter, P.; Goodisman, J.; Vollano, J. F.; Norden, B.; Dabrowiak, J. C. Biochemistry 1994, 33, 417–426. (40) Jasuja, R.; Jameson, D. M.; Nishijo, C. K.; Larsen, R. W. J. Phys. Chem. B 1997, 101, 1444–1450. (41) Pasternack, R. F.; Bustamante, C.; Collings, P. J.; Giannetto, A.; Gibbs., E. J. J. Am. Chem. Soc. 1993, 115, 5393–5399. (42) Lei, J.; Ju, H.; Ikeda, O. Electrochim. Acta 2004, 49, 2453–2460. (43) Naue, J. A.; Toma, S. H.; Bonacin, J. A.; Araki, K.; Toma, H. E. J. Inorg. Biochem. 2009, 103, 182–189.

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Figure 1. Chemical structure of TMPyP.

of the flow dynamics coupled with solution of the convection-diffusion/binding process provided considerable kinetic insight. While a monolayer of TMPyP was readily formed at the quartz surface, this was found to be desorbed when exposed to solutions of CT-DNA. The desorption process could again be readily followed in real time using EW-CRDS. In this configuration, the surface-immobilized TMPyP serves as an indicator for CT-DNA and represents an interesting possible sensor format for DNA analysis generally.

2. Materials and Methods 2.1. Materials. All chemicals were used as received. These were sodium phosphate dibasic (SigmaUltra, minimum 99.0%, Sigma-Aldrich), sodium phosphate monobasic monohydrate (98.0-102.0% from Sigma-Aldrich), methyl alcohol (99.9% from Acros), poly-L-lysine hydrobromide (mol wt 30 000-70 000 from Sigma), and sodium chloride (99þ% from Aldrich). Ultrapure water was obtained from a Milli-Q plus 185 system from Millipore (resistivity of 18.2 MΩ cm at 25 °C). 5,10,15,20-Tetrakis(1-methyl-4-pyridinio)porphyrin tetra(ptoluenesulfonate) (TMPyP 3 TTS) was purchased from Fluka (BioChemika, for fluorescence, g95% (N)) and used without further purification. Porphyrin stock solution was made by dissolving the solid TMPyP 3 TTS in phosphate buffer solution (PBS, 1 mM, pH 7.4.) and the resulting solution was stored at 4 °C in the dark to prevent photodegradation. Concentrations of the TMPyP solutions were determined via spectrophotometry at a wavelength of 422 nm, using an extinction coefficient, ε422, of 2.26  105 M-1 cm-1.44 Deoxyribonucleic acid (DNA) sodium salt from calf thymus (CT) was obtained from Sigma (Type I fibers). This highly polymerized DNA (prepared from male and female calf thymus tissue) contains both double-stranded and single-stranded forms. The single-to-double-stranded DNA ratio in PBS was determined to be 19:81 by the analysis of the experimental DNA melting curve (data not shown). The molecular weight was stated to be between 10 and 15 million Da. DNA from calf thymus is 41.9 mol % guanine-cytosine and 58.1 mol % adenine-thymine.45 To prepare the CT-DNA stock solution, CT-DNA was dissolved in PBS at 4 °C for 48 h with occasional stirring to ensure the formation of a homogeneous solution. Concentrations, expressed in moles of base pairs per liter, were deduced spectrophotometrically using an ε260 = 1.32  104 M-1 cm-1 for CT-DNA.46 (44) Kalyanasundaram, K.; Neumann-Spallart, M. J. Phys. Chem. 1982, 86, 5163–5169. (45) Marmur, J.; Doty, P. J. Mol. Biol. 1962, 5, 109–118. (46) Pasternack, R. F.; Gibbs, E. J.; Villagranca, J. J. Biochemistry 1983, 22, 2406–2414.

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Figure 2. Schematic of the experimental apparatus for EW-CRDS. Light from a broadband diode laser (λ = 405 nm, 1 nm fwhm, 5 kHz) is injected into a ring cavity formed by two high reflectivity mirrors and the total internal reflection surface of a right-angled prism. The exponential decay of the light intensity in the cavity after extinguishing the light source is recorded using a photomultiplier tube (PMT). A Teflon nozzle arranged perpendicular to the TIR interface directly over the area probed by the evanescent field is used to deliver solution to the interface in a controlled manner.

In all experiments, the porphyrin and DNA solutions were freshly prepared from stock solutions before spectral analysis and were protected from light until they were added into the cell compartments. All measurements were at room temperature (20 ( 1 °C). 2.2. UV-vis Absorption Measurements. UV-vis absorption experiments were performed on a Lambda 25 UV-vis spectrometer (Perkin-Elmer Instruments) using a 1 cm quartz cuvette. The effect of NaCl concentration on porphyrin adsorption from PBS (1 mM, pH 7.4) to silica (glass vial) surfaces was monitored at different time scales by UV-vis spectroscopy. In these experiments, each of the porphyrin solutions with different NaCl content was prepared in a glass vial (25.43 mm diameter) and incubated for a certain time and then added to the quartz cuvette for immediate UV-vis analysis of the solution concentration. In addition, the equilibrium binding interaction of TMPyP with CT-DNA in solution was studied via UV-vis absorption measurements. 2.3. EW-CRDS Apparatus. Kinetic measurements of TMPyP adsorption to silica prism surfaces and the interaction of CTDNA with the resulting TMPyP-modified surface were performed with an EW-CRDS configuration similar to that described in detail elsewhere24 and is shown schematically in Figure 2. Two high-reflectivity concave mirrors (R = 99.997% at 405 nm, Los Gatos Research) with radii of curvature of 1 m and a 90° fused silica bending prism (CVI) formed a stable triangular ring cavity. The output of a pulsed diode laser (Power Technology Inc., 405 nm, 50 mW maximum output) operating at 5 kHz (TTi pulse generator, TGP110) was introduced into the cavity through the front mirror using a 5 μs pulse (2 ns rise/fall time), sufficient for the light intensity in the cavity to achieve a steady value. The light was then switched off, and the decay in the light intensity circulating within the cavity was monitored. Optical extinction at the prism/solution interface as well as mirror transmission losses, losses from the coated prism surfaces, and scattering within the prism result in an exponential decay in the light intensity, which is characterized by the ring-down time (the time taken for the intracavity light level to decrease by a factor 1/e). The light intensity in the cavity was measured via specular reflection at the incident prism surface using a photomultiplier tube (PMT, 4006 DOI: 10.1021/la903438p

Electron Tubes, 9781B). The PMT signal was digitized on a 12-bit 400 MS/s oscilloscope card (Gage CS12400), and the cavity ringdown time from each transient was calculated with the fast Fourier transform method47 using custom-written LabVIEW software. Typical ring-down times were between 50 and 600 ns. The interface of interest is probed by absorption/scattering within the evanescent field established within solution during the total internal reflection (TIR) of light at the hypotenuse of the prism. The area probed was estimated as 8 mm2. The other two surfaces of the prism were coated with a standard antireflection coating (AR, R < 0.5%, = 350-532 nm for normal incidence) to minimize optical losses. The total length of the cavity was 56.4 cm, and the penetration depth of the evanescent field in the geometry used was calculated to be 242 nm using the angle of incidence at the TIR interface, which was 68.4°.48 This angle is close to, but greater than, the critical angle, 66°, for the silica-water interface. A short cylinder of Plexiglas (inner diameter 1.5 cm) was secured to the prism hypotenuse to form a cell and allow the prepared solutions to be flowed over the prism. The cell was adhered to the prism surface with the use of contact pressure and an O-ring machined into the bottom of the Plexiglas cell wall. This setup was employed both for studies of porphyrin adsorption and the subsequent influence of CT-DNA on the resulting TMPyPmodified silica surface. Controlled flow conditions were achieved with a syringe pump (KD Scientific) connected to a Teflon nozzle (inner diameter: 2 mm; outer diameter: 8 mm) positioned directly over the prism surface in the region probed by EW-CRDS (see Figure 2). The separation between the end of the nozzle and the silica surface was controlled in three dimensions using a micropositioner and was typically located a few hundred micrometers above the interface. A syringe of 100 mL volume (diameter 3.26 cm) was used for the kinetic study of TMPyP adsorption at the silica-water interface, while that used for the effect of CT-DNA on the TMPyP-silica surface was 10 mL (diameter 1.47 cm). The former gave volume flow rates up to 10.6 mL min-1 and the latter (47) (a) Mazurenka, M.; Wada, R.; Shillings, A. J. L.; Butler, T. J. A.; Beames, J. M.; Orr-Ewing, A. J. Appl. Phys. B: Lasers Opt. 2005, 81, 135–141. (b) Everest, M. A.; Atkinson, D. B. Rev. Sci. Instrum. 2008, 79, 023108. (48) de Fornel, F. Evanescent Waves: From Newtonian Optics to Atomic Optics; Springer-Verlag: Berlin, 1997.

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up to 2.2 mL min-1. A pipe connected to the top of the cell ensured the solution did not overflow onto the prism. Previous work has shown that flow under similar conditions is well-defined.49 For adsorption studies, all the containers and tubing were presaturated by flowing through them a solution containing 300 μM TMPyP in PBS (1 mM, pH 7.4). This prevented any decrease of TMPyP concentration due to adsorption to the walls of the containers, which was found to occur with the low concentrations of TMPyP used if this precaution was not taken. With this precaution, the decrease in the porphyrin concentration as measured by UV-vis spectroscopy after flowing through all the containers was less than 5%.

2.4. EW-CRDS Interfacial Absorbance Measurements. Before each experiment, the prism surface was cleaned in an oxygen plasma asher for 3 min at 100 W (Emitech, K1050X) with the antireflection-coated surfaces protected. The prism surface was then wiped with methanol five times, and the cell was carefully assembled on the prism surface. The optical cavity was then aligned so as to maximize the ring-down lifetime, τ0 (typically ca. 400-600 ns) with 1 mM PBS present within the cell. Different concentrations of TMPyP solution, typically of 2.9, 2.0, 1.6, and 0.5 μM, were prepared in PBS (1 mM, pH = 7.4), drawn into the glass syringe, and then added to the cell by a syringe-driven constant flow. Adsorption of TMPyP at the silica/solution interface causes a reduction in the characteristic ring-down time (to τ) as a result of the additional cavity losses arising from the adsorbate. The absolute interfacial absorbance per pass through the sample, A, was calculated using20   L 1 1 A ¼ 0:4343 c τ τ0

3. Simulations and Modeling 3.1. TMPyP Adsorption Kinetics. The kinetics of porphyrin adsorption at the silica-water interface and the CT-DNA interaction with a TMPyP-modified surface were modeled using the commercially available software package, COMSOL Multiphysics 3.5.50 Specifically, the finite element method was used to solve for flow through the pipe coupled to the convection-diffusion equation for the solution and the chemical process occurring at the silica-water interface. The fluid flow was assumed to be axially symmetric in the region under the nozzle. The geometry for the fluid flow problem is shown in Figure 3a. In order to obtain the velocities, u and v, in the r and z directions, respectively, the time-independent incompressible Navier-Stokes equation was solved at each mesh point in the domain shown in Figure 3a. No-slip boundary conditions were applied to the walls of the pipe and the silica surface, and the following boundary conditions applied at the inlet of the pipe:

ð1Þ

where L is the cavity length (56.4 cm in this setup) and c is the speed of light. The factor 0.4343 (= log10{e}) converts the absorbance to the conventional decadic logarithmic scale. The effect of ionic strength on the adsorption of TMPyP at the silica-water interface was investigated by EW-CRDS at a chosen concentration of 1.2 μM TMPyP in 1 mM PBS, with different NaCl content: 0, 0.05, 0.2, and 0.6 M. For these experiments, a 0.5 mL solution volume of TMPyP with each different NaCl concentration was quickly added into the cell with a pipet, and the interfacial absorbance monitored during adsorption from static solution. 2.5. Bulk Solution Absorbance. The bulk optical absorbance by the buffer within the evanescent wave region at 405 nm was obtained by EW-CRDS for 1 mM PBS at pH = 7.4 above a bare silica prism. Additionally, the bulk absorbance due to nonadsorbed TMPyP was determined by measuring the EWCRDS signal for 7.38 μM of TMPyP in 1 mM PBS (pH = 7.4) with a poly-L-lysine (PLL)-modified prism surface and subtracting the absorbance due to 1 mM PBS on a PLL-modified prism alone. The latter, positively charged functionalized surface, which inhibits the adsorption of the porphyrin, was formed by coating 0.2 mL of PLL (1 mg mL-1 in 1 mM PBS) on a clean fused silica prism for 30 min, rinsing with 1 mM PBS, and then drying with a stream of nitrogen gas.

2.6. Interaction of CT-DNA with TMPyP-Modified Surfaces. TMPyP-coated silica surfaces were produced by drop-

ping 0.5 mL of 1.2 μM TMPyP (in 1 mM PBS, pH = 7.4) onto clean fused silica prisms as the adsorption behavior was monitored via the EW-CRDS signal. After ∼45 s, monolayer adsorption was achieved, the solution of TMPyP was removed, and the prism was washed five times with 0.5 mL of 1 mM PBS. During the washing, no noticeable change in the interfacial absorbance was observed, indicating that the TMPyP layer is irreversibly adsorbed. The EW-CRDS signal was then recorded while a (49) Rudd, N. C.; Cannan, S.; Bitziou, E.; Ciani, L.; Whitworth, A. L.; Unwin, P. R. Anal. Chem. 2005, 77, 6205–6217.

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CT-DNA solution, at known concentration and ionic strength, was introduced into the cell, using the flow system described, at a flow rate of 1 mL min-1 and nozzle-to-prism separation of 500 μm. The effect of CT-DNA on the TMPyP surface was further investigated by quickly adding 0.5 mL of CT-DNA solution (in 1 mM PBS with 10 mM NaCl, pH = 7.4) into the cell with a pipet, while monitoring the desorption of TMPyP via the EW-CRDS signal.

u ¼ 0,

! 2vf r2 1- 2 , v ¼ πrin 2 rin

z ¼ H,

0ererin

ð2Þ

where vf is the flow rate in m3 s-1 and rin is the pipe inner radius. There was assumed to be no pressure at the outlet (r = rout, 0 e z e d). The use of eq 2 assumes fully developed laminar (Poiseuille) flow in the pipe (before the outlet) and is reasonable for the flow rates and the nozzle used (diameter 2 mm; length 5 cm). The solution of the convection-diffusion equation for the concentration of porphyrin, cp, in the adsorption model was obtained in the subdomain shaded in gray in Figure 3a. No-flux boundary conditions were applied to the pipe walls, and a convective flux boundary condition was applied to the outlet (r = w, 0 e z e d). The boundary condition at the solution inlet (z = h, 0 e r e rin) was that the solution concentration was equal to that of bulk, cp = cp*. Previous studies on the adsorption of porphyrins at the silicawater interface did not determine any preferred orientation for surface adsorbed porphyrin molecules under similar conditions to those used here.51 In addition, since there are also likely to be significant surface-coverage-dependent adsorbate-adsorbate interactions as well as surface potential effects from the charged interface and charged adsorbate,52,53 we use an Elovich-type equation to model the adsorption process which can be considered to incorporate these various factors in a semiempirical manner. The Elovich equation has proven successful in describing the kinetics of a wealth of adsorption and desorption processes,54 including adsorption at heterogeneous surfaces55,56 and the (50) http://www.comsol.com/. (51) Bos, M. A.; Werkhoven, T. M.; Kleijn, J. M. Langmuir 1996, 12, 3980–3985. (52) Burt, D. P.; Cervera, J.; Mandler, D.; Macpherson, J. V.; Manzanares, J. A.; Unwin, P. R. Phys. Chem. Chem. Phys. 2005, 7, 2955–2964. (53) Slevin, C. J.; Unwin, P. R. J. Am. Chem. Soc. 2000, 122, 2597–2602. (54) Low, M. J. D. Chem. Rev. 1960, 60, 267–312. (55) Jaroniec, M. J. Catal. 1979, 57, 187–190. (56) Rudzinski, W.; Panczyk, T. Surfactant Sci. Ser. 1999, 78, 355–389.

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irreversible adsorption model was justified by the fact that silica surfaces modified by TMPyP could be rinsed with water with negligible detectable desorption (as evidenced by EW-CRDS). 3.2. Modeling DNA-Assisted TMPyP Desorption. To obtain the rate constant for DNA-assisted TMPyP desorption from the silica surface, a similar model to that described above was adopted, except that the convection-diffusion equation was solved for the concentration of DNA base pairs in solution, cbp, and the boundary condition at the prism surface was changed to describe the removal of porphyrin from the prism surface by CTDNA. For similar reasons as for the model above, the following Elovich-type boundary condition was applied at the prism surface: DDNA

0 Dcbp ¼ -k0 cbp Nθeλ θ Dz

0 dθ ¼ -k0 cbp θeλ θ dt

ð4aÞ ð4bÞ

where DDNA is the diffusion coefficient of the CT-DNA (calculated to be 1.2  10-8 cm2 s-1 according to the literature61), λ0 is a parameter which defines the surface-coverage dependence of the desorption coefficient value, and k0 is the desorption constant. The parameters N and θ again refer to the coverage of the silica surface by TMPyP. The initial conditions were θ = 1 and cbp = cbp*. An example flow profile and a concentration profile derived from these simulations are shown in parts b and c of Figure 3, respectively, after 1 s for a flow rate of 5 mL min-1 and nozzle-toprism separation of 500 μm for the TMPyP adsorption process, with [TMPyP] = 1 μM and k = 0.035 cm s-1. Note that since the nozzle outer diameter is larger than the footprint of the laser at the TIR interface, we assume the flux profile in the region probed by EW-CRDS is fairly uniform. This was a deliberate consequence of the nozzle design, which was such that the stagnation zone of the impinging jet was probed where mass transport to the interface was reasonably uniform.62

4. Results and Discussion

where Dp is the diffusion coefficient of porphyrin in solution, θ is the fractional surface coverage of porphyrin, N is monolayer surface coverage of porphyrin, λ is a factor describing the change in the apparent adsorption rate coefficient with surface coverage (for θ = 0), and k is the initial adsorption rate constant. The initial conditions were θ = 0 and cp = cp*. The adoption of an

4.1. Solution Properties of TMPyP, CT-DNA, and the TMPyP-(CT-DNA) Complex. UV-vis spectroscopy was employed to identify the solution properties of TMPyP and related complexes. Optical absorption spectra were recorded over the wavelength range 350-800 nm for TMPyP for concentrations between 5  10-7 and 1  10-5 M in 1 mM PBS at pH 7.4 (Figure 4). The Soret band appears at 422 nm, and the maximum obeyed the Beer-Lambert law (Figure 4 inset) in that there was no deviation from linearity, which indicates that there was no selfaggregation of the porphyrin. From the slope of the inset in Figure 4, the extinction coefficient of TMPyP was determined to be ε422 nm = 2.26  105 M-1 cm-1, in agreement with previous findings.44 The extinction coefficients and positions of the other four peaks were also in agreement with the literature, indicating the purity of the sample. The purity of DNA samples is commonly assessed by comparing the ratio of the optical absorbances at 260 and 280 nm.63 A CT-DNA solution of 0.05 mg mL-1 in 1 mM PBS gave a ratio of 1.84:1 (see inset of Figure 5), indicating there was no protein

(57) Elkhatib, E. A.; Mahdy, A. M.; Saleh, M. E.; Barakat, N. H. Int. J. Environ. Sci. Technol. 2007, 4, 331–338. (58) Hellal, F. A.; Amer, A. K.; Zaghloul, A. M. J. Appl. Biol. Sci. 2008, 2, 79–86. (59) Nafiu, A.; Agbenin, J. O.; Raji, B. A. Soil Sci. 2008, 173, 837–844. (60) Yang, Y.; Liang, L.; Wang, D. J. Environ. Sci. 2008, 20, 1097–1102.

(61) Nkodo, A. E.; Garnier, J. M.; Tinland, B.; Ren, H.; Desruisseaux, C.; McCormick, L. C.; Drouin, G.; Slater, G. W. Electrophoresis 2001, 22, 2424–2432. (62) Bitziou, E.; Rudd, N. C.; Edwards, M. A.; Unwin, P. R. Anal. Chem. 2006, 78, 1435–1443. (63) Sambrook, J.; Russell, D. Molecular Cloning: A Laboratory Manual, 3rd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 2001.

Figure 3. (a) Geometry used for simulating adsorption and desorption using the pipe-flow system. (b) Velocity profile from the simulation with flow rate of 5 mL min-1. (c) Typical concentration profile from the simulation for a porphyrin concentration of 1 μM, k = 0.035 cm s-1, and λ = 3 after 1 s.

desorption of ions from soil samples.57-60 The boundary condition at the prism surface for the adsorption model was Dcp ¼ -kcp ð1 -θÞe -λθ Dz dθ ¼ kcp ð1 -θÞe -λθ N dt

Dp

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ð3aÞ ð3bÞ

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For intercalation of TMPyP with CT-DNA, the hydrophobic effect, the van der Waals interactions with the DNA bases, and Coulombic interactions-involving the positively charged pyridiniumyl substituents-all promote uptake.33 From the spectrum in Figure 5, the extinction coefficient ε435 of the TMPyP-(CTDNA) complex was calculated to be 9.81  104 M-1 cm-1. Finally, it is also clear from Figure 5 that at 405 nm (the wavelength for EW-CRDS measurements) there is a decrease by a factor of 3.35 in the absorbance of porphyrin upon DNA complexation. 4.2. EW-CRDS Bulk Absorbance Measurements. In general, the optical absorbance, A, recorded using EW-CRDS will contain two contributions: Figure 4. UV-vis absorption spectra of TMPyP at different concentrations in 1 mM PBS at pH 7.4: 10, 5, 2.5, 1.8, 1.2, 0.8, and 0.5 μM (from top to bottom). The inset is the absorbance at the Soret band as a function of concentration for TMPyP over the concentration range between 0.5 and 10 μM.

Figure 5. Absorption spectra of 2.5 μM TMPyP without (black

line) and with (red line) CT-DNA of 0.025 mg mL-1 in 1 mM PBS (pH 7.4). The inset shows the absorbance of CT-DNA in 0.05 mg mL-1 in 1 mM PBS at pH 7.4.

contamination. The base pair concentration of CT-DNA was determined to be 5.5  10-5 M from absorbance measurements using ε = 1.32  104 M-1 cm-1 at the maximum near 260 nm.46 Assuming a molecular weight of (1-1.5)  107 g mol-1 (11-16.5 kbp), the molecular concentration of CT-DNA in this solution was calculated to be 3.3-5 nM. The absorption spectra of 2.5 μM TMPyP without (black line) and with (red line) 0.025 mg mL-1 CT-DNA (2.8  10-5 M base pairs concentration) are shown in Figure 5. A spectrophotometric titration showed that this is a sufficient excess of DNA to complex all of the available porphyrin under these conditions. To ensure that the TMPyP-(CT-DNA) system was at equilibrium, the spectrum was collected 5 min after adding the CT-DNA solution and was found to be time-invariant on longer time scales. The formation of the TMPyP-(CT-DNA) complex produces a 13 nm bathochromic shift of the Soret maximum from 422 to 435 nm and a hypochromic effect of 53%. This is consistent with intercalation of the porphyrin into the DNA base pairs.39,64 DNA groove binding is typically characterized by no (or minor) changes of the UV-vis spectrum, and outside binding to DNA is accompanied by a red shift in the Soret band, Δλ e 8 nm, and weak hypochromism (typically ΔH e 10% of the Soret band). (64) Lugo-Ponce, P.; McMillin, D. R. Coord. Chem. Rev. 2000, 208, 169–191.

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A ¼ As þ Ab

ð5Þ

where As is the absorbance due to surface adsorbed species and Ab is the absorbance from bulk solution. In order to determine As (and thus the surface concentration of adsorbed porphryin) from the EW-CRDS signal, it was necessary to subtract the bulk absorbance, Ab. To obtain Ab, the EW-CRDS signal was recorded for a solution of 7.38 μM TMPyP in 1 mM PBS (pH 7.4) on a PLL-modified surface. The TMPyP was expected not to adsorb to the PLL as a result of the strong electrostatic repulsion between the positively charged porphyrin molecules and the positively charged surface. This was confirmed by the morphology of the EW-CRDS transients, as shown in our previous work.25 When species are adsorbing to the prism surface, the EW-CRDS transient is time-dependent. In contrast, when no adsorption process occurs, there is a step-change in the ring-down time upon addition of the analyte to the prism surface. The absorbance measured was therefore expected to be solely due to TMPyP in the bulk and was expected to take the same value as absorbance due to TMPyP in the bulk over a bare-silica prism surface (concentration gradients over the two different surfaces are assumed to be negligible as the length of the diffuse layer is small compared to the effective path length probed, ∼1 μm). The bulk absorbance of 7.38 μM TMPyP was 4.9  10-4. The optical absorbance of TMPyP bulk solution followed the Beer-Lambert law, so all other bulk absorbances could be calculated accordingly, allowing As to be determined in subsequent measurements. 4.3. Adsorption of TMPyP to Silica Surfaces. Initial studies of the effect of NaCl on the adsorption of 1.2 μM TMPyP to silica (glass vial) surfaces from PBS (1 mM, pH 7.4) were carried out using UV-vis spectroscopy. For the results summarized in Figure 6a, all spectroscopic measurements were taken within 2 min of solution preparation in a glass vial and then transferred to a quartz cuvette. Even so, a strong electrolyte effect on the magnitude of the absorbance is evident, which can be attributed to different degrees of porphyrin adsorption from solution to the glass vial surface. As can be seen from the inset of Figure 6a, as the ionic strength increases, the TMPyP Soret band absorbance increases, as the adsorption of TMPyP on the vial surface is prevented and the concentration of TMPyP in solution does not change. UV-vis absorption spectra of 1.2 μM TMPyP (1 mM PBS, pH 7.4) were measured at three different time scales (2 min, 3 h, and 70 h) for 0, 50, and 200 mM NaCl. The relative absorbances at the Soret band for these spectra are displayed in Figure 6b. As the time increases from 2 min to 70 h, the absorbance of TMPyP dramatically decreases for each of the three different NaCl solutions, again showing that the adsorption of TMPyP to the silica (glass vial) surface can be effectively inhibited by increasing the concentrations of NaCl in solution. DOI: 10.1021/la903438p

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Figure 7. EW-CRDS signal for 1.2 μM TMPyP adsorption to silica at various NaCl concentrations in buffer (1 mM PBS, pH 7.4): 0, 50, 200, and 600 mM (from top to bottom).

Figure 6. (a) Effect of NaCl concentration on TMPyP adsorption. The UV-vis absorption spectra of 1.2 μM TMPyP in PBS (1 mM, pH 7.4) stored in a glass vial for 2 min with different NaCl concentrations (from top to bottom: 600, 200, 50, 10, 0 mM). The inset shows the dependence of the absorbance at 422 nm on the NaCl concentration. The increase in absorbance with increasing NaCl was attributed to Naþ competing with the porphyrin to bind at the glass surface. (b) Competitive binding of TMPyP and Naþ to a glass vial as a function of time and [NaCl].

To complement the above experiments, the effect of ionic strength on the adsorption of 1.2 μM TMPyP (1 mM PBS, pH 7.4) to the silica prism surface was also followed in situ by EWCRDS. To this end, solutions of TMPyP in different NaCl concentrations were dropped onto the prism surface and the EW-CRDS response was monitored in real time (see Figure 7). Adsorption of TMPyP was clearly inhibited by the presence of NaCl. Salt concentrations much higher than those of TMPyP (g50 mM NaCl) result in a lower concentration of surfaceadsorbed TMPyP and thus a lower value of As. This effect could be attributed to competitive binding between the Naþ ion and the TMPyP, or screening of the surface charge by the Naþ ion, or screening of the charge on the TMPyP ion by the Cl- ions. These data are qualitatively consistent with the UV-vis absorption data in Figure 6, but in Figure 7, the interfacial adsorption of TMPyP is measured directly, in situ and in real time (see second y-ordinate). The formation of a monolayer of TMPyP on the silica surface is controlled by the assembly of the individual TMPyP units in response to the surface charge and the ionic strength of the bulk solution. The silica has a surface potential at pH 7.4 of the order -75 mV.65 This potential arises from the dissociation of the silanol groups on the silica surface which leaves the surface partially charged. There are two types of groups with pKa of 4.5 (65) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and Applications, 2nd ed.; Wiley and Sons: New York, 2001.

4010 DOI: 10.1021/la903438p

and 8.5.15 At pH 7.4, 99.7% of [-SiOH] groups are deprotonated in solution, giving a -SiO- surface density of ∼0.93 nm-2 and hence a surface charge density of ∼-0.15 C m-2. TMPyP adsorption takes place onto this surface, but Naþ can influence the binding at these deprotonated sites. 4.4. Kinetics of TMPyP Adsorption. To investigate the kinetics of porphyrin adsorption from solution (1 mM PBS pH 7.4) to the silica-water interface, EW-CRDS combined with the flow cell system was used to monitor in situ the absorbance signal. The solution was introduced from the nozzle to the surface at a volume flow rate of 5 mL min-1 so that the rapid adsorpion process could be captured. Figure 8a shows the absorbance due to surface-adsorbed TMPyP for different concentrations of the bulk solution. As the concentration of TMPyP in solution increases from 0.5 to 2.9 μM, the adsorption rate increases markedly. For the larger concentrations, following a rapid initial increase, the TMPyP adsorption reaches a plateau within ca. 20 s, at which point the silica surface is saturated with TMPyP. The surface concentration of TMPyP (right-hand axis in Figure 8a) can be obtained using the extinction coefficient at 405 nm (ε405 nm = 8.71  104 M-1 cm-1), and the limiting value of the optical absorbance (∼0.0090) corresponds approximately to full monolayer coverage ((6.2 ( 0.4)  1013 molecules/cm2).51 The data in this figure are noisy at absorbances of around 0.009 due to deterioration of the antireflective coatings on the prisms used for these kinetic runs, decreasing the background ring-down time for this particular run to around 320 ns, which limited the maximum optical absorbance that can be measured. Despite this, the limiting monolayer coverage of 0.009 was consistent with EW-CRDS experiments performed on better quality prisms where TMPyP was simply dropped onto the prism surface rather than delivered using the impinging jet flow cell and previous reports.51 The charge on TMPyP (4þ, see Figure 1) prevents a second layer of molecules depositing on the first. In addition, the tetraphenyl moieties may prevent the stacking of TMPyP for multilayer adsorption. The initial adsorption rate constant for the attachment of TMPyP to a clean silica surface was k = (4.1 ( 0.6)  10-2 cm s-1, and the whole data set could be modeled satisfactorily for all surface coverages and solution concentrations with the Elovich model described earlier with λ = 3. To illustrate the sensitivity of the modeling to k, Figure 8b shows the data for 2.9 μM TMPyP adsorption with different k demonstrating that the kinetics can be resolved; i.e., the response is not simply mass transport controlled. The red line is the best fit to the data with k = 0.05 cm s-1 (λ = 3). The blue line shows the results of the simulation for k = 0.07 cm s-1 and λ = 3. The model was also sensitive to the parameter λ. Langmuir 2010, 26(6), 4004–4012

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Figure 9. EW-CRDS measurements of TMPyP-modified silica surface after exposure to solutions of CT-DNA with concentrations of 0.3 mg mL-1 (black), 0.1 mg mL-1 (red), and 0.05 mg mL-1 (green) in buffer (1 mM PBS, 10 mM NaCl, pH 7.4). In each case 0.5 mL of solution was dropped into the cell with a pipet.

Figure 8. (a) Typical EW-CRDS optical absorbance (and equivalent surface adsorption) transients and simulations for TMPyP at the silica-water interface at different concentrations in 1 mM PBS (pH 7.4): 2.9 μM (red), 2.0 μM (green), 1.6 μM (blue), and 0.5 μM (magenta). The data have been analyzed using the model outlined with k = (4.1 ( 0.6)  10-2 cm s-1 and λ = 3. (b) Data for 2.9 μM (red) TMPyP adsorption to the silica surface. The dotted line is the best fit to the data with k = 0.05 cm s-1 (λ = 3). The solid black lines show the results of the simulation for k = 0.07 cm s-1 and λ = 3 to demonstrate that finite kinetics can be resolved. In all cases, the flow rate was 5 mL min-1 and the nozzle-to-prism distance was 500 μm.

4.5. Interaction of DNA with TMPyP-Functionalized Silica Surfaces. Figure 9 shows how the EW-CRDS response changes when a TMPyP-functionalized silica surface is exposed to different concentration CT-DNA solutions. The absorbance due to surface-adsorbed TMPyP decreases significantly upon introduction of the DNA solution as a result of TMPyP desorption. It was demonstrated earlier (Figure 5) that a TMPyP-(CT-DNA) complex forms readily in solution, and this drives the desorption of TMPyP from the silica surface. The absorbance signal tends to zero at long times, indicating that the TMPyP-(CT-DNA) complex desorbs from the silica surface into the solution rather than binding of the DNA to the surface. This is probably due to the large electrostatic repulsion between the negatively charged CT-DNA66 and the silica surface. To confirm that no DNA remained on the surface, in situ AFM experiments were carried out which showed the surface remained clear (data not shown). To study the kinetics of DNA-induced porphyrin desorption in detail, the flow cell system was used to introduce CT-DNA solution with the flow rate of 1 mL min-1. The TMPyP-functionalized silica surface was again prepared as before, and the CTDNA solution was then flowed over this surface as the interfacial absorbance was monitored via the EW-CRDS signal. As shown in Figure 10, the desorption rate was, as expected, dependent on (66) Rodriguez-Pulido, A.; Aicart, E.; Llorca, O.; Junquera, E. J. Phys. Chem. B 2008, 112, 2187–2197.

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Figure 10. EW-CRDS measurements of a TMPyP-functionalized silica prism during exposure to different CT-DNA concentrations 0.3 mg mL-1 (blue), 0.2 mg mL-1 (green), 0.1 mg mL-1 (red), and 0.05 mg mL-1 (cyan) in buffer (1 mM PBS, 10 mM NaCl, pH 7.4). The flow rate was 1 mL min-1, and the nozzle-to-prism distance was 500 μm. The black lines are the fits to the model with k0 = (1.9 ( 0.1)  10-4 cm3 mol-1 s-1 with λ = 9.9 ( 0.6.

the concentration of CT-DNA with the surface coverage decreasing more rapidly as the DNA concentration increased from 0.05 to 0.3 mg mL-1. Using the model outlined earlier, the average desorption rate of TMPyP from the surface due to binding with CT-DNA was found to be k0 = (1.9 ( 0.1)  10-4 cm3 mol-1 s-1 with λ = 9.9 ( 0.6. The average was taken from the four data sets shown in Figure 10. From these parameters the initial binding rate constant was determined to be k0 = (4.3 ( 0.2) cm3 mol-1 s-1. These data indicate that the kinetics of the desorption process are strongly affected by the DNA concentration in solution and such an approach may be interesting for the quantitative analysis of DNA in solution.

5. Conclusions The adsorption kinetics of TMPyP at the silica-water interface (1 mM PBS, pH 7.4) has been probed using a fast flow impinging (confined) jet system and, on a clean surface, is characterized by a first order heterogeneous rate constant, k = (4.1 ( 0.6)  10-2 cm s-1. This has been achieved using a new methodology that combines the surface sensitivity of EW-CRDS with a fast flow delivery system for which mass transport can be readily calculated. The limiting value of the optical absorbance of adsorbed DOI: 10.1021/la903438p

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TMPyP corresponded approximately to full monolayer coverage (6.2  1013 molecules/cm2). Competitive adsorption of the TMPyP ion and Naþ has been observed at the silica/aqueous interface and quantified using both UV-vis spectroscopy and EW-CRDS. Further studies have investigated the effect of CT-DNA on a TMPyP-functionalized silica surface. Efficient DNA-assisted desorption of TMPyP from the silica surface was observed with an initial rate constant of 4.3 ( 0.2 cm3 mol-1 s-1. This suggests that TMPyP-functionalized surfaces, and related functionalized surfaces, coupled to a surface-sensitive spectroscopic technique such as EW-CRDS may be useful as a DNA sensor. The studies herein provide further evidence of the sensitivity (in terms of both time and surface concentration) of EW-CRDS as a technique for probing kinetics at condensed phase interfaces. Such processes are widespread, spanning chemistry, the life sciences, and materials science, but are often difficult to characterize. We anticipate that EW-CRDS will find increasing application in this area, particularly when combined with fluidic and microfluidic systems, electrochemical methods, and other means of controlling the delivery of analyte to an interface. It is important to point out that the silica prism used in this study can readily be modified, for example with lipid bilayers, polymer films, nanostructured materials, etc., which will greatly diversify

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the range of processes open to study. Furthermore, other cavity arrangements will expand the types of interfaces which can be studied to include, for example, liquid/liquid and liquid/gas environments. Dynamic processes on the time scale highlighted herein, or better, will be open to study. Acknowledgment. This work was supported by a Marie Curie Incoming International Fellowship (040126: “SECM-CRDS”) from the sixth Framework Programme on Research, Technological Development and Demonstration of European Commission (MZ). H.V.P. is grateful for a PhD studentship under the MOAC doctoral training centre (EPSRC). This work was funded by the UK Engineering and Physical Sciences Research Council under grant EP/C00907X and an Advanced Research Fellowship to S. R.M. The authors thank Prof. Julie Macpherson and Mathias Schnippering for helpful advice and Jonathan Armond at Warwick for performing the in situ AFM experiments. Some of the equipment used in this research was provided through Birmingham Science City: Innovative Uses for Advanced Materials in the Modern World (West Midlands Centre for Advanced Materials Project 2), with support from Advantage West Midlands (AWM) and part funded by the European Regional Development Fund (ERDF).

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