Evanescent Wave Cavity Ringdown Spectroscopy: A Platform for the

Jan 24, 2012 - 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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Technical Note pubs.acs.org/ac

Evanescent Wave Cavity Ringdown Spectroscopy: A Platform for the Study of Supported Lipid Bilayers Hayley V. Powell,† Michael A. O’Connell,† Meiqin Zhang,† Stuart R. Mackenzie,‡ and Patrick R. Unwin*,† †

Department of Chemistry, University of Warwick, Coventry CV4 7AL, United Kingdom Department of Chemistry, University of Oxford, Physical and Theoretical Chemistry Laboratory, Oxford OX1 3QZ, United Kingdom



S Supporting Information *

ABSTRACT: Evanescent wave cavity ringdown spectroscopy (EW-CRDS) is advocated as an approach for monitoring the formation of supported lipid bilayers (SLBs) on quartz substrates in situ and for the quantitative study of fast molecular adsorption kinetics at the resulting modified biomimetic surface. This approach is illustrated using SLBs of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). Complementary atomic force microscopy (AFM) and quartz crystal microbalance with dissipation (QCM-D) measurements confirm the formation of bilayers on quartz. The subsequent interaction of the porphyrin, 5,10,15,20-tetraphenyl-21H,23H-porphinep,p′,p″,p‴-tetrasulfonic acid tetrasodium hydrate (TPPS) with the cationic bilayer-modified silica surface has been studied using EW-CRDS combined with an impinging-jet to deliver analyte to the surface in a well-defined manner. The adsorption of TPPS to the bilayer was kinetically controlled and the adsorption rate constant was found to be 1.7 (±0.6) × 10−4 cm s−1 from finite element modeling of the jet hydrodynamics and associated convective− diffusion equation, coupled to a first-order surface process describing adsorption. These proof-of-concept studies provide a platform for the investigation of molecular processes at biomembranes using EW-CRDS for chemical species showing optical absorbance in the visible and ultraviolet range.

S

proteins into the SLB by forming proteoliposomes.15 Furthermore, the SLB formation process can be followed in situ which is valuable in verifying that a continuous SLB has been formed. Quartz crystal microbalance with dissipation (QCM-D) is a particularly well-established technique to follow the kinetics of SLB formation via the VFM.16−23 Using this technique, the two pathways of bilayer formation mentioned above have been identified.19 The QCM-D response to bilayer formation by each pathway is characteristic: a decrease in frequency which plateaus with little or no change in the dissipation (instantaneous rupture) or a decrease in frequency which is accompanied by an increase in dissipation, followed by an increase in frequency and decrease in dissipation (critical surface coverage pathway). Surface plasmon resonance (SPR),17,18 atomic force microscopy (AFM),24,19,20 attenuated total reflectance infrared spectroscopy,25 ellipsometry,20 and fluorescence17 have also been used to follow the formation of SLBs. Characterization of the SLBs, once formed, can be achieved by a wide-range of techniques, including neutron reflectivity and X-ray scattering12,26 and sum frequency vibrational spectroscopy.10 Scanning probe techniques, including spatially resolved mass spectrometry, have been used to determine lipid distribution in bilayers,27 and AFM13 can be used to characterize the topology of bilayers.

ince the first demonstration of a planar phospholipid bilayer by Mueller et al. in 1962,1 biomembrane mimics have received considerable attention for fundamental studies of biophysical processes. Solid-supported lipid bilayers (SLBs) are particularly attractive, as they constitute a robust platform, to which various techniques can be applied. They have consequently been used extensively as models for cellular membranes.2,3 A key aspect of research in this area is the ability to probe and confirm SLB formation and subsequently study dynamic processes. Herein, we demonstrate evanescent wave cavity ringdown spectroscopy (EW-CRDS) as a potentially powerful technique for addressing these aspects. We focus herein on SLBs formed using cationic phospholipids,4 which have received considerable attention due to their applicability to drug delivery,5 transfection,6 gene silencing,7 and the ability to form complexes with DNA.8 The methodology should, however, be widely applicable to many types of SLBs. Popular methods used to form SLBs include the vesicle fusion method (VFM),9 Langmuir−Blodgett and Langmuir− Schaeffer techniques10,11 and, to a lesser extent, spin coating12,13 and air bubble collapse.14 The principle behind the VFM is that, under appropriate conditions, small unilamellar vesicles (SUVs) of phospholipids in solution adsorb to a solid substrate and subsequently rupture (either instantly or once a critical surface coverage has been achieved) to form a bilayer on the substrate. The popularity of the VFM arises from its simplicity and because it is relatively straightforward to incorporate membrane © 2012 American Chemical Society

Received: November 5, 2011 Accepted: January 24, 2012 Published: January 24, 2012 2585

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However, the use of a single technique to both characterize the formation of SLBs and measure adsorption to the resulting surface is largely limited to QCM-D systems combined with flow modules. Even so, fast adsorption rates are not generally monitored via QCM-D; even when combined with ellipsometry, only mass-transport controlled adsorption has been monitored on the most rapid time scale available.28 Quantification of rapid binding kinetics of biologically relevant species to SLBs is an important aspect of SLB research. The ability to unambiguously prove SLB formation and measure rapid and sensitive adsorption kinetics of biologically relevant species to this surface in situ represents a powerful experimental configuration for SLB studies. EW-CRDS is a highly sensitive, and rapidly developing, technique for probing the solid/liquid interface and that has been used to provide kinetic information on a range of processes.29−42 Because the quartz prism surface inherent in EW-CRDS can be functionalized in different ways, a recent trend has been to diversify the range of interfaces open to study.35,36,43,44 Given that SLBs can readily be formed on quartz, it is natural to determine whether EW-CRDS is a potentially powerful probe of SLBs. We show that this is indeed the case and, significantly, that the EW-CRDS signature during SLB formation on quartz is highly characteristic. Furthermore, once the SLB is formed, the same setup can be used quickly and easily to follow physicochemical processes at the SLB, as demonstrated through studies of the binding kinetics of the porphyrin TPPS to the SLB. The approach described herein is thus potentially broad in scope for studying SLB processes, considering the wide range of tunable and diode lasers available and limited largely by the availability of high-quality mirrors in the deep UV range.45

UV−Visible Spectroscopy. UV−visible spectra of TPPS and DOTAP vesicles were measured using a Lambda 25 UV− visible spectrometer (Perkin-Elmer Instruments) using a 1 cm quartz cuvette. SUV Preparation. Part of the stock solutions of DOTAP was dried in a nitrogen stream and vacuum desiccated for at least 4 h to remove the chloroform. The lipids were then resuspended in buffer A to a concentration of 0.1 mg mL−1, sonicated for 2 min to ensure complete resuspension, freeze− thawed five times, before finally being extruded through 100 nm polycarbonate membranes (GC Technology). SUV solutions were used within 4 h of preparation. Monitoring SLB Formation Using EW-CRDS. The EWCRDS setup and impinging jet system used is shown schematically in Figure 2 and is described in detail elsewhere.38,42

EXPERIMENTAL SECTION Materials. 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) was purchased as the chloride salt from Avanti Polar Lipids, and stock solutions were prepared and stored in chloroform at −20 °C. 5,10,15,20-Tetraphenyl-21H, 23Hporphine-p,p′,p″,p‴-tetrasulfonic acid tetrasodium hydrate (TPPS, shown in Figure 1) was purchased from Sigma-Aldrich,

Figure 2. Schematic of the EW-CRDS apparatus with an integrated impinging jet flow system for washing and exchange of solution. PMT indicates photomultiplier tube.



In the present study a fast-modulated diode laser (Power Technology Inc., λ = 405 nm, maximum output 50 mW, spot size ∼1 mm diameter) was used to inject 5 μs light pulses into the optical cavity and the ringdown time, the time taken for the light within the cavity to fall to 1/e of its initial value, was measured. The background ring-down time, τ0, was recorded with buffer in the cell. The buffer was removed and the EWCRDS signal recorded as a function of time as 500 μL of SUV solution was dropped into the cell using a plastic pipet. The resulting SLB formed from the DOTAP solution was rinsed using the impinging jet setup. The nozzle−prism separation was chosen to be 500 ± 100 μm, giving well-defined hydrodynamics46 and ensuring that the nozzle did not penetrate the evanescent field (extending into the solution 60 s). The inset shows the response over a longer time during rinsing of the bilayer with buffer A using the flow cell, with rinsing commencing at t = 300 s. Cartoons shown not to scale. 2588

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on the adsorption rate for a bulk TPPS concentration of 0.5 μM was studied. Typical data are shown in Figure 7a, plotted as the

homogeneous and stable bilayer can be formed for EW-CRDS experiments with flow. TM-AFM Characterization of SLBs. To determine the quality of the bilayer that had formed upon adsorption of DOTAP to silica, complementary in situ TM-AFM experiments were performed. SLBs were formed on single crystal quartz by adsorbing SUVs from 100 μL of 0.1 mg mL−1 DOTAP in buffer A for 15 min and then rinsing the surface with 3 mL of buffer A. A representative in situ TM-AFM image of the surface is shown in Figure 6a, which is similar to the images obtained in previous

Figure 7. (a) Effect of flow rate on the adsorption of TPPS to the bilayer-modified prism surface. The data are shown as the optical absorbance for the interfacial excess after correction for bulk optical absorbance. Flow rates were 0.5 mL min−1 (red), 0.75 mL min−1 (green), and 1 mL min−1 (blue). (b) EW-CRDS response to the adsorption of TPPS to the bilayer with linear fits according to the model used (see text for values) for TPPS concentrations of 0.5 μM (black), 1 μM (red), 2 μM (green), 3 μM (blue), 4 μM (cyan), and 5 μM (orange).

interfacial optical absorbance due to the adsorbed TPPS vs time. Note that when using EW-CRDS to study adsorption processes,34,41 it is necessary to account for the contribution to the EW-CRDS signal from the optical absorbance by bulk solution (since the effective path length is ∼1 μm41). To determine this contribution, the EW-CRDS response was monitored upon the addition of TPPS to the cell. At pH 7.4, the silica surface is negatively charged28 meaning that the negatively charged TPPS would not be expected to adsorb significantly to silica on electrostatic grounds. To determine the absolute absorbance by TPPS in bulk, the initial ringdown time, τ0, with buffer A on the prism was first measured. The steady-state value of the optical absorbance with TPPS in buffer A in the EWCRDS setup was then measured. For example, the bulk absorbance for 5 μM TPPS was A = 3.2 × 10−4. The bulk absorbance scales linearly with concentration and represents a small correction. An alternative method to correct for the bulk was to recognize that the initial value of optical absorbance upon the introduction of TPPS flow is dominated by the bulk (rather than the surface) TPPS. Both methods of treating the bulk optical absorbance gave consistent data. The EW-CRDS response in Figure 7a shows only a small effect due to mass transport for the flow rates of 0.5, 0.7, and 1 mL min−1, indicating that the process is largely surfacecontrolled with these mass transport rates. The measurement of interfacial absorbance allows direct output of surface concentration, Γ, and it can be seen that TPPS accumulates on the surface linearly with time reaching a concentration of

Figure 6. (a) Typical 1 μm × 1 μm TM-AFM image of a bilayercovered quartz surface after the adsorption of 0.1 mg mL−1 DOTAP for 15 min. (b) TM-AFM image of the same area as shown in part a but after the surface had been deformed using contact mode AFM. Scale bars denote 200 nm.

reports.19 In all areas imaged, the surface was uniform with the exception of a few 1−2 nm pits and bulges, and there was no evidence of adsorbed vesicles. Since the surface morphology is very similar to that of the underlying silica, these images do not necessarily provide unambiguous information about SLB formation. Furthermore, the fluid nature of a SLB prevents complete and permanent removal of small areas of lipids from the surface. Hence, considerable mechanical perturbation of the SLB is required to disrupt the SLB. CM-AFM was thus also used to cause deformation of the surface, which was subsequently imaged in tapping mode. The change in morphology after such an experiment (Figure 6b) indicated that a deformable, thin layer was present on the surface. These observations, coupled with the characteristic EW-CRDS response and the QCM-D experiments in the Supporting Information, indicated that a SLB had, indeed, formed on the surface. EW-CRDS of TPPS on Silica and SLB-Modified Silica. We were first interested in observing whether there was any significant contribution from mass transport in the adsorption kinetics of TPPS at an SLB. To this end, the effect of flow rate 2589

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∼2.5 × 10−11 mol cm−2 at the end of the experiment. Note that the apparent noise on the values of interfacial absorbance (Γ) at long times is because the ring down time approaches the shortest measurable value. Figure 7b shows the bulk-corrected EW-CRDS response as a function of time. Studies were made with TPPS concentrations of 0.5, 1, 2, 3, 4, and 5 μM at a rate of 0.5 mL min−1. The second y-ordinate in Figure 7b is again the concentration of TPPS at the bilayer−solution interface calculated using the extinction coefficient, ε405 = 1.15 × 105 dm3 mol−1 cm−1, determined by UV−visible spectroscopy. The transients were again essentially linear with time for all concentrations supporting the assumption of pseudo first-order adsorption kinetics. The data were analyzed to deduce rate constants for each concentration giving 0.5 mM (k = 1.6 × 10−4 cm s−1), 1 mM (k = 2.8 × 10−4 cm s−1), 2 mM (k = 1.6 × 10−4 cm s−1), 3 mM (k = 1.4 × 10−4 cm s−1), 4 mM (k = 1.1× 10−4 cm s−1), and 5 mM (k = 1.5 × 10−4 cm s−1) for the binding of TPPS to the DOTAP SLB. This gives a mean rate constant of k = 1.7 (±0.6) × 10−4 cm s−1.

the MOAC doctoral training centre (EPSRC), M.A.O’C. for a Ph.D. studentship funded by Syngenta and the EPSRC, M.Z. for a Marie Curie Incoming International Fellowship (Grant 040126: “SECM-CRDS”), and S.R.M. for an Advanced Research Fellowship (EPSRC). The authors thank Tahani Bawazeer for advice and assistance on SLB formation. 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 partly funded by the European Regional Development Fund (ERDF).





CONCLUSIONS We have demonstrated EW-CRDS as a powerful technique for monitoring the formation of a SLB via the method of vesicle fusion and for the study of molecular adsorption kinetics at the resulting SLBs. Notably, the EW-CRS time signature is itself highly characteristic of the bilayer formation process. To confirm that SLBs were formed from the DOTAP vesicles, complementary AFM experiments were performed. The in situ TM-AFM images showed that the resulting SLB was uniform and free from defects. Significantly, the DOTAP SLB was essentially optically transparent to allow the study of the adsorption of the porphyrin, TPPS, in real-time to the SLBmodified surface. The adsorption process was modeled as a first-order heterogeneous process using the finite element method, and the rate constant for the adsorption of TPPS to the SLB was found to be 1.7 (±0.6) × 10−4 cm s−1. The ability to form a SLB on the quartz prism inherent to EW-CRDS and to monitor the interactions of biomolecules with the SLB in situ and in real time expands the applicability of EW-CRDS to the study of biomolecules in biologically relevant environments. The studies herein demonstrate that EW-CRDS is a powerful and straightforward technique to probe physicochemical phenomena at SLBs, and we envisage a wide range of processes that could be investigated.



ASSOCIATED CONTENT

S Supporting Information *

Details of complementary studies of SLB formation monitored using QCM-D. This material is available free of charge via the Internet at http://pubs.acs.org.



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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This work was supported by the Engineering and Physical Sciences Research Council (EPSRC) under Grant EP/ C00907X. H.V.P. is grateful for a Ph.D. studentship under 2590

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