Combined Reflectometric Interference Spectroscopy and Quartz

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Combined reflectometric interference spectroscopy and quartz crystal microbalance detect differential adsorption of lipid vesicles with different phase transition temperatures on SiO2, TiO2, and Au surfaces Taisuke Kojima Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04105 • Publication Date (Web): 22 Nov 2017 Downloaded from http://pubs.acs.org on November 29, 2017

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Analytical Chemistry

Combined reflectometric interference spectroscopy and quartz crystal microbalance detect differential adsorption of lipid vesicles with different phase transition temperatures on SiO2, TiO2, and Au surfaces Taisuke Kojima†* †Department of Biomedical Engineering, Georgia Institute of Technology, 950 Atlantic Drive NW, Atlanta, GA 30332, United States.

KEYWORDS: quartz crystal microbalance, reflectometric interference microscopy, protein adsorption, vesicle adsorption

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Abstract Quantitative analysis of biomolecular adsorption on a substrate is crucial for understanding biomolecular interactions. A quartz crystal microbalance (QCM) is a highly sensitive device to detect such interactions based on mass. However, the physicochemical analysis by the QCM alone often leads to overestimation of the actual adsorbed mass. Here a combined reflectometric interference spectroscopy (RIfS) and QCM is developed to simultaneously analyze adsorption of biomolecules. The RIfS detects the adsorbed mass based on the reflectance and predicts the adsorbed condition by modeling the reflection spectra using the transfer matrix method. In contrast, the QCM detects physicochemical characteristics of the adsorbed molecules along with the adsorbed mass. The combined RIfS-QCM successfully detected adsorption of proteins with different surface properties and lipid vesicles with different phase transition temperatures. The initial stage of adsorption revealed distinct individual properties of the adsorbates. Moreover, the RIfS-QCM revealed differential adsorption of the vesicles on silicon dioxide, titania, and gold surfaces, and the differences in adsorption were further interrogated by atomic force microscopy. The results demonstrate that the RIfS-QCM serves as a useful tool to quantitatively analyze molecular adsorption on various surfaces.


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Introduction Biosensors often utilize quantitative analysis of adsorption of biomolecules on a solid substrate to reveal the underlying biomolecular interactions and properties. The quartz crystal microbalance (QCM), a label-free and highly sensitive mass detection device, has been widely used to analyze biomolecular interactions in aqueous solution.1-3 In liquid phase, the frequency shifts correlate not only with the adsorbed mass but also with other factors such as viscoelasticity,4 solvation,5 and surface roughness6 of the adsorbates. Such properties involve stabilities7, molecular recognition,8 and enzymatic activities9 of biomolecules. The properties can be properly interrogated when the actual adsorbed mass is isolated by other analytical methods. The simultaneous integration of QCM and various optical methods on the same sensing surface has been implemented with surface plasmon resonance,10 reflectometry,11, 12 anomalous reflection,13 and X-ray photoelectron spectroscopy14. Notably, adsorption events of various lipid vesicles have been studied to interrogate vesicle-substrate interactions.15-17 The lipid vesicles are known to change adsorption modes depending on the substrate properties and form a supported lipid bilayer (SLB) under the strong vesicle-substrate attraction.18-20 The SLB serves as a model membrane

platform21

where

subsequent

molecule-membrane

interactions

have

been

investigated.22-26 However, the simultaneous measurements often require complicated and delicate setup and most studies employed limited lipid types on either gold (Au), titania (TiO2), or silicon dioxide (SiO2) surfaces. Surface effects on adsorption of lipid vesicles are deeply involved in biological events such as cell attachment-growth27, 28 and bacterial infection.29, 30 Thus, a comprehensive study of the vesicle adsorption on the various surfaces with a simple experimental setup should be demonstrated. Here a novel method combining reflectometric interference spectroscopy (RIfS)31-33 and QCM in an open module system is developed. A QCM unit with a network analyzer generates


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the acoustic mass (F) and the energy dissipation (D) reflecting the physical and viscoelastic properties of the adsorbed film. Meanwhile a RIfS unit, simply collecting light reflection on the sensing surface, provides the adsorbed mass (R) and estimates the physical thickness of the adsorbed layer by the transfer matrix method.34 This setup eliminates complicated optical adjustment and allows convenient integration. RIfS and QCM responses were calibrated by SLB formation to use the same mass unit (ng cm-2). Adsorption of proteins (BSA and lysozyme) on a TiO2-QCM surface was characterized and adsorption of lipid vesicles comprised of 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC) / 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was quantified. A comparison of the detected mass of RIfS and QCM enables the characterization of the viscoelastic properties. The vesicle adsorption was expanded to SiO2 and Au surfaces and the vesicles on those surfaces were further characterized by AFM. The optical estimation by RIfS and viscoelastic characterization by QCM were discussed.


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Materials. All general chemicals and reagents were purchased from commercial sources: 1,2dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and polycarbonate membrane (30 nm, 50 nm, and 100 nm pore size) from Avanti Polar Lipids. Copper (Cu) coil, chloroform, methanol, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), 1M sodium hydroxide (NaOH), 20% hydrogen peroxide (H2O2), sulfuric acid (H2SO4), sodium chloride (NaCl), calcium chloride (CaCl2), ethylenediaminetetraacetic acid (EDTA), lysozyme (14 kDa), and bovine serum albumin (BSA: 66kDa) were from SigmaAldrich. NanoSphere size standards (polystyrene nanoparticles: 60 ± 4 nm) was from Thermo Scientific. 6-[4-[(1,6-dioxo-1,6-bis(tetradecyloxy)hexan-2-yl)carbamoyl]phenoxy]-N,N,Ntrimethylhexan-1-ammonium bromide (calibration lipid) was a gift from Dr. Takayoshi Kawasaki.

Experimental Section. Cleaning protocol of SiO2-, TiO2-, and Au-QCM surfaces. QCM sensor chips with a Au surface (QCM27C-AU, ULVAC, JP) were treated with piranha solution (H2O2 : H2SO4 = 1 : 3) for 3 min x 3 times. QCM sensor chips with a SiO2 surface (QCM27C-SIO2, ULVAC, JP) and anodized QCM sensor chips with a TiO2 surface (Supporting Information) were cleaned for 25 min by UV-Ozone irradiation. These cleaning processes were performed immediately before measurement.

QCM-RIfS setup. A 27 MHz QCM (AFFINIX QN-Pro, ULVAC, JP) was combined with a prototype spectrometer (JASCO, JP) using an optical fiber in a stainless steel tube to monitor reflection spectra on a QCM sensor simultaneously with RIfS (Scheme 1). A QCM chip was installed in a 5mL glass


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container with a magnetic stir bar (QCMMGS, ULVAC, JP) where temperature is machinecontrolled (± 0.1 °C). A QCM network analyzer collects time-lapse frequency (∆F) and D-value (∆D) every second where RIfS records time-lapse reflection spectra (∆R) in hundreds of milliseconds. The data points of RIfS were synchronized with the data points of QCM using a moving average to obtain one data point per second.

Adsorption of proteins on the TiO2-QCM surface. Lysozyme (final concentration of 25 µg mL-1) was injected into a water-filled sample container and BSA (final concentration of 25 µg mL-1) was then dispensed after ∆F, ∆D, and ∆R reached a plateau. The reflection spectra were compared to the simulated spectra with the fixed refractive index as a general organic layer (n = 1.45) in order to estimate a layer thickness.

Preparation of multilamellar lipid vesicles. A powder mixture of DOPC : DPPC (mole ratio: 0 : 1, 1 : 1, and 1 : 0) was added in a 50 mL round-bottom glass flask and dissolved in chloroform or a chloroform-methanol mixture to be a 5 mg mL-1 mixture. An aluminum foil was used to shield ambient light. The solvent was evaporated by a rotary evaporator over 3 hours and dried in a desiccator over 3 hours. Argon gas was blown off to remove residual solvents. Degassed HEPES buffer (10 mM HEPES, 100 mM NaCl, pH 7.4) was added to the flask for a 5 mg mL-1 mixture and vortexed. The flask was sonicated for 15 min. One mL aliquots of the obtained multilamellar vesicles (MLV) solutions were made under Argon gas and stored at -40 °C (Figure S-4).

Preparation of small unilamellar lipid vesicles.


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A MLV stock solution was thawed at room temperature, vortexed, and sonicated for 10 min. An extruder (Mini Extruder, Avanti Polar Lipids, US) was placed on top of a heat block where the temperature was set above transition temperature of the lipids (DOPC: Tm = -20 °C, 1:1 DOPCDPPC: Tm = 37 °C, and DPPC: Tm = 41 °C).35, 36 The extrusion process was repeated more than 20 times in each filter size (100 nm, 50 nm, and 30 nm). The 5 mg mL-1 solution of small unilamellar vesicles (SUVs) was stored at 4 °C and used within 4 days.

Dynamic light scattering measurement of SUVs. One mL of filtered HEPES buffer and 10 µL of the SUV solution were added in a disposable plastic cuvette (PS cuvette, BrandTech, US) to get a standard concentration for DLS measurement (Zetasizer Nano-ZS, Malvern, DE). Size distribution in volume % and average size of the particles were obtained by 10 times integration at 25 °C.

Surface zeta potential measurement of SiO2-, TiO2, and Au-QCM surfaces. One mL of filtered HEPES buffer and a few droplets of a reference solution containing polystyrene nanoparticles were added in the disposable cuvette. SiO2-, TiO2, and Au-QCM substrates were cut into pieces and attached on a surface zeta potential cell (Surface Zeta Potential Cell Kit, Malvern, DE). The cell was inserted in the cuvette and surface zeta potential at 25 °C was measured at a distance of 125 µm, 250 µm, 375 µm, 500 µm, and 1000 µm (reference) from the surface. All values were expressed as mean ± standard deviation (n = 3).

Adsorption of SUVs on SiO2-, TiO2-, and Au-QCM surfaces. Four mL of HEPES-CaCl2 buffer (10 mM HEPES, 100 mM NaCl, 2 mM CaCl2, pH 7.4) or HEPES-EDTA buffer (10 mM HEPES, 100 mM NaCl, 2 mM EDTA, pH 7.4) was added to a


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sample container and the QCM-RIfS signal was recorded to obtain a stable baseline. The SUV stock solution was injected in the sample container (final concentration of 0.1 mg mL-1). All values were expressed as mean ± standard deviation (n = 4).

AFM measurement of SUVs on SiO2-, TiO2-, and Au-QCM surfaces. SiO2-, TiO2- and Au-QCM surfaces were physically isolated from the chips. Each surface was cleaned by the cleaning protocol before measurement. The surfaces were first scanned across 3 µm x 3 µm area in air tapping mode (Nanoscope IIIa, Veeco, US) using a gold-coated probe (k = 0.02 N m-1: OMCL-TR400PSA-1, Olympus, JP) to characterize surface roughness expressed as average ± RMS (nm). Next, 40 µL of HEPES-CaCl2 buffer were added on the surface and the laser alignment was performed in liquid tapping mode. After stabilization, the SUV solution (final concentration of 0.1 mg mL-1) was injected and each scan was immediately recorded. A cleaved mica surface was used as an alternative to the SiO2 surface for the vesicle characterization. All values were expressed as mean ± standard deviation (n = 3).

Theoretical Section Simulation of reflection spectra by the transfer matrix method Reflection spectra of a multilayer surface can be simulated by the transfer matrix method. The details are described in Supporting Information. Briefly, the reflectance R is given by

 η −Y   η0 − Y  R = r × r* =  0   (1)  η0 +Y   η0 + Y  *

where η0 is the complex refractive index of the medium, Y is the optical admittance, and r is the reflection coefficient. The optical constants of each layer were readily available online and summarized in Table S-1. Based on the simulation, the reflectance at 470 nm showed a large


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change in intensity among other wavelengths on the TiO2-QCM surface. Thus, the reflectance change at 470 nm (∆R470nm) was adopted.

Mass and viscoelastic contributions in the QCM measurement When solid and hydrophobic materials are immobilized on a surface of a QCM chip, they behave as an elastic film as described by the Sauerbrey equation given by

−∆ F =

2F02 A ρqµ q

∆ m (2)

where ∆F is the frequency shift (Hz), ∆m is the mass change (g), F0 is the fundamental frequency, A is the electrode area (cm2), ρq is the density of quartz (g cm-3), and µq is the shear modulus of quartz (dyn cm-2). However, ∆F in liquid phase is often affected by viscoelastic properties of soft substances together with the actual adsorbed mass. A network analyzer detects the viscoelastic properties as the energy dissipation (D) given by

E  D =  Dissipated  (3)  2π EStored  where EDissipated is the energy dissipated during one oscillatory cycle, and EStored is the energy stored in the oscillating system. In this work, RIfS (∆R) is utilized to isolate the adsorbed mass and evaluate the viscoelastic contributions from QCM responses (∆F and ∆D). The solvation indicates the degree of hydration of the adsorbate (ref 5) given by

∆ m  QCM −∆ mRIfS  (4) Solvation =  ∆ mRIfS   where ∆mQCM and ∆mRIfS are the adsorbed mass determined by QCM and RIfS, respectively. The mass conversion from ∆F and ∆R was carried out by lipid calibration (Figure S-2).


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Results and Discussion RIfS-QCM setup A TiO2-QCM surface was fabricated by anodic oxidation of a commercially available TiQCM chip (Figure S-1A). Reflectance at 470 nm was adopted based on a linear increase in the theoretical reflectance as a function of the adsorbed layer thickness (n = 1.45) (Figure S-1B). The frequency shift (∆F) and reflectance change at 470 nm (∆R470nm) were converted to mass (∆F to ∆mQCM and ∆R470nm to ∆mRIfS, respectively) through calibration by SLB formation on SiO2-, TiO2-, Au-QCM sensors (Figure S-2). Interestingly, the sensitivity on the TiO2-QCM surface increased four-fold compared to the SiO2- and Au-QCM surfaces (Table 1). AFM measurement revealed that the TiO2 surface increased surface roughness compared to the SiO2 and Au surfaces (Figure S-3). The enhanced sensitivity can be attributed to the combined surface roughness and distinct optical properties of the TiO2 surface according to the previous study.37 To validate the RIfS-QCM system, the TiO2-QCM surface was explored first. Protein adsorption on a TiO2-QCM surface Adsorption of lysozyme and BSA on a 35 nm TiO2-QCM surface was demonstrated (Figure 1). Given the size of lysozyme (4.5 x 3 x 3 nm3)38 and BSA (14 x 4 x 4 nm3),39 the expected height of each adsorbed layer is around 4 nm each when the proteins sit on the surface. The modeled reflection spectra agreed well with the measured reflection spectra and estimated the layer thickness around 4 and 8 nm after lysozyme and BSA adsorption, respectively (Figure 1A). The TiO2-QCM surface with the increased sensitivity enabled a fine comparison of the protein adsorption (Figure 1B). The obtained adsorbed mass of lysozyme detected by RIfS (∆mRIfS = ~200 ng cm-2) was slightly smaller than the mass detected by QCM (∆mQCM = ~260 ng cm-2). The measured energy dissipation was almost negligible in lysozyme (∆D = ~0), indicating the protein layer behaved as an elastic film. In contrast, the subsequent BSA adsorption showed


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similar ∆mRIfS to the lysozyme adsorption (∆mRIfS = ~200 ng cm-2) and elevated ∆mQCM (∆mQCM = ~600 ng cm-2). The measured energy dissipation suggested that the accumulated protein layer became viscous (∆D = ~2 x 10-6). The observed trend was illustrated by a linear slope (∆mQCM against ∆mRIfS per data point) at the initial phase of adsorption (Figure 1C). The slope represents the viscoelastic characteristics of each protein. The steeper slope during BSA adsorption correlates to more hydration than lysozyme. The results were in good agreement with the previous report comparing the individual protein adsorption on a SiO2 surface.40 Note that, with the small energy dissipation (∆D < 2 x 10-6), QCM alone failed to quantify the property difference. Vesicle adsorption on the TiO2-QCM surface Next the technique was expanded to adsorption of lipid vesicles with different transition temperatures. At the experimental condition of 25 °C, DOPC vesicles were in liquid phase while DPPC vesicles were in gel phase.41 I hypothesized that RIfS-QCM would distinguish such a property difference during adsorption. DOPC and DPPC vesicles were prepared by the standard extrusion method42 and the size distribution of each vesicle was confirmed to be identical (average diameter: 65 ± 5 nm) by DLS measurement (Figure S-4). The adsorption of DPPC vesicles showed a substantially larger discrepancy between RIfS and QCM (Figure 2A) compared to the adsorption of DOPC vesicles (Figure 2B). The mass detected by RIfS was relatively similar to each other (DPPC / DOPC: ∆mRIfS = ~980 ng cm-2 / ∆mRIfS = ~780 ng cm-2). However, the mass detected by QCM was substantially different between the DOPC and DPPC vesicles (DPPC / DOPC: ∆mQCM = ~5500 ng cm-2/ ∆mQCM = ~3200 ng cm-2). The energy dissipation was also distinguishable (DPPC / DOPC: ∆D = 120 x 106

/ ∆D = 90 x 10-6). Moreover, RIfS-QCM could detect a subtle difference between DOPC-DPPC

vesicles at a 1:1 molar ratio in gel phase43 and a mixture of DOPC and DPPC vesicle solutions at


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a 1:1 volume ratio (Figure S-5). The results imply that the QCM responses reflected the difference between the gel and liquid phase of the vesicles. The simulated reflection spectra during the initial phase of adsorption were in good agreement with the measured reflection spectra of adsorbed DPPC and DOPC vesicles when the theoretical thickness was fixed at 60 nm (Figure 2C) and 50 nm (Figure 2D), respectively. The model indicates that the vesicles were adsorbed in a different manner and the adsorbed thickness is smaller than the measured vesicle diameter. Note that, as opposed to the protein adsorption (Figure S-6A), the refractive index was varied instead of the layer thickness due to the unique nature of the vesicle structure (Figure S-6B and Eq S-2). A comparison of the initial and final stages of adsorption elucidates the differences in vesicle adsorption (Figure 3 and S-7). ∆mQCM and ∆D were normalized by ∆mRIfS and expressed as solvation and unit energy dissipation, respectively. The solvation corresponds to containing and surrounding water of the adsorbed vesicles (Eq 4) while the unit energy dissipation reflects the viscoelastic properties of the vesicles. At the initial stage of adsorption, a clear difference of the solvation and unit energy dissipation was observed between the lipid vesicles (Figure 3A). In contrast, at the final stage of adsorption, all the data points shifted toward the origin and the difference became negligible (Figure 3B). Interestingly, the unit energy dissipation remarkably changed compared to the solvation, implying the viscous property was more influential than the solvation. Given the maximum coverage of the vesicles (Eq S-3),44 the surface coverage was estimated as a function of the measured mass by RIfS (Figure S-8). I assumed that the individual property of the lipid vesicles in either gel or liquid phase were distinguishable at ~10% coverage while the properties as an ensemble layer of the lipid vesicles became more dominant at ~70% coverage. It has been shown that the vesicles in liquid phase are susceptible to deformation under shear stress


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compared to the vesicles in gel phase.45 The results together imply that DOPC vesicles in liquid phase were more deformed by the vesicle-surface attraction than DPPC vesicles in gel phase. Since increasing temperature above the transition temperature would make the QCM unit unstable, the substrate effect was more reasonable to test. To interrogate the vesicle-surface effect, the RIfS-QCM platform was expanded to other surfaces. Vesicle adsorption on SiO2- and Au-QCM surfaces Adsorption of the DOPC and DPPC vesicles was investigated on SiO2- and Au-QCM surfaces (Figure S-9 and Table 2). ∆mRIfS and ∆mQCM substantially increased as opposed to decreased ∆D upon adsorption of DPPC vesicles on the SiO2 surface (Figure S-9A). Conversely, adsorption of DOPC vesicles on the surface showed a typical sign of rupture and subsequent SLB formation (Figure S-9B).46 It was most likely due to strong vesicle-surface interaction that prompted adsorption of DPPC vesicles in gel phase and caused rupture of DOPC vesicles in liquid phase. In contrast, ∆mRIfS significantly reduced while ∆mQCM substantially increased upon adsorption of DPPC and DOPC vesicles on the Au-QCM surface (Figure S-9C and D). Negligible adsorption of the vesicles in the absence of calcium ions was observed on all of the surfaces (data not shown). Since the vesicles were slightly negatively charged under similar conditions as previously demonstrated,47 the data indicated that the vesicle-surface attraction was deeply involved for the adsorption manner. The initial stage of adsorption reveals differential adsorption among the surfaces (Figure 4). The solvation and unit energy dissipation of DPPC vesicles increased on SiO2- < TiO2- < AuQCM surfaces in this order. DOPC vesicles followed the same trend as DPPC vesicles. However, solvation and unit energy dissipation became relatively smaller compared to DPPC vesicles. The results highlight the difference between gel and liquid phase of the vesicles and illustrate the surface effect against the vesicle adsorption. Given the isoelectric point of each surface (SiO2: 2,


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TiO2: 3 – 6, Au: not applicable),48 the surface potential should substantially differ between the surfaces in the experimental condition (pH 7.4). Surface zeta potential measurements validated that the surface charge indeed negatively increased on Au- < TiO2- < SiO2-QCM surfaces in this order (Figure S-10 and Table 1). Under such condition, the vesicles should experience strong electrostatic attraction on the SiO2 and TiO2 surfaces and perhaps deform or rupture. Meanwhile, the vesicles would remain spherical on the less charged Au surface. AFM characterization of vesicle adsorption on the SiO2-, TiO2-, and Au-QCM surfaces To confirm the assumption, AFM in liquid measurement was performed on each surface during vesicle adsorption (Figure S-11, S-12, and Table 3). Time-lapse AFM imaging of the vesicles on the SiO2 surface verified the aforementioned discussion of the vesicle coverage (Figure 3, S-8, and S-11). DOPC vesicles on the SiO2 surface exhibited the typical height of the SLB (~5 nm)49 and supported the previously studies50, 51 that the vesicles spontaneously ruptured and formed SLBs (Figure S-11A). In contrast, some DPPC vesicles on the surface remained intact and some formed SLBs while SLBs became dominant toward the final stage of adsorption (Figure S-11B). In both cases, a few vesicles smaller than the critical size were observed in SLB crevasses at the final stage. The results support that DOPC vesicles in liquid phase are more prone to rupture on the SiO2 surface compared to DPPC vesicles in gel phase. The data were consistent with the RIfS-QCM data and the previous studies using silica-based substrates.52, 53 Moreover, the topological data of DPPC vesicles on other surfaces indicated that the vesicles were more crushed on the SiO2 and TiO2 surfaces compared to the Au surface (Figure S-12A-D and Table 3). Altogether the results confirmed that DOPC and DPPC vesicles were adsorbed in a distinct manner depending on the surface. The RIfS-QCM detected the phase difference of the vesicles and the surface effect upon vesicle adsorption based on the solvation and unit energy dissipation.


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Conclusions In this work, a combination of RIfS and QCM is presented to quantify protein and vesicle adsorption. RIfS-QCM with a TiO2 surface detected protein adsorption and solvation in a quantitative manner. Modeling reflection spectra to align with actual reflection spectra enabled estimation of the adsorbed condition based on the layer thickness and refractive index. RIfSQCM with the TiO2 surface also quantified adsorption of DOPC and DPPC vesicles with the same mean size and successfully distinguished the two during the initial phase of adsorption using the solvation and unit energy dissipation. The simulated reflection spectra suggested different modes of adsorption. Since RIfS-QCM is open to other surfaces, adsorption of the vesicles on SiO2 and Au surfaces was further investigated. The results obtained by RIfS-QCM indicated differential adsorption on the surface and AFM confirmed that the vesicles were deformed in accordance with the surface-vesicle attraction. The high sensitivity of RIfS-QCM allows further investigation of membrane-protein interactions and the versatility of the surface envisions interrogation of surface-biomolecule interactions such as RNA, DNA, and polysaccharides in the future study. RIfS-QCM would provide a unique opportunity to quantitatively interrogate the interactions in a facile setup.


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Figures

Scheme 1. A combined unit of RIfS and QCM. (A) A schematic illustration of simultaneous monitoring of frequency (∆F) and D-value (∆D) by a 27 MHz QCM unit and reflection spectra (∆R) between 450 nm and 650 nm by a RIfS unit. (B) An actual setup of RIfS and QCM equipped with an admittance mode.


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Table 1. Characterization of SiO2-, TiO2-, and Au-QCM surfaces. Surface

Optical Sensitivitya (ng cm-2 / %)

Surface Roughnessb (nm)

Surface Zeta Potential (mV)

Au

-258 ± 43

7.3 ± 1.3

-29 ± 1.6

TiO2

-65 ± 4.7

18 ± 5.9

-34 ± 1.8

SiO2

-304 ± 62

10 ± 1.0

-51 ± 1.2

a) ∆m / ∆R470 nm determined by lipid calibration. b) average height ± RMS in a 3 µm x 3 µm scan area determined by AFM. Table 2. A summary of adsorption of lipid vesicles on SiO2-, TiO2-, and Au-QCM surfaces. Surface

SiO2

Vesicle

DOPC

∆mRIfS 497 ± 58 (ng cm-2) ∆mQCM 476 ± 18 (ng cm-2) ∆D (x 10-6)

0.6 ± 0.5

TiO2

Au

DPPC

DOPC

DPPC

DOPC

DPPC

1583 ± 97

799 ± 37

1000 ± 8

116 ± 20

1177 ± 27

3290 ± 322

3017 ± 47

5503 ± 84

1111 ± 55

5281 ± 13

36 ± 11

91 ± 3

118 ± 5

65 ± 3

107 ± 2

Table 3. The topology of the vesicles adsorbed on SiO2-, TiO2-, and Au-QCM surfaces. Surface

Height - AFM (nm)

Width - AFM (nm)

Ellipticitya

SiO2-DOPC

4.7 ± 0.092

> 100

< 0.1

SiO2-DPPC

29 ± 5.6

152 ± 22

0.19 ± 0.085

TiO2-DPPC

42 ± 1.7

197 ± 4.0

0.21 ± 0.044

Au-DPPC

46 ± 3.8

103 ± 13

0.45 ± 0.030

a) ellipticity: height divided by width.


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Figure 1. Adsorption of lysozyme and BSA on a TiO2-QCM surface at 25 C°. (A) Measured reflection spectra (dots) at t = 0 (light blue), 500 sec (blue) after lysozyme adsorption, and 1500 sec (magenta) after BSA adsorption. Simulated reflection spectra (solid line) on a 40 nm TiO2 / Ti surface where 0 nm (light blue), 4 nm (blue), and 8 nm (magenta) adsorbed film with the fixed refractive index (n = 1.45) formed. (B) Adsorbed mass detected by QCM and RIfS. ∆F (Hz) and ∆R470nm (%) were converted to mass (ng cm-2) as ∆mQCM and ∆mRIfS, respectively, by calibration (Figure S2). (C) A comparison of adsorbed mass detected by RIfS and QCM. Data points were plotted at the initial stage of protein adsorption (0 – 100 ng cm-2): Lysozyme (blue) and BSA (orange). The dotted line represents one-to-one correspondence of ∆mQCM and ∆mRIfS.


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Figure 2. Adsorption of DPPC and DOPC vesicles on a TiO2-QCM surface at 25 C°. Adsorption of (A) DPPC and (B) DOPC vesicles detected by RIfS-QCM: ∆mQCM (red) and ∆mRIfS (blue), and ∆D (green). Black arrows indicate sample injection. Measured reflection spectra (dot) upon adsorption of (C) DPPC and (D) DOPC vesicles at t = 0 – 5 min. Simulated reflection spectra (solid line) on a 35 nm TiO2 / Ti surface where adsorbed film with the fixed layer thickness of 60 nm for DPPC adsorption and 50 nm for DOPC adsorption, respectively, varying the refractive index (n = 1.333 – 1.363).


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Figure 3. A comparison of adsorption of DOPC and DPPC vesicles on a TiO2-QCM surface at 25 °C. Data points adopted (A) during the initial stage of adsorption (coverage ≈ 10%: ∆mRIfS ≈ 200 ng cm-2) and (B) during the final stage of adsorption (coverage ≈ 70%: ∆mRIfS ≈ maximum). A line of best fit (solid line) estimated by linear least-squared method. X-axis: unit energy dissipation / Y-axis: solvation


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Figure 4. A comparison of adsorption of DOPC and DPPC vesicles on SiO2-, TiO2- and AuQCM surfaces at 25 °C. Data points adopted during the initial stage of adsorption (coverage < 10%: ∆mRIfS < 200 ng cm-2). The vesicles (DPPC: top and DOPC: bottom) on the SiO2 (red closed square), TiO2 (blue closed circle), and Au (yellow closed triangle) surfaces. A line of best fit (solid line) estimated by linear least-squared method. X-axis: unit energy dissipation / Y-axis: solvation


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Supporting Information Available: This material is available free of charge via the Internet at http://pubs.acs.org. The supporting information includes the supplemental methods, the simulation of the reflectance by the transfer matrix method, calculation of the refractive index of the lipid vesicle, calculation of the close-packed coverage and maximum mass of the vesicles, adopted optical constants, schematic illustration of anodic oxidation, calibration of RIfS-QCM surfaces by lipid bilayer formation, AFM images of SiO2-, TiO2-, and Au-QCM surfaces, preparation of small unilamellar vesicles, a comparison of DOPC-DPPC vesicles on a TiO2QCM surface, different models in RIfS simulation, a comparison of vesicle adsorption at the initial stage, surface coverage as a function of the adsorbed mass, a comparison of the vesicle adsorption on the SiO2- and Au-QCM surfaces, surface zeta potential of SiO2-, TiO2-, and AuQCM surfaces, and AFM images of the vesicles adsorbed on the surfaces.


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Corresponding Author Dr. Taisuke Kojima 950 Atlantic Drive NW Atlanta, GA, USA 30332 *E-mail: [email protected]

Notes The author declares no competing financial interest.

Acknowledgment This work was supported by MEXT, Grant-in-Aid for Scientific Research. RIfS-QCM and some AFM experiments were conducted in the department of biomolecular engineering at Tokyo Institute of Technology. The author deeply appreciates guidance and advice from Dr. Takayoshi Kawasaki and Dr. Yoshio Okahata. This article is dedicated to them.

Abbreviations QCM, quartz crystal microbalance; RIfS, reflectometric interference spectroscopy; DPPC, 1,2dipalmitoyl-sn-glycero-3-phosphocholine;

DOPC,

1,2-dioleoyl-sn-glycero-3-phosphocholine;

SUV, small unilamellar vesicle; SLB, supported lipid bilayer; DLS, dynamic light scattering; AFM, atomic force microscopy.


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TOC Graphic

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A combined unit of RIfS and QCM. (A) A schematic illustration of simultaneous monitoring of frequency (∆F) and D-value (∆D) by a 27 MHz QCM unit and reflection spectra (∆R) between 450 nm and 650 nm by a RIfS unit. (B) An actual setup of RIfS and QCM equipped with an admittance mode. 82x145mm (300 x 300 DPI)


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Adsorption of lysozyme and BSA on a TiO2-QCM surface at 25 C°. (A) Measured reflection spectra (dots) at t = 0 (light blue), 500 sec (blue) after lysozyme adsorption, and 1500 sec (magenta) after BSA adsorption. Simulated reflection spectra (solid line) on a 40 nm TiO2 / Ti surface where 0 nm (light blue), 4 nm (blue), and 8 nm (magenta) adsorbed film with the fixed refractive index (n = 1.45) formed. (B) Adsorbed mass detected by QCM and RIfS. ∆F (Hz) and ∆R470nm (%) were converted to mass (ng cm-2) as ∆mQCM and ∆mRIfS, respectively, by calibration (Figure S2). (C) A comparison of adsorbed mass detected by RIfS and QCM. Data points were plotted at the initial stage of protein adsorption (0 – 100 ng cm-2): Lysozyme (blue) and BSA (orange). The dotted line represents one-to-one correspondence of ∆mQCM and ∆mRIfS. 82x226mm (300 x 300 DPI)



Adsorption of DPPC and DOPC vesicles on a TiO2-QCM surface at 25 C°. Adsorption of (A) DPPC and (B) DOPC vesicles detected by RIfS-QCM: ∆mQCM (red) and ∆mRIfS (blue), and ∆D (green). Black arrows indicate sample injection. Measured reflection spectra (dot) upon adsorption of (C) DPPC and (D) DOPC vesicles at t = 0 – 5 min. Simulated reflection spectra (solid line) on a 35 nm TiO2 / Ti surface where adsorbed film with the fixed layer thickness of 60 nm for DPPC adsorption and 50 nm for DOPC adsorption, respectively, varying the refractive index (n = 1.333 – 1.363). 175x172mm (300 x 300 DPI)


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A comparison of adsorption of DOPC and DPPC vesicles on a TiO2-QCM surface at 25 °C. Data points adopted (A) during the initial stage of adsorption (coverage ≈ 10%: ∆mRIfS ≈ 200 ng cm-2) and (B) during the final stage of adsorption (coverage ≈ 70%: ∆mRIfS ≈ maximum). A line of best fit (solid line) estimated by linear least-squared method. X-axis: unit energy dissipation / Y-axis: solvation 84x221mm (300 x 300 DPI)



A comparison of adsorption of DOPC and DPPC vesicles on SiO2-, TiO2- and Au-QCM surfaces at 25 °C. Data points adopted during the initial stage of adsorption (coverage < 10%: ∆mRIfS < 200 ng cm-2). The vesicles (DPPC: top and DOPC: bottom) on the SiO2 (red closed square), TiO2 (blue closed circle), and Au (yellow closed triangle) surfaces. A line of best fit (solid line) estimated by linear least-squared method. Xaxis: unit energy dissipation / Y-axis: solvation 84x96mm (300 x 300 DPI)


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Combined Reflectometric Interference Spectroscopy and Quartz

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