Surface Immobilization of Bio-Functionalized Cubosomes: Sensing of

Nov 15, 2011 - ... G. Hartley‡, Anastasios Polyzos*‡, and Frances Separovic†. †School of Chemistry, Bio21 Institute, The University of Melbour...
0 downloads 8 Views 4MB Size
ARTICLE pubs.acs.org/Langmuir

Surface Immobilization of Bio-Functionalized Cubosomes: Sensing of Proteins by Quartz Crystal Microbalance Scott J. Fraser,†,‡ Xavier Mulet,‡ Lisandra Martin,§ Slavica Praporski,§ Adam Mechler,|| Patrick G. Hartley,‡ Anastasios Polyzos,*,‡ and Frances Separovic† †

School of Chemistry, Bio21 Institute, The University of Melbourne, Melbourne, VIC 3010, Australia CSIRO Materials Science and Engineering, Bayview Avenue, Clayton South, VIC 3169, Australia § School of Chemistry, Monash University, Clayton, VIC 3800, Australia Department of Chemistry, LaTrobe University, Bundoora, VIC 3083, Australia

)



ABSTRACT: A strategy for tethering lipid liquid crystalline submicrometer particles (cubosomes) to a gold surface for the detection of proteins is reported. Time-resolved quartz crystal microbalance (QCM-D) was used to monitor the cubosome protein interaction in real time. To achieve specific binding, cubosomes were prepared from the nonionic surfactant phytantriol, block-copolymer, Pluronic F-127, and a secondary biotinylated lipid, 1,2-distearoyl-sn-glycero-3-phosphoethanolamineN-[biotinyl(polyethyleneglycol)-2000], which enabled attachment of the particles to a neutravidin (NAv)alkanethiol monolayer at the gold surface of the QCM sensor chip. A second set of cubosomes was further functionalized with addition of the glycolipid (GM1) to facilitate a specific binding uptake of the protein, cholera toxin B subunit (CTB), from solution. QCM-D confirmed the specificity of the cubosomeNAv binding. The analysis of titration experiments, also performed with QCM, suggests that an optimal concentration of cubosomes is required for the efficient packing of the particles at the surface: high cubosome concentrations lead to chaotic cubosome binding onto the surface, sterically inhibiting surface attachment, or require significant reorganization to permit uniform cubosome coverage. The methodology enabled the straightforward preparation of a complex nanostructured edifice, which was then used to specifically capture analyte proteins (cholera toxin B subunit or free NAv) from solution, supporting the potential for development of this approach as a biosensing platform.

’ INTRODUCTION The spontaneous self-assembly of lipids at solid surfaces provides an excellent model for the two-dimensional cell membrane. The fluidity of the bilayer derived from the lateral diffusion and rotation of lipid molecules makes them well suited to fundamental studies of surface membrane processes such as ligand protein interaction, protein adsorption, and cellular signal transduction processes.1 An elegant application of solid-supported lipid bilayer membranes is within the emerging area of nanostructured sensor platforms.2 In this approach, an analyte specific receptor or protein is immobilized within a surface-deposited bilayer, and binding with an analyte molecule can be detected using a variety of methods, including surface-sensitive techniques such as ellipsometry and surface plasmon resonance (SPR).3 Furthermore, microarrays of supported lipid membranes provide a powerful method for presenting large addressable, combinatorial libraries of biological macromolecules,2d,4 enabling the rapid screening and identification of target proteins in drug discovery programs. A key element of these applications is the localization of the sensitive detection molecules of interest within the bilayer. The lipid layer can contribute to the retention of the detection molecule functionality as the natural cell membrane is mimicked at the sensor surface. r 2011 American Chemical Society

The architecture of supported lipid bilayer membranes has generally been restricted to two-dimensional (lamellar-type) structures in sensor applications. This has evolved from the use of lipids, which adopt a lamellar or planar geometry on hydration, such as phospholipids. However, this approach imposes a serious limitation on the analytical sensitivity of sensor devices: the moderate surface area of the planar assemblies restricts the achievable surface density of receptor molecules. Extended three-dimensional lipid structures are known but, however, remain unexplored for sensor applications. Higher order lipid architectures such as the inverse bicontinuous cubic (QII) phase afford a lipid bilayer system with a threedimensional structure and large surface area.5 These bicontinuous cubic phases have drawn considerable attention due to their long-range order, thermodynamic and thermal stability, and large surface area (typically 400 m2/g).6,7 Structurally, the cubic phase consists of continuous curved bilayers, which partition two separate, nonintersecting aqueous domains.8 The two aqueous channels allow water-soluble substrates to penetrate through the entire nanostructure and bind to both sides of the bilayer membrane. Received: August 22, 2011 Revised: November 14, 2011 Published: November 15, 2011 620

dx.doi.org/10.1021/la2032994 | Langmuir 2012, 28, 620–627

Langmuir The nanostructure of the material therefore offers significantly larger surface for the presentation of ligands to capture analyte molecules and a corresponding interfacial area for substrate binding as compared to planar lipid bilayers or curved liposomes. Three lyotropic liquid crystalline inverse bicontinuous cubic phases are typically observed, the gyroid (G), diamond (D), and primitive (P), corresponding to the crystallographic space groups Ia3d, Pn3m, and Im3m, respectively. The incorporation of chemical and biological functionality to cubic phase lipidic nanostructures represents an opportunity to employ these systems in chemical and biosensor devices as high surface area bilayer scaffolds.9 Pioneering work by Angelova et al.10 established that cubic phases of the surfactant glycerolmonoleate (MO) could be functionalized with a secondary surfactant (comprising a reactive maleimide headgroup that can form covalent bonds with thiol (cysteine) on peptides and proteins), without disruption of the cubic phase at room temperature. However, these studies did not examine the immobilization of cubic phases on surfaces for sensor development. Previous work in our laboratories has developed the introduction of chemical and biological functionality to lyotropic liquid crystalline inverse bicontinuous cubic phases through the incorporation of secondary lipids.5,6,11,12 Such an approach has been adopted toward the development of polyvalent inhibitors against cholera toxin. It has been demonstrated that bulk cubic phases of the nonionic surfactant phytantriol readily integrate the glycolipid monosialoganglioside GM1 (GM1), the natural cell surface receptor for cholera toxin (CT).6 The carbohydrate residues of the GM1 headgroup, which are displayed on the bilayer surface, bind to CT, and GM1-functionalized cubic phases showed a strong inhibitory response against CT with a high specificity for the toxin. These studies also demonstrate that the lattice parameter of the phase can be altered by variation of the fraction of GM1 present, imparting a degree of control over the lipid assembly. Colloidally stable, submicrometer dispersions of the inverse cubic phase, also referred to as cubosomes, are formed by the fragmentation of the QII phase in excess water.13 The resulting dispersions have a significantly reduced viscosity and greater surface area relative to the bulk material. The dispersion of cubic phases becomes important in many biological or clinical applications of cubic phases such as controlled release drug delivery, which are incompatible with a viscous material.14,15 Within the context of sensors, cubosomes are easier to handle relative to bulk cubic phases, providing the opportunity to fabricate sensor surfaces using established deposition techniques.2d,16 Recent work has demonstrated that intact cubosomes can be tethered to a protein monolayer surface via specific ligandprotein interaction.11 A classical biotinstreptavidin binding interaction was employed to immobilize the bifunctional cubosomes containing both biotin and the glycolipid monosialoganglioside-GM1 (GM1). Biological analysis revealed the retention of biological activity of the GM1 within the immobilized cubosome bilayer. This work established that cubosomes could be fabricated into composite surfaces for biosensor applications. QCM-D involves the measurement of the resonance frequency of quartz crystals, which provides information about the mass of adsorbed material in a time-resolved fashion. The combination of the acoustic parameters, the resonant frequency (f) and the dissipation factor (D), permits an estimation of the thickness and viscoelasticity of the adsorbed layer, allowing insight into the stablity and surface coverage of adsorbed cubosomes. An important feature of QCM-D is that the accuracy of frequency

ARTICLE

measurements allows for the monitoring and determination of the affinity of biological molecules to appropriately functionalized surfaces. QCM-D has been used to investigate the physical properties of surface bound lamellar particles (e.g., liposomes, etc.).17 In particular, studies by Brochu and Vermette utilized a multilayer strategy to capture liposomes on a chemically modified QCM sensor surface while retaining liposomes as stable particles.18 This study is of particular importance to the work presented herein, as it supports earlier observations that cubosomes could be tethered to a chemically modified surface without collapse into a planar lipid bilayer.11 As such, QCM-D was used as an appropriate method to evaluate the interaction of cubosomes with a surface and their relative temporal stability. Importantly, the technique provides an opportunity to probe biochemical interactions of proteins with tethered cubosomes. In this Article, the surface immobilization of cubosomes comprised of the synthetic surfactant, phytantriol, and the nonionic block-copolymer, Pluronic F-127, is presented. The addition of the PEG-biotin lipid (bDSPE) to the cubosome matrix enabled specific tethering to QCM Au sensor surfaces modified with neutravidin (NAv). A second series of cubosomes were prepared as above, with the addition of the CT receptor GM1. The biotinylated cubosomes formed a complex with the AuNAv surface, and then the secondary binding interaction with cholera toxin B subunit (CTB) was evaluated. These preliminary studies demonstrate that tethered cubosomes specifically bind proteins from solution. As functionalization of the cubosomes with secondary and ternary lipids may alter their nanostructure, the integrity of particle internal geometry was verified using synchrotron small-angle X-ray scattering (SAXS) and cryo-transmission electron microscopy (cryo-TEM).

’ MATERIALS AND METHODS Materials. Cholera toxin B subunit, monosialoganglioside GM1 (sodium salt, bovine brain, 95%), 11-mercaptoundecanoic acid (MUA, 99%), 6-mercapto-1-hexanol (97%), N-ethyl-N0 -(3-imethylaminopropyl) carbodiimide) (EDC), and N-hydroxysulfosuccin-imide (NHS) were purchased from Sigma Aldrich (US). Antibody (IgG) from rabbit serum (g95%, reagent grade), bovine serum albumin (BSA, g98%), and phosphate buffered saline (PBS, BioReagent, pH 7.4) were purchased from Sigma Aldrich (Castle Hill, Australia). 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (ammonium salt, 100%) (bDSPE) was purchased from Avanti Polar Lipids Inc. (US). Neutravidin was purchased from Thermo-Fischer Scientific (US). 3,7,11,15-Tetramethyl-1,2,3-hexadecanetriol (phytantriol) (96%) was purchased from Aldrich. Pluronic F127 was purchased from Sigma. Dichloromethane (HPLC grade), hydrogen peroxide (30% GR for analysis ISO), and ethanol (100%) were purchased from Merck (UK). Ammonia solution (28% analytical Univar Reagent) was purchased from Ajax Finechem (Seven Hills, Australia). Milli-Q grade (0.5 μS cm1 at 25 °C) water was purified through a Millipore system (Sydney, Australia) and used throughout this study. Cubosome Preparation. GM1/phytantriol, bDSPE/phytantriol, or GM1/bDSPE/phytantriol mixtures were prepared for cubosome production according to the method described by Polyzos et al.6 with functional moieties each added at concentrations of 0.5% (w/w). Briefly, phytantriol, GM1, and bDSPE were dissolved in an appropriate volume of CH2Cl2/MeOH (1:1). Volumes corresponding to 0.5% (w/w) of each functional additive were added to a 1.5 mL Eppendorf tube together with a volume of dissolved phytantriol to give a total mass of 0.05 g after solvent removal. The solvent was then removed by rotary evaporation, and pluronic F127 (7.0% w/w) in water was added to the 621

dx.doi.org/10.1021/la2032994 |Langmuir 2012, 28, 620–627

Langmuir

ARTICLE

microcentrifuge tube, resulting in the formation of a viscous phase. The bulk lyotropic phase was dispersed by probe ultrasonication using an S-4000 sonicator (Misonix Inc., Australia), 10 s intervals for 20 min using a 5 mm probe, affording cubosomes with a mean diameter of ∼150 nm. Particle Size Measurements. The particle diameter distribution was obtained from triplicate dynamic light scattering (DLS) measurements using a Nano-ZS Zetasizer (Malvern Instruments, UK) at 25 °C, assuming a viscosity of pure water. The polydispersity index (PDI) taken to be the width of the particle size distribution is calculated from a Cumulants analysis of the DLS measured intensity autocorrelation function using the Malvern software. Cryo-Transmission Electron Microscopy (Cryo-TEM). Samples were prepared for imaging by Cryo-TEM according to the method of Adrian et al.19 A custom-built vitrification system was used, allowing humidity to be kept close to 90% during sample plunging and vitrification. 45 mL of sample was applied to a 200 mesh copper TEM grid coated with lacy carbon film (ProSciTech, Thuringowa, Australia) and was allowed to settle for 30 s. The grid was manually blotted for 1015 s, and the resulting thin film was then vitrified by plunging into liquid ethane. Grids were stored in liquid nitrogen before transferring into a Gatan 626-DH Cryo-holder. Imaging was carried out using a FEI Tecnai 12 TEM, operating at 120 kV, with the sample at a temperature of 180 °C. Images were recorded using a MegaView III CCD camera equipped with AnalySis imaging software (Olympus Soft Imaging Solutions, Japan), using standard low-dose procedures to minimize radiation damage. Synchrotron Small-Angle X-ray Scattering (SAXS). All SAXS measurements were undertaken on the SAXS beamline of the Australian Synchrotron, Melbourne, Australia. Samples were loaded into 1.5 mm special glass capillaries (Hampton Research, US) before being mounted into a bespoke sample holder. Sample temperature was set using a custom designed Peltier driven cell with temperature control of (0.1 °C. The X-ray beam was set to a wavelength of 0.8266 Å with a typical flux of 1013 photon/s. Diffraction patterns were collected in 2-D on a Pilatus 1 M detector, which was offset to access a greater q-range. All measurements were calibrated against silver behenate (a = 58.38 Å), which diffracts X-ray in the low-angle range. Analysis of diffraction images was carried out using the IDL-based AXcess software package20 with measured lattice parameter spacings accurate to 0.1 Å. Quartz Crystal Microbalance (QCM-D). Preparation and Surface Modification of QCM Sensor Chips. The commercially available QCM gold sensor chips were comprised of a bare gold surface (Q-Sense SX-301, Q-SENSE, Sweden). These were rinsed with ethanol and dried under a gentle stream of N2 gas, after which they were placed in a 1:1:3 mixture of ammonia (28%), hydrogen peroxide (30%), and Milli-Q water, at ∼75 °C for 1520 min. Subsequently, chips were thoroughly rinsed with Milli-Q water and ethanol, then dried under a steady stream of N2 gas and immediately inserted into the QCM chamber ready to use. For experiments involving the addition of a self-assembled monolayer (SAM) onto the QCM chip surface, following cleaning the chips were immersed in a binary mixture of 2.5 mM 11-mercaptoundecanoic acid (MUA) and 7.5 mM mercaptohexanol in absolute ethanol for 24 h. Surfaces were then rinsed in ethanol, dried under a gentle stream of N2 gas, and immediately assembled into the QCM chamber. Neutravidin (NAv) was immobilized to the sensor surfaces via covalent attachment to the SAM. Briefly, the binary 11-MUA/mercapto-hexanol SAM surfaces were activated in situ by treating the SAM-modified crystals with 46 mM of EDC/NHS for 1 h to produce a carboxylterminated surface (Figure 1). After activation, surfaces were immediately modified by flowing a solution of PBS pH 7.4 containing NAv (1 mM), a nonglycosylated, re-engineered avidin, at 25 °C. Samples were then rinsed in PBS buffer pH 7.4 before being tested for any further assembly. Quartz Crystal Microbalance (QCM) with Dissipation Monitoring. QCM analysis was carried out using a Q-SENSE E4 system (Q-SENSE,

Figure 1. Schematic diagram of neutravidin (NAv) modification of QCM sensor surfaces using a binary mercaptohexanol and 11-mercaptoundecanoic acid (MUA) self-assembled monolayer (SAM). Sweden). The sensor crystals used were 5 MHz, AT-cut, polished quartz discs (chips) with electrodes deposited on both sides (Q-SENSE). The resonance frequency and energy dissipation were measured simultaneously at four odd harmonics (5, 15, 25, 35 MHz). In the results, the fundamental frequency of the crystal is called the first harmonic. The values reported throughout for Δf and ΔD are measured at the arbitrarily chosen fifth and seventh harmonics, respectively, unless otherwise stated.21 The working temperature was 25 °C. Raw data were analyzed with OriginPro 8.0 (Origin-Lab, US) and QTools software (Q-Sense). The estimation of the mass of each respective layer deposited (ng/cm2) and the layer thickness (nm) were calculated where applicable using the Sauerbrey22 eq 1: 2f02 Δm Δf ¼  pffiffiffiffiffiffiffiffiffiffiffi A Fq μ q

ð1Þ

where f0 is the resonant frequency of the chip (Hz), Δf is the frequency change (Hz), Δm is the mass change (g), A is the piezoelectrically active area of the crystal surface (cm2), Fq is the density of quartz (2.648 g/cm3), and μq is the shear modulus of quartz for an AT-cut crystal (2.947  1011), which assumes a rigid, tightly coupled, low dissipating, and uniform layer on the surface of the chip. To calculate the thickness from the Sauerbrey equation, a variation of the equation, given below as eq 2,23 was employed: def f ¼

Δm Fef f

ð2Þ

where deff is the effective thickness, Δm is the calculated change in mass from eq 1, and Feff is the effective density of the adhering layer. In instances where the surfaces did not behave according to the assumptions required for use of the Sauerbrey model, we approximated the surface as a homogeneous viscoelastic body. Assuming a parallel action of forces responsible for elastic and a viscous (dissipating) deformation, the dissipative forces will act in a frequency-dependent manner, and thus the viscosity and shear moduli of the surface layer might be modeled if measurements are performed at a set of higher harmonics of the sensor resonance. Separating viscoelastic effects then permits a more accurate calculation of the mass. The modeling was performed using the Voigt model available within the QTools software. Voigt modeling provides insight into the elastic and inelastic properties of the medium adsorbed/ absorbed onto the QCM surface. The model relates the dampening of the crystal oscillation, measured as dissipation (eq 3), to the shear 622

dx.doi.org/10.1021/la2032994 |Langmuir 2012, 28, 620–627

Langmuir

ARTICLE

Table 1. Dynamic Light Scattering (DLS) Data for the Different Cubosome Populationsa mass %

mean particle

polydispersity

additive

size (nm)

index (PDI)

phytantriol phytantriol/bDSPEb

0.5

175 ( 12 180 ( 9

0.4 0.2

phytantriol/GM1b

0.5

185 ( 13

0.3

188 ( 9

0.2

cubosome type

phytantriol/bDSPEb/GM1b 0.5/0.5 a

Each population was measured in triplicate, after an initial equilibration step of 2 min at 25 °C. b 0.5% w/w bDSPE and/or GM1.

Figure 2. A QCM-D trace for binding of NAv, cubosomes, and CT. Traces show both change in frequency (Δf) and dissipation (ΔD) for the seventh harmonic of a given experiment flowed over an Au sensor surface at 25 °C. At point (1), NAv is flowed into the chamber, at point (2), cubosomes are flowed into the chamber, and at point (3), the secondary analyte is flowed into the chamber. The symbol “*” indicates a washing step with PBS buffer at pH 7.4. (Note: (Δf) and (ΔD) for the fifth harmonic are not shown on this trace for clarity.)

Figure 3. Cryo-TEM images of cubosomes comprised from (A) b-DSPE/phytantriol system and (B) the GM1/phytantriol.

Table 2. Synchrotron SAXS Data for Different Cubosome Populations at 25 °C

modulus and viscosity of the material adsorbed/absorbed on the QCMD sensor surface. Dissipation is defined as:24 Elost D¼ 2πEstored

cubosome type

ð3Þ

where D is the dissipation of the film, Elost is the energy lost during one oscillation cycle, and Estored is the total energy stored in the oscillation. The modeling software makes use of the measurement of the difference in the frequency and dissipation values due to velocity-dependent viscous energy loss at multiple frequency overtones.21 Analyses were performed by flowing analyte through the QCM cell at a flow rate of 30 μL/min at each addition step. After each addition, the flow was stopped and the baseline was allowed to stabilize. The cell was then rinsed with PBS solution (pH 7.4) at a flow rate of 30 μL/min. The order of analyte addition was NAv, bDSPE/phytantriol cubosomes, then Cholera toxin B subunit (CTB) or NAv (or additional control proteins: BSA and IgG, which were dissolved in 0.1 M PBS to a final concentration of 0.10 mg/mL of each protein, respectively). A typical trace showing each of the steps in data collection, for Δf and ΔD, is shown in Figure 2. All experiments were triplicate runs, and data reported are the average of three separate runs.

a

phase

lattice spacing

crystallographic

(Å)

space group

phytantriol

cubic

69.8 ( 0.03

Pn3m

phytantriol/bDSPEa

cubic

70.3 ( 0.04

Pn3m

phytantriol/GM1a phytantriol/bDSPEa/GM1a

cubic cubic

70.9 ( 0.04 71.4 ( 0.07

Pn3m Pn3m

0.5% w/w bDSPE and/or GM1.

phology, as the patterning and size are comparable to those reported elsewhere.11,27 To confirm cubic morphology, we employ SAXS measurements. Table 2 shows the synchrotron small-angle X-ray data for the different cubosome preparations. The data indicate that the morphology of cubosomes in the phytantriol/water cubosome system was Pn3m with a lattice parameter of 69.8 Å, which correlates well with previously reported values28 for the bulk inverse cubic phase of phytantriol cubosomes containing 10% (w/w) pluronic F127. We observed that the GM1 and bDSPE had a very small but measurable effect on the lattice parameter at low (0.1 mol %) mole equivalents. The addition of GM1, bDSPE, or a combination of the two resulted in slight increase of the lattice spacing or “swelling” of the cubic phase (70.3, 70.9, and 71.4 Å for GM1, bDSPE, and GM1/bDSPE systems, respectively) relative to the phytantriol/water cubosomes (69.8 Å). We attribute this to a decrease in the cubic phase interfacial curvature29 induced by the larger headgroup of the additives relative to phytantriol. Surface Modification of the QCM-D Au Sensors. The gold quartz crystal microbalance sensor surfaces were modified by covalent attachment of NAv to a thiol self-assembled monolayer (see Materials and Methods for details), and the deposition of the NAv to the monolayer was interrogated by QCM-D analysis (Table 3). To determine a similar deposition density for NAv

’ RESULTS AND DISCUSSION Cubosome Characterization. Dynamic light scattering (DLS) results (Table 1) indicated a mean cubosome size of 180 nm for bDSPE/phytantriol cubosomes, 185 nm for GM1/phytantriol cubosomes, 188 nm for bDSPE/GM1/phytantriol cubosomes, and 175 nm for phytantriol cubosomes, respectively. The polydispersity index (PDI) for the cubosome samples, also obtained by DLS, was approximately 0.3, which is comparable to reported values for related systems.25,26 Cryo-TEM images of the various cubosome formulations (Figure 3) qualitatively confirm their existence in a cubic mor623

dx.doi.org/10.1021/la2032994 |Langmuir 2012, 28, 620–627

Langmuir

ARTICLE

Table 3. QCM-D Data for Au Sensor Surfaces Modified with NAva areal mass surface

thickness (nm)

(ng/cm2)

Δf (Hz)

ΔD (106)

SAMNAv NAv

18.4 ( 1.5 16.2 ( 1.2

18.2  102 16.2  102

82.4 ( 5.4 79.0 ( 2.4

3.6 ( 0.35 3.2 ( 0.29

a

Raw data were modeled using the Voigt model. Data were collected at 25 °C.

Table 4. QCM-D Data for the Changes in Frequency and Dissipation of the NAv Surface in the Presence of Cubosomes of Different Compositiona layer thickness cubosome preparation phytantriol

Δf (Hz)

ΔD (106)

(nm)

2.2 ( 2.2

2.4 ( 0.1

0.93 ( 0.40

18.5 ( 3.7

3.6 ( 2.2

1.00 ( 0.16

192.5 ( 32.0

22.7 ( 3.1

62.4 ( 11.7

phytantriol/bDSPE/GM1b 192.5 ( 21.7

22.7 ( 1.8

57.4 ( 3.77

phytantriol/GM1b phytantriol/bDSPEb

Figure 4. QCM-D traces of Δf (A) and ΔD (B) for different concentrations of bDSPE/phytantriol cubosomes. The surface was washed (*), and NAv was flowed into the chamber (1). The surface was washed again (*), and the cubosomes were flowed into the chamber (2) at concentrations of 0.05 mg/mL (green), 0.10 mg/mL (blue), 0.25 mg/mL (purple), and 0.50 mg/mL (orange). Note: The Δf versus time runs were performed in triplicate, and data from a single representative experiment are shown to aid clarity.

a

Data were modeled using the Voigt model. Data were collected at 25 °C. b 0.5% w/w bDSPE and/or GM1. The concentration of cubosomes was 0.10 mg/mL.

the phytantriol/GM1 cubosomes devoid of bDSPE. For both cubosomes samples containing bDSPE, a 10-fold increase in the magnitude of the frequency change was observed for each, indicative of greatly increased particlesurface interaction with the modified surface, as shown in Figure 4. It is of particular note that the Δf and ΔD for the bDSPE cubosomes are identical, suggesting a commensurate binding to the surface for each system. To assess the level of surface coverage, the cubosome concentration was varied from 0.05 to 0.5 mg/mL. Interestingly, the highest cubosome concentration did not yield the largest Δf or the most stable dissipation. The 0.20 and 0.50 mg/mL cubosome sample solutions exhibited rapid binding kinetics with the protein surface but did not afford the highest Δf. Moreover, the lowest concentration of cubosomes tested (0.05 mg/mL) did not reach the maximum Δf observed for the higher cubosome concentrations and displayed a slow interaction with the NAv-modified surface, indicating that total surface coverage had not been obtained. The optimum concentration for deposition was found to be a solution containing 0.10 mg/mL of cubosomes, which showed the largest Δf of approximately 300 Hz. The dissipation sensorgram collected in parallel with the frequency sensorgram did not display a peak and was also smooth, indicating that the surface was stable and noticeably lacking the layer reorganization processes that were present for the QCM time-resolved traces of the higher cubosome concentrations. The maximum dissipation for the 0.10 mg/mL cubosome sample was also slightly higher than that for the larger cubosome concentrations at 42  106. These data suggest that an optimal concentration of cubosomes exists that permits the efficient packing of particles onto the modified surface. We propose that high cubosome concentrations coupled with the rapid binding kinetics of the avidin biotin interaction lead to chaotic cubosome binding onto the surface, leaving small areas in which cubosomes cannot attach or that require a significant cubosome layer reorganization to permit stable cubosome coverage. This is evidenced by the decreased Δf for the higher cubosome concentrations, and the reorganization

deposited via the alkanethiol monolayer and directly to a gold surface, the initial layer thickness, frequency shift, and dissipation, both methods were compared. Both data sets were similar; however, the Δf was increased slightly for the SAMNAv layers corresponding to the added mass of the alkanethiol SAM. For both surfaces, Sauerbrey and Voigt models30 were used to characterize the surface behavior; however, due to the large dissipative values obtained for the adsorbed layer (>2.0  106 units), Voigt modeling was used for the remainder of the measurements. Interestingly, for the initial surface modification of the sensors, the Voigt model and Sauerbrey model did not differ significantly in their values for thickness, with the Sauerbrey model giving thicknesses of 16.0 ( 0.4 and 17.6 ( 0.5 nm for the NAv and SAMNAv surfaces, respectively. Dissipation values for both layers are similar, indicating that the SAM layer does not influence the overall viscoelasticity of the NAv layer when compared to adsorbed NAv, and both methods for NAv modification of the QCM-chip surface result in layers that behave similarly. For both methods of surface modification, the NAv layer appeared to be a dense, rigid continuum, as indicated by the minimal difference in Δf values for consecutive higher sensor harmonics. This is in good agreement with results reported elsewhere for avidin-based systems31 and other similar proteins.3234 Formation of CubosomeProtein Complex at the Au QCM-D Sensor Surface. Following immobilization of NAv to Au-sensor surfaces, functionalized cubosomes were flowed into the QCM chamber to tether the cubosomes to the modified surface via the biotinylated-DSPE lipid. Table 4 shows the frequency and dissipation changes of the NAv surface in the presence of cubosomes of different composition. As a control measurement, phytantriol cubosomes without any bDSPE were flowed over the NAv layer, and these did not appreciably interact with the surface as demonstrated by the low frequency shift (Δf ≈ 12 Hz) that was interpreted to represent minimal nonspecific adsorption. Similar nonspecific behavior (Δf ≈ 18 Hz) was observed for 624

dx.doi.org/10.1021/la2032994 |Langmuir 2012, 28, 620–627

Langmuir

ARTICLE

contact AFM measurements or ellipsometry36 would provide a reliable estimate of the thickness of the cubosome layer. Protein Interaction with the CubosomeProteinAu Surface Complex. To assess if the immobilized cubosomes captured proteins from solution, a series of protein solutions were flowed into the QCM chamber to interact with the cubosomes (0.10 mg/mL) containing bDSPE or GM1, as described in the Materials and Methods. Table 5 shows the frequency and dissipation data for a fixed concentration (100 nM) of protein analytes flowed against each of the cubosome preparations. As expected, NAv significantly interacted with both cubosome surfaces (Δf ≈ 13.5 Hz), indicating that NAv readily bound to the available biotin groups in the phytantriol/GM1/bDSPE and phytantriol/ bDSPE cubosomes, respectively. Cholera Toxin B subunit, CTB, binding to the phytantriol/ GM1/bDSPE cubosomes was indicated by the moderate frequency shift (Δf ≈ 13.3 Hz). From the raw data shown in Figure 5A, there is a discernible shift in frequency upon CTB interaction with the cubosome surface at the seventh harmonic. Conversely, appreciable binding to the phytantriol/bDSPE cubosome surface (without GM1) was not observed. The nonspecific control analytes, IgG and BSA, did not interact significantly with the cubosome complex, typically resulting in a small positive change in the observed frequency (Table 5). This absence of appreciable nonspecific binding of proteins to lipid cubic phases supports similar results previously reported for MO cubic phases.10 An important feature of these results centers on the preservation of the functionality of NAv and GM1 within the immobilized cubosomes. This suggests that the phytantriol cubosomes provide an appropriate environment to maintain the activity of the introduced ligands and may be extended to molecules of greater complexity such as proteins, peptides, and DNA. Noteworthy is the consistently low frequency shifts for the analytes examined, along with a concomitant decrease in the dissipation with addition of the CTB or NAv. The dissipation decrease in general may be associated with a loss of viscous character, which is the result of restricted movement within the material. This may be attributed to the increased rigidity of the surface layer, and this is consistent with CTB or NAv binding to the corresponding receptors (GM1 and biotin, respectively)

step in the dissipation sensorgrams at higher concentrations (causing a peak in ΔD). At lower concentrations (0.05 and 0.10 mg/mL of cubosomes), however, the signals were smoother for both Δf and ΔD, indicating a better organization of the cubosomes on the surface and thus greater deposition of cubosomes at the surface. Viscoelastic modeling (Voigt model) of the data was performed to extract further details about the deposition process (Table 4). The model predicts a thickness of approximately 60 nm. Neto and co-workers35 have demonstrated by atomic force microscopy that glycerol monooleate cubosomes deposited on muscovite mica results in a thickness of 23 nm. The mean particle size of the corresponding cubosomes in water was approximately 254 nm by dynamic light scattering (DLS) experiments. Similarly, the mean particle size of phytantriol/bDSPE and Phytantriol/bDSPE/GM1 cubosomes was approximately 187 nm by DLS measurements, suggesting that phytantriol cubosomes undergo a similar shape deformation (without collapse to a planar lipid bilayer) on surface immobilization. It is noteworthy that the Voigt model used in this work assumes a homogeneous layer (i.e., thickness and height) in addition to complete surface coverage, which may be incompatible with cubosome packing at the surface, further contributing to the surface inhomogeneity. Indeed, direct Table 5. QCM-D Data (Δf and ΔD) for Protein Analytes Interacting with Immobilized Cubosomes analyte cubosomesa phytantriol/bDSPEb

phytantriol/bDSPE/GM1b

(100 nM)

Δf (Hz)

ΔD (106)

NAv

13.7 ( 0.2

1.2 ( 0.4

CTB

+1.4 ( 0.3

2.7 ( 0.3

BSA

+2.1 ( 0.4

2.9 ( 2.1

IgG

+4.8 ( 0.8

5.3 ( 1.4

NAv

13.5 ( 0.5

1.3 ( 0.3

CTB

13.3 ( 5.9

1.5 ( 0.7

BSA IgG

+2.2 ( 0.6 +5.0 ( 1.0

3.1 ( 1.9 5.5 ( 1.2

a +7% w/w pluronic. b 0.5% w/w bDSPE and/or GM1. The concentration of cubosomes was 0.10 mg/mL.

Figure 5. (A) Typical raw data for frequency and dissipation versus time, and (B) modeled data for mass and thickness versus time, for the interaction of surface bound GM1 containing cubosomes with CTB at the seventh harmonic. The surface was cleaned and then modified with NAv (1). The surface was then washed, and GM1/bDSPE/phytantriol cubosomes were flowed over the surface (2). The surface was washed, and then CTB was flowed over the surface (3). The “*” indicates a brief wash step with PBS pH 7.4. 625

dx.doi.org/10.1021/la2032994 |Langmuir 2012, 28, 620–627

Langmuir within the structure. This increases the mechanical bulk and reduces the mobility of the cubosome surface. Because the measured frequency signal carries components from both the pure mass and the viscoelastic character of the surface layer, the changing viscoelasticity can account for frequency changes, and thus the dissipation trend might be seen as a better proof of the specific binding than the frequency change. Because of the complexity of the surface, a quantitative assessment of the analyte binding affinity and total analyte mass adsorption cannot be made; however, the retained analyte binding capacity, its specificity, and selectivity are clearly demonstrated.

ARTICLE

’ REFERENCES (1) (a) Kiessling, V.; Domanska, M. K.; Murray, D.; Wan, C.; Tamm, L. K.; Begley, T. P. Supported Lipid Bilayers. Wiley Encyclopedia of Chemical Biology; John Wiley & Sons, Inc.: New York, 2007. (b) Tanaka, M.; Sackmann, E. Nature 2005, 437, 656–663. (2) (a) Shahal, T.; Melzak, K. A.; Lowe, C. R.; Gizeli, E. Langmuir 2008, 24, 11268–11275. (b) Jonsson, M. P.; J€ onsson, P.; H€ o€ok, F. Anal. Chem. 2008, 80, 7988–7995. (c) H€o€ ok, F.; Stengel, G.; Dahlin, A. B.; Gunnarsson, A.; Jonsson, M. P.; J€onsson, P.; Reimhult, E.; Simonsson, L.; Svedhem, S. Biointerphases 2008, FA108. (d) Castellana, E. T.; Cremer, P. S. Surf. Sci. Rep. 2006, 61, 429–444. (3) (a) Williams, T. L.; Jenkins, A. T. A. J. Am. Chem. Soc. 2008, 130, 6438–6443. (b) Jonsson, M. P.; J€onsson, P.; Dahlin, A. B.; H€o€ok, F. Nano Lett. 2007, 7, 3462–3468. (4) (a) Majd, S.; Mayer, M. Angew. Chem., Int. Ed. 2005, 44, 6697–6700. (b) Yee, C. K.; Amweg, M. L.; Parikh, A. N. J. Am. Chem. Soc. 2004, 126, 13962–13972. (5) Fraser, S.; Separovic, F.; Polyzos, A. Europ. Biophys. J. 2009, 39, 83–90. (6) Polyzos, A.; Alderton, M. R.; Dawson, R. M.; Hartley, P. G. Bioconjugate Chem. 2007, 18, 1442–1449. (7) Luzzati, V.; Tardieu, A.; Gulikkrz., T; Rivas, E.; Reisshus, F. Nature 1968, 220, 485–488. (8) Clogston, J.; Caffrey, M. J. Controlled Release 2005, 107, 97–111. (9) Nazaruk, E.; Bilewicz, R.; Lindblom, G.; Lindholm-Sethson, B. Anal. Bioanal. Chem. 2008, 391, 1569–1578. (10) Angelova, A.; Ollivon, M.; Campitelli, A.; Bourgaux, C. Langmuir 2003, 19, 6928–6935. (11) Fraser, S. J.; Dawson, R. M.; Waddington, L. J.; Muir, B. W.; Mulet, X.; Hartley, P. G.; Separovic, F.; Polyzos, A. Aust. J. Chem. 2011, 64, 46–53. (12) Fraser, S. J.; Rose, R.; Hattarki, M. K.; Hartley, P. G.; Dolezal, O.; Dawson, R. M.; Separovic, F.; Polyzos, A. Soft Matter 2011, 7, 6125–6134. (13) Garg, G.; Saraf, S. Biol. Pharm. Bull. 2007, 30, 350–353. (14) Drummond, C. J.; Fong, C. Curr. Opin. Colloid Interface Sci. 1999, 4, 449–456. (15) Boyd, B. J.; Whittaker, D. V.; Khoo, S. M.; Davey, G. Int. J. Pharm. 2006, 309, 218–226. (16) Tien, H. T.; Ottova, A. L. Colloids Surf., A 1999, 149, 217–233. (17) (a) Tarasova, A.; Griesser, H. J.; Meagher, L. Langmuir 2008, 24, 7371–7377. (b) Vermette, P.; Gengenbach, T.; Divisekera, U.; Kambouris, P. A.; Griesser, H. J.; Meagher, L. J. Colloid Interface Sci. 2003, 259, 13–26. (c) Vermette, P.; Meagher, L.; Gagnon, E.; Griesser, H. J.; Doillon, C. J. J. Controlled Release 2002, 80, 179–195. (d) Volodkin, D. V.; Schaaf, P.; Mohwald, H.; Voegel, J. C.; Ball, V. Soft Matter 2009, 5, 1394–1405. (18) Brochu, H.; Vermette, P. Langmuir 2007, 23, 7679–7686. (19) Adrian, M.; Dubochet, J.; Lepault, J.; McDowall, A. W. Nature 1984, 308, 32–36. (20) Seddon, J. M.; Squires, A. M.; Conn, C. E.; Ces, O.; Heron, A. J.; Mulet, X.; Shearman, G. C. Philos. Trans. R. Soc., A 2006, 364, 2635–2655. (21) Praporski, S.; Ng, S. M.; Nguyen, A. D.; Corbin, C. J.; Mechler, A.; Zheng, J.; Conley, A. J.; Martin, L. L. J. Biol. Chem. 2009, 284, 33224–33232. (22) Sauerbrey, G. Z. Phys. 1959, 155, 206–222. (23) Kruss, S.; Wolfram, T.; Martin, R.; Neubauer, S.; Kessler, H.; Spatz, J. P. Adv. Mater. 2010, 22, 5499–5506. (24) (a) Hook, F.; Kasemo, B.; Nylander, T.; Fant, C.; Sott, K.; Elwing, H. Anal. Chem. 2001, 73, 5796–5804. (b) Hook, F.; Rodahl, M.; Brzezinski, P.; Kasemo, B. Langmuir 1998, 14, 729–734. (25) Sagnella, S. M.; Conn, C. E.; Krodkiewska, I.; Drummond, C. J. Soft Matter 2009, 5, 4823–4834. (26) Popescu, G.; Barauskas, J.; Nylander, T.; Tiberg, F. Langmuir 2007, 23, 496–503. (27) Rizwan, S. B.; Dong, Y. D.; Boyd, B. J.; Rades, T.; Hook, S. Micron 2007, 38, 478–485.

’ CONCLUSION In this study, methodology is described, which enables the controlled deposition and binding of protein receptor-functionalized cubosomes on a QCM-D crystal sensor. The incorporation of a secondary biotinylated lipid into the inverse cubic phase of phytantriol cubosomes permitted specific tethering of the lipid particles to the sensor surface via a neutravidin (NAv) interlayer, thereby creating a complex sensing matrix. Analyte capture was assessed by examining the binding of cholera toxin B subunit (CTB) or NAv to the surface immobilized cubosomes functionalized with GM1 (phytantriol/GM1/bDSPE) or unbound biotinylated lipid (phytantriol/bDSPE). The frequency data showed that both CTB and NAv significantly interacted with both cubosome preparations that contained the complementary binding partner. Importantly, control measurements determined that the cubosomes layer retained specificity for the selected analytes, and nonspecific adsorption was not observed. Concurrently, structural studies of unbound particles show that the functionalization of the phytantriol cubosomes with secondary and ternary lipids does not compromise the integrity of the particle internal geometry, and the nanostructure remains intact. This significant feature enables a high degree of chemical or biological complexity to be introduced into the cubosome bilayer without perturbation of the cubic scaffold, with retention of activity of the added functional lipids. Titration experiments determined an optimal concentration for cubosomes to promote the formation of a continuum of cubosomes on the attachment surface with minimal rearrangement of cubosomes after immobilization. Work is presently underway to elucidate the precise structure of the immobilized cubosome layer and the internal cubic phase. This work now paves the way for the introduction of extended threedimensional bilayer structures (cubosomes) with multiple functionalities, as high surface area scaffolds for the functionalization of sensor devices. ’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

’ ACKNOWLEDGMENT We thank CSIRO OCE PhD Scheme for funding (S.J.F.). We acknowledge Lynne Waddington for her valued help in obtaining the Cryo-TEM images. L.M. would like to acknowledge the financial support received from the Australian Research Council. S.J.F. would like to thank the David Lachlan Hay Memorial Fund (Postgraduate Writing-Up Award) for their generous financial support. 626

dx.doi.org/10.1021/la2032994 |Langmuir 2012, 28, 620–627

Langmuir

ARTICLE

(28) Dong, Y. D.; Larson, I.; Hanley, T.; Boyd, B. J. Langmuir 2006, 22, 9512–9518. (29) (a) Kulkarni, C. V.; Tang, T. Y.; Seddon, A. M.; Seddon, J. M.; Ces, O.; Templer, R. H. Soft Matter 2010, 6, 3191–3194. (b) Li, X. F.; Kunieda, H. Langmuir 2000, 16, 10092–10100. (c) Wang, Z. N.; Zheng, L. Q.; Inoue, T. J. Colloid Interface Sci. 2005, 288, 638–641. (30) Voinova, M. V.; Rodahl, M.; Jonson, M.; Kasemo, B. Phys. Scr. 1999, 59, 391–396. (31) Wolny, P. M.; Spatz, J. P.; Richter, R. P. Langmuir 2010, 26, 1029–1034. (32) Hook, F.; Rodahl, M.; Kasemo, B.; Brzezinski, P. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 12271–12276. (33) Caruso, F.; Rodda, E.; Furlong, D. N.; Haring, V. Sens. Actuators, B 1997, 41, 189–197. (34) Nezu, T.; Taira, M.; Saitoh, S.; Sasaki, K.; Araki, Y. Int. J. Biol. Macromol. 2010, 46, 396–403. (35) Neto, C.; Aloisi, G.; Baglioni, P.; Larsson, K. J. Phys. Chem. B 1999, 103, 3896–3899. (36) Vandoolaeghe, P.; Tiberg, F. K.; Nylander, T. Langmuir 2006, 22, 9169–9174.

627

dx.doi.org/10.1021/la2032994 |Langmuir 2012, 28, 620–627