Development of Cubosomes as a Cell-FreeBiosensing Platform

Scott J. Fraser,A,C Raymond M. Dawson,B Lynne J. Waddington,C

BenW. Muir,C Xavier Mulet,C Patrick G. Hartley,C Frances Separovic,A

and Anastasios PolyzosC,D,E

ASchool of Chemistry, Bio21 Institute, The University of Melbourne, Melbourne,

Vic. 3010, Australia.BDSTO Melbourne, Defence Science and Technology Organisation, PO Box 4331,

Melbourne, Vic. 3001, Australia.CCSIRO Materials Science and Engineering, Bayview Avenue, Clayton South,

Vic. 3169, Australia.DDepartment of Chemistry, University of Cambridge, Lensfield Road,

Cambridge CB2 1EW, United Kingdom.ECorresponding author. Email: tash.polyzos@csiro.au

The parallel between the lipidic microenvironments of the inverse bicontinuous cubic phase and the biological membranedistinguishes cubic phases as an attractive option for development of cell-free biosensors containing protein or glycolipid

receptors. Herein we describe a novel strategy toward the creation of a biosensing platform derived from the surfaceattachment of a colloidally stable inverse cubic structure (cubosomes).We report the preparation of cubosomes composedof the amphiphile phytantriol, themembrane glycolipid receptormonosialoganglioside-GM1 and the biotin-functionalizedamphiphile 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethyleneglycol)-2000] (bDSPE). The

tethering of cubosomes to the various surfaces was mediated through bDSPE binding to streptavidin- and avidin-modifiedsurfaces. Allylamine plasma polymer surface modification enhanced the surface immobilization of avidin, whichincreased the density of bound cubosomes. The resultant polymer–protein–cubosome complex was imaged by cryo-

transmission electron microscopy analysis and the cubosome structure was impressively preserved within the complex.Cholera toxin binding to cubosomes containing GM1 was used to assess the performance of the cubosomes, subsequent tosurface attachment, via a modified enzyme-linked immunosorbent assay. Specific immobilization of complex protein–

receptor–cubosome systems paves the way for development of a structurally complex, heterogeneous platform for sensingapplications.

Manuscript received: 1 October 2010.

Manuscript accepted: 22 November 2010.

Introduction and Aims

The development of high-throughput nanodevices capableof detecting small molecule–protein or protein–proteininteractions, while concomitantly minimizing the consump-tion of valuable materials in biological screening, is of great

interest to the pharmaceutical industry and life-scienceresearch. As the majority of drug targets include surface-bound or integral membrane proteins, cell-free biosensing

devices are reliant on the surface attachment of a targetreceptor in a macromolecular environment that mimics thenatural biological environment of the receptor.[1] This is

particularly applicable to integral membrane proteins where amembrane-like environment is essential for the retention ofactivity for this sensitive class of biological receptor. Recent

advances in surface chemistry and molecular biology haveallowed the incorporation of important receptors, such asG-protein coupled receptors (GPCRs),[2] membrane pro-teins[3] and DNA,[4] to an appropriate surface via several

different media. These scaffolds include lipid bilayers,[5,6]

immobilized vesicles[7–9] and liposomes,[10] high-density

lipoprotein particles,[11] nanodiscs,[12] polyelectrolyte cap-sules,[13] and macromolecular assemblies.[14]

An alternative scaffold for the presentation of biologicalmolecules is the inverse bicontinuous cubic phase (QII), which

is a three-dimensional structure of cubic symmetry formed bythe self-assembly of particular amphiphiles in water.[15–17]

The three-dimensional structure (Fig. 1b) affords a self-

assembled material with a remarkably high surface area, upto 400m2 g�1, and extensive porosity.[18] The QII phase is alsoresistant to osmotic or mechanical rupture, in contrast to the

properties of liposomes or micelles,[19] and is thermodynami-cally stable in excess water over a broad temperaturerange.[20–21] High viscosity, however, inhibits the application

of bulk QII phase materials to fabricated biosensing devices.This can be overcome by the preparation of a stable dispersionof submicron-sized particles, termed cubosomes, that retains

CSIRO PUBLISHING

Aust. J. Chem. 2011, 64, 46–53 www.publish.csiro.au/journals/ajc

� CSIRO 2011 10.1071/CH10361 0004-9425/11/010046

Full Paper

RESEARCH FRONT

the internal structure of the originating cubic phase and

possesses a viscosity approximately equal to that of water.[22]

As part of our ongoing interest in the application of cubicphases to present biologically active molecules,[17] we have

undertaken a study to examine methods for the immobilizationof cubosomes to a surface, along with an evaluation of theirpropensity to bind a target protein once surface-bound. We

chose to use phytantriol (3,7,11,15,-tetramethyl-1,2,3,-hexade-canetriol) (Fig. 1a) as the amphiphile to form the inverse cubicphase scaffold. Although monoolein is the traditional choice of

amphiphile for cubic phase structures, the greater chemicalstability of phytantriol coupled with the broad thermal stabilityof the phase in excess water (20–408C) provides an alterna-tive.[23] One ligand and a receptor were incorporated into the

phytantriol scaffold: (i) the ligand, the biotinylated-lipid1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethyleneglycol)-2000] (bDSPE, Fig. 1b right), and (ii) the

receptor monosialoganglioside-GM1 (GM1, Fig. 1b left), theglycolipid receptor for cholera toxin (CT). The bDSPE wasincorporated to facilitate the immobilization of cubosomes at

surfaces that were modified with either streptavidin, an avidinderived from Streptomyces avidinii, or Neutravidin (NAv)(a commercially available, genetically modified avidin with anet neutral charge). Both have a dissociation constant (Kd) with

respect to biotin similar to that of avidin (Kd ,10�15M[24]).A key aspect of this investigation is the characterization of

cubosomes bound to a surface. Cryo-transmission electron

microscopy (cryo-TEM) analysis was used to obtain images ofcubosomes bound to an avidin surface layer to determine ifthe microstructure of cubosomes was preserved on binding.

Essential to the success of this experiment was the surface

modification of the grid surface using a plasma polymer film.

Treatment of the carbon TEM surfacewith an allylamine plasmapolymer film should promote a stronger interaction betweenthe NAv and the TEM grid surface, consistent with other studies

that have shown greater interaction of NAv with net positivelycharged surfaces.[25] Additional studies examined the binding ofCT by GM1-phytantriol cubosomes using enzyme-linked immu-

nosorbent assay (ELISA) on a streptavidin-modified 96-wellplate. The ELISA data complemented the cryo-TEM analysisand, by comparison with characterization of cubic mesophases

using ELISA,[26,27] provided quantitative information about thebinding capacity of such complex systems and their potential asa new class of biosensing surfaces.

Experimental

Materials

All materials were used as provided from the supplier unlessotherwise noted. Phytantriol was obtained from DSM Nutri-tional Products (Parsippany, USA). Cholera toxin from Vibrio

cholerae (Inaba 569B, azide-free) was obtained from ListBiological Laboratories (Campbell, USA). Pluronic F127 waspurchased from Sigma (St Louis, MO, USA). 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene-

glycol)-2000] (ammonium salt, 100%) (bDSPE) was pur-chased from Avanti Polar Lipids Inc. (Alabastar, AL, USA).Neutravidin was purchased from Thermo Fisher Scientific

(Waltham, MA, USA). Dichloromethane (HPLC grade),hydrogen peroxide (30%), and ethanol (100%) were purchasedfrom Merck (Hull, UK). Allylamine (98% purity) was pur-

chased from Sigma–Aldrich (St Louis) and was used as a

HOOH

200 nm

(a)

(c) (d) (e)

(b)

100 nm 200 nm

OH

Fig. 1. (a) Stick and space-filling representations of the surfactant phytantriol. (b) A representation of the Pn3m (diamond) cubic structure highlighting the

integration of GM1 (monosialoganglioside-GM1) (left) and bDSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethyleneglycol)-

2000]) (right) into the phytantriol bilayers. Cryo-transmission electron microscopy (TEM) images of bDSPE/GM1/phytantriol cubosomes (c) and (d), and

phytantriol cubosomes (e) taken using an AA (allylamine)-modified electron microscopy (EM) grid. NAv (Neutravidin) was adsorbed onto the AA-modified

(yellow arrow, d), carbon grid (red arrow, d) to test for interaction with bDSPE in cubosomes.

Cubosomes as Cell-Free Biosensors 47

RESEARCH FRONT

monomer without further purification. Nunc-Immuno Max-isorp 96-well plates (cat. no. 439454) and Nunc immobilizer

streptavidin 96-well plates (cat. no. 436014) were obtainedfrom Invitro Technologies (Melbourne, Vic.) and ThermoFisher Scientific Instruments (Melbourne, Vic.) respectively.Purified polyclonal goat anti-cholera toxin was a product of

Biogenesis (Poole, UK). Rabbit anti-goat alkaline phosphataseconjugate, monosialoganglioside-GM1 (495% sodium salt)and all other consumables (e.g. buffer ingredients) were pur-

chased from Sigma–Aldrich (Sydney, NSW). MilliQ grade(0.5 mS cm�1 at 258C) water was purified through a Milliporesystem (Sydney, NSW) and used throughout this study.

Cubosome Preparation and Characterization

GM1/phytantriol, bDSPE/phytantriol or GM1/bDSPE/phytantriolmixtures were prepared for cubosome production according to

the methods described by Polyzos et al.[27] and Mulet et al.[28]

with 0.005mol per mol GM1 and bDSPE. Briefly, phytantriol,GM1 and bDSPE were dissolved in CH2Cl2/MeOH, where

the total mass of phytantriol, GM1 and bDSPE was 0.5 g. Theratio between the functional lipids (GM1 or bDSPE) was0.005molmol�1. The solvent was then removed by rotary

evaporation and aqueous Pluronic F127 (0.7%w/v) solutionwasadded up to a final volumeof 500mL to themicrocentrifuge tube.The viscous appearance of the hydrated material was qualita-tively attributed to the formation of an inverse cubic phase.

Dispersion of the bulk cubic phase was accomplished by probeultrasonication in a Misonix S-4000 sonicator (Qsonica, LLC,Newtown, CT, USA) (10-s intervals for 20min, at 7-mWamplitude using a 5-mm probe), affording an opaque solution of

dispersed materials, with an observable viscosity approximatelyequal to water.

A measurement of particle size distribution (diameter)was obtained in triplicate using a Nano-ZS Zetasizer (MalvernInstruments, Malvern, UK) at 258C assuming a viscosity of purewater. These measurements confirmed the preparation of parti-

cles with a mean diameter of ,150 nm. The crystallographiccubic structure of the particles was confirmed using the small-angle X-ray scattering (SAXS) beam-line of the Australian

Synchrotron, Melbourne, Vic. (Fig. 2b and 2c). Samples wereloaded into 1.5-mm X-ray special glass capillaries (HamptonResearch, NC, USA) before being mounted into a bespoke

sample holder. Sample temperature was set using a custom-designed Peltier-driven cell with temperature control of�0.18C.The X-ray beam was set to a wavelength of 0.8266 A with a

typical flux of 1013 photon s�1. Diffraction patterns werecollected in 2-D on a Pilatus 1M detector, which was offset toassess a greater range for acquisition of scattering vectors (q-range). All measurements were calibrated against silver behe-

nate (lattice parameter (a)¼ 58.38 A). Analysis of diffractionimages was carried out using the interface definition language(IDL) based AXcess software package,[29] with measured X-ray

diffraction spacings accurate to 0.1 A.

Cryo-transmission Electron Microscopy

Plasma Polymer Deposition onto Cryo-TEM Grids

Radio frequency glow discharge (RFGD) plasma polymer-izations were carried out in a custom-built reactor described byMuir and coworkers.[30] Briefly, the cylindrical reactor chamber

is defined by a height of 35 cm and a diameter of 17 cm. Within

NAvCubosomes

Parafilmmq H2O

Carbon TEM grid

3000 bDSPE/GM1/phytantriol cubosomes

(a)

(b)(c)

bDSPE/phytantriol cubosomes

GM1/phytantriol cubosomes2700

2400

2100

1800

1500

1200

I/I0

900

600

300

0

[110][111][200][211][220]

0.10 0.15 0.20

Q [�1]

0.25 0.30

NH2

Plasmapolymerization

Allylaminemonomer

Dropletpreparation on

parafilm

Equilibration ofgrid with droplets

30 min 10 min 10 min 30 min 10 min 30 min

Side viewTop view

Allylamineplasma polymer

Side view

Fig. 2. (a) Schematic flow chart for the modified cryo-transmission electron microscopy (TEM) assay; (b) the SAXS (small-angle X-ray scattering)

diffraction pattern for phytantriol cubosomes, typical of a Pn3m morphology, and (c) the SAXS diffractograms of the three cubosome populations studied

herein. The allowed Bragg reflections for the Pn3m space group corresponding to the Miller indices, hkl¼ 110, 111, 200, 211, 220 are indicated by arrows.

48 S. J. Fraser et al.

RESEARCH FRONT

this are placed two circular copper electrodes 10.3 cm indiameter, spaced 15 cm apart. The 200-mesh copper TEM gridcoated with Lacey carbon film (ProSciTech, Thuringowa, Qld)was placed on the lower grounded electrode and a continuous

radio-frequency glow discharge was generated at the upperelectrode. The allylamine (AA) monomer vapour streams weredirected to the reactor chamber from the liquid reagents con-

tained in a round-bottom flask via a stainless steel line and amanual valve for fine control of the flow. The neat AAmonomerliquid was degassed before plasma deposition with the flask

cooled in ice during the experiment.The parameters used for RFGD deposition of the AA film

were a frequency of 200 kHz, a load power of 20W, a monomer

pressure of 20 Pa and a treatment time of 25 s. Prior to plasmadeposition, the reactor was evacuated to a base pressure of lessthan 0.1 Pa. After deposition, the reactor was immediatelypumped down to base pressure before venting. The grids were

stored in clean tissue-culture-grade Petri dishes under ambientconditions until required for further surface modification withcubosomes.

Sample Preparation and Imaging

Samples were prepared for cryo-TEM imaging using a modifiedmethod of Adrian et al.[31] Accordingly, NAv in solution and

cubosomes were allowed to passively interact with AA-mod-ified cryo-TEM grids. Droplets of NAv solution, cubosomesolution and washing buffer (phosphate buffered saline (PBS) at

pH 7.4) were placed on a piece of Parafilm (Fig. 2a). AA-modified cryo-TEM grids were gently rested atop the NAvdroplet for 30min to allow for physisorption of NAv onto thegrid. The grid was transferred to the top of a MilliQ H2O droplet

for 10min, then gently transferred to a cubosome-containingdroplet for 30min. A final resting period of 30min on top of anmqH2O droplet was used to remove weakly bound cubosomes

from the surface. The grids were then transferred to the vitrifi-cation system. The gridwasmanually blotted for 10–15 s and the

resulting thin film then vitrified by plunging into liquid ethane.A laboratory-built vitrification system was used, allowinghumidity to be maintained at 90% during sample plunging andvitrification. The grids were stored in liquid nitrogen before

transferring into a Gatan 626-DH Cryo-holder. Imaging wascarried out using an FEI Tecnai 12 TEM, operating at 120 kV,with the sample at a temperature of �1808C. Images were

recorded using a MegaView III CCD camera equipped withAnalySis imaging software (Olympus Soft Imaging Solutions,Munster, Germany), using standard low-dose procedures to

minimize radiation damage.

Enzyme-linked Immunosorbent Assay (ELISA)

The assay of CT bound to GM1 immobilized onto Maxisorp

plates has been previously described.[26] A similar procedurewas used with the streptavidin plates, except that the incubationwith blocking buffer preceded addition of cubosomes (Fig. 3).Briefly, the wells of the plate were initially incubated for 1 h

with 360mL blocking buffer (0.25% bovine serum albumin inPBS/0.05%Tween 20) to block non-specific binding sites. Afterthis and subsequent incubations, the plate was washed three

times with PBS/Tween. The wells were then incubated with100mL of bDSPE/GM1/phytantriol cubosomes, followed by100mL CT (1–100 ngmL�1), 100mL goat anti-CT antibody

(Ab) and 100mL rabbit anti-goat alkaline phosphatase (Apase)conjugate, each in blocking buffer. These incubations wereperformed at room temperature for 1 h. Finally, the plate

received an additional washwith distilled water, followed by theaddition of 100mL alkaline phosphatase substrate (p-nitrophe-nyl phosphate) in Tris buffer. The rate of change of absorbanceat 405 nm was measured over 10min using a Bio-Tek Synergy

HT plate reader (Bio-Tek, Winooski, VT, USA). Inhibition ofthe binding of CT to immobilized GM1 on normal Maxisorpplates by Pluronic F127 was determined by incubating with CT

(total volume 130mL) for 1 h in disposable glass test tubes withgentle rocking on a rocking platform mixer (Ratek Instruments,

Streptavidin

Block; cubosomes

Wash;cholera toxin

Apase substrate

Wash; assay#

Cholera toxin (CT)

Streptavidin-coated 96-wellplate surface

GM1 bDSPE

Rabbit anti-goat Apase

Goat anti-CT Ab

Fig. 3. Schematic representation of the ELISA (enzyme-linked immunosorbent assay) used for measurement of CT (cholera toxin) bound to streptavidin-

modified 96-well plates. All experiments were carried out at 208C and in triplicate. #Awash with buffer (phosphate buffered saline with 0.5% (w/w) Tween-20

(PBST) at pH 7.4, with 0.25% w/w bovine serum albumin (BSA)) was carried out between each addition step. The abbreviations Ab and Apase correspond to

antibody and alkaline phosphatase respectively. The cubosome representation was modified from Spicer et al.[32]

Cubosomes as Cell-Free Biosensors 49

RESEARCH FRONT

Boronia, Vic.) during the incubation of the 96-well plate with

360 mL blocking buffer. A 100-mL aliquot from each test tubewas then added to wells of the washed plate, after which theassay proceeded as normal.

Data analysis was performed by constructing calibrationcurves for the net assay absorbance per hour (abs h�1) versusthe known [CT]. Data were fitted using the Langmuir equation:

abs h� 1 ¼ k1k2½CT�=ð1þ k2½CT�Þ ð1Þ

This rearranges to give:

½CT�=ðabs h� 1Þ ¼ 1=k1k2 þ ½CT�=k1 ð2Þ

where k1 is the rate constant for formation of the CT/GM1

complex, and k2 is the rate constant for dissociation of thecomplex. A plot of [CT]/(abs h�1) versus [CT] should, therefore,be linear and this was found to be the case. The amount of CT

that was not bound to immobilized GM1 (‘free CT’) wasdetermined by reference to net rates of change of absorbancein control wells (CT only) using Eqn 2. Data are quoted as

mean� standard error.

Results and Discussion

Preparation and Characterization of Cubosomes

Cubosomes composed of phytantriol, bDSPE/phytantriol,

GM1/phytantriol, or GM1/bDSPE/phytantriol were all preparedsuccessfully. Particle size distributions obtained by dynamiclight scattering (DLS) measurements indicated that the mean

diameter of these cubosomes was 175, 180, 185 and 188 nmrespectively (Table 1), with a polydispersity index in the order of0.15, which is in good agreementwith values reported elsewhere

for similar cubosomes systems.[33,34]

Synchrotron SAXS experiments verified cubic symmetry forall different cubosomes, with all samples maintaining a double

diamond morphology (Pn3m space group) (Fig. 2 and Table 1).The allowed Bragg reflections for a Pn3m space group corre-spond to theMiller indices, hkl¼ 110, 111, 200, 211, 221. Fromthe scattering vector positions (qhkl) of the Bragg reflection

maxima, the lattice spacing (a) of the cubic phase can bedetermined using the relationship in Eqn 3:

qhkl ¼ ð2pðh2 þ k2 þ l2Þ1=2Þ=a ð3Þ

An increase in the value of a indicates a decrease in negativeinterfacial curvature, and thus a tendency towards ‘flatter’ (less

negatively curved) structures. We anticipated that addition ofGM1 and bDSPE to the phytantriol/water system may favour aphase transition away from inverse cubic phase behaviour

toward the formation of a lamellar phase at higher

concentrations (i.e. decreased negative interfacial curva-

ture).[27] This was expected when we considered the overallmolecular geometric shape of the added lipids (Fig. 1b).[35]

However, at such low concentrations of the GM1 and bDSPE

(0.001molmol�1), minimal effect on phase behaviour wasobserved. The GM1/phytantriol (70.3 A), bDSPE/phytantriol(70.2 A), and GM1/bDSPE/phytantriol (71.4 A) cubosomes allshowed only a slight increase in lattice spacing a, and, therefore,a minimal decrease in negative interfacial curvature whencompared with the phytantriol cubosomes (69.3 A) while retain-ing Pn3m morphology (Fig. 2).

Data derived from the cryo-TEM analysis provide additionalinsight into cubosome interaction with surfaces. Visible in theseimages (Fig. 1c, 1d and 1e) are the plasma polymer layers

(yellow arrow) with a uniform average thickness of 14� 1 nmfrom the surface of the cryo-TEM grid (red arrow). In Fig. 1e,the phytantriol cubosomes lacking the bDSPE lipid appeared

to have no measurable affinity for the NAv-AA surface. Theimages generated for this system indicated that there was nosignificant interaction of cubosomes with the grid surface. InFig. 1c and 1d, however, for phytantriol cubosomes containing

bDSPE, the attachment to the NAv-AA surface of the grid isclearly visible. The cubosome particles retained a cubic shapeand displayed the textured surface that is typical of the cubic

structure as seen in other TEM images of cubosomes.[36]

Furthermore, on close inspection of Fig. 1d, a narrow layer ofwhat appears to be electron-beam damage is discernible along

the surface of the AA polymer layer – this is most probably theNAv, which may undergo irreversible radiation damage inelectron microscopy (EM) imaging, as is the case with mostproteins.[37]

The cubosomes in Fig. 1c are well separated at the gridsurface and show no signs of aggregation (with the exception ofa few regions of the grid that are highly curved). Several studies

have suggested such phenomena involving cubosome–surfaceinteractions using techniques such as quartz crystal microba-lance[38] and atomic force microscopy.[39] While each study has

provided insight into the potential behaviour of cubosomes atsurfaces, to the best of our knowledge, previous studies have notimaged cubosomes when incorporated into a protein–polymer

complex at a surface.

Cubosome–Protein Binding Interaction

The binding of the protein toxin CT to the cubosomes containingbDSPE, GM1 and phytantriol was investigated. We used an

ELISA technique to measure CT binding to bDSPE/GM1/phytantriol cubosomes, which were immobilized to 96-wellplates via a streptavidin coating. Similarly to the cryo-TEM

assay where cubosomes containing bDSPE attached to NAvphysisorbed to the grid surface, in this modified ELISA, thebiotin-modified cubosomes were available to interact with the

Table 1. Synchrotron small-angle X-ray scattering (SAXS) and DLS data for different cubosome populations at 258C

Dynamic light scattering (DLS) measurements are averages of five measurements, with an equilibration time of 3min between each reading and each reading

comprising 12 scans. bDSPE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethyleneglycol)-2000]; GM1, monosialoganglioside-GM1

Cubosome type Phase Lattice spacing [A] Space group Mean diameter (and polydispersity index)

Phytantriol Cubic 69.3� 0.044 Pn3m 175 nm (0.135)

Phytantriol/bDSPE Cubic 70.2� 0.066 Pn3m 180 nm (0.123)

Phytantriol/GM1 Cubic 70.3� 0.017 Pn3m 185 nm (0.187)

Phytantriol/bDSPE/GM1 Cubic 71.4� 0.068 Pn3m 188 nm (0.164)

50 S. J. Fraser et al.

RESEARCH FRONT

streptavidin surface to create a cubosome–protein complex for

CT sensing (Fig. 3). A solution of CTwas delivered to the boundcubosomes with intent to achieve an equilibrium bound and freeCT. The concentration of free CT was calculated from the assay

by reference to a control plot of rate of change of absorbance v.concentration of CT.

Several different cubosomes were evaluated for CT binding:bDSPE/GM1/phytantriol cubosomes (final solution concentra-

tion 16 nm GM1); GM1/phytantriol cubosomes (final solutionconcentration 75 nm GM1); and bDSPE/phytantriol cubosomes.Phytantriol cubosomes, assay buffer only and GM1 (75 nM) in

PBS were also evaluated as controls. The results (abs h�1),whereby the rate of change of absorbance is a function of theamount of CT bound at the surface, are shown in Fig. 4a for a

CT concentration of 100 ngmL�1. The largest measurablebinding was detected for bDSPE/GM1/phytantriol cubosomes(1.22� 0.020 abs h�1). The larger signal observed for thesecubosomes is derived from the higher concentration of CT on

the plate after incubation. This strongly suggests that there mustbe a higher concentration of GM1 on the surface, which must befrom the immobilized cubosomes. The importance of the

biotinylated lipid (bDSPE) in anchoring the bDSPE/GM1/phytantriol cubosomes to the plate is further evidenced by thelower signal recorded for the assays in which it was absent. This

was despite a 4.7-fold higher concentration of GM1 (GM1 in PBSor GM1/phytantriol cubosomes) in the other systems.

In order to optimize the assay conditions (i.e. maximum

signal to minimum noise), a time-course study was conducted.The time-dependence for CT binding by GM1 cubosome-coatedplates immobilized with streptavidin is shown in Fig. 4b. Unex-pectedly, the relationship between bound CT (as derived from

ELISA abs h�1 measurements) and incubation time was linearand not hyperbolic as reported by Dawson, who found that theassay reached amaximum after 24 h using aMaxisorp plate with

GM1 physisorbed to the surface.[26] The linear relationship found

here suggests that binding equilibrium has not been reached andconsiderably longer incubation periods may be required under

these conditions. This may be related to the large surface area ofthe cubosomes, along with the tortuous structure of the aqueouschannels, which limits the rate of diffusion of protein towardsaccessible GM1 receptors within the interior of the cubosome.

Indeed, the absence of equilibrium binding may additionally berelated to the turnover rate of the GM1-CT complex at the bilayersurface within the internal aqueous domains, and the lateral

diffusion of GM1 to regions where GM1-CT complex formationmay be hindered. Further studies are required to investigate theorigin of the linear behaviour (bound CT versus time), including

an exploration of the influence of the aqueous channel diameteron diffusion of CTwithin the cubic phase alongwith the ensuingbinding kinetics.

A similar linear behaviour is seen in Fig. 4c for thedependence of the CT uptake on [CT] using a 10-fold higherconcentration of bDSPE/GM1/phytantriol cubosomes (160 nM).This linear relationship indicates that saturation of this system

has not been reached and longer incubation times are required.Additionally, Fig. 4c shows that bDSPE/phytantriol cubosomes(no GM1) also bind CT non-specifically, but at a concentration

approximately three orders of magnitude higher than that ofbDSPE/GM1/phytantriol cubosomes (160 nM cf. 150mM for thebDSPE/phytantriol cubosomes).

Furthermore, even at relatively high and low concentrationsof bDSPE/GM1/phytantriol cubosomes (160 nm GM1 or 16 nmGM1), the rate of change of absorbance remained equal (1.20 and

1.22 abs h�1 respectively). Coupled with the fact that the rate ofchange of absorbance for bDSPE/GM1/phytantriol cubosomesusing streptavidin plates with 100 ngmL�1 CT (1.22 abs h�1) is

only 9% of that for the corresponding control measurement forGM1 in PBS alone (13.96 abs h�1),[26] this suggests that CT maynot be completely binding to the cubosomes under these

1.4(a)

(b)

(c)

b-GM1cubosomes

b-GM1 cubosomes(10 nM)

160 nM b-GM1 cubosomes

150 �M b-PT cubosomes

GM1 in PBS(75 nM)

GM1 cubosomes(75 nM)

Time [h]

Cholera toxin [ng mL�1]

b-PTcubosomes

PTcubosomes Buffer

only

GM1cubosomes

GM1 inPBS

1.2

1.0

0.8

0.6

0.4

0.2

0.0

3.0

2.5

2.0

1.5

1.0

0.5

0.00 5 10 15 20 25

0 50 100 150 200 250

abs

h�1

abs

h�1

abs

h�1

2.5

2.0

1.5

1.0

0.5

0.0

Fig. 4. ELISA (enzyme-linked immunosorbent assay) results for (a) CT

(cholera toxin) binding (abs h�1) to different cubosome mixtures that have

been pre-incubated with the streptavidin plate; (b) absorbance rate versus

time for different cubosomemixtures interactingwith the streptavidin plates.

Note here that the fit is approximately linear for all surfaces; and (c)

absorbance rate versus [CT] for cubosomes with and without GM1. The fits

are approximately linear in each case. For clarity, the following abbrevia-

tions apply: b-GM1, bDSPE/GM1/phytantriol cubosomes; b-PT, bDSPE/

phytantriol cubosomes; GM1 cubosomes, GM1/phytantriol cubosomes; and

PT cubosomes, phytantriol cubosomes. (All error bars shown are standard

errors of the mean (s.e.m.). bDSPE, 1,2-distearoyl-sn-glycero-3-phosphoetha-

nolamine-N-[biotinyl(polyethyleneglycol)-2000]; GM1, monosialoganglio-

side-GM1.)

Cubosomes as Cell-Free Biosensors 51

RESEARCH FRONT

conditions. The bDSPE contains a phosphate group that links the

biotin-PEG to the head group of the DSPE molecule. Thealkaline phosphatase step in the assay may be hydrolyzing thislinkage and releasing the cubosomes from the surface, thereby

generating the decreased absorbance rate values for the strepta-vidin plate assay compared with the control measurements.However, some cubosomes must be attached to the plates forthe signal to be generated, particularly when a comparison is

drawn between the bDSPE/GM1/phytantriol system and the non-biotinylated cubosomes shown in Fig. 4a. In this case, the signalfor the bDSPE/GM1/phytantriol system is six times greater

than the remaining cubosome preparations, and twice that ofGM1 in PBS.

CT Binding Inhibition by Pluronic F127

It is also possible that the stabilizing agent, a block copolymersurfactant polaxamer (Pluronic F127), also affects the binding ofcubosomes to the surface. Using a classical inhibition assay with

standard Maxisorp plates (no bDSPE present), we measured theconcentration of Pluronic F127 required to inhibit the binding ofCT by 50% (IC50). The concentration of Pluronic F127 was

based on an average molecular weight of 12500 g mol�1. To our

surprise, we found that Pluronic F127, when incubated withCT before addition to the assay plate, reduced CT binding. Anexample is shown in Fig. 5a, where the Pluronic F127 con-

centration for 50% inhibition was found to be ,12.5 mM andvaried significantly with [CT] in the range of 2–70 ngmL�1,with higher potency at higher [CT] (Fig. 5b). Although thepolymer was diluted to very low concentrations with the cubo-

somes, the Pluronic F127may have adsorbed to exposed regionsof the plate, and resulted in some inhibition of binding of CT tothe plate surface. This is supported by an assay where Maxisorp

plates with immobilized GM1 were incubated for 1 h withPluronic F127, after which the Pluronic F127 was washed outand CT only was added to the plate. The assay then proceeded as

normal with addition of antibody, buffer, etc. Reduced CTbinding was indeed observed, as shown in Fig. 5c (pooled datafrom two to three experiments). Compared with the experimentswhen Pluronic F127 was incubated with CT before addition to

the plate (Fig. 5a), there was considerably more experimentalvariability and the measured binding was three times greater.Despite this, we observed that the bDSPE/GM1/phytantriol

10(a) (b)

(c) (d)

15

10

5

0

8

6

4

2

00 5 10 15 20

Pluronic F127 [�M]

Free

cho

lera

toxi

n [n

g m

L�1 ]

IC50

for

inhi

bitio

n of

bin

ding

by

F12

7 [�

M]

25 30 35 40 0 10 20 30 40

Cholera toxin [ng mL�1]

50 60 70 80

40

30

20

0

10

IC50

for

inhi

bitio

n of

bin

ding

[�M

]

0 10 20 30 40

Cholera toxin [ng mL�1]

50 60 70 80

45

10

8

6

4

2

00 20 40

F127 [�M]

Free

cho

lera

toxi

n [n

g m

L�1 ]

60 80 100

Fig. 5. ELISA (enzyme-linked immunosorbent assay) results for the inhibition of binding of CT (cholera toxin) to immobilized GM1 (monosialoganglioside-

GM1) by Pluronic F127 including half-maximal inhibitory concentration (IC50) values. (a) The Pluronic F127was incubatedwith 10 ngmL�1 CT for 1 h before

adding to the plate. Error bars represent the standard error of the mean from four replicates. (b) The effect of CT concentration on inhibition by Pluronic F127

of the binding of CT to immobilized GM1. (c) Binding of 10 ngmL�1 CT to immobilized GM1 after the plate had been incubated with Pluronic F127 of

concentration shown and then washed out before addition of CT. Error bars represent the standard error of the mean from two to three replicates. (d) Effect

of CT concentration on the apparent inhibition of binding of CT to immobilized GM1 by incubation of the plate with Pluronic F127 before addition of CT to the

washed plate.

52 S. J. Fraser et al.

RESEARCH FRONT

cubosomes bound to the streptavidin plates gave the largest

absorbance rate values (Fig. 4a) and therefore suggest that asignificant population of the cubosomes remains attached to thesurface and the GM1 is still available to bind CT.

The ELISA data in combination with the cryo-TEM resultsshow that it is indeed possible to tether cubosomes incorporatinga specific biological or chemical functionality to a complemen-tary surface. To the best of our knowledge, we have been able for

the first time to image intact cubosomes bound to a surface viaspecific ligand–protein interaction. We have also successfullydemonstrated that a classical biotin–streptavidin binding inter-

action can be used to immobilize cubosomes to a streptavidin-coated plate with retention of biological activity of the incorpo-rated receptor (GM1). Although the ELISA results indicate that

optimization of experimental conditions is required, the useof a biotinylated lipid with non-hydrolyzable linker to replacethe phosphate group in bDSPE may well improve the bindingresults. The linear behaviour of the rate of absorbance versus CT

assay data suggests that the high surface area of cubosomes mayresult in non-equilibrium binding of receptor–analyte interac-tion. We are currently investigating methods to control the

aqueous channel size of cubosomes, along with reducing thesize of the particles to evaluate if these factors will improvethe binding kinetics. On this basis, we anticipate that cubosomes

may be incorporated into biosensor devices where the specificinteraction of an immobilized protein with an analyte (proteinor small molecule) can be accurately measured and character-

ized. We have already extended this technology to commer-cially available, non-labelled sensing techniques, such assurface plasmon resonance and we intend to report these studiesshortly.

Acknowledgements

This research was undertaken in part on the small-angle X-ray scattering

beamline at the Australian Synchrotron, Vic., Australia. S.J.F. thanks

CSIRO for a PhD studentship.

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Cubosomes as Cell-Free Biosensors 53

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