Biomedical Engineering Reference
In-Depth Information
(a)
(b)
α
β
γ
α
OR
Odorant
Tag
Ab
SAM
Au
Glass
Prism
Light
Detector
Figure 10.3
(See companion CD for color igure.)
. Examples. of. ORs. functionalized. to. surface. plasmon. resonance. (SPR).
sensors..(a).Puriied.OR.protein.is.linked.via.antibody.(Ab).binding.to.a.terminal.tag.(Cook.et.al..2009;.Kaiser.
et.al..2008)..(b).Nanosomes.containing.the.OR.and.heterotrimeric.G-protein.complex.are.functionalized.using.
a. self-assembling. monolayer. (SAM).. Binding. of. odorant. leads. to. dissociation. of. the. Gα. protein. (Vidic. et. al.,.
2006,.2007)..The.plasmon.wave.of.the.gold.(Au).ilm.is.detected.using.total.internal.relection.mode,.where.the.
relected.light.intensity.at.a.given.angle.is.reduced.due.to.the.plasmon.wave..Binding.of.ORs.shifts.the.angle.of.
the.reduced.light.intensity.region.
Some. proteins. are. self-assembling,. such. as. the. pore. gramicidin,. and. spontaneously.
insert.when.added.to.a.lipid.bilayer.(Zagnoni.2012)..ORs.are.dificult.to.reconstitute.into.
artiicial.lipid.bilayers,.and.most.studies.have,.therefore,.relied.on.immobilizing.crude.
or. puriied. membrane. fractions. from. the. heterologous. expression. system. containing.
the. recombinantly. overexpressed. protein. (Table. 10.1).. This. also. largely. prevents. the.
issue. of. integrating. the. receptor. proteins. into. artiicial. lipid. bilayers. in. the. incorrect.
orientation.
Lipid.bilayers.can.be.immobilized.onto.solid.surfaces.in.a.number.of.ways..The.most.
commonly.used.techniques.are.vesicle.fusion.and.the.Langmuir-Blodgett.method..Both.
methods.take.advantage.of.the.self-assembling.nature.of.surfactants..Small.vesicles.in.
solution. rupture. once. adsorbed. onto. a. surface. and. fuse. to. form. a. lat. lipid. bilayer..
The. Langmuir-Blodgett. method. is. primarily. used. for. artiicial. lipid. bilayers. and.
exploits.the.fact.that.phospholipid.molecules.orient.themselves.at.a.water-air.interface..
A.number.of.different.subsequent.steps.can.then.be.used.to.assemble.the.two.layers.of.
the.bilayer..For.details.on.these.techniques.we.refer.the.reader.to.a.number.of.review.
articles.and.studies.referenced.within.(Castellana.and.Cremer.2006,.Hirano-Iwata.et.al..
2008,.Zagnoni.2012).
The.lipid.bilayer.may.be.directly.adsorbed.onto.the.physical.sensor.surface,.tethered.to.it,.
usually.by.self-assembling.monolayers.(SAM),.or.deposited.onto.a.“cushion”.polymer.sup-
port.(examples.are.shown.in.Figures.10.2.and.10.3).(Castellana.and.Cremer.2006,.Saito.et.al..
2009,.Zagnoni.2012)..Tethering.(e.g.,.by.thiolipids.and.silanelipids).and.polymer.supports.
(e.g.,. agarose). are. often. preferred. for. transmembrane. proteins. (Castellana. and. Cremer.
2006)..Eliminating.direct.contact.of.the.underside.of.the.bilayer.with.the.solid.surface.can.
help.in.maintaining.structural.integrity.of.the.membrane.protein..For.example,.compared.
to. direct. adsorption. of. cell. membranes. onto. solid. sensor. surfaces,. Wu. et. al.. found. that.
afinity. of. ODR-10. toward. its. natural. ligand. diacetyl. increased. 10-fold. when. membrane.
fractions.containing.the.receptor.were.immobilized.onto.a.surface.acoustic.wave.sensor.
