Biomedical Engineering Reference
In-Depth Information
(a)
(b)
(c)
Odorant
OR
His
Cys
P3CA
Ni-NTA
Source
Drain
CNT
SiO
2
Si
Figure 10.2
(See companion CD for color igure.)
.Examples.of.ORs.coupled.to.carbon.nanotube.ield.effect.transistors.
(CNT-FET)..(a).Direct.adsorption.of.ORs.within.lipid.bilayers.(Kim.et.al..2009)..(b).Covalent.linking.of.OR.
cysteine.residues.to.carboxylated.polypyrrole.CNTs.(Yoon.et.al..2009)..(c).Histidine.(His).tagged.OR.bind-
ing.to.Ni-NTA.linked.CNTs.(Goldsmith.et.al..2011)..CNTs.are.deposited.on.silicon.oxide.(SiO
2
).and.placed.
in. contact. with. source. and. drain. electrodes.. The. OR. mobilized. to. the. CNT. changes. its. current-voltage.
signature.
Ultimately,. to. enable. manufacture. of. GPCR. incorporated. biosensors,. there. will. be. a.
need. to. standardize. protein. expression. and. puriication. techniques. that. will. result. in.
large-scale. production,. keeping. in. mind. reductions. in. cost. and. labor. intensiveness. of.
said.technique..This.may.be.achieved.by.the.use.of.mammalian.cell.culture.bioreactors..
Human.OR17-4.was.effectively.expressed.in.this.manner.using.an.inducible.expression.
system.in.HEK293.cells..These.ORs.were.puriied.using.immunoafinity.and.size.exclu-
sion. chromatography,. yielding. a. solubilized. receptor. that. was. able. to. bind. its. model.
ligand.when.coupled.to.a.surface.plasmon.resonance.(SPR).sensing.platform.using.anti-
body. conjugation. (Cook. et. al.. 2009).. It. remains. to. be. seen. if. this. receptor. retains. the.
ability.to.elicit.downstream.signaling.events.without.the.hydrophobic.nature.of.the.lipid.
bilayer.
A.further.challenge.to.using.GPCRs.on.biosensor.platforms.is.the.inherent.instability.
of. expressed. proteins.. Progress. has. been. made. in. genetically. engineering. GPCRs. to.
enhance.thermostability.(Robertson.et.al..2011);.however,.this.effort.has.not.been.extended.
greatly.into.the.OR.ield..Other.common.modiications.to.recombinant.OR.proteins.are.the.
incorporation.of.terminal.tags,.such.as.histidines.or.myc,.allowing.for.protein.puriication.
steps.or.tethering.to.surfaces.(Leifert.et.al..2005,.Vidic.et.al..2007,.Cook.et.al..2009).(Figures.
10.2.and.10.3)..It.is.important.to.assess.that.this.modiication.does.not.signiicantly.inluence.
protein.structure.or.function.
10.4.2 immobilization of Lipid-Bilayers and Membrane-
Bound Proteins on Sensor Surfaces
The. dificulty. in. using. membrane. proteins. for. biosensor. application. is. the. need. for.
the. hydrophobic. nature. of. the. lipid. bilayer,. which. ensures. correct. structural. protein.
conformation. and. function.. Therefore,. most. cell-free. biosensor. applications. involving.
GPCRs.utilize.a.supported.lipid.bilayer.(Castellana.and.Cremer.2006,.Maynard.et.al..2009).
(Figures.10.2.and.10.3).
Ideally,. the. lipid. bilayer. is. synthesized. from. phospholipids,. applied. to. the. physical.
transducer.and.the.protein.of.choice.inserted.into.this.membrane..This.assembly.ensures.
a. consistent. distribution,. known. protein. concentration. and. lack. of. interference. from.
other.membrane.proteins.found.in.membrane.fractions.of.cellular.expression.systems..
