Biomedical Engineering Reference
In-Depth Information
specimens,.the.color.of.the.solution.changed.from.red.to.blue.within.1.min..Also,.a.red.
shift.and.a.broadening.of.the.absorbance.peak.of.AuNPs.were.observed..The.assay.has.a.
sensitivity.of.92%.and.a.speciicity.of.90%..The.assay.detection.limit.was.50.copies/reac-
tion..The.developed.assay.is.highly.sensitive,.has.a.turnaround.time.of.30.min,.and.elimi-
nates.the.need.for.thermal.cycling.and.detection.instruments..This.assay.could.be.further.
developed.for.HCV.viral.load.monitoring.and.genotyping.
3.5.2.1.3 Förster Resonance Energy Transfer (FRET)
FRET.is.the.nonradiative.transfer.of.excitation.energy.emitted.from.a.donor.and.absorbed.
by.an.acceptor.via.dipole-dipole.interactions..This.can.only.occur.if.the.distance.between.
the.donor.and.acceptor.is.less.than.the.Förster.distance,.roughly.less.than.10.nm,.where.the.
energy.transfer.eficiency.between.the.donor.and.acceptor.is.at.50%..Moreover,.the.donor's.
emission.spectrum.must.correspond.with.the.acceptor's.absorption.spectrum.
Due.to.their.large.molar.extinction.coeficient,.AuNPs.serve.as.excellent.acceptors..When.
dealing.with.metal.nanoparticles,.the.FRET.phenomenon.is.referred.to.as.nanometal.sur-
face.energy.transfer.(NSET).
2
.Metal.nanoparticles.have.higher.energy.transfer.eficiency.
due.to.the.presence.of.free.conduction.bands.of.electrons,.leading.to.more.dipole-dipole.
interactions,. which. can. occur. at. longer. distances. compared. to. FRET.. AuNPs. can. also.
accept.a.wide.range.of.emission.frequencies,.from.visible.to.NIR,.which.makes.them.excel-
lent.quenchers.
Compared.to.conventional.luorescent.dyes,.AuNPs.have.stood.their.ground,.as.they.are.
not.susceptible.to.photobleaching.and.have.superior.absorption.and.scattering.properties..
Not.only.are.they.more.eficient.quenchers.than.luorescent.dyes,.but.their.unique.optical.
properties.allow.for.the.detection.of.zeptomolar.nucleic.acid.concentrations.with.a.sensi-
tivity.that.is.ive.times.higher.than.that.obtained.using.luorescent.dyes.
139
Dubertret.et.al.
140
.targeted.DNA.using.ssDNA.in.a.hairpin.conformation.with.an.AuNP.
attached.at.one.end.and.a.luorescent.dye.attached.at.the.other.(Figure 3.10)..Because.the.
AuNP.and.dye.are.in.close.proximity.in.this.conformation,.the.AuNP.is.able.to.quench.the.
dye's.luorescent.emission..When.the.target.complementary.DNA.is.present,.the.hairpin.
structure. opens. and. hybridization. occurs.. In. this. open. form,. the. distance. between. the.
AuNP.and.dye.exceeds.the.NSET.distance,.and.the.unquenched.luorescence.of.the.dye.
can.be.detected..Compared.to.the.use.of.organic.quenchers,.AuNPs.increase.the.sensitivity.
of.the.test.100-fold,.and.increase.the.ability.to.detect.single-base.mismatches.8-fold.
Grifin. et. al.
141
. used. luorophore-labeled. ssRNA. and. AuNPs. to. detect. synthetic. HCV.
RNA.targets..In.this.assay,.ssRNA.was.attached.at.one.end.to.the.AuNP.and.at.the.other.
end. to. a. luorophore. which. adsorbed. onto. the. same. AuNP,. forming. a. loop.. When. the.
complementary.target.RNA.hybridizes.with.the.RNA.probe,.they.form.a.dsRNA.that.is.
released.into.solution..In.this.case,.two.observations.can.be.made:.the.change.of.solution.
color.from.red.to.blue.due.to.the.aggregation.of.AuNPs,.and.the.unquenching.luorescent.
signal.of.the.dye,.the.strength.of.which.is.directly.proportional.to.the.concentration.of.the.
target..The.size.of.the.AuNPs.was.also.found.to.be.of.signiicance,.with.an.increase.in.sen-
sitivity.as.well.as.quenching.eficiency.up.to.1,000-fold,.as.the.AuNP.size.increased.from.5.
to.70.nm..At.110.nm.AuNP.size,.the.limit.of.detection.(LOD).reached.300.fM.of.RNA.
3.5.2.2 Immunoassays
AuNPs.can.be.coated.with.proteins/antibodies.and.aggregate.in.the.presence.of.the.tar-
get.antigen..Thanh.and.Rosenzweig
126
.used.10.nm.AuNPs.coated.with.protein.A.antigen.
to.detect.anti-protein.A.antibodies.in.aqueous.solutions.and.serum..The.presence.of.the.
