Biomedical Engineering Reference
In-Depth Information
the.solutions.onto.a.white.C18.reverse-phase.thin-layer.chromatography.(TLC).plate,.we.
can.better.observe.and.record.the.change.in.color..This.is.a.relatively.fast,.easy,.and.low-
cost.method.compared.to.others,.such.as.those.based.on.the.use.of.luorophores.
2
Multiplexing.can.be.achieved.by.changing.the.composition.of.the.AuNPs,.such.as.using.
gold.nanoshells.containing.a.silver,. copper,. or.platinum.core.
130
.The.silver/gold. (Ag/Au).
nanoshells.shift.in.color.from.yellow.to.brown.upon.aggregation.following.target.hybrid-
ization..This.has.been.used.by.Cao.et.al.,
131
.where.spherical.AuNPs.were.functionalized.
with. speciic. oilgonucleotides. to. recognize. the. wild. type. sequence,. while. Ag/Au. nano-
shells.were.functionalized.to.recognize.a.mutant.with.a.single.nucleotide.polymorphism.
(SNP).. The. wild. type. target. was. added. to. the. two. solutions,. each. containing. one. probe.
type,.and.the.solution.temperature.was.raised.from.20.to.70°C..At.different.temperatures,.
aliquots. of. the. solutions. were. then. spotted. on. a. C18. reverse-phase. TLC. plate.. The. gold.
solution.in.the.AuNP.probe.system.exhibited.a.higher.melting.temperature.(T
m
),.detected.
by.a.color.change.from.blue.to.red,.than.the.Ag/Au.core-shell.probe.system.designed.to.
detect.the.mutant.target..In.a.second.experiment,.when.the.mutant.target.was.added.to.
the.same.two.solutions,.the.opposite.results.were.obtained,.where.the.Ag/Au.core-shell.
probe.system.showed.a.higher.T
m
.than.the.AuNP.probe.system..In.this.method,.the.Ag/Au.
core-shell.system.served.as.a.control.to.ensure.accurate.results;.however,.a.similar.strategy.
could.also.be.used.for.the.simultaneous.detection.of.two.different.DNA.targets.”
3.5.2.1.1.2 Non-Cross-Linking (Single-Probe) Method
Citrate-capped.AuNPs.possess.a.neg-
ative.charge.that.can.be.neutralized.by.the.addition.of.a.salt,.which.allows.them.to.aggre-
gate.and.change.from.red.to.blue..Hybridization.of.oligonucleotide-functionalized.AuNPs.
increases. their. negativity,. thereby. requiring. higher. salt. concentrations. for. aggregation.
to. occur. (Figure 3.8).. The. single-probe. method. is. easier. and. faster. than. the. two-probe.
method.
132
Chakrabarti. and. Klibanov
133
. used. AuNPs. functionalized. with. short,. six-base. peptide.
nucleic.acids.(PNAs),.which.have.a.single-base.mismatch.selectivity.ive.times.greater.than.
DNA-functionalized.AuNPs..When.the.PNA-AuNPs.hybridize.with.the.target.DNA,.their.
resistance.to.aggregation.in.the.presence.of.high.salt.concentrations.increases..In.the.event.
that.the.DNA.target.contains.a.mutation,.the.PNA-AuNPs.do.not.hybridize.perfectly.to.the.
target.and.aggregate.at.low.salt.concentrations,.changing.in.color.to.blue..Sato.et.al.
134
.used.
this.method.with.AuNPs.functionalized.with.one.type.of.oligonucleotide..In.the.presence.
of.NaCl.at.a.concentration.higher.than.0.5.M,.addition.of.target.DNA.led.to.the.aggregation.
of.the.AuNPs.and.their.color.changed.to.purple.
Multiplex.detection.was.done.by.Kim.and.Lee
135
.by.using.gold.nanoclusters.instead.of.
single.AuNPs..The.nanoclusters.were.synthesized.in.the.presence.of.dithiothreitol.(DTT).
and. monothiol. DNA,. where. each. AuNP. was. 15. nm. in. diameter. and. the. 19-base. DNA.
sequence.used.was.6.nm.long..As.the.amount.of.DTT.increased,.the.size.of.the.gold.nano-
clusters.increased..Four.solutions.were.selected:.red,.purple,.violet,.and.blue,.which.con-
tained.35,.42,.49,.and.115.nm.DNA-AuNP.clusters,.respectively..Upon.DNA.hybridization.
with.the.target,.red.turned.to.purple,.purple.solution.turned.to.pale.violet,.violet.solution.
turned.to.pale.blue,.and.the.blue.solution.turned.to.pale.indigo..A.spot.test.would.allow.
for.better.differentiation.between.color.changes..Moreover,.an.even.wider.variety.of.col-
ors.can.be.obtained.by.synthesizing.gold.nanoclusters.with.different.core.compositions..
The.cluster.sizes.could.be.controlled.by.the.DTT.and.monothiol.DNA.ratios..Quantitation.
was.done.by.conducting.melting.experiments,.and.the.limit.of.detection.of.the.test.was.
1. nM,. which. is. ive. times. lower. than. that. using. single. DNA-AuNPs. having. the. same.
sequence.
