Biomedical Engineering Reference
In-Depth Information
3.5.2.1.2 Unmodiied AuNPs' Colorimetric Method
As.mentioned.above,.addition.of.NaCl.to.citrate-capped.AuNPs.leads.to.neutralization.of.
their.negative.charge,.which.causes.them.to.aggregate..ssDNA.stabilizes.AuNPs.by.increas-
ing.their.negativity,.thereby.preventing.their.aggregation.upon.addition.of.salt..Adsorption.
of.ssDNA.onto.AuNPs.occurs.as.a.result.of.the.partial.uncoiling.of.ssDNA,.which.exposes.
its.bases,.and.the.resulting.van.der.Waals.attractions.lead.to.adsorption.onto.AuNPs..This.
adsorption.is.further.strengthened.due.to.electrostatic.attractions.between.the.ssDNA.and.
AuNPs,.and.between.them.and.counter-ions.in.solution..In.contrast,.the.phosphate.back-
bone. of. dsDNA. prevents. its. adsorption. onto. AuNPs. due. to. repulsion. between. the. two.
negatively.charged.molecules.
This.principle.can.be.used.in.colorimetric.nucleic.acid.detection,.where.addition.of.ssDNA.
to.a.salt.solution.containing.denatured.complementary.target.DNA.leads.to.hybridization..
When.AuNPs.are.added.to.this.mixture,.no.adsorption.or.stabilization.of.AuNPs.occurs.
due.to.hybridization.of.ssDNA.to.the.target.DNA..The.ssDNA.is.therefore,.no.longer.free.
to.become.adsorbed.onto.the.AuNPs.and.the.salt.in.solution.causes.the.AuNPs.to.aggre-
gate.and.their.color.to.change.from.red.to.blue..On.the.other.hand,.if.the.target.DNA.is.not.
present,.hybridization.does.not.occur.and.the.ssDNA.remains.free.in.solution..The.addi-
tion.of.AuNPs.leads.to.their.stabilization.due.to.adsorption.of.ssDNA,.thereby.preventing.
the. aggregation. of. the. AuNPs,. and. their. color. remains. red.. The. main. advantage. of. this.
method.is.that.no.functionalization.is.required.
A.modiication.of.this.method.can.be.done.by.using.probes.carrying.luorescent.tags..In.
the.absence.of.the.target.DNA,.the.probes.are.adsorbed.onto.the.AuNPs.and.the.luores-
cence.is.quenched,.giving.no.signal..On.the.other.hand,.when.the.target.DNA.is.present,.
no.adsorption.occurs.and.a.luorescent.signal.indicates.the.presence.of.the.target..These.
methods.have.been.used.for.both.DNA.and.RNA.detection.
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Our.research.group
137
.reported.the.irst.assay.for.hepatitis.C.virus.(HCV).using.unmodi-
ied.AuNPs.targeting.HCV.RNA.performed.on.clinical.samples..In.this.study,.HCV.RNA.
was. extracted. from. patient. sera. and. ampliied. using. reverse. transcriptase. (RT). PCR.. A.
hybridization.buffer.was.added.containing.two.primers.each.complementary.to.the.sense.
and.antisense.stands.of.the.dsDNA.amplicon..Citrate-capped.colloidal.AuNPs.were.then.
added. to. the. mixture.. In. HCV. positive. specimens,. the. AuNPs. aggregated. and. the. solu-
tion.color.changed.from.red.to.blue..In.HCV.negative.specimens,.no.change.in.color.was.
observed.due.to.the.stabilization.of.the.AuNPs'.surface.by.the.probes..In.this.method,.the.
use.of.AuNPs.has.allowed.the.detection.of.HCV.after.only.15.PCR.cycles.rather.than.40.
cycles,.thus.reducing.cost.and.time..The.detection.limit.was.3.femtomolar.
In. another. experiment,. we. used. unmodiied. AuNPs. for. the. detection. of. unampliied.
full-length.HCV.RNA.in.serum.samples
138
.(Figure 3.9)..In.this.study,.a.colloidal.solution.of.
AuNPs.with.a.diameter.of.15.nm.was.prepared.by.citrate.reduction.of.hydrogen.tetrachlo-
roaurate.(III)..The.size.and.extinction.spectra.of.the.prepared.AuNPs.were.characterized.
using.ield.emission.scanning.electron.microscopy.and.spectrophotometry,.respectively.
Serum.samples.were.collected.from.healthy.volunteers.(n.=.20).and.chronic.HCV.patients.
(n.=.25;.positive.by.HCV.antibody.and.RT-PCR.tests)..The.assay.was.performed.by.mixing.
the.extracted.RNA.and.hybridization.buffer.containing.phosphate-buffered.saline.(PBS).
and. NaCl,. and. a. primer. targeting. a. sequence. in. the. 5'UTR. of. HCV. speciic. to. all. geno-
types.and.subtypes..The.mixture.was.denatured.at.95°C.for.30.s,.annealed.at.59°C.for.30 s,.
and. then. cooled. to. room. temperature. for. 10. min.. Colloidal. AuNPs. were. then. added. to.
the mixture. and. the. color. was. observed.. Serial. dilutions. of. HCV. positive. samples. with.
known.viral.load.were.used.to.determine.the.detection.limit.of.the.assay..In.HCV.positive.
