Biomedical Engineering Reference
In-Depth Information
relative.dye.orientation.from.changes.in.distance,
92a
.limited.observation.time.of.a.few.tens.
of.seconds.due.to.blinking.and.rapid.photobleaching.of.luorescence,.and.an.upper.dis-
tance. limit. of. ~10. nm.. Silver. and. gold. NPs. have. LSPR. in. the. visible. range. and. do. not.
blink.or.bleach..Alivisatos,.Liphardt,.and.co-workers.have.exploited.them.as.a.new.class.of.
molecular.ruler.to.monitor.the.distance.between.ssDNA.linked.single.pairs.of.Au.and.Ag.
NPs,
89b
.overcoming.the.limitations.of.organic.luorophores.
93
.They.irst.attached.a.strepta-
vidin-functionalized.NP.to.the.bovine.serum.(BSA).BSA-biotin-coated.glass.surface.and.
then.introduced.a.second.NP.modiied.by.a.thiol-ssDNA-biotin.bifunctional.linker.to.be.
attached.to.the.irst.NP.via.biotin-streptavidin.binding.(Figure.5.5a.and.c)..Light.scatter-
ing.was.measured.by.transmission.dark.ield.microscopes.(Figure.5.5a)..For.both.gold.and.
silver.NPs,.they.observed.the.signiicant.color.change.and.spectral.shift.between.a.single.
isolated.NP.and.a.pair.of.adjacent.NPs.(Figure.5.5b-e)..The.LSPR.shift.was.used.to.follow.
the.directed.assembly.of.gold.and.silver.nanoparticle.dimers.in.real.time..The.team.also.
used.the.ruler.to.study.the.kinetics.of.single.DNA.hybridization.events.by.monitoring.the.
LSPR.shift.(Figure.5.5f).due.to.the.resulting.2.nm.distance.increase.between.the.pairs.of.
the.adjacent.NP..These.“plasmon.rulers”.make.it.possible.to.continuously.monitor.sepa-
rations. of. up. to. 70. nm. for. >3,000. s. and. become. an. alternative. to. dye-based. FRET. for. in.
vitro. single-molecule. experiments,. especially. for. applications. demanding. long. observa-
tion.times.without.dye.bleaching.
Chen,.Lee,.and.co-workers.demonstrate.another.molecular.ruler.in.which.double-stranded.
DNA.is.attached.to.a.20.nm.Au.nanoparticle.through.the.thiol-Au.chemistry.
20
.Instead.of.
monitoring.the.LSPR.between.a.pair.of.DNA-linked.metal.NPs,.they.monitored.the.LSPR.of.
individual.Au-DNA.conjugates.cleaved.by.various.endonuclease.enzymes..The.team.found.
that.the.LSPR.λ
max
.increases.with.the.increased.length.of.the.attached.double-strand.DNA.
(dsDNA).(Figure.5.6)..An.average.λ
max
.red.shift.of.approximately.1.24.nm.is.observed.per.
DNA.base.pair.
They.also.used.this.nanoplasmonic.molecular.ruler.to.monitor,.in.real.time,.DNA.being.
digested. by. an.enzyme..Therefore,. this. system. allows. for.a. label-free,. quantitative,. real-
time.measurement.of.nuclease.activity.with.a.time.resolution.of.one.second.due.to.high.
quantum.eficiency.of.Rayleigh.scattering.compared.with.luorescence.or.Raman.scatter-
ing..They.can.also.serve.as.a.new.DNA.footprinting.platform.that.can.accurately.detect.
and.map.the.speciic.binding.of.a.protein.to.DNA,.which.is.essential.to.genetic.information.
processing.
5.5.2 Case 2: Multiplexed LSPR Detection
Plasmonic. nanobiosensors. are. potentially. ideal. biosensors. for. the. high-throughput.
screening. applications. in. the. proteomics. and. drug. discovery.. One. of. the. requirements.
to. introduce. them. to. a. wider. proteomics. and. drug. discovery. community. is. to. develop.
the.substrates.that.are.well.compatible.with.current.high-throughput.platforms..Recently,.
Endo. and. co-workers. developed. a. promising. multiarray. LSPR-based. nanochip. biosen-
sor. suitable. for. screening. biomolecular. interactions.
94
. This. LSPR. nanobiosensing. array.
provides. rapid,. label-free. detection. of. protein. concentration. in. small. sample. volumes..
The.plasmonic.parts.of.the.biochip.are.fabricated.using.nanosphere.lithography.to.form.
three-layer.structures:.the.bottom.layer.is.a.gold.ilm.deposited.onto.a.glass.substrate,.the.
middle.is.the.silica.nanosphere.self-assembled.monolayer.(SAM),.and.the.top.is.the.gold.
shell.coated.over.silica.nanosphere.cores.to.form.a.SiO
2
-Au.core-shell.array..Subsequently,.
the.bifunctional.thiol.linker.(4,4'-dithiodibutyric.acid).SAM.is.formed.over.the.gold.shells..
