Nanopr

obesf
orAnal
yti
calAppl
i
cat
ions:ANewMet
hodol
ogyi
nMedi
cal
Di
agnosi
sandBi
otechnol
ogy

C20 Sev
eralpr
obeshav
ebeenuseddur
ingt
hepastdecadest
o under
standt
he
mechani
smsofbi
ologi
calpr
ocesses.Thepr
obesar
enor
mal
l
ymol
ecul
ari
nnat
ure,whi
ch
ar
eei
theri
ncor
por
atedwi
thaf
luor
escentt
agorl
abel
l
edwi
thar
adi
oact
ivei
sot
ope.I
nor
der
t
ounder
standt
hebi
ologi
calact
ivi
tyofat
argetmol
ecul
e,t
hesamei
slabel
l
edwi
thei
thera
f
luor
escentt
agorr
adi
oact
ivei
sot
opeandi
nject
edi
ntot
hebi
ologi
calsy
stem f
ort
raci
ngt
he
pat
hwayoft
hemol
ecul
eofi
nter
est
.Asar
esul
toft
hemal
i
gnanci
esori
nfect
ionsi
nthe
bi
ologi
calsy
stems,
someoft
hepr
otei
nsar
eov
er-
expr
essed.Theseov
er-
expr
essedpr
otei
ns
ar
ecal
l
ed‘
biomar
ker
s’f
oreachmal
i
gnancyordi
sor
der
.Theant
ibodyoft
hebi
omar
keri
s
usedasamol
ecul
arpr
obewi
thappr
opr
iat
elabel
s.Asdescr
ibedear
li
er,t
hei
nor
gani
c
nanopar
ti
clesexhi
bitsi
zecompat
ibi
l
ityandhav
eexcel
l
entopt
icalf
eat
uresi
nthev
isi
ble
r
egi
on.Thet
rendofusi
ngnanopar
ti
clesf
orpr
obi
ngbi
ologi
calpr
ocessesi
sver
yrecent
.The
nanopar
ti
cles exhi
bit
ing di
ff
erentpr
oper
ti
es such as l
i
ghtscat
ter
ing,f
luor
escence or
magnet
ism ar
eusedf
orpar
ti
cul
arappl
i
cat
ions.

Fer
ri
magnet
ic Au-
Fe3O4 nanocomposi
te par
ti
cles ar
e used as pr
e-concent
rat
ion
pr
obesf
orpept
idesatext
remel
ylow concent
rat
ions(
10–8–10–7M)
,whi
char
efor
medby
t
hedi
gest
ionofcy
tochr
omecbyenzy
mes.Themassspect
romet
ri
canal
ysi
soft
heenzy
me
di
gest
sofpr
otei
nsi
samet
hodol
ogyi
npr
oteomi
csf
ort
heel
uci
dat
ionofami
no aci
d
sequence.Butt
hechancesofobt
aini
ngt
hemasssi
gnat
ureoft
hesepept
idesi
nMALDI
-TOF
MSanal
ysi
sar
esl
i
m duet
othel
argeconcent
rat
ionofi
oni
cimpur
it
iesi
nbr
othobt
ainedaf
ter
enzy
medi
gest
ion.Thedi
gestcont
ainssev
erali
mpur
it
iessuchasphosphat
e,chl
ori
de,
sodi
um dodecy
lsul
phat
eandur
ea.Thesei
mpur
it
iesar
eabl
etosuppr
esst
hemol
ecul
ari
on
peaki
nMALDI
-TOFMSanal
ysi
s.Theel
ect
rost
ati
cadsor
pti
onofeachpept
ideonAu-
Fe3O4
seedi
sdr
ivenbyt
hesur
faceposi
ti
vechar
geoft
hepept
ide.Thi
sposi
ti
vechar
gedensi
ty
dependsont
hepHoft
hemedi
um andt
hei
ntr
insi
cpIv
alueofeachpept
ide,asdi
scussed
ear
li
er.Theseseedsar
eincubat
edov
erni
ghti
nthedi
gestandsepar
atedwi
tht
heai
dof
appl
i
edmagnet
icf
iel
d.Thei
oni
cimpur
it
iesandur
eaar
eremov
edbywashi
ngwi
thwat
er.
TheMALDI
-TOFMSanal
ysi
sshowsmasssi
gnat
uresofeachpept
idecomponent
.Anot
her
r
epor
tdescr
ibes t
he ut
il
it
yofmagnet
ic nanopar
ti
cles f
ort
he det
ect
ion ofpat
hogeni
c
bact
eri
ainbi
ologi
calf
lui
dsbyt
hesel
ect
iveadhesi
onofi
mmunogl
obul
i
nG (
IgG)
-capped
Fe3O4 nanopar
ti
clesont
hecel
lwal
loft
hebact
eri
um.TheMALDI
-TOFMSanal
ysi
soft
he
r
esi
dueobt
ainedbymagnet
icsepar
ati
onshowst
hemasssi
gnat
uresoft
hepat
hogens
secr
etedbyt
hebact
eri
a.Thi
spr
otocolshowst
heef
fi
cientdet
ect
ionf
rom ur
inesampl
esof
pat
hogensatv
eryl
ow concent
rat
ions.Thedet
ect
ionpr
otocolofpat
hogensusi
ngI
gG-
cappedmagnet
it
e(Fe3O4)nanopar
ti
clescanbeusedi
nthecl
i
nicaldet
ect
ionofmany
di
seasesi
nthei
rear
lyst
ages.

Thesur
faceenhancementofRamansi
gnal
sbynobl
emet
alnanopar
ti
cleshasbeenknown
f
orsev
eraly
ear
s.The use ofnobl
e met
alnanopar
ti
cles as sur
face-
enhanced Raman
scat
ter
ing(
SERS)pr
obesi
nbi
ologi
calappl
i
cat
ionswasf
ir
sti
nvest
igat
edbyt
her
esear
ch
gr
oupofMi
rki
n.Thei
rsal
i
entf
eat
uresar
egi
venbel
ow.

1.Met
alnanost
ruct
ures ar
e usef
ulf
orf
abr
icat
ing sensi
ti
ve opt
icalpr
obes,based on
enhancedspect
roscopi
csi
gnal
s.

2.Ther
emar
kabl
eef
fecti
sconnect
edwi
tht
heconf
inedopt
ical
fiel
dsi
sSERS.

3.SERSenhancedf
eat
urescanat
tai
nlar
geri
ntensi
ty,whi
chpr
oducessi
gnal
stoal
evel
compar
abl
etoorev
enbet
tert
hanf
luor
escence.

4.I
ncont
rastt
ofl
uor
escence,whi
chcr
eat
escompar
ati
vel
ybr
oadbands,Ramanscat
ter
ing
cr
eat
esadi
sti
nct
ivespect
rum composedofanumberofnar
row spect
rall
i
nes,ensui
ngi
n
wel
l
-not
iceabl
espect
ra.

5.SERSpr
obesof
ferel
evat
edspect
ral
sel
ect
ivi
ty,
andar
eresi
stantt
owar
dsphot
o-bl
eachi
ng.

6.Met
alnanost
ruct
urescappedwi
thRamanact
ivemol
ecul
escanbeusedasi
ntr
acel
l
ular
SERSpr
obes.7.Si
l
vernanopar
ti
clescappedwi
th4–mer
capt
obenzoi
caci
dwer
eusedas
SERSpr
obesf
orsensi
ngt
hepHoft
hecy
topl
asm ofChi
nesehamst
erov
arycel
l
s.

8.Gol
dnanopar
ti
clecappedwi
thi
ndocy
ani
negr
eeni
susedasani
ntr
acel
l
ularSERSpr
obe
f
orr
atpr
ost
rat
ecar
cinomacel
ll
ine.

9.TheSERS pr
obef
ori
mmunoassayi
sfabr
icat
edbyt
hei
ntegr
ati
onofanobl
emet
al
nanopar
ti
cle,aRamanact
ivedy
eandat
argetsel
ect
ivebi
omol
ecul
e.Thet
argetsel
ect
ive
bi
omol
ecul
eisanchor
edt
othenanopar
ti
clet
hrought
hedy
emol
ecul
ebycov
alentbondi
ng.
The anal
yte mol
ecul
eisadsor
bed on t
he gol
d coat
ed gl
asspl
atef
unct
ional
i
zed wi
th
di
thi
obi
s(succi
nimi
deundecanoat
e)(
DSU)
.Thesucci
nimi
demoi
etyofDSUr
eact
swi
tht
he
anal
ytemol
ecul
efor
minganami
del
i
nkagewi
tht
hemer
capoundecanoy
lgr
oupont
hegol
d
coat
edgl
asspl
ate.Thust
hecov
alent
lyf
ixedanal
ytemol
ecul
eismar
kedbyt
hebi
oconj
ugat
e
f
ormat
ionasshowni
nScheme11.
3(Pl
ate9)
.TheRamandy
esusedf
orSERSexper
iment
s
ar
ebi
funct
ional
,whi
chi
ncl
udedi
sul
phi
desf
orchemi
sor
pti
ont
othenanopar
ti
clesur
faceand
succi
nimi
desf
orf
ormi
ngami
del
i
nkagewi
thmonocl
onalant
ibodi
es.I
nthi
swaypr
obe
f
abr
icat
ion wi
l
lconf
ine t
he dy
e mol
ecul
es i
nthe opt
icalf
iel
d oft
he nobl
e met
al
nanopar
ti
clesandconf
inementoft
heanal
ytef
ort
heenhancementofRamanmodesoccur
s.
Thust
hehy
bri
dizat
ionoft
hemonocl
onalant
ibodyt
ailofSERS pr
obewi
tht
heanal
yte
mol
ecul
eont
heAucoat
edgl
asssubst
rat
ewi
l
lresul
tint
headhesi
onofpr
obeont
he
subst
rat
e.Thesubst
rat
eiswashedandanal
yzedf
ort
heSERSi
ntensi
tyf
rom t
hedy
e.Ther
e
ar
eear
li
err
epor
tssi
mil
art
otheant
igenant
ibodyhy
bri
dizat
ionpr
otocol
;aDNAst
randwi
tha
knownsequencecanbeusedi
nst
eadofamonocl
onalant
ibodyi
ntheSERSpr
obe,i
nor
der
t
odet
ectt
argetDNA(
Ref
.29)
.Thedet
ect
ionofext
remel
ysmal
lquant
it
iesofbi
omar
keri
s
sel
dom achi
evedbyusi
ngt
henor
malmet
hodol
ogi
esofi
mmunoassay
s.Pr
ost
ate-
Speci
fi
c
Ant
igen(
PSA)
,a33-
kDagl
ycopr
otei
n,abi
omar
kerf
orpr
ost
atecancer
,ispr
esenti
nbl
ood
pl
asma,i
nconcent
rat
ionsr
angi
ngbet
ween4and10ng/
mLf
oraheal
thyadul
tmal
e.Asa
r
esul
tofani
ncr
easei
ntheoccur
renceofpr
ost
rat
ecancer
,thechancesoft
hecompl
ex
f
ormat
ionofPSA andα-
ant
ichy
mot
rypsi
nar
eel
evat
ed.Thi
sphenomenondecr
easest
he
amountoff
reePSAi
ncancerpat
ient
s.Thust
heconcent
rat
ionoff
reePSAi
simpor
tantf
or
t
hedet
ermi
nat
ionoft
heext
entofpr
ost
rat
ecancer
.Apr
otocolhasbeendev
elopedr
ecent
ly
f
orusi
ngSERS-
basedi
mmunoassayusi
ngAunanopar
ti
clepr
obef
orf
emt
omol
ardet
ect
ion
ofPSA (
Ref
.31)
.5,5´
-di
thi
obi
s(succi
nimi
dyl
-2-
nit
robenzoat
e)(
DSNB)wasusedast
he
bi
funct
ionalRamandy
etoconnectAu(
32nm)nanopar
ti
cleandt
hemonocl
onalant
ibodyof
PSA.The i
mmobi
l
izat
ion ofPSA was done on an Au-
coat
ed gl
ass sur
face whi
ch i
s
f
unct
ional
i
zedwi
thdi
thi
obi
s(succi
nimi
deundecanoat
e)(
DSU)
.ThePSAsol
uti
oni
nhuman
ser
um wast
akeni
naconcent
rat
ionbet
ween1µg/
mL(
30nM)t
o1pg/
mL(
30f
M)andwas
usedf
orf
ixi
ngont
heDSUf
uct
ional
i
zedAucoat
edgl
asspl
ate.Af
tert
hef
ixat
ionofPSAat
di
ff
erentconcent
rat
ionsont
hesubst
rat
e,i
twasal
l
owedt
ohy
bri
dizewi
thSERSpr
obesas
showni
nScheme11.
3.TheSERSpr
obeconsi
stsofasel
f-
assembl
edmonol
ayerof5-
mer
apt
o2-
nit
robenzami
deat
tachedt
oamonocol
onal
ant
ibody
.Theant
ibodywasanchor
ed
t
othe5-
mer
acapt
o2-
nit
robenzoy
lgr
oupwi
thapept
idel
i
nkage,whi
chwasf
ormedbyt
he
condensat
ionoft
heami
nogr
oupoft
heant
ibodyandt
hesucci
nimi
dyl2-
nit
robenzoat
e
gr
oup.TheRamanscat
ter
ingi
ntensi
tyoft
hesy
mmet
ri
cst
ret
chi
ngoft
heni
tr
ogr
oupoft
he5
-
mer
acapt
o2-
nit
robenzoy
lmoi
etyat1338cm–1wast
akenast
hemakersi
gnalf
ort
he
i
mmunoassay
.InFi
g.11.
10(
a),t
hespect
rawer
eacqui
redusi
ng60-
sint
egr
ati
onofSERS-
based i
mmunoassay
.As ment
ioned abov
e,t
he Raman f
eat
ures oft
he DSNB-
label
ed
nanopar
ti
clesat1338cm–1demonst
rat
eagr
adualenhancementasaf
unct
ionoft
he
concent
rat
ionofPSA.Theconcent
rat
ionl
evelshowni
nFi
g.11.
10i
scr
it
icalf
orpr
ost
ate
cancerdi
agnosi
s(Ref
.32)
.Thet
racel
abel
edbl
ankr
epr
esent
stheRamanspect
rum oft
he
sampl
eint
heabsenceofPSAont
hesubst
rat
e.Addi
ti
onali
nfor
mat
ioncanbeobt
ainedf
rom
t
hedose-
responsecur
veshowni
nFi
g.11.
10(
b).Thi
scur
vewascr
eat
edbypl
ott
ingt
he
i
ntensi
tyoft
hesy
mmet
ri
cst
ret
chi
ngoft
heni
tr
ogr
oupat1338cm–1v
ersusl
ogar
it
hm of
t
heconcent
rat
ionofPSAi
npg/
mLusedf
oranal
ysi
s.Thi
srepor
tdemonst
rat
est
hepot
ent
ial
oft
hegol
dnanopar
ti
cle-
basedSERSpr
obef
ort
heear
lydet
ect
ionofpr
ost
rat
ecanceri
na
quant
it
ati
vef
ashi
on.Thi
sal
sounv
eil
stheut
il
it
yofnanopar
ti
cle-
basedSERSpr
obesi
na
v
ari
etyofappl
i
cat
ionsi
nthef
iel
dsofmedi
cal
diagnosi
sandbi
otechnol
ogy